acta periodica technologica - Tehnoloski fakultet Novi Sad

Transcription

UNIVERSITY OF NOVI SAD
FACULTY OF TECHNOLOGY NOVI SAD
УНИВЕРЗИТЕТ У НОВОМ САДУ
ТЕХНОЛОШКИ ФАКУЛТЕТ НОВИ САД
ACTA PERIODICA
TECHNOLOGICA
APTEFF, 43, 1-342 (2012)
ACTA PERIODICA TECHNOLOGICA (formerly Zbornik radova Tehnološkog
fakulteta and Proceedings of Faculty of Technology) publishes articles from all
branches of technology (food, chemical, biochemical, pharmaceutical), process engineering and related scientific fields.
Articles in Acta Periodica Technologica are abstracted by: Chemical Abstracts,
Columbus, Ohio, Referativnyi zhurnal -Khimija, VINITI, Moscow, listed in Ulrich’s International Periodical Directory, and indexed in the Elsevier Bibliographic
databases – SCOPUS.
YU ISSN 1450 – 7188
UDC 54:66:664:615
CODEN: APTEFF
Publisher
University of Novi Sad, Faculty of TechnologyNovi Sad
Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
For Publisher
Prof. Dr. Zoltan Zavargo, Dean
Editor-in-Chief
Prof. Dr. Sonja Đilas
Editorial Board
From Abroad
Prof. Dr. Živko Nikolov
Texas A and M University, Biological and Agricultural Engineering
Department, College Station, TX, USA
Prof. Dr. Erika Békássy-Molnár
University of Horticulture and Food Industry, Budapest, Hungary
Prof. Dr. Željko Knez
University of Maribor,
Faculty of Chemistry and Chemical Technology, Maribor, Slovenia
Dr. T.S.R. Prasada Rao
Indian Institute of Petroleum, Dehra Dun, India
Prof. Dr. Đerđ Karlović
Margarine Center of Expertise, Kruszwica, Poland
Dr. Szigmond András
Research Institute of Hungarian Sugar Industry, Budapest, Hungary
Dr. Andreas Reitzmann
Institute of Chemical Process Engineering, University Karlshruhe, Germany
From Serbia
Prof. Dr. Vlada Veljković
Prof. Dr. Spasenija Milanović
Prof. Dr. Vladimir Srdić
Prof. Dr. Slobodan D. Petrović
Prof. Dr. Jonjaua Ranogajec
Dr. Anamarija Mandić
ACTA PERIODICA TECHNOLOGICA
APTEFF, 43, 1-342 (2012)
CONTENT
FOOD TECHNOLOGY
Etelka B. Dimić, Vesna B. Vujasinović, Olga F. Radočaj and Oršolja P. Pastor
CHARACTERISTICS OF BLACKBERRY AND
RASPBERRY SEEDS AND OILS ............................................................................... 1
Mirela D. Iličić, Spasenija D. Milanović, Marijana Đ. Carić, Katarina G. Kanurić,
Vladimir R. Vukić, Dajana V. Hrnjez and Marjan I. Ranogajec
VOLATILE COMPOUNDS OF FUNCTIONAL DAIRY PRODUCTS ................... 11
Marija R. Jokanović, Natalija R. Džinić, Biljana R. Cvetković, Slavica Grujić
and Božana Odžaković
CHANGES OF PHYSICAL PROPERTIES OF COFFEE BEANS
DURING ROASTING ................................................................................................ 21
Zorica D. Knežević-Jugović, Andrea B. Stefanović, Milena G. Žuža, Stoja L.
Milovanović, Sonja M. Jakovetić, Verica B. Manojlović and Branko M. Bugarski
EFFECTS OF SONICATION AND HIGH-PRESSURE CARBON
DIOXIDE PROCESSING ON ENZYMATIC HYDROLYSIS OF
EGG WHITE PROTEINS .......................................................................................... 33
Snežana Ž. Kravić, Zvonimir J. Suturović, Ana D. Đurović, Tanja Ž. Brezo,
Spasenija D. Milanović, Radomir V. Malbaša and Vladimir R. Vukić
DIRECT DETERMINATION OF CALCIUM, SODIUM AND POTASSIUM
IN FERMENTED MILK PRODUCTS ...................................................................... 43
Radomir V. Malbašа, Jasmina S. Vitas, Eva S. Lončar and Spasenija D. Milanović
PHYSICAL AND TEXTURAL CHARACTERISTICS
OF FERMENTED MILK PRODUCTS OBTAINED BY
KOMBUCHA INOCULUMS WITH HERBAL TEAS ............................................. 51
Marija S. Milašinović-Šeremešić, Milica M. Radosavljević and Ljubica P. Dokić
STARCH PROPERTIES OF VARIOUS ZP MAIZE GENOTYPES ........................ 61
Dragan V. Palić, Kedibone Y. Modika, Andre Oelofse and Marijana B. Sakač
MODIFICATION OF THE METHOD FOR DETERMINING
PROTEIN SOLUBILITY OF HEAT TREATED FULL-FAT
SOYBEANS USING EXTRACTION IN POTASSIUM HYDROXIDE:
INTER-LABORATORY STUDY .............................................................................. 69
Slavica A. Sredanović, Jovanka D. Lević, Rade D. Jovanović and
Olivera M. Đuragić
THE NUTRITIVE VALUE OF POULTRY DIETS CONTAINING
SUNFLOWER MEAL SUPPLEMENTED BY ENZYMES...................................... 79
Slađana M. Stajčić, Aleksandra N. Tepić, Sonja M. Djilas, Zdravko M. Šumić,
Jasna M. Čanadanović-Brunet, Gordana S. Ćetković, Jelena J. Vulić and Vesna T.
Tumbas
CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF
BERRY FRUITS ........................................................................................................ 93
Slavica M. Vesković Moračanin, Slavko Mirecki, Dejana K. Trbović,
Lazar R. Turubatović, Vladimir S. Kurćubić and Pavle Z. Mašković
TRADITIONAL MANUFACTURING OF WHITE CHEESES IN BRINE
IN SERBIA AND MONTENEGRO - SIMILARITIES AND DIFFERENCES ...... 107
Marija M. Škrinjar, Žarko M. Petrović, Nevena T. Blagojev and
Vladislava M. Šošo
SUNFLOWER SEED FOR HUMAN CONSUMPTION AS A
SUBSTRATE FOR THE GROWTH OF MYCOPOPULATIONS.......................... 115
Vladislava M. Šošo, Marija M. Škrinjar and Nevena T. Blagojev
INFLUENCE OF ECOPHYSIOLOGICAL FACTORS ON THE PRESENCE
OF OCHRATOXIN A IN DRIED VINE FRUITS: A REVIEW ............................. 123
Danica B. Zarić, Biljana S. Pajin, Ivana S. Lončarević,
Dragana M. Šoronja Simović and Zita I. Šereš
THE IMPACT OF THE MANUFACTURING PROCESS ON THE
HARDNESS AND SENSORY PROPERTIES OF MILK CHOCOLATE .............. 139
CHEMICAL TECHNOLOGY AND PROCESS ENGINEERING
Mirjana G. Antov, Branimir Z. Jugović, Milica M. Gvozdenović
and Zorica D. Knežević Jugović
PARTITIONING OF CELLULOLYTIC ACTIVITY IN THE
POLYETHYLENE GLYCOL/DEXTRAN TWO-PHASE SYSTEMS ................... 151
Nevena M. Krkić, Vera L. Lazić and Danijela Z. Šuput
COMPARISON OF LIFE CYCLE ASSESSMENT FOR
DIFFERENT VOLUME POLYPROPYLENE JARS .............................................. 159
Jelena Đ. Marković, Nataša Lj. Lukić, Jelena D. Ilić,
Branislava G. Nikolovski, Milan N. Sovilj and Ivana M. Šijački
USING THE ANSYS FLUENT FOR SIMULATION OF TWO-SIDED
LID-DRIVEN FLOW IN A STAGGERED CAVITY ............................................. 169
M. P. Nagarkar, R.N. Zaware and S.G. Ghalmea
FINITE ELEMENT SIMULATION OF SINK PASS
ROUND TUBES USING ANSYS ........................................................................... 179
Jelena M. Petrović, Darko M. Ljubić, Marina R. Stamenović, Ivana D. Dimić
and Slaviša S. Putić
TENSION MECHANICAL PROPERTIES OF RECYCLED
GLASS-EPOXY COMPOSITE MATERIAL .......................................................... 189
Milan N. Sovilj
HYDRODYNAMICS OF GAS-AGITATED LIQUID-LIQUID
EXTRACTION COLUMNS .................................................................................... 199
Vesna M. Vasić, Marina B. Šćiban, Aleksandar I. Jokić,
Jelena M. Prodanović and Dragana V. Kukić
MICROFILTRATION OF DISTILLERY STILLAGE:
INFLUENCE OF MEMBRANE PORE SIZE .......................................................... 217
Zita I. Šereš, Ljubica P. Dokić, Biljana S. Pajin, Dragana M. Šoronja Simović,
Drago Šubarić, Jurislav Babić and Aleksandar Z. Fišteš
INFLUENCE OF THE OPERATING PARAMETERS ON
THE FLUX DURING MICROFILTRATION OF THE STEEPWATER
IN THE STARCH INDUSTRY................................................................................ 225
BIOCHEMICAL AND PHARMACEUTICAL ENGINEERING
Gordana R. Dimić, Sunčica D. Kocić-Tanackov, Olivera O. Jovanov,
Dragoljub D. Cvetković, Siniša L. Markov and Aleksandra S. Velićanski
ANTIBACTERIAL ACTIVITY OF LEMON, CARAWAY
AND BASIL EXTRACTS ON LISTERIA SPP. ....................................................... 239
Sunčica D. Kocić-Tanackov, Gordana R. Dimić, Dušanka J. Pejin,
Ljiljana V. Mojović, Jelena D. Pejin and Ilija J. Tanackov
ANTIFUNGAL ACTIVITY OF THE BASIL (Ocimmum basilicum L.)
EXTRACT ON Penicillium aurantiogriseum, P. glabrum, P. chrysogenum,
AND P. brevicompactum .......................................................................................... 247
Zoran Z. Kukrić, Ljiljana N. Topalić-Trivunović, Biljana M. Kukavica,
Snježana B. Matoš, Svetlana S. Pavičić, Mirela M. Boroja and Aleksandar V. Savić
CHARACTERIZATION OF ANTIOXIDANT AND ANTIMICROBIAL
ACTIVITIES OF NETTLE LEAVES (Urtica dioica L.) ......................................... 257
Sanja O. Podunavac-Kuzmanović, Lidija J. Jevrić, Strahinja Z. Kovačević and
Nataša D. Kalajdžija
CHEMOMETRIC APPROACH FOR PREDICTION OF
ANTIFUNGAL ACTIVITY OF SOME BENZOXAZOLE
DERIVATIVES AGAINST Candida albicans ........................................................ 273
Pouneh Ebrahimi, Akram Mirarab-Razi and Abbas Biabani
COMPARATIVE EVALUATION OF THE ESSENTIAL OIL
TERPENOIDS IN THE STEM AND LEAF OF Ziziphora Clinopodioides
IN THE REGIONS OF ALMEH AND SOJOUGH OF GOLESTAN
PROVINCE, IRAN ................................................................................................... 283
Vesna T. Tumbas, Jelena J. Vulić, Jasna M. Čanadanović-Brunet,
Sonja M. Djilas, Gordana S. Ćetković, Slađana S. Stajčić, Dubravka I. Štajner
and Boris M. Popović
ANTIOXIDANT AND SENSORIAL PROPERTIES OF
ACACIA HONEY SUPPLEMENTED WITH PRUNES .......................................... 293
Aleksandra S. Velićanski, Dragoljub D. Cvetković and Siniša L. Markov
SCREENING OF ANTIBACTERIAL ACTIVITY OF RASPBERRY
(Rubus idaeus L.) FRUIT AND POMACE EXTRACTS ......................................... 305
Senka S. Vidović, Zoran P. Zeković, Žika D. Lepojević, Marija M. Radojković,
Stela D. Jokić and Goran T. Anačkov
OPTIMIZATION OF THE Ocimum basilicum L. EXTRACTION
PROCESS REGARDING THE ANTIOXIDANT ACTIVITY................................ 315
Vesna M. Vučurović and Radojka N. Razmovski
ETHANOL FERMENTATION OF MOLASSES BY Saccharomyces
cerevisiae CELLS IMMOBILIZED ONTO SUGAR BEET PULP ......................... 325
Marina B. Šćiban, Jelena M. Prodanović and Radojka N. Razmovski
BIOSORPTION OF COPPER(II) AND CHROMIUM(VI)
BY MODIFIED TEA FUNGUS ............................................................................... 335
INSTRUCTION FOR MANUSCRIPT PREPARATION
ACTA PERIODICA TECHNOLOGICA
APTEFF, 43, 1-342 (2012)
САДРЖАЈ
ПРЕХРАМБЕНА ТЕХНОЛОГИЈА
Етелка Б. Димић, Весна Б. Вујасиновић, Олга Ф. Радочај
и Оршоља П. Пастор
КАРАКТЕРИСТИКE СЕМЕНА И УЉА КУПИНЕ И МАЛИНЕ ........................... 1
Мирела Д. Иличић, Спасенија Д. Милановић, Маријана Д. Царић, Катарина Г.
Канурић, Владимир Р. Вукић, Дајана В. Хрњез и Марјан И. Раногајец
ИСПАРЉИВЕ КОМПОНЕНТЕ АРОМЕ ФУНКЦИОНАЛНОГ
МЛЕЧНОГ ПРОИЗВОДА ........................................................................................ 11
Марија Р. Јокановић, Наталија Р. Џинић, Биљана Р. Цветковић,
Славица Грујић и Божана Оџаковић
ПРОМЕНЕ ФИЗИЧКИХ СВОЈСТАВА ЗРНА КАФЕ ТОКОМ ПЕЧЕЊА .......... 21
Зорица Д. Кнежевић-Југовић, Андреа Б. Стефановић, Милена Г. Жужа,
Стоја Л. Миловановић, Соња М. Јаковетић, Верица Б. Манојловић и
Бранко М. Бугарски
УТИЦАЈ СОНИКАЦИЈЕ И ПРЕТРЕТМА ВИСОКИМ ПРИТИСКОМ
НА ЕНЗИМСКУ ХИДРОЛИЗУ ПРОТЕИНА БЕЛАНЦЕТА ................................ 33
Снежана Ж. Кравић, Звонимир Ј. Сутуровић, Ана Д. Ђуровић,
Тања Ж. Брезо, Спасенија Д. Милановић, Радомир В. Малбаша
и Владимир Р. Вукић
ДИРЕКТНО ОДРЕЂИВАЊЕ КАЛЦИЈУМА, НАТРИЈУМА И
КАЛИЈУМА У ФЕРМЕНТИСАНИМ МЛЕЧНИМ ПРОИЗВОДИМА ................ 43
Радомир В. Малбаша, Јасмина С. Витас, Ева С. Лончар и
Спасенија Д. Милановић
ФИЗИЧКЕ И ТЕКСТУРАЛНЕ КАРАКТЕРИСТИКЕ
ФЕРМЕНТИСАНИХ МЛЕЧНИХ ПРОИЗВОДА ДОБИЈЕНИХ
ПОМОЋУ КОМБУХЕ ГАЈЕНЕ НА БИЉНИМ ЧАЈЕВИМА............................... 51
Марија С. Милашиновић-Шеремешић, Милица М. Радосављевић
и Љубица П. Докић
ОСОБИНЕ СКРОБА РАЗЛИЧИТИХ ЗП ГЕНОТИПОВА КУКУРУЗА .............. 61
Драган В. Палић, Кедибоне Модика, Андре Оелофсе и Маријана Б. Сакач
МОДИФИКАЦИЈА МЕТОДЕ ЗА ОДРЕЂИВАЊЕ РАСТВОРЉИВОСТИ
ПРОТЕИНА У ТЕРМИЧКИ ТРЕТИРАНОЈ ПУНОМАСНОЈ СОЈИ
КОРИШЋЕЊЕМ ЕКСТРАКЦИЈЕ У КАЛИЈУМ ХИДРОКСИДУ:
МЕЂУ-ЛАБОРАТОРИЈСКО ИСПИТИВАЊЕ ...................................................... 69
Славица А. Средановић, Јованка Д. Левић, Раде Д. Јовановић
и Оливера М. Ђурагић
ХРАНЉИВА ВРЕДНОСТ ОБРОКА ЗА ЖИВИНУ КОЈИ САДРЖЕ
СУНЦОКРЕТОВУ САЧМУ ДОПУЊЕНУ ЕНЗИМИМА ..................................... 79
Слађана М. Стајчић, Александра Н. Тепић, Соња М. Ђилас,
Здравко М. Шумић, Јасна М. Чанадановић-Брунет, Гордана С.
Ћетковић, Јелена Ј. Вулић и Весна Т. Тумбас
ХЕМИЈСКИ САСТАВ И АНТИОКСИДАТИВНА АКТИВНОСТ
БОБИЧАСТОГ ВОЋА .............................................................................................. 93
Славица М. Весковић Морачанин, Славко Мирецки, Дејана К. Трбовић,
Лазар Р. Турубатовић, Владимир С. Курћубић и Павле З. Машковић
ТРАДИЦИОНАЛНА ПРОИЗВОДЊА БЕЛИХ СИРЕВА У САЛАМУРИ
У СРБИЈИ И ЦРНОЈ ГОРИ – СЛИЧНОСТИ И РАЗЛИКЕ................................. 107
Марија М. Шкрињар, Жарко М. Петровић, Невена Т. Благојев
и Владислава М. Шошо
СЕМЕ КОНЗУМНОГ СУНЦОКРЕТА КАО СУПСТРАТ
ЗА РАСТ МИКОПОПУЛАЦИЈА .......................................................................... 115
Владислава М. Шошо, Марија M. Шкрињар и Невена Т. Благојев
УТИЦАЈ ЕКОФИЗИОЛОШКИХ ФАКТОРА НА ПРИСУСТВО
ОХРАТОКСИНА А У СУВОМ ГРОЖЂУ ........................................................... 123
Даница Б. Зарић, Биљана С. Пајин, Ивана С. Лончаревић,
Драганa M. Шороња Симовић и Зита И. Шереш
УТИЦАЈ ПОСТУПКА ПРОИЗВОДЊЕ НА ЧВРСТОЋУ
И СЕНЗОРНЕ КАРАКТЕРИСТИКЕ МЛЕЧНЕ ЧОКОЛАДЕ ............................. 139
ХЕМИЈСКА ТЕХНОЛОГИЈА И ПРОЦЕСНО ИНЖЕЊЕРСТВО
Мирјана Г. Антов, Бранимир З. Југовић, Милица М. Гвозденовић
и Зорица Д. Кнежевић Југовић
РАСПОДЕЛА ЦЕЛУЛОЛИТИЧКЕ АКТИВНОСТИ У ДВОФАЗНИМ
СИСТЕМИМА ПОЛИЕТИЛЕНГЛИКОЛ/ДЕКСТРАН ...................................... 151
Невена М. Кркић, Вера Л. Лазић и Данијела З. Шупут
ПОРЕЂЕЊЕ УТИЦАЈА ЖИВОТНИХ ЦИКЛУСА
ПОЛИПРОПИЛЕНСКИХ КУТИЈИЦА РАЗЛИЧИТИХ
ЗАПРЕМИНА НА ЖИВОТНУ СРЕДИНУ .......................................................... 159
Јелена Ђ. Марковић, Наташа Љ. Лукић, Јелена Д. Илић,
Бранислава Г. Николовски, Милан Н.Совиљ и Ивана М. Шијачки
СИМУЛАЦИЈА ТОКА У ДВОСТРАНО ВОЂЕНОМ ПОКРЕТНОМ
КАНАЛУ ПОМОЋУ ANSYS FLUENT ПРОГРАМСКОГ ПАКЕТА ................. 169
M. P. Nagarkar, R.N. Zaware и S.G. Ghalmea
СИМУЛАЦИЈА ПРОЦЕСА ИЗРАДЕ ОКРУГЛИХ ЦЕВИ ПОМОЋУ
КОНАЧНИХ ЕЛЕМЕНАТА ПРИМЕНОМ ANSYS СОФТВЕРА ...................... 179
Јелена М. Петровић, Дарко M. Љубић, Марина Р. Стаменовић,
Ивана Д. Димић и Славиша С. Путић
МЕХАНИЧКA СВОЈСТАВА РЕЦИКЛИРАНОГ
СТАКЛО-ЕПОКСИ КОМПОЗИТНОГ МАТЕРИЈАЛА ...................................... 189
Милан Н. Совиљ
ХИДРОДИНАМИКА ЕКСТРАКЦИОНИХ КОЛОНА
ТЕЧНО-ТЕЧНО АГИТОВАНИХ ГАСОМ ........................................................... 199
Весна М. Васић, Марина Б. Шћибан, Александар И. Јокић,
Јелена М. Продановић и Драгана В. Кукић
УТИЦАЈ ПРЕЧНИКА ПОРА НА МИКРОФИЛТРАЦИЈУ ЏИБРЕ ................... 217
Зита И. Шереш, Љубица П. Докић, Биљана С. Пајин, Драгана
М. Шороња Симовић, Драго Шубарић, Јурислав Бабић
и Александар З. Фиштеш
УТИЦАЈ РАДНИХ ПАРАМЕТАРА НА ФЛУКС ПЕРМЕАТА ТОКОМ
МИКРОФИЛТРАЦИЈЕ ВОДЕ ОД МОЧЕЊА У ИНДУСТРИЈИ СКРОБА ...... 225
БИОХЕМИЈСКО И ФАРМАЦЕУТСКО ИНЖЕЊЕРСТВО
Гордана Р. Димић, Сунчица Д. Коцић-Танацков, Оливера О. Јованов,
Драгољуб Д. Цветковић, Синиша Л. Марков и Александра С. Велићански
АНТИБАКТЕРИЈСКА АКТИВНОСТ ЕКСТРАКАТА ЛИМУНА,
КИМА И БОСИЉКА НА LISTERIA SPP. ............................................................. 239
Сунчица Д. Коцић-Танацков, Гордана Р. Димић, Душанка Ј. Пејин,
Љиљана В. Мојовић, Јелена Д. Пејин и Илија Ј. Танацков
АНТИФУНГАЛНА АКТИВНОСТ ЕКСТРАКТА БОСИЉКА (Ocimmum
basilicum L.) НА РАСТ Penicillium aurantiogriseum, P. glabrum, P.
chrysogenum И P. brevicompactum ......................................................................... 247
Зоран З. Кукрић, Љиљана Н. Топалић-Тривуновић, Биљана М. Кукавица,
Сњежана Б. Матош, Светлана С. Павичић, Мирела М. Бороја и
Александар В. Савић
КАРАКТЕРИЗАЦИЈА АНТИОКСИДАТИВНЕ И АНТИМИКРОБНЕ
АКТИВНОСТИ ЛИСТА КОПРИВЕ (Urtica dioica L.) ........................................ 257
Сања О. Подунавац-Кузмановић, Лидија Ј. Јеврић, Страхиња З. Ковачевић,
и Наташа Д. Калајџија
ХЕМОМЕТРИЈСКИ ПРИСТУП У ПРЕДВИЂАЊУ
AНТИФУНГАЛНЕ АКТИВНОСТИ НЕКИХ ДЕРИВАТА
БЕНЗОКСАЗОЛА ПРЕМА Candida albicans ....................................................... 273
Pouneh Ebrahimi, Akram Mirarab-Razi и Abbas Biabani
УПОРЕДНA КАРАКТЕРИЗАЦИЈА ТЕРПЕНОИДА ЕТЕРИЧНОГ
УЉА СТАБЛА И ЛИСТА БИЉКЕ Ziziphora Clinopodioides ИЗ
РЕГИОНА АЛМЕХ И СОЏОГ ПРОВИНЦИЈЕ ГОЛЕСТАН, ИРАН ................ 283
Весна Т. Тумбас, Јелена Ј. Вулић, Јасна М. Чанадановић-Брунет,
Соња М. Ђилас, Гордана С. Ћетковић, Слађана С. Стајчић,
Дубравка И. Штајнер и Борис М. Поповић
АНТИОКСИДАТИВНЕ И СЕНЗОРНЕ КАРАКТЕРИСТИКЕ
БАГРЕМОВОГ МЕДА СА ДОДАТКОМ СУВИХ ШЉИВА.............................. 293
Александра С. Велићански, Драгољуб Д. Цветковић и Синиша Л. Марков
СКРИНИНГ АНТИБАКТЕРИЈСКЕ АКТИВНОСТИ
ЕКСТРАКAТА ВОЋА И ТРОПА МАЛИНЕ (Rubus idaeus L.) .......................... 305
Сенка С. Видовић, Зоран П. Зековић, Жика Д. Лепојевић,
Марија Радојковић, Стела Јокић и Горан Аначков
ОПТИМИЗАЦИЈА ЕКСТРАКЦИЈЕ Ocimum basilicum L.
У ОДНОСУ НА АНТИОКСИДАТИВНУ АКТИВНОСТ ................................... 315
Весна М. Вучуровић и Радојка Н. Размовски
АЛКОХОЛНА ФЕРМЕНТАЦИЈА МЕЛАСЕ ПОМОЋУ ЋЕЛИЈА
Saccharomyces cerevisiae ИМОБИЛИСАНИХ НА РЕЗАНЦИМА
ШЕЋЕРНЕ РЕПЕ .................................................................................................... 325
Марина Б. Шћибан, Јелена М. Продановић и Радојка Н. Размовски
БИОСОРПЦИЈА Cu(II) И Cr(VI) МОДИФИКОВАНОМ ЧАЈНОМ ГЉИВОМ ..... 335
УПУТСТВО ЗА ПИСАЊЕ РАДА
FOOD TECHNOLOGY
APTEFF, 43, 1-342 (2012)
DOI: 10.2298/APT1243001D
UDC: 665.345
BIBLID: 1450-7188 (2012) 43, 1-9
Original scientific paper
CHARACTERISTICS OF BLACKBERRY AND RASPBERRY SEEDS AND OILS
Etelka B. Dimića*, Vesna B. Vujasinovićb, Olga F. Radočajc and Oršolja P. Pastora
a
b
University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
College of Professional Studies in Management and Business Communication, Mitropolita Stratimirovića 110,
21205 Sremski Karlovci, Serbia
c
OLTRAD Consulting Inc., 2669 Inlake Court, Mississauga, L5N 2A6 Ontario, Canada
This study is concerned with the determination of technological quality characterristics of dried pomaces, i.e. blackberry and raspberry seeds, along with the quality parameters, content of total carotenoids and chlorophyl and transparency of crude extracted
oil (using organic solvent). Blackberry seeds (Rubus fruticosus L.) were obtained from a
domestic variety Čačanska bestrna, while the raspberry seeds (Rubus idaeus L.) were of
the variety Willamette. Oil content of the blackberry pomace was 13.97 and 14.34%,
while the oil content of the raspberry pomace was 13.44 and 14.33% on dry basis (d.b.).
In regard to technological characteristics of the pomaces, i.e. volumetric and specific
weight, no considerably difference was found. However, a weight test for 1000 seeds
showed a significant difference in weight: 3.5 g (d.b.) for the blackberry pomace and 1.5
g for the raspberry pomace (d.b.). Proximate analysis of blackberry seed oil showed that
this oil had better quality since the FFA value was 3.43% (sample B1) and 3.53% (sample B2), while the peroxide value was 8.89 and 11.16 mmol/kg, respectively. Raspberry
seed oil had higher FFA (8.59 and 8.83% for sample R1 and R2) and peroxide values
(13.99 and 13.84 for sample R1 and R2) than the blackberry seed oil. Crude extracted
blackberry seed oil had a brown-greenish color due to the high total chlorophyll content
(around 3000 mg/kg dissolved in cyclohexane). Raspberry seed oil had a dark yellowishorange color, due to lower chlorophyll content (around 200 mg/kg) compared to the
blackberry seed oil, while the content of total carotenoids was slightly higher in this oil
(around 40 mg/kg) compared to the blackberry seed oil (33 mg/kg).
KEY WORDS: blackberry and raspberry seeds, seeds' technological quality characterristics, pigments
INTRODUCTION
Our country is known as a significant producer of berries, primarily blackberries and
raspberries. Serbia exported 78460 tons of fresh raspberries and 28313 tons of blackberries in 2011. However, significant amounts of berries are processed in the country.
* Corresponding author: Etelka B. Dimić, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
1, 21000 Novi Sad, Serbia, e-mail: [email protected]
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APTEFF, 43, 1-342 (2012)
DOI: 10.2298/APT1243001D
UDC: 665.345
BIBLID: 1450-7188 (2012) 43, 1-9
During the processing, predominantly in the fruit and beverage industry for juice and
jam production, a large amount of their by-product (pomace, consisting mostly of the
seeds) is produced. Since blackberry and raspberry seeds contain lipids, these by-products are very interesting as a raw material for oil manufacturing in small facilities. Namely, berry oils are considered to be specialty oils and have been in demand on the market. These oils have a unique fatty acid profile and they possess interesting minor components (1).
Berry fruits are rich in phenolic compounds such as phenolic acids, flavonoids, and
anthocyanins. The phenolic compounds in berries have been reported to have an antioxidant, anticancer, antiinflamatory, and antineurodegenerative biological properties (2). Tosun et al. (3) have shown that raspberries are a significant source of phenolic compounds
and ascorbic acid, while antioxidant activity was high and varied greatly amongst differrent genotypes. Godjevac et al. (4) have investigated blackberry seed extracts and showed
that three isolated polyphenolic compounds exhibited considerable protective effects on
human lymphocytes DNA.
The study by Parry and Yu (5) confirmed that the presence of n-3 fatty acid and antioxidants suggests potential value-added utilization of black raspberry seeds for preparing
functional foods or supplemental products. Reyes-Carmona et al. (6) have investigated a
range of blackberries of different cultivars and determined high antioxidant capacity
values that were highly correlated with total phenols and anthocyanin content.
The objective of this paper was to investigate the technological quality: moisture, volatile matter, oil and impurities content in seed, weight of 1000 seeds, specific weight and
weight per liter of the blackberry and raspberry pomace, obtained as by-product of the
industrial processing of berries, as well as the color quality parameters (acid and peroxide
value), content of total carotenoids and chlorophyl, and transparency of the extracted oil.
EXPERIMENTAL
Materials and methods
Blackbery and raspberry seeds. Blackberry seeds (Rubus fruticosus L.) were of the
domestic variety Čačanska beztrna, while raspberry seeds (Rubus idaeus L.) were of the
Willamette variety. Fresh berries were frozen and stored in plastic freezer bags for 8
months, after which they were processed to extract the juice. After pressing the berries,
the pomace (average water content 50.02% - blackberry and 48,38% - raspberry) was
collected and dried under these conditions:
Blackberry seeds:
Sample B1, the pomace was dried in a thin layer (thickness of approx. 1 cm) on
paper at ambient temperature (22 ºC) for 72 h, with occasional stirring.
Sample B2, the pomace was dried in a thin layer in the oven in two stages: first
at 63±2ºC for 20 h and then at 103±2ºC for 20 h.
Raspberry seeds:
Sample R1, the pomace was dried in a thin layer (thickness of approx. 1 cm) on
paper at ambient temperature (22ºC) for 72 h, with occasional stirring.
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APTEFF, 43, 1-342 (2012)
DOI: 10.2298/APT1243001D
UDC: 665.345
BIBLID: 1450-7188 (2012) 43, 1-9
Sample R2, the pomace was dried in a thin layer in the oven in two stages: first
at 63±2ºC for 6 h and then at 103±2ºC for 4 h.
Blackberry and raspberry seed oil. Oil from dried blackberry and raspberry pomace
was obtained using a standard laboratory method, extraction with hexane for 8 h. Upon
completion of the extraction process the solvent was removed using a rotary vacuum
evaporator at 70ºC.
Methods for seeds characterization. The quality of seeds was determined according
to standard methods: moisture and volatile matter content in oilseeds (7), oil content in
seeds (8) and impurities content (9). The weight of 1000 seeds was determined using a
gravimetric method used for grains (10). Specific weight of seeds was determined by
measuring the volume of the clean seed’s weight. For volume determination, 60% ethanol
solution was used. Specific seed volume (γ, g/ml) was calculated using the following
equation: γ = m/Vo-V, where m was weight of seeds (g), Vo beaker’s volume (ml), and V
ethanol volume added to the beaker (ml). Weight per liter of seeds (kg/l) was determined
using Shopper-s scale (10).
Methods for oil characterization. The peroxide value (PV), expressed in mmol/kg,
was determined by the reaction of oil and 3:2 (chloroform to acetic acid) with potassium
iodide in darkness. The free iodine was titrated with a thiosulfate solution (11). The acid
value (AV), expressed in mg KOH/g, as well as free fatty acid content (FFA) expressed
as % oleic acid, were determined by the titration of oil dissolved in a 1:1 ethanol to ether
solution with an ethanolic solution of potassium hydroxide (12). Acidity was determined
in the oil which was extracted by cold and hot extraction (13). Total carotenoid content
was determined using a spectrophotometric method (14), by measuring the absorption of
the oil dissolved in cyclohexane (1% solution) at 445 nm. Total chlorophyl content was
determined by measuring the absorption of the oil solution in cyclohexane (1% solution)
at 630 nm, 670 nm and 710 nm. In addition, oil absorption in chloroform (1% solution)
was determined at 667 nm (15). In order to determine parameters for color characterization, transparency of oil solutions (1%) in chlorophorm and cyclohexane was measured at
455 nm. Spectrophotometric measurements were conducted using UV/VIS spectrophotometer, model T80+ (PG Instruments Limited, London).
Color determination. CIE L*a*b* and CIE Y-xy graph color coordinates were determined using Minolta Chroma Meter CR-400 (Minolta Co., Ltd., Osaka, Japan) in D-65
lighting, with a 2 angle and 8 mm opening. L* - value is lightness, a* - value represents
red and green hue, while b* - value represents yellow and blue hues. In CIE Y-xy tristimolous system, color characteristics are presented as a dominant wavelength -  (nm) and
color purity - P (%). Values: L*, a*and b* were determined by reading, while the dominant wavelength was calculated based on the xy coordinates using a chromatic diagram.
All measurements were made in triplicates and the results were expressed as mean value ± standard deviation.
RESULTS AND DISCUSSION
Basic technological quality parameters of blackberry and raspberry seeds are shown
in Table 1.
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APTEFF, 43, 1-342 (2012)
DOI: 10.2298/APT1243001D
UDC: 665.345
BIBLID: 1450-7188 (2012) 43, 1-9
Table 1. Technological quality of blackberry and raspberry seeds
Parameter
Water content (%)
Oil content (%)
- tel quel (as is)
- on dry basis
Impurities content (%)
Pure seeds content (%)
Weight of 1000 seeds* (g)
- tel quel (as is)
- on dry basis
Specific weight (g/ml)
- pure seeds*
- tel quel seeds (as is)
Weight per 1liter (g/l):
- pure seeds*
- tel quel seeds (as is)
Blackberry seeds
B1
B2
6.59 ± 0.07
5.24 ± 0.04
Raspberry seeds
R1
R2
8.53 ± 0.02
6.08 ± 0.06
13.05±0.05
13.97±0.05
4.68 ± 0.98
95.32± 0.98
13.59 ± 0.09
14.34 ± 0.09
4.36 ± 1.11
95.64 ± 1.11
12.30 ± 0.13
13.44 ± 0.13
10.06 ± 0.98
89.94 ± 0.98
13.46 ± 0.22
14.33 ± 0.22
11.16 ±1.12
88.84 ± 1.12
3.21 ± 0.05
3.45 ± 0.05
3.32 ± 0.06
3.50 ± 0.06
1.50 ± 0.07
1.64 ± 0.07
1.45 ± 0.08
1.54 ± 0.08
0.999±0.000
0.997±0.010
0.997 ± 0.010
0.993 ± 0.010
0.968 ± 0.010
0.964 ± 0.020
0.967 ± 0.000
0.963 ± 0.010
423.6 ± 0.4
384.8 ± 0.4
429.2 ± 3.2
394.0 ± 1.6
421.0 ± 1.0
375.8 ± 0.2
419.2 ± 2.0
397.8 ± 2.2
*pure seeds were separated from the dry pomace by hand
Considering storage and oil extraction, water content is the most important characteristic of seeds as raw materials. Water content in dried seeds was pretty similar, ranging
from 5.24 to 8.52%. It could be said that an equilibrium state of moisture was achieved
using the mentioned drying conditions.
The most valuable macro component of the berry seeds is the oil. Results have demonstrated that investigated domestic blackberry and raspberry varieties were very similar in terms of oil content.
The oil content in the blackberry seed samples B1 and B2 was 13.97 and 14.34 %
d.b., respectively, while in the raspberry seeds R1 and R2 it was 13.44 and 14.33 % d.b.,
respectively. Literature reviews have shown scarce data for oil content in blackberry
seeds; the only value found was published by Kiss (16) and it was 14.7%, which is very
similar to our results. Our results are in good agreement with published literature data for
raspberry seed oil content too. According to Lampi and Heinonen (17) oil content was
10.7-23.2 %; Oomah et al. (18) reported 10.7% (d.b.), while Kiss (16) obtained 14%, and
Šućurović et al. (19) 14.5% (at the water content of 9.7%).
The content of impurities in the blackberry seeds was low (4.5% of the total seeds
weight), where 4% was dust, and the rest was organic plant material (dry fruit parts,
thorns, membranes etc.), while the pure seeds content was about 95%. Raspberry seeds
had higher impurity levels, 10.06 and 11.16% (with more than 10% dust), while the organic material quantity was negligible. The content of pure raspberry seeds was approximately 90 %.
The parameters such as the weight of 1000 seeds, specific weight and weight of 1 liter
of seeds, are used to determine not only the seeds’ quality, but also to help in the determination of volumetric seed weight for storage purposes. The weight of 1000 blackberry
seeds was around 3.5 g, while for the raspberry samples the values were 1.54 and 1.64 g
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BIBLID: 1450-7188 (2012) 43, 1-9
(d.b.), which means that blackberry seeds were bigger in size. The ratio between the
weight of 1000 seeds of blackberries and raspberries was 2.29. Kiss (16) published similar results: 3.97 g for blackberry seeds and 1.42 g for raspberry seeds. According to
Oomah et al. (18), the weight of 1000 blackberry seeds was 1.8 g. The specific volumetric (1 l) weight of pure seeds and tel quel (as is) seeds were pretty similar for both types
of seeds. To the best of our knowledge, there are no published data on impurities content,
or specific and volumetric weight of these seeds.
The most important quality parameters of berry seed oils are given in Table 2.
Table 2. Some quality parameters of blackberry and raspberry seed oils
Parameter
Acid value (mg KOH/g)
- hot extraction
- cold extraction
FFA (% oleic acid)
- cold extraction
Peroxide value (mmol/kg)
- cold extraction
Blackberry seed oil
B1
B2
Raspberry seed oil
R1
R2
7.75 ± 0.22
6.85 ± 0.11
8.23 ± 0.25
7.05 ± 0.30
18.74 ± 0.26
17.18 ± 0.43
17.65 ± 0.33
17.86 ± 0.51
3.43 ± 0.11
3.53 ± 0.30
8.59 ± 0.43
8.83 ± 0.51
8.89 ± 0.12
11.16 ± 0.23
13.99 ± 0.44
13.84 ± 0.48
The acidity of the oils was determined after their hot extraction, as well as after cold
extraction. The acid value of blackberry oil after hot extraction was in the range from
7.75 to 8.23 mg KOH/g, while of the raspberry seed oil it was higher, ranging from 17.18
to 18.74 KOH/g. The increase of the acidity of both seed types can be due to longer
storage time of the berries. Namely, the frozen berries were stored for 8 months before
they were used, so that it was likely that the oil hydrolysis took place by the action of
lipase. Van Hoed et al. (1, 20) reported that blackberry seed oils had FFA values of 0.96
and 0.80%, while raspberry seed oils had FFA values of 0.49 and 0.69% (expressed as
oleic acid). However, the oils in their study were obtained from the seeds originating
from fresh berries. Šućurović et al. (19) investigated freeze-dried raspberry seeds and
found an FFA value of 1.32%. Peroxide values, as indicators of the primary oxidation
process, were higher in the raspberry (13.99 and 13.84 mmol/kg) than in blackberry seed
oils (8.89 to 11.16 mmol/kg). Although these values are high for crude plant oils, it could
be said that they are in the range of PV reported in other studies for berry seed oils (1, 20,
21). Based on the PV, it is shown that raspberry seed oil is more prone to oxidation than
blackberry seed oil.
The color perceptions of the extracted berry seed oils are presented in Table 3. Visual
presentation of oils is very important for their potential application in food and cosmetic
products.
Based on the visual color evaluation, blackberry seed oil had a dark brown-greenish
color with an orange hue (viewed as a thin oil layer). Raspberry seed oil was dark
yellowish-orange in color. Hence, both oils were pretty dark in color and not clear at
room temperature.
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DOI: 10.2298/APT1243001D
UDC: 665.345
BIBLID: 1450-7188 (2012) 43, 1-9
Table 3. Color characteristics of blackberry and raspberry seed oils
Oil type
Blackberry B1
B2
Raspberry R1
R2
CIE system
λ (nm)
P (%)
589.0
8.19
588.0
8.09
580.5
24.56
580.0
25.87
L* (%)
16.94±0.03
16.84±0.02
20.43±0.06
20.23±0.03
CIE L*a*b* system
a*
2.32±0.04
2.30±0.03
3.23±0.01
3.63±0.01
b*
0.99±0.09
0.90±0.04
7.01±0.01
6.99±0.02
λ – dominant wave-length; P – color purity; L* – lightness; a* – red hue; b* – yellow hue
Table 3 shows that the color purity (clarity) of the blackberry seed oil was very weak
(around 8%), where the color determined using the CIE L*a*b*system confirmed orange
instead of green hue. The discrepancy between the visual and instrumental determination
of the oil color was due to very weak clarity of the oil color. Color clarity of the raspberry
seed oil was three times higher (around 25%), while the dominant wavelength was in the
yellowish-orange hue range. With the rise in color clarity (purity), the hue became saturated, and the raspberry oil color determined by the CIE L*a*b* system was in agreement
with the color determined visually.
Visual color perception is mainly a function of the pigment quantity present in the oil.
Total content of carotenoids, chlorophyl, as well as the transparency of blackberry and
raspberry seed oil is shown in Table 4.
Table 4. Content of total carotenoids, chlorophyl and transparency of blackberry and
raspberry seed oil
Oil
Blackberry B1
B2
Raspberry R1
R2
a
Total
carotenoidsa
(mg/kg)
32.30±0.55
33.92±0.67
39.06±0.25
41.44±0.68
Total chlorophyl
(mg/kg)
cyclohexane
chloroform
3049.52±17.79 1505.78±12.59
3094.98±20.54 1583.62±11.66
208.50±1.44
129.97±4.06
199.88±2.45
120.78±3.66
Transparency
(%)
cyclohexane chloroform
25.84±0.68
19.28±0.27
24.33±0.34
18.87±0.23
12.46±0.49
9.83±0.65
11.08±0.33
9.54±0.21
expressed as β-caroten
Total carotenoids content, expressed as β-caroten, was pretty similar in all oil samples. Parry et al. (21) have used the HPLC method to determine total carotenoids content.
They obtained the values of 23.4 μmol/kg for the blackberry seed oil and 12.5 μmol/kg
for the raspberry seed oil. In the study of Oomah et al. (18), the total carotenoids content
(expressed as β-caroten) of 23 mg/100g was reported. The values for total carotenoids
content in this study are much lower compared to that found for the cold-pressed pumpkin seed oil, where the values were in the range from 138.67 to 218.67 mg/kg. Virgin
cold-pressed pumpkin oil had these values even higher, ranging from 240.18 to 526.22
mg/kg (22).
The total chlorophyl content was determined by dissolving oil samples in cyclohexane
and chloroform. It has to be noted that there was a difference in the values obtained by
using these two solvents. Namely, when the total chlorophyl content was determined by
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DOI: 10.2298/APT1243001D
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dissolving oil in cyclohexane, the results were two times higher compared to the values
obtained by dissolving oil in chloroform. Thus, the values obtained for the blackberry
seed oil were 3000 mg/kg (in cyclohexane) and 1500 mg/kg (in chloroform). The raspberry seed oil had much lower values: the total chlorophyl content of 200 mg/kg (in cyclohexane) and 120 mg/kg in chloroform solution. The total chlorophyl content of blackberry seed oil was around fifteen times higher than of the raspberry seed oil.
Transparency was also determined using both cyclohexane and chloroform solutions.
Regardless of the method used, the values for oil transparency of the blackberry seed oil
were approximately twice than those for the raspberry seed oil. This can be explained by
the fact that the raspberry seed oil has a much higher content of total carotenoids, while
the oil transparency was measured at 455 nm, i.e. at the wavelength where carotenoids
exhibit maximum absorption.
CONCLUSION
Results obtained in this study have shown that pomace, a by-product from berry
processing, specifically blackberry and raspberry juice, can be used as a potential raw
material for oil extraction. This is supported by the fact that dried pomace has around
14% of oil (d.b.). Although both seeds were very small in size, blackberry seeds were
twice the weight of the raspberry seeds. Conversely, due to the small seed size, there was
no considerable difference in the volumetric weight, which was around 420 g/l. The FFA
and PV values for the oils extracted from dried seeds are much higher than those found in
other plant oils. Further studies should encompass investigations of storage time on the
primary oil characteristics of frozen berry seed oil.
The crude blackberry seed oil extracted using organic solvent was dark brown-greenish in color, primarily due to the high total chlorophyl content (around 3000 mg/kg in
cyclohexane). The raspberry seed oil was dark yellowish-orange in color, due to lower
chlorophyl content (around 200 mg/kg) compared to the blackberry seed oils, while the
total carotenoides content was slightly higher (40 mg/kg) compared to the blackberry
seed oil (33 mg/kg).
Acknowledgement
These results are part of the project TR 31014 „Development of the new functional
confectionery products based on oil crops“, financially supported by the Ministry of
Education and Science of the Republic of Serbia.
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16. Kiss, A.: Bogyós gyümölcsök magvainak és magolajainak vizsgálata, Szakdolgozat,
Kertészeti és Élelmiszeripari Egyetem, Élelmiszeripari Kar, Budapest, 1999.
17. Lampi, A. and Heionen, M.: Berry seed and grapeseed oils, in Gourmet and health
promoting speciality oils. Eds. Moreau R. A. and Kamal-Eldin, A., AOCS Press,
Urbana, Illinois (2009) pp. 215-235.
18. Oomah, B.D, Ladet, S., Godfrey, D.V., Liang, J. and Girard, B.: Characteristics of
raspberry (Rubus idaeus L.) seed oil. Food Chem. 69, 2 (2000) 187-193.
19. Šućurović, A., Vukelić, N., Ignjatović, Lj., Brčeski, I. and Jovanović, D.: Physicalchemical characteristics and oxidative stability of oil obtained from lyophilized
raspberry seed. Eur. J. Lipid Sci. Technol. 111, 11 (2009) 1133-1141.
20. Van Hoed, V., Barbouche, I., De Clercq, N., Dewettinck, K., Slah, M., Leber, E. and
Verhe, R.: Influence of filtering of cold pressed berry seed oils on their antioxidant
profile. Food Chem. 127, 4 (2011) 1848-1855.
8
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21. Parry, J., Su, L., Luther, M., Zhou, K., Yurawecz, M. P., Whittaker, P. and Yu, L.:
Fatty acid composition and antioxidant properties of cold-pressed marionberry,
boysenberry, red raspberry and blueberry seed oils. J. Agric. Food Chem. 53, 3 (2005)
566-573.
22. Vujasinović, V.: Influence of thermal treatment of pumpkin seeds Cucurbita pepo L.
on nutritive value and oxidative stability of oil, Ph.D. Thesis, University of Novi Sad,
Faculty of Technology, Novi Sad, 2011.
КАРАКТЕРИСТИКE СЕМЕНА И УЉА КУПИНЕ И МАЛИНЕ
Етелка Б. Димића, Весна Б. Вујасиновићб, Олга Ф. Радочајв и Оршоља П. Пастора
а
Универзитет у Новом Саду, Технолошки факултет, Булевар цара Лазара 1, 21000 Нови Сад, Србија
б
Висока школа струковних студија за менаџмент и пословне комуникације, Митрополита
Стратимировића 110, 21205, Сремски Карловци, Србија
в
OLTRAD Consulting Inc., 2669 Inlake Court, Mississauga, L5N 2A6 Ontario, Canada
У овом раду су одређене технолошке карактеристике осушеног тропа, односно,
семена купине и малине, као и параметри квалитета, садржај укупних каротеноида
и хлорофила, транспаренција и боја сировог уља издвојеног из семена помоћу органског растварача. Семе купине (Rubus fruticosus L.) је добијено из плода домаће
сорте Чачанска безтрна, а семе малине (Rubus idaeus L.) из плода сорте Willamette.
Садржај уља у семену осушеног тропа купине износиo je 13,97 и 14,34%, а код малине 13,44 и 14,33% на суву материју (с.м.). По технолошким карактеристикама,
литарској и специфичној маси семена, нису нађене разлике, међутим маса 1000 семена је код купине износила око 3,5г, а код малине око 1,5 г на с.м. Oсновни хемијски квалитет уља семена купине је био знатно бољи у односу на уље малине, будући да је садржај слободних масних киселина (СМК) био 3,43 и 3,53%, а пероксидни
број (Пбр) 8,89 и 11,16 ммол/кг, док су код малине вредности СМК биле 8,59 i
8,83%, а Пбр 13,99 и 13,84 ммол/кг. Сирово екстраховано уље семена купине било
је тамне мрко-зелене боје, пре свега због високог садржаја укупних хлорофила, око
3000 мг/кг (у циклохексану). Уље малине је било тамне жућкасто-наранџасте боје,
будући да је садржај хлорофила био знатно мањи, око 200 мг/кг, у односу на уље
купине, док је садржај укупних каротеноида био нешто већи, око 40 мг/кг (код купине око 33 мг/кг).
Кључне речи: семе купине и малине, технолошке карактеристике семена, пигменти
Received: 03 September 2012
Accepted: 02 October 2012
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BIBLID: 1450-7188 (2012) 43, 11-19
VOLATILE COMPOUNDS OF FUNCTIONAL DAIRY PRODUCTS
Mirela D. Iličić*, Spasenija D. Milanović, Marijana Đ. Carić, Katarina G. Kanurić,
Vladimir R. Vukić, Dajana V. Hrnjez and Marjan I. Ranogajec
Volatile compounds, affecting flavour of traditional and probiotic fresh cheese, were
determined. Functional dairy product-fresh cheese was produced from milk of 2.5% fat
content and milk of 4.2% fat content, under the semi-industrial conditions. The traditional starter culture Flora Danica (FD) and a combination of probiotic starter ABT-1 and
FD (ABT-1:FD=1:1) were applied as starters. The volatile fractions were isolated by
employing the combined simultaneous distillation-extraction technique (SDE). The compounds were identified by gas chromatography – mass spectrometry (GC-MS) and quantified by using standard procedure. Following 19 compounds have been identified: 8 hydrocarbons (decane, undecane, tridecane, tetradecane, pentadecane, hexadecane, octadecane and 2, 6, 10, 14-tetramethyl hexadecane); 6 ketones (2-heptanone, 2-nonanone, 2undecanone, 2-pentadecanone, 2-heptadecanone and 2-tridecanone); 3 aldehydes (nonanal, tetradecanal and hexadecanal); 1 fatty acid (decanoic acid) and disulfide, bis (1-methylethyl). The highest levels were associated with hexadecanal, 2-pentadecanone, 2-tridecanone, and 2-undecanone in all examined samples, regardless to the starter culture
and type of milk used.
KEY WORDS: Fresh cheese, volatile flavour components, probiotics.
INTRODUCTION
Functional dairy product is highly valuable products, particularly if it contains probiotics. However, main obstacle to its broader consumption could be lower sensory characteristics, compared to the traditional fresh cheese. Very important factor, which affects
flavour, is great number of volatile compounds appearing as a result of an action of enzymes on the milk components: protein, fat, lactose, and citrate. Proteolytic enzymes
from lactic acid bacteria cause the degradation of casein and peptides, leading to production of free amino acids that contribute directly to the basic taste of cheese and indirectly to cheese flavour, as the precursors for other catabolic reactions (1, 2, 3). These
reactions and side-chain modification may yield keto-acids, ammonia, amines, aldehydes,
acids and alcohols, which are essential contributors to cheese taste and aroma. For exam* Corresponding author: Mirela D. Iličić, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
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ple, bitterness is due to hydrophobic peptides, rancidity to fatty acids, and fruitiness to
esters (3).
Volatile fatty acids in fresh cheese are the products of various metabolic pathways,
mostly microbial. Their further degradation leads to a generation of very important group
of compounds - aldehydes and ketones. Besides ß-oxidation of fatty acids, they can be
synthesized by direct oxidation of hydrocarbons (4). Panseri et al. (number ) investigating
flavour of the Italian mountain cheese- Bitto have detected aldehyde hexadecanal, which
is associated with the waxy, floral aroma; 2-pentadecanone, giving delicate musk aroma;
2-undecanone, giving citrus, rose and iris aroma and n-nonanal, giving floral, citrus and
green aroma (5). Many authors have found 2-undecanone, 2-heptanone and 2-nonanone
as flavour compounds in semi fat and fat cheeses, exposed to a certain period of ripening
(6-9). Nogueira et al. (10) identified 2-undecanone, which gives fruity, oral notes to Minas cheese, while Beuvier and Buchin detected 2-heptanone in Emmental, Gruyere and
Padano cheese (10, 11).
Important flavour compounds are esters, formed by condensation of an acid and an alcohol either spontanously or mediated by microbial esterases. Sulphur compounds are
particularly present in mould- or smear-surface cheese, and provide typical cabbage or
garlic flavours (11-14). Cheeses can contain also a great number of hydrocarbons, which
belong to a family of secondary products of lipid antioxidants (15). They do not have a
major contribution to aroma in cheese, but may serve as precursors for the formation of
other aromatic compounds (16).
Starter culture is an important factor which affects flavour of the final product. When
starters are new systems, as probiotics, very little knowledge about their aroma impact
exists. Recently, an investigation was performed by Kourkoutas et al. (17). The investigators immobilized Lb. casei cells on fruit (apple and pear) peaces and used them as adjunct
culture in probiotic cheese making. Sensory evaluation revealed the fruity taste of the obtained probiotic cheese. It was found that the commercial Feta cheese has a more sour
taste, whereas no significant differences concerning two cheeses flavour were reported. A
significant content of n-hexadecanoic acid was found in both products. Other investigation dealt with the use of freeze-dried kefir coculture as a starter in the production of
Feta-type cheese (18). The main active microbial associations were members of the genera Pseudomonas and Lactococcus. The effect of the starter culture on the production of
aroma-related compounds was studied. Among 18 carbonyl compounds, 2-undecanone
and hexadecanone were identified. Broadbent et al. (19) made the reduced-fat Cheddar
cheeses with Lc. lactis starter only, starter plus Lb. casei ATCC 334, and starter plus Lb.
casei ATCC 334 transformed with pTRKH2: dhic (19). They found that the culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols
and esters, after 3 months of ripening. Among other volatile compounds, hexadecanal
ranging 0.22-0.52 μg/g as well as 2-undecanone varying 0.01-0.03 µg/g were quantified.
Very limitted data on fresh cheese are available when probiotics as the starters are applied. The aim of this study was to investigate the volatile compounds in functional dairy
products-probiotic fresh cheese produced with combination of probiotics and traditional
cultures from partially skimmed and full-fat milk.
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MATERIAL AND EXPERIMENTAL
Starter Culture
Traditional culture Flora Danica - FD (Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris, Ln. mesenteroides subsp. cremoris, Lc. lactis subsp. lactis biovar diacetylactis) was
applied for production of control samples, while the 1:1 combination of FD and probiotic
starter culture ABT-1 (Lb. acidophilus-5, Bifidobacterium-12, Str. thermophilus) was
used for production of probiotic cheese. Both starters are commertially available (Chr.
Hansen, Denmark).
Cheese manufacture
Two series of functional dairy products - fresh cheese samples with different fat content were manufactured under semi-industrial conditions. Milk was pasteurized at 71C,
during 15s, and cooled to 28C.
Starter culture (0.01%) and 0.005% enzyme for coagulation were added into milk at
28C. Coagulation lasted 18 hours (pH= 4.6). After that, coagulum was cut, pasteurized
by gently stirring at 60C (5 min) and quickly cooled and drained. Cheese samples were
homogenized by mixing and packed in cups of 1.8 dl volume.
Series I was produced from milk of 2.5% fat; it consisted of the traditional fresh
cheese - I.FD and probiotic fresh cheese - I.ABT-1: FD=1:1. Series II was produced
from milk of 4.2% fat; it consisted of the traditional fresh cheese - II.FD and probiotic
fresh cheese - II.ABT-1: FD=1:1.
Chemical analyses
Milk composition and quality of fresh cheese samples were analysed by standard methods. Dry matter content was measured in milk (20) and cheese by drying at 105oC (21).
Fat content in milk and cheese (F) were analysed according to Gerber (22) and Van Gulik
(23), respectively. Total nitrogen content (TN) was determined according to Kjeldahl method (24), while total proteins (TP) were calculated by multiplying TN by 6.38%. Ash
content was determined by ignition at 550oC (AOAC, 2000), while pH value was measured by pH-meter Iskra, MA 5713, Kranj, Slovenia.
All results are based on 3 to 5 measurements of each parameter.
Analyses of volatile flavour compounds
The volatile flavour components were isolated from fresh cheese samples employing
the combined simultaneous distillation-extraction procedure widely used in cheese analysis (25).
10 g of cheese sample was poured into the distillation vessel with 40 mL distilled water. Distilled water (1.5 mL) and 1 ml n-pentane were mixed in the extractor. Extraction
was performed for 2 hours. The obtained extract was evaporated and reconstituted to 100
µL with ethyl-acetate. The concentrated extracts were analysed by Agilent Technologies
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G1777A Gas Chromatograph, equipped with flame ionization detector and capillary column DB-5 30m x 0.25mm. The oven temperature was programmed: 50°C, 1 min; from
50 to 100°C at 5°C/min, from 100 to 200°C at 9°C/min, for 2.9 min. The carrier gas was
nitrogen (2mL/min). The injected volume was 3μL. The injector and detector temperature
was set at 250°C. The volatile components were identified by gas chromatography - mass
spectrometry (GC-MS).
Chemical composition of functional dairy products-fresh cheese samples is presented
in Figure 1.
30
Total solids
Fat
Total proteins
Lactose
Quality parameters (%)
25
20
15
10
5
0
I.FD
I.ABT-1:FD = 1:1
II.FD
II.ABT-1:FD = 1:1
Fresh cheese samples
Figure 1. Composition of fresh cheese samples
This analysis shows that some differences between cheeses produced by traditional
starter culture application: I.FD and II.FD and starter combination: I. ABT-1:FD=1:1 and
II. ABT-1:FD=1:1 exist. Probiotic cheese I.ABT-1:FD=1:1 produced in series I, from
milk with 2.5% fat, contains less dry matter than the traditional cheese. On the contrary,
probiotic cheese produced from milk with 4.2% fat in series II, has greater content of dry
matter. In cheeses manufactured from partially skimmed milk content of proteins is greater than content of fat, while in the cheeses produced from full-fat milk content of fat
overcomes the content of proteins. Nutritive value of obtained cheeses is high and all
cheeses can be suggested as valuable food under the condition that their sensory characteristics are acceptable.
Due to analyses of aromatic fractions of the manufactured traditional and probiotic
cheeses, the SDE-procedure was applied.
Figure 2 and 3 shows the obtained capillary gas hromatogram of the SDE volatile
fraction of cheeses produced from partially skimmed milk in series I and from full-fat
milk in series II, respectively.
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88 (2012) 43, 11-19
Origin
nal scientific paper
Figure 2. Chrom
matogram of the SD
DE fraction of cheeese from milk of 2.5% fat:
a) traditional fresh cheese;
c
b) fresh chheese produced byy application of sttarter culture
combinattion ABT-1:FD=1:1
Figure 3. Chrom
matogram of the SD
DE fraction of cheeese from milk of 4.2 % fat:
a)) traditional fresh cheese b) fresh chheese produced byy application of staarter culture
combinattion ABT-1:FD=1:1
In
n the traditional frresh I.FD cheese m
manufactured from
m skimmed milk, 77 compounds
were identified by GC
C-MS; 79 compounnds were identifieed in the probioticc I.ABT-1 : FD
= 1 : 1 cheese, produced from the samee milk. Quantitivelly, 18 compoundss were determined by
b internal standarrd procedure in booth cases (Table 1)).
In
n the traditional frresh II.FD cheese produced from fuull-fat milk, 71 co
ompounds were
deteccted by GC-MS; 58 compounds w
were detected in the probiotic II.A
ABT-1:FD=1:1
cheesse, manufactured from the same miilk. Quantitively, 19 compounds were
w
determined
by in
nternal standard prrocedure (Table 11) in traditional cheese and 15 com
mpounds of the
probiiotic cheese.
The obtained valuees of the quantifiedd compounds are shown as the relaative concentrations (%). Following 19
1 compounds havve been identifiedd: 8 hydrocarbons (decane, n-undecan
ne, tridecane, tetraadecane, pentadeccane, hexadecane,, octadecane and 2,6,10,14-tetra2
methy
yl hexadecane); 6 ketones (2-heptaanone, 2-nonanonee, 2-undecanone, 2-pentadecano2
ne, 2-heptadecanone and
a 2-tridecanone)); 3 aldehydes (noonanal, tetradecan
nal and hexadecanall); 1 fatty acid (decanoic acid) and ddisulfide, bis (1-m
methylethyl).
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The main compounds are hexadecanal, 2-pentadecanone, 2-tridecanone and 2-undecanone, present in all examined samples (Table 1).
Table 1. The quantified volatile components detected in SDE fraction of functional dairy
samples
Volatile compounds
I.FD
RTa
(min)
5.61
8.50
SERIES I
I.ABT-1:FD=1:1
RTa
RCb
RCb
(%)
(%)
(min)
0.12
5.61
0.17
0.03
8.50
0.06
2-Heptanone
Decane
Disulfide, bis
9.08
0.04
9.08
(1-methylethyl)
2-Nonanone
11.1
0.52
11.1
Undecane
11.38
0.05
11.37
Nonanal
11.51
0.03
11.5
2-Undecanone
15.66
0.81
15.65
Tridecane
15.8
0.4
15.8
Decanoic acid
16.88
0.21
16.86
Tetradecane
17.49
2-Tridecanone
18.98
1.48
18.95
Pentadecane
19.02
0.05
19.02
Hexadecane
20.41
0.27
20.42
Tetradecanal
20.6
0.61
20.6
2-Pentadeca-none
21.7
1.59
21.7
Octadecane
23.02
0.59
23.03
2, 6, 10, 14-Tetra23.12
0.15
methyl hexadecane
Hexadecanal
23.29
7.41
23.28
2-Heptadeca-none
24.66
0.34
24.66
a
RT – Retention time; b RC – Relative concentration
RTa
(min)
5.62
8.51
SERIES II
II.ABT-1:FD=1:1
RTa
RCb
RCb
(%)
(min)
(%)
0.21
5.61
0.12
0.07
-
0.06
9.08
0.08
-
-
0.43
0.07
0.04
0.75
0.45
0.14
0.08
1.35
0.06
0.1
0.4
1.36
0.7
11.1
11.38
11.51
15.65
15.8
16.88
17.49
18.94
19.02
20.41
20.6
21.7
28.02
0.47
0.08
0.11
0.63
0.48
0.43
0.09
1.31
0.08
0.09
0.29
1.44
0.4
11.09
11.37
15.65
15.79
16.85
17.49
18.94
19.01
20.41
20.1
21.69
23.02
0.57
0.08
1.02
0.52
0.05
0.08
2.01
0.06
0.09
0.05
2.2
0.34
-
23.11
0.09
23.11
0.09
2.9
0.22
23.55
24.66
3.7
0.36
23.28
-
2.1
-
II.FD
The highest level of hexadecanal (7.4%) was found in the traditional I.FD cheese.
Also, very high level (3.7%) was detected in the traditional II.FD cheese. The probiotic
cheeses contain lower levels of hexadecanal; probiotic I.ABT-1:FD=1:1 cheese 2.9% and
probiotic II.ABT-1:FD=1:1 cheese 2.1%. Second important volatile compound is 2-pentadecanone. Its level is the highest (2.2%) in the probiotic II.ABT-1: FD=1:1 cheese,
which is followed by the traditional I.FD cheese (1.5%) and the other traditional II.FD
cheese (1.44%). The lowest level was found in the probiotic I.ABT-1: FD=1:1 cheese
(1.36%). Very similar results were obtained in the case of the third compound present in
all samples, 2-tridecanоne. The fourth volatile compound, present in all samples, was 2undecanone. It reached 1.02% in the probiotic II.ABT-1:FD=1:1 cheese, 0.81% in the traditional I.FD cheese, 0.75% in the probiotic I.ABT-1:FD=1:1 cheese and 0.63% in the
traditional II.FD cheese.
Based on the analysis of values presented in Table 1, it can be concluded that probiotic cheeses in both series contained less quantity of the main volatile compound (hexadecanal) than the traditional cheeses. Some authors (5) associated the presence of hexadecanal with the waxy, floral aroma of cheese. The remaining three important compounds (2pentadecanone, 2-tridecanone and 2-undecanone) were present in probiotic cheeses pro16
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duced in series I and series II even at a higher level than in the traditional cheeses, especially when full-fat milk was used (Table 1). Presence of 2-pentadecanone can be related
to the delicate musk aroma of cheese (5). Many authors have found 2-undecanone as a
compound giving citrus, rose and iris aroma flavour to semi fat and fat cheeses (5-10).
Unfortunately, there are not literature date about aroma of fresh cheeses particularly cheeses produced with probiotics application.
Volatile compounds, present in smaller quantities in investigated cheeses, are: tetradecanal, octadecanal, 2-nonanone, tridecane, 2-heptadecanone, hexadecane and decanoic
fatty acid. These compounds were detected in all samples, except 2-heptadecanone in
probiotic cheese II.ABT-1:FD=1:1. Their quantity varied from 0.7% to 0.1%. They were
identified by other authors, but in semi-hard and hard cheeses. So, 2-heptanone and 2-nonanone were identified by Moio et al. (1998), Fernandez-Garcia et al. (2002) and Lanciotti et al. (2006) (6,8,9). Panseri et al. (5) associated presence of n-nonanal with floral,
citrus and green flavour of Italian Bitto cheese (5).
Finally, a great number of n-alkanes, such as decane, undecane, tridecane, tetradecane, pentadecane, hexadecane, octadecane etc., were present in all fresh cheese samples.
These compounds also were found as volatile flavour components of buffalo Mozzarella
cheese in a study of Moio et al. (26). Although the hydrocarbons do not have a major
contribution to aroma in cheese, they may serve as precursors for the formation of other
aromatic compounds (15).
CONCLUSION
On the basis of the comparative study carried out it can be concluded that there is an
acceptable level of similarity between aroma of traditional fresh cheese and probiotic
fresh cheese. Consequently, the functional dairy product-probiotic cheeses manufactured
from milk of 2.5% fat content and full-fat milk (4.2 % fat) by applying the combination
of the traditional and probiotic starter culture in ratio 1:1, possess good characteristics,
regarding to their taste and flavour.
Acknowledgements
The financial support from the Ministry of Education and Science (Project No. 46009)
of Serbia is gratefully acknowleged. The authors thank also MTC, Sombor, Serbia for
supply of probiotic starters Chr. Hansen, Denmark and AD Imlek, Division Novi Sad
Dairy, Serbia for cooperation in experiments in their plant.
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(1971) 383-395.
5. Panseri, S., Giani, I., Mentasti, T., Bellagamba, F., Caprino, F. and Moretti, V.M.:
Determination of flavour compounds in a mountain cheese by headspace sorptive extraction-thermal desorption-capillary gas chromatography-mass spectrometry. LWT Food Sci. Technol. 41 (2008) 185-192.
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8. Fernandez-Garcia, E., Carbonell, M. and Nunez, M.: Volatile fraction and sensory
characteristics of Manchego cheese. 1. Comparison of raw and pasteurized milk cheese. J. Dairy Res. 69 (2002) 579-593.
9. Lanciotti, R., Vannini, L., Patrignani, F., Iucci, L., Vallicelli, M., Ndagijimana, M.
and Guerzoni, M.E.: Effect of high pressure homogenisation of milk on cheese yield
and microbiology, lipolysis and proteolysis during ripening of Caciotta cheese. J.
Dairy Res. 73 (2006) 216-226.
10. Nogueira, C.M.L., Lubachevsky, G., Scott A. and Rankin, S.A.: A study of the volatile composition of Minas cheese. J. Food Sci. Tech. 38 (2005) 555-563.
11. Beuvier, E. and Buchin, S.: Raw milk Cheeses in Cheese, Chemistry, Physics and
Microbiology, General Aspects. Eds. Fox, P.F., McSweeney, Cogan, T.M., Guinee,
T.P. Elsevier, Academic Press (2005) pp. 319-345.
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Fundamentals of Cheese Science, Aspen publishers Inc., Gaithersburg, Maryland
(2000) pp. 282-303.
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Components of Manchego Cheese. J. Dairy Res. 58 (1991) 239-246.
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R.: Study of the volatile fraction of Parmesan cheese. J. Agric. Food Chem. 42 (1994)
1170-1176.
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748-752.
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Probiotic Cheese Production Using Lactobacillus casei Cells Immobilized on Fruit
Pieces. J. Dairy Sci. 89 (2006) 1439-1451.
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A.: Evaluation of Freeze-Dried Kefir Coculture as Starter in Feta-Type Cheese Production. Appl. Environ. Microb. 72, 9 (2006) 6124-6135.
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Dairy Products, Faculty of Technology, Novi Sad (2000) p.204.
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IDF Standard 20. Brussels, Belgium: International Dairy Federation (1962).
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analysis. J. Chromatography 203 (1981) 325-335.
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water buffalo Mozzarella cheese. Ital. J. Food Sci. 5 (1993) 57-68.
ИСПАРЉИВЕ КОМПОНЕНТЕ АРОМЕ ФУНКЦИОНАЛНОГ МЛЕЧНОГ
ПРОИЗВОДА
Мирела Д. Иличић, Спасенија Д. Милановић, Маријана Д. Царић,
Катарина Г. Канурић, Владимир Р. Вукић, Дајана В. Хрњез и Марјан И. Раногајец
Испарљиве компоненте ароме су одређене у традиционалном и пробиотском
свежем сиру-функционалном млечном производу. Свежи сир произведен је из
млека са 2,5% и 4,2% млечне масти уз коришћење традиционалне стартер културе
Flora Danica (FD) и комбинације пробиотске стартер културе ABT-1 и традиционалне културе FD у односу 1:1. Фракције испарљивих компонената ароме сира
изоловане су применом симултане дестилације и екстракције (СДЕ). Компоненте
ароме сира идентификоване су применом гасне хроматографије и масене спектрометрије (GC-MS) и квантификоване коришћењем стандардне процедуре. Идентификовано је 19 компонената: 8 угљоводоника (декан, н-ундекан, тридекан, тетрадекан, пентадекан, хексадекан, октадекан и 2,6,10,14-тетраметил хексадекан); 6 кетона (2-хептанон, 2-нонанон, 2-ундеканон, 2-пентадеканон, 2-хептадеканон и 2-тридеканон,); 3 алдехида (нонанал, тетрадеканал и хексадеканал); 1 масна киселина
(деценска киселина) и дисулфид, бис (1-метилетил). У свим узорцима сира најзаступљенији су хексадеканал, 2-пентадеканон, 2-тридеканон и 2-ундеканон.
Кључне речи: Свеж сир, испарљиве компоненте ароме, пробиотици
Received: 26 June 2012
Accepted: 10 September 2012
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CHANGES OF PHYSICAL PROPERTIES OF COFFEE BEANS DURING
ROASTING
Marija R. Jokanovića*, Natalija R. Džinića, Biljana R. Cvetkovićb, Slavica Grujićc and
Božana Odžakovićc 
a
b
University of Novi Sad, Institute for food technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
c
University of Banja Luka, Faculty of Technology, Stepe Stepanovića 73, 78000 Banja Luka,
Republic of Srpska, Bosnia and Herzegovina
The effects of heating time on physical changes (weight, volume, texture and colour)
of coffee beans (Outspan and Guaxupe coffee) were investigated. The roasting temperature of both samples was 170°C and samples for analysis were taken at the intervals of
7 minutes during 40 minutes of roasting. Total weight loss at the end of the roasting
process was 14.43 % (light roasted) and 17.15 % (medium to dark roasted) for Outspan
and Guaxupe coffee beans, respectively. Significant (P < 0.05) changes in the coffee bean
breaking force values were noted between the 7th and 14th minutes, and statistically not
significant (P > 0.05) between the 35th and 40th minutes of the roasting. According to the
L* colour parameter as a criterion for the classification of roasted coffee colour (light,
medium, dark), the Outspan sample was medium and Guaxupe sample was dark roasted.
KEY WORDS: coffee bean, roasting, texture, colour
INTRODUCTION
Coffee is one of the most widely consumed beverages in the world. The high acceptability of coffee is due to many factors, one of the most contributory factors being its
flavour (1). Commercial coffee beverage is made from arabica or robusta beans or blends
of them (2). The quality of coffee used for beverages is strictly related to the chemical
composition of the roasted beans, which is affected by the composition of the green beans
and post-harvesting processing conditions (drying, storage, roasting and grinding) (3).
Green coffee is devoid of the pleasant aroma and flavour appreciated worldwide in
roasted coffee. The desired aroma and flavour of coffee beans used for beverage preparation are developed during the roasting process, where the beans undergo a series of reactions leading to the desired changes in the chemical and physical composition (4). So, in
order to obtain a good quality cup of coffee with specific organoleptic properties (flavour,
* Corresponding author: Marija R. Jokanović, M.Sc., University of Novi Sad, Faculty of Technology, Bulevar
cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]
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aroma and colour), the step of roasting is very important (5). The characteristic flavour
and aroma of coffee result from a combination of hundreds of chemical compounds
produced by the reactions that occur during the roasting (3). This implies controlling the
roasting time and temperature so that they are sufficient for the required chemical reactions to occur, without burning the beans and compromising the flavour of the final beverage. In general, in conventional roasting process the temperature is in the range from 200
to 230C, and the process time is ranging from 12 to 20 minutes. However, these values
can vary greatly, depending on the degree of roast required (light, medium or dark), on
the type of roaster used, and also on the variety, age, moisture content, etc. of the coffee
beans (6). The roasting process can be divided into three consecutive stages: drying,
roasting or pyrolysis and cooling (3).
The degree of roast can be monitored by the colour of the beans, the loss of mass, the
developed flavour and aroma or by the chemical changes of certain components (5, 6).
Control of temperatures and duration of roasting, in industry, are only effective if the
quality of the raw material does not vary (5).
Therefore, the objective of this study was to evaluate the changes of different physical
characteristics such as total weight loss, breaking force, and colour of Outspan and
Guaxupe coffee beans during roasting.
EKSPERIMENTAL
Sample preparation
Two green (crude) coffee samples of Rio Minas, Outspan and Guaxupe, used for the
production of commercial blends, were provided by a local industrial coffee roaster.
Beans of each variety (20 kg/batch) were roasted separately using an oven with direct
heating. The roasting conditions were the same; the highest roasting temperature was
170C, and roasting time was 40 min. For each sampling step (0, 7, 14, 21, 28, 35, 40
min) the coffee beans (100 g) were taken from the oven, and the following determinations were carried out:
Determination of moisture, protein, carbohydrate and ash content
Moisture content was measured based on the sample weight-loss after oven-drying at
105C for 16 h (3). The nitrogen (N2) content of the coffee sample was determined on a
Kjeldahl Digestion System. Protein content was calculated as nitrogen x 6.25 (3). Ash
content was calculated from the weight of the sample after burning at 580C for 17 h (3).
Carbohydrate content was estimated by volumetric method of Luff-Schoorl, which is
based on the reduction of alkaline Cu2+-complex (7). Results are expressed as the mean
value of three measurements.
Total weight loss (WT)
Total weight loss is expressed as g/100 g, and is calculated by weighing coffee
samples before (WI) and after roasting (W), as follows: WT=100 (WI-W)/WI (8). The
results are expressed as the mean value of three measurements.
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Mass, volume and bulk density
Mass was measured for 100 beans. Volume was measured for 100 beans in a 50 mL
graduated recipient.
Bulk density was evaluated as the ratio between the weight and volume of the 100
beans sample in a 50 mL graduated recipient (8). The results are expressed as the mean
value of three measurements.
Mechanical testing
An Instron Universal Testing Machine, equipped with a 25 kN load cell was used.
The uniaxial compression was carried out at a rate of 50 mm/min. For the measurements,
20 beans of each sample were taken at random. Each bean was positioned on its longest
side and with the flat side up between two parallel metal plates. A compression force was
applied until failure occurred; the working temperature was 23C. The breaking force (N)
corresponded to the force at the major failure event. It was considered as an empirical
measure of the strength (8). The results are expressed as the mean value.
Colour analysis
Colour was analysed by using a tristimulus colorimeter MINOLTA CP410. Standard
CIE conditions with illuminate were used. The configuration included the illuminant D65
and an angle of 10. The readings were made using the CIELAB system (L*, a*, b*), and
presented as L* value (colour brightness).
Colour was evaluated for ground coffee beans placed in a suitable tank. The results
are expressed as the mean value of five measurements.
Statistical analyses
Analysis of variance (one-way ANOVA) was used to test the hypothesis about the
differences among the mean values. The software package STATISTICA 8.0 (9) was
used for the analysis.
Table 1 presents the composition of raw Outspan and Guaxupe coffee beans. The data
show that the raw Outspan and Guaxupe coffee beans, according to their chemical
composition, are of the same quality category.
Table 1. Chemical composition of raw Outspan and Guaxupe coffee beans
Parameters
Moisture (%)
Protein (%)
Mono and disaccharide (%)
Total ash (%)
Outspan
10.43±0.02
13.66±0.07
7.45±0.09
4.06±0.12
Guaxupe
9.59±0.06
13.41±0.06
5.16±0.11
3.92±0.07
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The contents of moisture and total ash of both samples are in agreement with Serbian
national legislation (10). The protein contents of the analysed coffee samples (13.66 %
for Outspan and 13.41 % for Guaxupe) are in the range for green coffee reported in the
literature: 11.0–16.5 g/100 g (11). Franca et al. (11) reported that the higher quality
coffee samples have higher protein levels, but there is no evidence suggesting that the
protein contents in the varieties of different quality or even of different species (arabica
vs. robusta) should be noticeably different.
The changes in the weight, volume, bulk density and total weight loss (measured for
100 beans) of Outspan and Guaxupe coffee beans during roasting are presented in Tables
2 and 3, respectively.
Table 2. Changes in weight, volume, density and total weight loss of Outspan coffee
beans during roasting (100 beans)
Heating time
(min)
0
7
14
21
28
35
40
Weight
(g)
14.86±0.04
14.43±0.07
13.81±0.07
13.64±0.04
13.63±0.04
12.98±0.04
12.71±0.04
Volume
(mL)
23.03±0.06
27.10±0.36
28.13±0.15
28.10±0.17
29.10±0.10
31.07±0.12
34.10±0.10
Bulk density
(g/mL)
Weight
loss (%)
0.53±0.009
0.49±0.000
0.49±0.002
0.47±0.000
0.42±0.000
0.37±0.002
2.88
7.09
8.23
8.29
12.64
14.43
Table 3. Changes in weight, volume, density and total weight loss of Guaxupe coffee
beans during roasting (100 beans)
Heating time
(min)
0
7
14
21
28
35
40
Weight
(g)
12.62±0.04
12.10±0.09
11.69±0.03
11.46±0.04
11.35±0.03
11.37±0.03
10.45±0.03
Volume
(mL)
19.13±0.12
20.47±0.06
21.07±0.12
22.10±0.10
24.13±0.15
25.10±0.10
28.57±0.12
Bulk density
(g/mL)
0.66±0.006
0.59±0.003
0.56±0.004
0.52±0.001
0.47±0.002
0.45±0.003
0.37±0.001
Weight
loss (%)
4.11
7.32
9.16
9.90
10.03
17.15
Protein contents of analysed coffee samples (13.66 % for Outspan and 13.41 % for
Guaxupe) are in the range for green coffee reported in the literature: 11-16.5 g/100 g
(11). Franca et al. (11) reported that the higher quality coffee samples present higher protein levels, but there is no evidence suggesting that the protein contents in varieties of different qualities or even of different species (arabica vs. robusta) should be noticeably different.
24
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Changes in weight, volume, bulk density and total weight loss (measured for 100 beans) of Outspan and Guaxupe coffee beans during roasting are presented in Tables 2 and
3, respectively.
At the beginning of the heating process, the total mass of the 100 beans was 14.86 g
for Outspan sample, and 12.62 g for Guaxupe coffee. During the heating, the processed
coffee beans lose mass due to the water loss and loss of volatile materials (11). The total
weight loss at the end of the roasting process was 14.43 % and 17.15 % for Outspan and
Guaxupe coffee beans, respectively. The total weight loss of green coffee beans after
roasting can be one of the criteria for determining the degree of roasting. According to
Oosterveld et al. (12), the weight losses of 11 %, 15 % and 22 % represent light, medium,
and dark roasted coffee beans, respectively.
The increase in the beans volume at the end of the heating process was 48 % for
Outspan coffee beans, and 50 % for Guaxupe beans. The results of Franca and co-workers (11) for the volume increase of samples of different quality were 40 – 65 %, where
the volume increase of non-defective beans was higher than for black beans. Also, these
authors reported that the beans which swell less should be roasted more slowly.
Further, the changes in the density are noticeable during the roasting. These changes
are caused by the simultaneous increase of the volume and internal gas formation, products of the heat-induced reactions (mainly water vapour and carbon dioxide, and pyrolysis reaction products), and the decrease in the mass (due to the loss of volatiles) (8).
Bulk density changes are implied in bean expansion and in the formation of a characteristic porous structure of the roasted coffee bean (13). The variations in the bean density
and volume probably reflect the bean porosity and compressibility of ground coffee, thus
being a consequence of commercial percolation (11). If the changes of density are detected, they can determine eligible roasting degree. An adequate roasting degree is needed
for coffee beans to be fragile and breakable, and as such proper for grinding and making
coffee beverages with pleasant sensory properties (8).
The results of breaking force for Outspan and Guaxupe coffee beans after different
heating time are presented in Table 4.
Table 4. Breaking force of Outspan and Guaxupe coffee beans during roasting (n=20)
Heating time
(min)
7
14
21
28
35
40
a,b,c
Outspanns
Breaking force (N)
96.76±18.86 a
69.58±15.49 b
64.13±18.75 b
63.03±16.99 b
52.12±15.14b,c
37.82± 5.67 c
Guaxupens
Breaking force (N)
94.75±15.87 a
69.30±18.00 b
68.43±17.79 b
66.10±15.66 b
52.78±15.61 b
35.39±9.50 b
Means within a column with different superscripts differ (P < 0.05)
ns
Means within a row no significant difference (P > 0.05)
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The textural characteristics of roasted beans could be related to the effects of some
chemical changes induced on the raw bean components by the severe thermal process
(13). The braking force values after 7 minutes of heating were similar (P > 0.05) for both
samples, 96.76 N for Outspan and 94.75 N for Guaxupe. According to Pittia et al. (13), a
higher breaking force value for raw coffee beans could be attributed, partly, to the
presence of a certain amount of some structural polysaccharides. Also, the force needed
to break the bean depends on the content of water in the bean. When the content of water
is low, the bean has more fragile and breakable structure, and when the content of water
is high, the bean is no longer crunchy, and becomes viscose and plastic (8). In both cases,
for Outspan and Guaxupe coffee, as the heating time went by, the force at failure tended
to decrease, reaching, again, similar values (37.82 N and 35.39 N) at the end of the
process. The reduction of the breaking force indicates a progressive reduction in the
strength of the bean.
(a)
7
100
Breaking force (N)
6
Moisture (%)
120
Moisture (%)
80
5
4
60
3
40
2
Breaking force (N)
8
20
1
0
0
0
10
20
30
Heating time (min)
40
50
(b)
Density (g/mL)
Breaking force (N)
Density (g/mL)
0.50
120
100
0.40
80
0.30
60
0.20
40
0.10
20
0.00
Breaking force (N)
0.60
0
0
10
20
30
Heating time (min)
40
50
Figure 1. Changes in moisture (a), bulk density (b) and breaking force value of Outspan
coffee beans during roasting
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The breaking force can also show the degree of roasting, but to a lesser extent than the
water content can (13). The characteristic brittleness and fragility induced by roasting is
the primary attribute of roasted coffee beans. The reaching of a certain degree of brittleness is very important in the grinding process, which is carried out on roasted coffee
beans before the extraction of coffee brew (8).
(a)
100
Moisture (%)
Breaking force (N)
7
80
Moisture (%)
6
5
60
4
40
3
2
20
1
Breaking force (N)
8
0
0
0
10
20
30
40
50
Heating time (min)
(b)
0.60
Density (g/mL)
100
Density (g/mL)
Breaking force (N)
80
0.50
0.40
60
0.30
40
0.20
20
0.10
Breaking force (N)
0.70
0
0.00
0
10
20
30
Heating time (min)
40
50
Figure 2. Changes in moisture (a), bulk density (b) and breaking force value of Guaxupe
coffee beans during roasting
The influence of moisture content and bulk density on the breaking force values of
Outspan and Guaxupe coffee beans are shown in Fig. 1 and 2. The coffee beans with the
lowest moisture content and the lowest bulk density had the lowest breaking force values.
The water content has a great influence on the texture of roasted beans, and therefore
influence the work applied during the grinding process (13). According to the results of
Pittia et al. (13) the lowest breaking force values are reached by the samples of coffee
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beans having very low moisture content. Water, acting as plasticizer, is expected to
decrease the bean’s stiffness. Therefore, decreasing the moisture content should increase
the stiffness of the material up to the glassy state. When the moisture of coffee bean is
very low (1.5 - 2 g/100g), it permits a glassy-like structure to form, which is very easy to
break (13). As can be seen from Figs. 1 and 2, there is an initial (between 7th and 14th minutes of roasting) significant (P < 0.05) decrease in the breaking force that could be attributed to the higher water loss and higher density decrease caused at high temperatures.
This can be explained by the finding of Massini et al (14), who analysed coffee beans
under industrial roasting conditions (200 - 210C), and found formation of cavities and
cracks after 4 minutes of roasting in the internal and external bean surface due to the relevant increase in the internal pressure and volume of coffee bean. Pittia et al. (13) reported the relation of texture changes of coffee beans roasted at 170 and 200C to the changes of density.
60
Outspan
Guaxupe
50
L*
40
30
20
10
0
0
7
14
21
28
Heating time (min)
35
40
Figure 3. Changes in L* value of Outspan and Guaxupe coffee beans during roasting
In coffee, the characteristic colour, aroma and flavour are developed during roasting,
and thus it is necessary to adapt the roasting process to the type of coffee being roasted.
This implies controlling the roasting time and temperature so that they are sufficient for
the required chemical reactions to occur, without burning the beans and compromising
the flavour of the beverage (6). During roasting, due to the non-enzymatic browning and
pyrolysis reactions, changes in the coffee bean colour take place. So, beside the loss of
mass and the chemical changes of certain components which could serve as tools in the
control of the process, the effects of heating and the degree of roast can also be monitored
by the colour of the beans (5). Yellow-green colour of the raw bean changes to a brownblack roasted colour (13). Browning is, in turn, described by a decrease of L* as well as
of a* and b* parameters. These colour changes for samples of Outspan and Guaxupe
coffee beans are shown by the decrease of the L* value in Fig. 3. The initial L* value of
48.72 and 49.32 for raw coffee beans decreased during roasting to the final values of
26.77 and 24.45 for Outspan and Guaxupe, respectively. In their paper Pittia et al. (8)
stated the criteria for the classification of differently roasted coffee samples on the basis
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of the L* colour parameter: the samples are classified as light, medium or dark roasted
when L* value is 31.1, 26.0 and 24.3 respectively. According to these criteria, it can be
concluded that Outspan sample was medium and Guaxupe was dark roasted.
CONCLUSION
On the basis of obtained the results, it is possible to summarise that the moisture
content, and density changes, mainly affect the mechanical properties of coffee beans
during roasting. Bulk density values at the end of the heating process were the same for
both coffee samples, 0.37g/mL, and the mean values of bean's breaking force were also
very similar (P > 0.05), 37.82 N for Outspan and 35.39 N for Guaxupe. According to the
total weight loss of green coffee beans after roasting the Outspan coffee was medium
roasted, and Guaxupe coffee was between medium and dark roasted. According to the
values of L* colour parameter at the end of roasting process it can be concluded that the
Outspan sample was medium and Guaxupe dark roasted.
It is possible to control and standardize the quality of ground roasted coffee as the
final product by way of applying an adequate roasting procedure. In conventional
roasting, the temperature range and the time can vary greatly, depending on the degree of
roast required (light, medium or dark), on the type of used roaster, and also depend on the
variety, age, moisture content, and other quality characteristics of the coffee.
REFERENCES
1. Kumazawa, K., Masuda, H.: Investigation of the Change in the Flavor of a Coffee
Drink during Heat Processing. J. Agric. Food Chem. 51, (2003) 2674-2678.
2. Martin, M.J., Pablos, F., Gonzalez, A.G.: Discrimination between arabica and robusta
green coffee varieties according to their chemical composition. Talanta. 46, (1998)
1259-1264.
3. Franca, A. S., Mendonca, J. C. F., Oliveira, S. D.: Composition of green and roasted
coffees of different cup qualities. LWT. 38, (2005) 709–715.
4. Dutra, E. R., Oliveira, S., Franca, A. S., Ferraz, V. P., Afonso, R. J. C. F.: A preliminary study on the feasibility of using the composition of coffee roasting exhaust
gas for the determination of the degree of roast. Journal of Food Engineering. 47,
(2001) 241-246.
5. Hernandez, J. A., Heyd, B., Irles, C., Valdovinos, B., Trystram, G.: Analysis of the
heat and mass transfer during coffee batch roasting. Journal of Food Engineering. 78,
(2007) 1141-1148.
6. Mendes, C. L., De Menezes, C. H., Aparecida, M., da Silva, A. P.: Optimization of
the roasting of robusta coffee (C. canephora conillon) using acceptability tests and
RSM. Food Quality and Preference. 12, (2001) 153-162.
7. Pravilnik o metodama uzimanja uzoraka i vršenja hemijskih i fizičkih analiza radi
kontrole kvaliteta proizvoda od voća i povrća, Službeni list SFRJ 29/1983.
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8. Pittia ,P., Nicoli, M. C., Sacchetti, G.: Effect of moisture and water activity on textural properties of row and roasted coffee beans. Journal of Texture Studies. 38, (2007)
116-134.
9. StatSoft, Inc.: STATISTICA (data analysis software system), version 8.0, 2008. Available from: http://www.statsoft.com/.
10. Pravilnik o kvalitetu i drugim zahtevima za sirovu kafu, proizvode od kafe i surogat
kafe, („Sl.list SRJ“, br.35/2001 i 49/2001-ispr.i „Sl.list SCG“,br.56/2003 - dr.pravilnik i 4/2004 -dr.pravilnik).
11. Franca, A. S., Oliveira, S. L., Mendonca, C. F. J., Silva, A. X.: Physical and chemical
attributes of defective crude and roasted coffee beans. Food Chemistry. 90, (2005)
89-94.
12. Oosterveld, A., Harmsen, J. S., Voragen, H. A.: Extraction and characterization of
polysaccharides from green and roasted Cofee arabica beans. Carbohydrate Polymers. 52, (2003) 258-296.
13. Pittia, P., Dalla Rosa, M., Lerici, C. R.: Textural changes of coffee beans as affected
by roasting conditions. Lebensm.-Wiss.Technol. 34, (3) (2001) 168-175.
14. Massini, R., Nikoli, M. C., Cassara, A., Lerici, C. R.: Study on physical and physicochemical changes of coffee beans during roasting. Italian Journal of Food Science 2,
(1) (1990) 123-130.
ПРОМЕНЕ ФИЗИЧКИХ СВОЈСТАВА ЗРНА КАФЕ ТОКОМ ПЕЧЕЊА
Марија Р. Јокановића*, Наталија Р. Џинића, Биљана Р. Цветковићб, Славица
Грујићц и Божана Оџаковићц
а
Универзитет у Новом Саду, Институт за прехрамбене технологије, Булевар цара Лазара 1, 21000 Нови
Сад, Србија
ц
Универзитет у Бањој Луци, Технолошки факултет, Степе Степановића 73, 78000 Бања Лука, Република
Српска, Босна и Херцеговина
б
У овом раду испитане су промене физичких својстава (маса, запремина, текстура и боја) зрна кафе сорти Outspan и Guaxupe, које се користе за производњу комерцијалних мешавина, у различитим временским интервалима топлотне обраде.
Топлотна обраде обе соре била је индентична, примењена је максимална температура од 170C у току 40 минута. Узорци за анализе узиману су током просеца топлотне обраде у временским интервалима од 7 минута.
Укупан губитак масе зрна на крају процеса печења био је 14,43% и 17,15% за
Outspan и Guaxupe, редом и према том критеријуму узорак Outspan кафе био је
средње печен, док је узорак Guaxupe кафе био средње до тамно печен. Током топлотне обраде смањивала се и просечна вредност силе лома зрна кафе. Највеће, статистички значајне (P < 0,05) разлике вредности силе лома за оба узорка уочене су
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имеђу 7. и 14., као и нумеричке, али не и статистички значајне (P > 0,05) разлике
између 35. и 40. минута топлотне обраде.
L* вредност, као један од параметара за дефинисање степена печености зрна
кафе, током процеса печења се смањује, односно зрно постаје тамније. Према критеријумима за дефинисање степена печења на основу L* вредности може се закључити да је узорак Outspan (26,77) кафе средње, а узорак Guaxupe (24,45) кафе тамно
печен.
Кључне речи: зрно кафе, печење, текстура, боја
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EFFECTS OF SONICATION AND HIGH-PRESSURE CARBON DIOXIDE
PROCESSING ON ENZYMATIC HYDROLYSIS OF EGG WHITE PROTEINS
Zorica D. Knežević-Jugović*, Andrea B. Stefanović, Milena G. Žuža, Stoja L.
Milovanović, Sonja M. Jakovetić, Verica B. Manojlović and Branko M. Bugarski
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia
The objectives of this study were to examine the effect of sonication and high-pressure
carbon dioxide processing on proteolytic hydrolysis of egg white proteins and antioxidant activity of the obtained hydrolysates. It appeared that the ultrasound pretreatment resulted in an increase in the degree of hydrolysis of the enzymatic reaction while
the high-pressure carbon dioxide processing showed an inhibition effect on the enzymatic
hydrolysis of egg white proteins to some extent. The antioxidant activity of the obtained
hydrolysates was improved by ultrasound pretreatment of egg white proteins at the pH
8.3. Thus, the combination of ultrasound pretreatment at the pH 8.3 and subsequent enzymatic hydrolysis with alcalase at 50oC and pH 8.0 could offer a new approach to the improvement of the functional properties of egg white proteins and their biological activity.
KEY WORDS: Egg white proteins, alcalase, antioxidant activity, ultrasound pretreatment, high-pressure carbon dioxide processing
INTRODUCTION
Egg producers are faced with the problems of excess of egg white because the mayonnaise and bakery industries use relatively large amounts of egg yolk, and egg white is
the remainder. Although egg white proteins (EWPs) have unique functional characteristics such as excellent gelling and foaming and almost perfect amino acid composition,
their high viscosity and allergenicity are limiting factors for their widespread use in food
products, especially in the case of medical, dietary and infant foods (1).
Enzymatic hydrolysis of egg white proteins has been proven to be an effective approach to improve their properties such as increased solubility, emulsification, water holding
capacity, stability, digestibility, and to reduce protein allergenicity while still retaining
their nutrition value (2). Moreover, certain oligopeptides released during protein hydrolysis have been shown to possess distinctive physiological activities, such as anti-hypertensive activity, antioxidant activity, immunostimulating activity, and as such may contribute to enhanced biological activities and health benefits of the hydrolysate (3). However,
the production of these functional ingredients requires new and innovative technologies
* Corresponding author: Zorica D. Knežević-Jugović, University of Belgrade, Faculty of Technology and
Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia, e-mail: [email protected]
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because they are sensitive to a variety of environmental processing factors that may cause
the loss of nutritional quality and chemical degradation.
Major proteins in chicken egg white including ovalbumin and ovotransferin, in their
native forms have a low susceptibility to digestion by alcalase, trypsin or -chymotrypsin. Heat denatured ovalbumin however, shows an increased susceptibility to these proteases (4). However, thermal treatments are obviously the most critical steps able to impair protein functionalities, inducing various chemical reactions such as Maillard browning which may lead to nutritional, sensory and safety deterioration in egg white hydrolysates. Several papers reported a decrease in the nutritional properties of different natural protein sources due to thermal processing. Thus, a lot of research works have been
performed to investigate the effectiveness of replacing conventional thermal treatment
with several non-thermal alternative approaches for improvement of the overall process
performances of EWP hydrolysis and produce protein solutions with new functional
properties (4,5).
High-pressure processing (HPP) is popular as an alternative to heat pasteurisation for
various food systems because it can be used to obtain stable products with minimal effects on flavour, colour and nutritional value or to create novel texture and taste (6,7). To
date, there seem to be a limited number of commercial processes based on HPP, mainly
because the process has not yet been optimized with respect to yield and process cost and
the overall lack of knowledge of HPP effects on food systems, especially egg white proteins.
The purpose of this research was to study the effect of ultrasound pretreatment and
high-pressure carbon dioxide processing on the in vitro digestibility of egg white proteins
by alcalase. The influence of pretreatment parameters including pH, pressure, and holding time on enzymatic hydrolysis of pretreated EWPs was studied. The antioxidant activity of the obtained hydrolysates has also been determined and compared.
EXPERIMENTAL
Materials
Chicken egg white obtained from a local supermarket was separated from the yolk
and gently stirred without foam formation to provide homogeneous mixture. Alcalase
2.4L (proteinase from Bacillus licheniformis Subtilisin) was obtained from Sigma Aldrich (St Louis, MO, USA). The enzyme activity was ≥2.4 U/g Anson Units, where one
Anson unit is defined as the amount of enzyme which, under specified conditions, digests
urea-denatured hemoglobin at an initial rate such that there is liberated an amount of
TCA-soluble product per minute which gives the same colour with the Folin-Ciocalteu
Phenol reagent as one milliequivalent of tyrosine at 25°C at the pH 7.50. 2,2-Diphenyl-1picrylhydrazyl (DPPH) used for radical scavenging test was also purchased from Sigma
Aldrich (St Louis, MO, USA). Commercial carbon dioxide (99% purity) was supplied by
Messer-Tehnogas (Serbia). Other chemicals were of analytical grade.
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Sonication pretreatment
Ultrasonic denaturation was investigated in the pH range of 6.0 to 10.0 at 27°C in an
ultrasonic water bath. Prior to sonication, the pH of the 10% (w/w) egg white solution
was adjusted to 6.0-10.0 using 0.1 M HCl or 0.1 M NaOH. The ultrasound treatments
were performed for 15, 30, 60, and 180 min under a power setting of 30 kHz. The samples were half-immersed in an ice-water bath to avoid temperature increase during sonication. Each treatment was conducted in duplicate.
High-pressure carbon dioxide processing
The experiments of denaturation of EWPs were conducted in the Autoclave Engineers
Screening System with the configuration previously described (8), shown in Figure 1.
The extractor vessel (150 ml) was filled with 10% (w/w) egg white solution, and temperature was risen to 35°C. Heaters were supplied on the extractor vessel for temperature
elevation. A thermocouple, connected to a temperature controller, was used to control
and maintain a constant temperature.
PI
TI
V2
TIC
PC
BPR2
S
V3
CO2
E
PI
V1
C
LP
PC
BPR1
CO2 TANK
Figure 1. Scheme of the autoclave engineers screening system - C: cryostat; LP: high
pressure liquid pump; BPR: back pressure regulators; E: extractor vessel; S: separator
vessel (8).
Liquid CO2 was supplied from a CO2 cylinder by a siphon tube. The liquid CO2 was
cooled in a cryostat between the cylinder outlet and the pump. The pump operated at a
maximum output pressure of 41.3 MPa and an adjustable flow rate from 38 to 380 mL/h.
The CO2 was pumped into the system until the required pressure was obtained. Back
pressure regulators were used to set the system pressure (in the extractor and separator).
The sample was exposed to SC CO2 at 35°C and 10 MPa, 20 MPa and 30 MPa for 20
min in a batch vessel. After 20 min of exposure, the pressure was decreased very fast to
atmosferic conditions. During the decompression, the SC CO2 flew through the extractor
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and entered the separator vessel (500 ml) with treated egg white solution. The CO2 continued to flow out of the separator through the flowmeter/totalizer and out to atmosphere.
The pressure and temperature were controlled to an accuracy of ±0.4 MPa and ±0.5°C,
respectively.
Samples of the treated egg white solution were taken by opening the ball valve located at the bottom of the separator vessel.
Enzymatic hydrolysis of EWPs
The pretreated aqueous egg white solutions (360 mL, 10 mg of protein/mL, pH 8.0)
were hydrolysed at 50 oC by adding 2.3 Units of alcalase. The pH was kept at 8.0 by
adding 0.01 M NaOH, using a pH-stat (Metrohm, Basel, Switzerland) with automatic
dosage of the base. The degree of hydrolysis (DH) was used as a parameter to measure
the effect of ultrasound and high-pressure pretreatment on the susceptibility of egg white
proteins to enzymatic hydrolysis. After 4 h of incubation, the reaction was terminated by
heating during 10 min on a boiling water bath. The DH was calculated by the pH stat
method according to Adler-Nissen (9), using the following equation:
DH 
h
1 1
1
 Nb  B   
100
htot
 mp htot
[1]
where h is the number of equivalents of peptide bonds hydrolysed at the time t; htot is the
total number of peptide bonds in protein substrate in mmol/gprotein; B is the base consumption in mL; Nb is the base normality;  is the average degree of dissociation of the
-NH groups, and mp is the mass of protein in g.
Antioxidant activity measured by DPPH assay
The antioxidant activity of EWP hydrolysates was measured by their ability to scavenge DPPH radical, which was monitored via the decrease of the absorbance at 517 nm,
as described elsewhere (10). A volume of 200 µL of samples was mixed, in spectrophotometric cuvette, with 1800 µL of methanolic DPPH solution (0.1 mM), vortexed and left
in dark and after 30 min absorbance was measured on 517 nm. The calculation was done
as follows:
  A  A0 
RSA %   1  s

Ab


[2]
where As is the absorbance of the tested sample; A0 is the absorbance of the sample in methanol, and Ab is the absorbance of the DPPH solution without the sample.
Sonication pretreatment
As sonication-induced protein denaturation strongly depends on the temperature and
pH at which pretreatment occurs, the effect of ultrasound pretreatment was studied at
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constant temperature and at three pH values: 6.0, 8.3 and 10.0. The aim of optimization
was to find the conditions that would lead to irreversible denaturation of the alcalase inhibitor, but would not result in the formation of dense and compact precipitates, which are
poor substrate for proteolysis.
As shown in Figure 2, it appeared that for all cases studied, the ultrasonic pretreatment changed the proteolytic pattern of EWPs, resulting in an increase in their digestibility by alcalase. This ultrasound-dependent increase in the susceptibility to enzymatic
hydrolysis is in accordance with the results of Su et al. (11), who observed an increase of
susceptibility to alcalase hydrolysis of egg white after 120 min treatment at 40 kHz. It is
considered that the sonication produces hemolytic water molecule cleavage, generating
high-energy intermediates such as hydroxyl and hydrogen free radicals, and therefore,
causing structural changes of proteins.
Figure 2. Comparison of the DH profiles of EWPs hydrolysis by alcalase at the pH 8.0
and 50oC. The labels indicate samples with different sonication pretreatment (0/10 means
alkali pretreatment at the pH 10 without ultrasound treatment; 1/10 means 1 h sonication
at the pH 10.0; 0/8.3 means alkali pretreatment at pH 8.3 without ultrasound treatment;
1/8.3 means 1 h sonication at pH 8.3; 0/6 means acid pretreatment at the pH 6.0 without
sonication; and 1/6 means 1 h sonication at the pH 6.0).
In contrast, an increase in the sensitivity of ovotransferrin to proteolysis by using
thermolysin after sonication was not observed by Lei et al. (4). However, these authors
studied the effect of ultrasound pretreatments on model egg white protein – ovotransferrin. In this case, the reaction mixture seemed to be free from proteases’ inhibitors such as
ovomucoid. It is one of the major egg white protein accounting for 11% in egg white,
well known as a strong inhibitor of alcalase. The increase in the susceptibility of egg
white solutions to enzymatic hydrolysis after sonication could also be due to the ovomucoid denaturation.
The strongest increase in the susceptibility of egg white solution to enzymatic hydrolysis was observed after ultrasound treatment at an acidic or alkaline pH. The partially
unfolded conformation of the major EWPs formed by sonication may be attributed to the
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increase of the susceptibility but this also could be the result of disulfide exchange and
formation of non-native disulfide bonds in ovomucoid, resulting in its irreversible denaturation. It appeared that its effective denaturation was attained by combined alkali/acid
and ultrasonic treatment.
A longer treatment time (up to 60 min) resulted in a higher subsequent susceptibility
to enzymatic hydrolysis (Figure 3).
Figure 3. Time-dependent changes in the susceptibility of egg white solutions due to
ultrasound pretreatment at the pH 10 lasting 0 min ( ); 15 min (), 30 min (▲), 60 min
(▼), and 180 min (). Hydrolysis condition: 2.3 Units of alcalase, 10% w/w egg white,
pH 8.0, 50 oC.
The prolonged exposure to ultrasound of 60 min at the pH 10.0 seemed to have a
negative effect on the EWPs hydrolysis. Under this condition, a turbid suspension of protein aggregates could be observed, which might lower the accessibility of the unfolded
proteins to the alcalase.
High-pressure carbon dioxide processing
As shown in Figure 4, the high-pressure carbon dioxide processing showed an inhibitory effect on the enzymatic hydrolysis of EWP. The highest degree of hydrolysis was
observed under the pretreatment at 30 MPa, but it was still lower than that achieved without pretreatment. Hayashi et al. (12) reported an extensively improved digestibility by
subtilisin of homogenized pressure-induced egg white gels as compared to raw egg white.
Although some other researchers showed that carbon dioxide treatment could inactivate pectin esterase, lipoxygenase, polyphenol oxidase and peroxidase either in pure enzymatic solutions or in real food systems, the understanding of the synergetic effect between ultrasounds and enzymes is still far from being completely understood.
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Figure 4. Comparison of the DH profiles of EWPs hydrolysis by alcalase at the pH 8.0
and 50 oC. The samples were pretreated under different high pressure carbon dioxide
conditions: () 10 MPa; (▲) 20 MPa; (▼) 30 MPa.
Antioxidant activities of sonication-pretreated alcalase hydrolysates
The release of bioactive peptides from their parent proteins is affected by various
factors such as temperature, pH, enzyme, pressure, sonication, and others. The production
of bioactive peptides was monitored after the hydrolysis. The antioxidant activities of
samples treated under different ultrasound conditions followed by alcalase hydrolysis are
compared in Figure 5.
Radical scavenging activity, RSA (%)
45
40
35
30
25
20
15
10
5
0
1/8.3
0/8.3
1/10.0
0/10.0
1/6.0
0/6.0
0/0
Sample
Figure 5. Effects of sonication on the radical scavenging activity of egg white hydrolysates obtained with or without sonication treatments. Data were the results of three individual determinations. Labels of 0/0 means raw egg white, 0/6.0 means acid egg white treatment without sonication treatment, 0/10 means alkali but no sonication treatment, 1.6.0,
1/8.3, and 1/10.0 means 1 hours sonication at the pH 6.0, 8.3, and 10.0, respectively.
Error bars represent the standard deviations of triplicate measurement.
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The results indicate that 1-hour sonication treatment at the pH 8.3 improved the antioxidant activity of raw egg white, while the sonication at the pH 6.0 or at 10.0 did not have a substantial effect on radical scavenging activity.
CONCLUSION
The study addressed the effect of the ultrasound pretreatment at 27oC and at atmospheric pressure and of high-pressure carbon dioxide processing in the range of 10-30
MPa on the enzymatic hydrolysis of EWPs with alcalase. As the pH seemed to strongly
affect the ultrasound induced protein denaturation, the effect of pH during sonication in
the range 6.0-10.0 on the egg white susceptibility to enzymatic hydrolysis was investigated. The antioxidant activity of obtained hydrolysates was also tested. The ultrasound pretreatment of egg white proteins resulted in an increase in the degree of hydrolysis of the
enzymatic reaction, while the high-pressure carbon dioxide processing showed an inhibition effect on the enzymatic hydrolysis of EWPs to some extent. The antioxidant activity
of the obtained hydrolysates was improved by ultrasound pretreatment of EGPs at the pH
8.3. Thus, the combination of ultrasound pretreatment at the pH 8.3 and subsequent enzymatic hydrolysis with alcalase at 50oC and pH 8.0 could offer a new approach to improve
the functional properties of EWPs and their biological activity. The sonication procedure
is simple, rapid and efficient, and could be useful for the industrial production of functional products from egg white.
Acknowledgement
This work was supported by the Ministry of the Education and Science of the Republic of Serbia, Grant number E!6750.
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415-420.
11. Su, Z., Li, X. and Jin, L.: Comparison Between Enzymatic Hydrolysis of Egg White
Proteins with Ultrasound and Without Ultrasound. Food Sci. Technol. (Chinese) 31,
12 (2006) 74-76.
12. Hayashi, R., Y. Kawamura, T. Nakasa and Okinaka, O.: Application of High Pressure
to Food Processing: Pressurization of Egg White and Yolk, and Properties of Gels
Formed. Agric. Biol. Chem. 53 (1989) 2935-2939.
УТИЦАЈ СОНИКАЦИЈЕ И ПРЕТРЕТМАНА ВИСОКИМ ПРИТИСКОМ НА
ЕНЗИМСКУ ХИДРОЛИЗУ ПРОТЕИНА БЕЛАНЦЕТА
Зорица Д. Кнежевић-Југовић, Андреа Б. Стефановић, Милена Г. Жужа, Стоја Л.
Миловановић, Соња М. Јаковетић, Верица Б. Манојловић и Бранко М. Бугарски
Универзитет у Београду, Технолошко-металуршки факултет, Карнегијева 4, 11000 Београд, Србија
Протеини беланцета спадају у веома квалитетне протеине због свог јединственог аминокиселинског састава. Међутим, већа комерцијална примена хидролизата
протеина беланцета је ограничена услед неадекватног процесног третмана при обради и стерилизацији беланцета термичким третманом као и хемијској хидролизи
протеина, који довoде до значајне промене боје, укуса, функционалности и нутритивних својстава производа. Циљ овог рада је био да се испита могућност примене
нетермичких третманима, као што су соникација и третман високим притиском, да
би се унапредила ензимска хидролиза протеина беланцета и омогућило добијање
хидролизата са антиоксидативном активношћу. Показано је да третирање протеина
беланцета ултразвуком под одређеним условима доводи до њихове касније побољшане хидролизе алкалазом, док процесирање беланцета високим притиском има
негативан утицај на активност алкалазе. Антиоксидативна активност добијених
хидролизата је повећана након претретмана ултразвуком на pH 8,3. Тако, комбинација претретмана протеина беланцета ултразвуком на pH 8,3 и њихова сукцесивна
хидролиза алкалазом на 50оC и при pH 8,0 се показала као ефикасна алтернативна
метода за добијање хидролизата протеина беланцета са унапређеним функционалним својствима и биолошком активношћу.
Кључне речи: протеини беланцета, алкалаза, антиоксидативна активност,
претретман ултразвуком, процес под високим притиском угљен-диоксидa
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DIRECT DETERMINATION OF CALCIUM, SODIUM AND POTASSIUM IN
FERMENTED MILK PRODUCTS
Snežana Ž. Kravić*, Zvonimir J. Suturović, Ana D. Đurović, Tanja Ž. Brezo, Spasenija D.
Milanović, Radomir V. Malbaša and Vladimir R. Vukić
The aim of this study was the investigation of the possibilities of direct determination
of calcium, sodium and potassium in the commercial and kombucha-based fermented
milk products by flame photometry. Two procedures were used for sample preparation:
simple dilution with water (direct method) and extraction with mineral acid. Calcium, sodium and potassium levels determined after mentioned sample preparation methods were
compared. The results showed that the differences between the values obtained for the
different sample treatment were within the experimental error at the 95% confidence level. Compared to the method based on extraction with mineral acid, the direct method is
efficient, faster, simpler, cheaper, and operates according to the principles of Green Chemistry. Consequently, the proposed method for the direct determination of calcium, sodium and potassium could be applied for the rapid routine analysis of the mineral content
in the fermented dairy products.
KEY WORDS: calcium, sodium, potassium, fermented milk product
INTRODUCTION
Milk and dairy products are composed of macronutrients (proteins, lipids and sugars)
contributing to their nutritional and biological values. They contain also micronutrients
like minerals and vitamins. These minerals and vitamins, which are quantitatively minor
compounds, are not sources of energy but are essential for the life because they contribute to multiple and different vital functions in the organism, like bone structure, homeostasis, muscular contraction, metabolism via the enzymatic systems, etc. The mineral fraction of milk (approximately 8–9 g/l) is composed of macroelements (Ca, Mg, Na, K, P
and Cl) and oligoelements (Fe, Cu, Zn and Se) (1, 2). Macroelements are differently distributed in the aqueous and micellar phase of milk, depending of their nature. The monovalent cations, Na+ and K+, are present mainly in the free form and only to a limited extent in the form of ion pairs. The divalent cations, Ca2+ and Mg2+, play the role in the
physicochemical properties of casein micelles, such as gelation induced by acid and rennet, heat stability, ethanol stability and sediment formation (3).

* Corresponding author: Snežana Kravić, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
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Calcium is an essential macronutrient for humans, which represents approximately
2% of body weight in an adult person (4). This element has mainly a structural function
in bones and teeth, along with the regulation of many vital biological functions. More recently, the interest in calcium has centered on its role in preventing osteoporosis. It is
known that the highest demands for this element occur during the periods of maximum
growth such as in childhood and adolescence, and also during lactation and in the elderly.
No less than 75% of calcium in many western diets originates from milk. The bioavailability of calcium and magnesium in milk is considered to be excellent since the intestinal
absorption of these nutrients is facilitated by lactose (5). Moreover, it has been demonstrated by in vitro and in situ experiments that phosphopeptides, released during the digestion of casein micelles, increase the concentration of soluble calcium in the intestine
and enhance calcium absorption.
Sodium and potassium concentrations in the body are 1.4 g/kg and 2 g/kg, respectively (6) Sodium is present mostly as an extracellular constituent and maintains the osmotic pressure of the extracellular fluid. In addition, it activates some enzymes, such as
amylase. From the nutritional standpoint, only the excessive intake of sodium is of importance because it can lead to hypertension. Milk is not a rich source of sodium, so that
the contribution of milk and dairy products to the intake of sodium is modest. Potassium
is most common cation in the intracellular fluid. It regulates the osmotic pressure within
the cell and is involved in cell membrane transport and also in the activation of a number
of glycolytic and respiratory enzymes (6). Potassium deficiency causes muscular weakness, mental confusion, and abnormalities in the electrocardiogram (5). Potassium is the
most abundant cation in eukaryotic cells and is thus amply supplied when intact or moderately altered tissues from plant or animal foods are consumed. Milk and yogurt, as
well as nuts, are also excellent sources of potassium.
Several analytical techniques have been used to quantify some minerals in milk and
derivates, mainly flame photometry (7), flame atomic absorption spectrometry (8), atomic emission spectrometry (9), and inductively-coupled plasma mass spectrometry (10).
Most of the analytical techniques used to determine the specific mineral content require
the minerals to be dissolved in an aqueous solution. For this reason, it is often necessary
to isolate the minerals from the surrounding organic matrix, prior to the analysis. This is
usually carried out by dry or wet decomposition of the samples in open or closed systems,
using thermal, ultrasonic, infrared, and microwaves energy. Sample preparations is time
limiting, requiring ca. 61% of the total time to perform the complete analysis, and is
responsible for 30% of the total analysis error (11). Nowadays, the goals to be reached
are the best results in the shortest time, with minimal contamination, low reagent and
energy consumption, and generation of minimal residue or waste. So, the main goal of
this study was to investigate the possibilities of direct determination of calcium, sodium
and potassium in the fermented milk products by flame photometry..
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EXPERIMENTAL
Chemicals
Hydrochloric acid 36.5% was purchased from Centrohem (Stara Pazova, Serbia), lanthanum (III) oxide and calcium carbonate were purchased from Merck (Darmstadt, Germany) while sodium chloride and potassium chloride were purchased from Alkaloid
(Skoplje, Macedonia). All used chemicals were of analytical reagent grade.
Apparatus
The determination of calcium, sodium and potassium was carried out by flame photometry using Evans Electroselenium LTD flame photometer (Halstead, Essex, England) in
air-butane flame.
Samples
Thirteen samples of fermented milk products were used. Nine of them were commercial products purchased in local market, while four samples were obtained by the fermentation of cow’s milk with kombucha cultivated on black, stinging nettle, mint tea and
winter savory tea, respectively, as previously described (12). The analyzed samples are
given in Table 1.
Table 1. Overview of analyzed samples
1.
Sample type
“AB” yogurt
2.
“Viva” yogurt
3.
“Balans +” yogurt
4.
„Zdravo“ yogurt
5.
„Dukat“ yogurt
6.
„Dukat b-aktiv LGG natur“ yogurt
7.
8.
„Balans +“ kefir
„Ella“ yogurt
9.
„Danone Activia“ yogurt
10.
11.
12.
13.
Kombucha based milk product 1
Starter cultures
Lactobacillus delbrueckii subp.bulgaricus
Streptococcus thermophilus
Lactobacillus acidophilus LA-5
Bifidobacterium BB-12
Bifidobacterium lactis HNO-19
Lactobacillus acidophilus LA-5
Bifidobacterium BB-12
Lactobacillus rhamnosus ATCC 53103
Mesophilic and probiotic kefir cultures
Selected yogurt cultures
Bifidus actiregularis
Inoculum of kombucha cultivated on black tea
Inoculum of kombucha cultivated on stinging nettle tea
Inoculum of kombucha cultivated on mint tea
Inoculum of kombucha cultivated on winter savory tea
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Sample preparation
In order to investigate the possibility of using the direct method for the determination
of calcium, sodium and potassium in the fermented milk products by flame photometry
two procedures were used for the sample preparation.
Direct method: After the homogenization by mixing, 2.5 g of the sample was transferred to a calibrated flask, 2 cm3 of 10% solution of lanthanum was added and diluted to
a final volume of 50 cm3 with distilled water.
Extraction with mineral acid was performed using modified methods of Blay &
Simpson (13). An amount of 2.5 g of the homogenized sample was boiled for 10 minutes
with 10 cm3 of 6 mol/dm3 hydrochloric acid with reflux. The solution was cooled to room
temperature and transferred to a calibrated flask. After addition of 2 cm3 of 10% solution
of lanthanum, the sample was diluted to the final volume of 50 cm3 with distilled water,
filtered, and the resulting solution was used for the analysis.
Quantitative determination
The quantitative determination of potassium, sodium and calcium were performed
using the method of calibration curve, defined for each element and for the two sample
preparation procedure. Since, the matrix of the samples prepared by two different methods is different, the standard solutions were prepared with and without hydrochloric acid.
The calibration curves were defined based on seven points. The concentration of potassium, sodium and calcium ranged within 2.769-110.76 mg/l, 2.482-99.28 mg/l and 4.763190.52 mg/l, respectively.
The characteristics of the obtained analytical curves are presented in Table 2.
Table 2. Characteristics of the calibration curves of calcium, sodium and potassium
Concentration
range (mg/l)
a
Ca
4.763-190.52
Na
2.482-99.28
K
2.769-110.76
Standard
solution
without HCl
with HCl
without HCl
with HCl
without HCl
with HCl
Dependencea
y=0.4085·c+0.1999
y=0.4031·c+0.9242
y=-0.0068·c2+1.4053·c+8.3862
y=-0.0063·c2+1.4331·c+4.0366
y=-0.0047·c2+1.1731·c+5.2530
y=-0.0037·c2+1.1486·c+0.1086
Correlation
coefficient
0.9999
0.9979
0.9951
0.9989
0.9986
0.9994
y - flame photometer reading, c- concentration in mg/l
The results for the determination of calcium, sodium and potassium in the samples of
fermented milk products obtained without sample pre-treatment (direct method) and after
the sample preparation by extraction with hydrochloric acid are given in Table 3.
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Table 3. Content of Ca, Na and K (mg/l) in fermented milk products
Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
Ca (mg/l)
DM
EX
1205.14 1295.14
1153.75 1243.75
1102.29 1205.62
1102.32 1205.30
1165.20 1219.60
1459.00 1542.20
1167.32 1249.20
1213.24 1290.74
1214.20 1290.00
1133.40 1149.20
1180.11 1218.92
1183.40 1219.60
1182.14 1219.44
Na (mg/l)
DM
EX
458.28 542.20
476.79 625.80
422.89 569.00
405.96 596.80
319.02 516.60
525.19 688.00
374.49 516.60
436.22 542.20
436.47 569.00
373.67 424.00
413.70 468.16
432.80 468.60
431.63 492.20
K (mg/l)
DM
EX
1488.22 1581.20
1371.85 1443.40
1334.82 1407.20
1371.85 1407.56
1441.16 1479.00
1682.00 1794.20
1445.70 1543.82
1441.26 1543.40
1441.16 1513.80
1486.06 1576.40
1463.86 1517.07
1464.06 1516.20
1509.68 1516.49
DM – direct method; EX – extraction with hydrochloric acid
The calcium, sodium, and potassium levels determined without sample pre-treatment
(direct method) in thirteen fermented milk samples were compared with those obtained
after the sample preparation by extraction with hydrochloric acid. A two-tailed t-test
showed that the obtained results were in agreement at 95% confidence level, meaning
that at the chosen significance level, the differences between the values obtained for the
different sample treatment were within the experimental error. Consequently, the direct
method as a faster, simpler and cheaper procedure could be applied for the rapid routine
analysis of the mineral content in the fermented dairy products.
The most abundant mineral in the analyzed samples was potassium, with a content
from 1334.82 to 1794.20 mg/l, the calcium content was in the range of 1102.32-1524.20
mg/l, whereas the sodium content was the lowest (319.02-688.00 mg/). The obtained results are in agreement with the previously published data (8). According to the results, the
fermented milk products are rich sources of Ca and K and a medium source of sodium.
From the health point of view, adequate intakes of potassium are needed to counter the
high intakes of sodium. Therefore, a dietary balance between potassium and sodium is
needed to maintain efficient cellular functions that require potassium ions. For the prevention and treatment of hypertension, attention has to be given to the importance of low
dietary Na/K ratio rather than to the Na restriction alone. It should be stressed in this regard that the Na/K ratio in fermented milk products is favorable. Moreover, fermented
milk products could act as an alternative source of mineral for sufferers of lactose intolerance.
The measured mineral contents were correlated among themselves. The results of
ANOVA test showed that sample 6, with the highest content of Ca, Na and K, was significantly different in terms of potassium and calcium content, at a 95% confidence level.
Considering the fact that different labeled starter were used in the manufacture of analy47
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zed commercial fermented milk products can be assumed that the reason was the used
starter cultures, Lactobacillus rhamnosus GG (ATCC 53103). It could be concluded that
the inoculums of kombucha cultivated on different kinds of tea used to manufacture the
fermented milk products did not influence the concentration of calcium, sodium and
potassium. However, in order to check the influence that the employment of different
probiotic microorganisms can exert on the mineral composition of fermented milk products, more extensive research has to be performed.
CONCLUSION
According to the results shown in this paper, the determination of calcium, sodium
and potassium in samples of fermented milk products by flame photometry could be done
after simple dilution with water. The proposed method for the direct determination of
macroelements content in fermented milk samples is simple, fast, efficient, and it operates according to the principles of Green Chemistry. As a consequence, the analytical costs
and reagents consumption are reduced.
Acknowledgement
This investigation is financially supported by the Ministry of Education and Science
of the Republic of Serbia (Grant III 46009).
REFERENCES
1. Gaucheron, F.: The minerals of milk. Reprod. Nutr. Dev. 45, 4 (2005) 473-483.
2. Gaucheron, F.: Milk and dairy products: a unique micronutrient combination. J. Am.
Coll. Nutr. 50, 5 Suppl 1 (2011) 400S-409S.
3. Gao, R., Temminghoff, E.J.M., van Leeuwen, H.P., van Valenberg, H.J:F:, Eisner,
M.D. and van Boekel, M.A.J.S.: Simultaneous determination of free calcium, magnesium, sodium and potassium ion concentrations in simulated milk ultrafiltrate and reconstituted skim milk using the Donnan Membrane Technique. Int. Dairy J. 19,
(2009) 431-436.
4. Petrovich, M.B., Filho, V.R.A. and Neto, J.A.G.: Direct determination of calcium of
milk by atomic absorption spectrometry using flow-injection analysis. Ecl. Quím. 32,
3 (2007) 25-30.
5. Renner, E.: Micronutrients in milk and milk-based food products, Elsevier Science
Publishers, England (1989) p.29.
6. Belitz, H.D., Grosch, W. and Schieberle, P.: Food Chemistry, Springer-Verlag, Berlin
Heidelberg (2009) p.421-423.
7. Ahmed, A.I., Mohamed, B.E. and Elkhatim, N.M.: Some minerals contents of fermented camel (Camelus dromedarius) milk (Gariss) from Khartoum state, Sudan.
Res. J. Agr. Biol. Sci. 7, 1 (2011) 47-51.
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8. de la Fuentea, M.A., Montes, F., Guerrero, G. and Juárez, M.: Total and soluble contents of calcium, magnesium, phosphorus and zinc in yoghurts. Food Chem. 80, 4
(2003) 573-578.
9. Rinaldoni, A.N., Campderrós, M.E., Pérez Padilla, A., Perino, A. and Fernández, J.E.:
Analytic determinations of minerals content by XRF, ICP and EEA in ultrafiltered
milk and yoghurt. Lat. Am. Appl. Res. 39, 2 (2009) 113-118.
10. Chen, K.L. and Jiang, S.J.: Determination of calcium, iron and zinc in milk powder by
reaction cell inductively coupled plasma mass spectrometry. Anal. Chim. Acta 470, 2
(2002) 223-228.
11. de Oliveira, E.: Sample preparation for atomic spectroscopy: Evolution and future
trends. J. Braz. Chem. Soc. 14, 2 (2003) 174-182.
12. Malbaša, R.V., Vitas, J.S., Lončar, E.S. and Kravić, S.Ž.: Influence of fermentation
temperature on the content of fatty acids in low energy milk-based kombucha products. APTEFF 42 (2011) 81-90.
13. Simpson, G.R. and Blay, R.A.: Rapid method for the determination of the metals
copper, zinc, tin, iron and calcium in foodstuffs by atomic absorption spectroscopy.
Food Trade Rev. 36, 8 (1966) 35-37.
ДИРЕКТНО ОДРЕЂИВАЊЕ КАЛЦИЈУМА, НАТРИЈУМА И
КАЛИЈУМА У ФЕРМЕНТИСАНИМ МЛЕЧНИМ ПРОИЗВОДИМА
Снежана Ж. Кравић*, Звонимир Ј. Сутуровић, Ана Д. Ђуровић, Тања Ж. Брезо,
Спасенија Д. Милановић, Радомир В. Малбаша и Владимир Р. Вукић
У оквиру овог рада испитана је могућност директног одређивања калцијума,
натријума и калијума пламеном фотометријом у комерцијалним ферментисаним
млечним производима и млечним производима на бази комбухе. Припрема узорака
за анализу је изведена на два начина: разблаживањем дестилованом водом (директна метода) и екстракцијом са минералном киселином. Садржаји калцијума, натријума и калијума одређени након примене наведених метода припреме узорка су међусобно статистички упоређени. Резултати су показали да су разлике између вредности добијених за различите поступке припреме узорка у оквиру експерименталне
грешке уз 95% вероватноћу. У поређењу са методом заснованом на екстракцији минералном киселином, директна метода је ефикасна, бржа, једноставнија и јефтинија, а такође задовољава принципе зелене хемије. Стога би се предложена метода за
директно одређивање калцијума, натријума и калијума пламеном фотометријом
могла применити за брзо рутинско одређивање садржаја макроелемената у ферментисаним млечним производима.
Kључне речи: калцијум, натријум, калијум, ферментисани млечни производи
Accepted:19 October 2012
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PHYSICAL AND TEXTURAL CHARACTERISTICS OF FERMENTED MILK
PRODUCTS OBTAINED BY KOMBUCHA INOCULUMS WITH HERBAL TEAS
Radomir V. Malbaša*, Jasmina S. Vitas, Eva S. Lončar and Spasenija D. Milanović
In this investigation, kombucha fermented milk products were produced from milk
with 1.6% milk fat using 10% (v/v) kombucha inoculums cultivated on the extracts of
peppermint and stinging nettle.
The fermentation process was conducted at temperatures of 37, 40 and 43°C. Fermentation was stopped when the pH value of 4.5 was reached. The fermentation process
was shortened with an increase of temperature. Physical characteristics of the fermented
products were determined by using standard methods of analysis. Textural characteristics were determined by texture profile analysis. The obtained products showed good
physical and textural characteristics, typical for the yoghurt-like products.
KEY WORDS: fermented milk products, kombucha, herbal tea, physical characteristics,
texture
INTRODUCTION
Kombucha is a symbiosis of several yeast species (genera Schizosaccharomyces,
Saccharomycodes, Saccharomyces, Zygosaccharomyces, Candida, Pichia, Kloeckera,
Brettanomyces and Torulopsis) and acetic acid bacteria (Gluconacetobacter xylinus (formerly known as Acetobacter xylinum), Acetobacter xylinoides, Bacterium gluconicum,
Acetobacter aceti, Acetobacter pasteurianus) (1, 2). Microbiological composition depends on the geographic origin of the culture. Kombucha metabolises on different substrates. Apart from traditional ones (sweetened black or green tea), it is capable for biotransformation of coca-cola, beer, coffee, Jerusalem artichoke, molasses, herbal tea, milk,
and others. Kombucha is traditional refreshing beverage and food supplement (3, 4).
The products obtained after fermentation of kombucha on milk are, by their physicochemical and sensory characteristics, similar to fermented milk products such as yoghurt
and kefir (5). These types of fermented milk products are widely consumed as functional
food due to their good sensory and nutritional properties, and beneficial effects to human
health (6, 7). Gel formation is the most important functional property of fermented milk
products. The physical and textural characteristics of this composite gel are governed by
* Corresponding author: Radomir V. Malbaša, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]
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milk composition, dry matter content, type and quantity of the starter culture that is used
to inoculate the milk, fermentation temperature, and the storage conditions of the final
product. In addition, the consistency and water-holding capacity of acidified milk gels are
strongly related to the quality of fermented milk products (7).
Previous studies showed that it is possible to obtain kombucha beverage on herbal tea
extract, and to use the obtained beverage in production of kombucha fermented milk
products (8). Peppermint and stinging nettle are well known medicinal herbs, with a
variety of positive effects to human health (9, 10).
The objective of this study was to investigate the physical and textural characteristics
of fermented milk products obtained by kombucha inoculums with peppermint and stinging nettle.
EXPERIMENTAL
Production of kombucha inoculums
The inoculum for the fermentation of milk was obtained by cultivating kombucha on
cooled tea, which was prepared as follows: to 1 L of boiling tap water was added 70 g
sucrose and 2.25 g of appropriate tea: peppermint (label P) and stinging nettle (label SN),
using herbal teas from a health food store. The tea extract was cooled to room temperature, filtered and then 100 mL of kombucha inoculum from a previous fermentation
(10%, v/v) were added. The glass container was covered with cheesecloth for air. Kombucha incubation was performed at room temperature for 7 days. The obtained kombucha
inoculums (marked as PI and SNI) were used for the fermentation of milk.
Production of fermented milk products
Fermented milk products were produced from pasteurized, homogenized milk with
1.6% milk fat (AD IMLEK Beograd, branch Novosadska mlekara, Novi Sad, Serbia), as
follows: to the 500 mL of milk, 10% (v/v) of the appropriate kombucha inoculum (PI or
SNI) was added. The fermentation was performed at 37, 40 and 43oC and it lasted until
the pH value of 4.5 was reached. Milk gel was then cooled to the temperature of 8°C, homogenized by mixer, and the samples were stored in refrigerator. The obtained products
were marked as P37, P40, P43, SN37, SN40 and SN43 in dependence of the used herbal
tea and the applied temperature.
Methods of analysis
The pH values were measured with a pH-meter (PT-70, Boeco, Germany).
The examined physical characteristics were whey syneresis (11) and water holding
capacity (WHC) (12).
Textural characteristics were analyzed using Texture analyzer TA.HDplus, Micro
Stable System, England (13).
All analyses were performed in triplicate. Statistical analyses were done using Microsoft Office Excel 2003.
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The average pH value of milk used for the production of kombucha fermented milk
products was 6.68, and therefore the milk was very slightly acidic. The determined pH
value of the milk was in accordance to the current Regulation (14).
The average pH value of kombucha inoculum obtained from peppermint extract and
stinging nettle extract was 3.27 and 2.84. It is evident that the measured pH values of the
inoculums were significantly lower compared to the pH value of the milk.
The fermentation of milk with inoculums PI and SNI at 37, 40 and 43°C is presented
in Figs. 1 and 2.
7.00
P37
P40
6.50
P43
pH
6.00
5.50
5.00
4.50
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
fermentation time (h)
Figure 1. Fermentation process of milk at 37, 40 and 43°C with PI
6.50
SN37
SN40
SN43
pH
6.00
5.50
5.00
4.50
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
fermentation time (h)
Figure 2. Fermentation process of milk at 37, 40 and 43°C with SNI
From the results presented in Figs. 1. and 2. it can be concluded that the fermentation
temperature of 43°C shortened the time needed to reach the pH 4.5, with both applied
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inoculums. The fermentation process was the longest for the obtaining of P37 (13.50
hours) and the shortest for P43 (9.45 hours). The shape of all fermentation curves given
in Figs. 1 and 2 is characteristic for the production of kombucha fermented milk products
(5, 8, 13).
The results of whey syneresis and water holding capacity (WHC) are given in Figs. 3.
and 4.
37.00
whey syneresis (mL)
36.00
35.00
34.00
33.00
32.00
31.00
30.00
29.00
P37
P40
P43
SN37
SN40
SN43
samples
Figure 3. Whey syneresis of the kombucha fermented milk products
Whey separation, i.e. the appearance of whey on the surface of a gel, is a common defect during storage of fermented milk products like yogurts and cream cheese. Manufacturers try to prevent whey separation by increasing the total solid content of milk by heating it prior to the fermentation and/or by adding stabilizers. Spontaneous syneresis is the
contraction of a gel without the application of any external force (e.g., centrifugation),
and is related to the instability of the gel network (i.e., large-scale rearrangements) resulting in the loss of the ability to entrap all the serum phase (15). During the syneresis,
whey passes through the protein matrix, which can be explained by the law of Darcy
(16).
The results given in Fig. 3. show that the values of whey syneresis of the obtained
products did not differ significantly. The highest value of syneresis had the sample P37
(35.00 mL) and this indicated its lower quality comparing to the other products.
The increase of fermentation temperature does not create the same trend for the WHC
of products with peppermint and stinging nettle. While WHC was increased with an increase of temperature for the kombucha fermented milk products containing peppermint,
the opposite behaviour for the products with stinging nettle was noticed (Fig. 4). The results show that the average values of WHC were higher for products obtained with SNI
(43.44%), which suggested its better quality in comparison to products obtained with PI
(36.82%).
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60.00
50.00
WHC (%)
40.00
30.00
20.00
10.00
0.00
P37
P40
P43
SN37
SN40
SN43
samples
Figure 4. Water holding capacity of the kombucha fermented milk products
The results of texture analyses (firmness, consistency, cohesiveness and index of viscosity) are presented in Figs. 5-8.
20.000
18.000
16.000
firmness (g)
14.000
12.000
10.000
8.000
6.000
4.000
2.000
0.000
P37
P40
P43
SN37
SN40
SN43
sample
Figure 5. Firmness of the kombucha fermented milk products
Sweetening agents, such as sucrose, high-fructose corn syrup or honey, are usually
added to stirred yoghurts to mask the acidity for acid-conscious consumers and, perhaps,
produce a firmer texture (17). It is important fact because kombucha inoculums contain
sucrose and fructose.
For yoghurt products, lower incubation temperatures (e.g. 40°C instead of 45°C) lead
to slightly longer gelation times but firmer more viscous gels are formed that are less prone to whey separation (18).
The firmness of the products obtained with SNI increased with the increase of temperature, while for the products obtained with PI, this trend was reversing (Fig. 5).
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500.000
450.000
consistency (gs)
400.000
350.000
300.000
250.000
200.000
150.000
100.000
50.000
0.000
P37
P40
P43
SN37
sample
SN40
SN43
Figure 6. Consistency of the kombucha fermented milk products
The consistency of the yoghurt base is enhanced by homogenisation, in that a portion
of the casein and whey proteins become attached to the fat globule surfaces, so effectively increasing the number of structure-building components in the system; native fat globule membranes do not interact with proteins in the same way (17).
The results given in Fig. 6 present that consistency of the products obtained with SNI
increased with the increase of temperature, while for the products obtained with PI, this
trend was opposite, but similar with firmness (Fig. 5).
sample
P37
P40
P43
SN37
SN40
SN43
0.000
cohesiveness (g)
-1.000
-2.000
-3.000
-4.000
-5.000
-6.000
-7.000
-8.000
-9.000
Figure 7. Cohesiveness of the kombucha fermented milk products
The cohesiveness indicates the maximum capacity of possible deformation of the
sample before the break and could be very important for the consumers. The best cohesiveness of the products obtained with PI had the sample produced at 43°C, while for products obtained with SNI, the best cohesiveness had the sample SN40 (Fig. 7).
The viscosity and the structure of the gel are influenced by several factors, including
the incubation temperature, casein concentration, heat treatment of the milk, acidity and
type of starter culture; as well as the temperature at which the measurements are made
(17).
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sample
P37
P40
P43
SN37
SN40
SN43
0.000
index of viscosity (gs)
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
-3.500
-4.000
Figure 8. Index of viscosity of the kombucha fermented milk products
Index of viscosity for products obtained with SNI increased with the decrease of temperature. The best index of viscosity of the products obtained with PI had sample P43
(Fig. 8).
CONCLUSION
This study examined the physical and textural characteristics of fermented milk products obtained at the fermentation temperatures of 37, 40 and 43°C, using milk with 1.6%
milk fat and kombucha inoculums cultivated on peppermint and stinging nettle. The
fermentation was stopped after reaching pH 4.5.
The values obtained for the physical properties were in the range characteristic for
that type of products. The values of whey syneresis and water holding capacity suggest
that the best quality has the sample produced with kombucha inoculum cultivated on
stinging nettle at the fermentation temperature of 37°C.
The highest joint value of firmness and consistency had the sample P37, the highest
value of cohesiveness had the sample SN40 and the best index of viscosity showed the
sample SN37.
Acknowledgement
Authors want to thank to the Ministry of Education, Science and Technological Development of the Republic of Serbia for financing the investigations presented in this article, Grant III-46009.
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products. Acta Period. Technol. 42 (2011) 81-90.
9. Akdogan, M., Ozguner, M., Kocak, A., Oncu, M. and Cicek, E.: Effects of peppermint teas on plasma testosterone, follicle-stimulating hormone, and luteinizing hormone levels and testicular tissue in rats. Urology, 64, 2 (2004) 394-398.
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11. Atamer, M., Carić, M., Milanović, S. and Gavarić, D.: The quality of yoghurt produced from UF milk. Zbornik Matice srpske za prirodne nauke, Matica srpska Novi Sad,
91 (1996) 19-26.
12. Guzman-Gonzalez, M., Morais, F., Ramons, M., and Amigo, L.: Influence of skimmed milk concentrate replacement by dry dairy products in a low fat set-type yoghurt
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13. Milanović, S.D., Iličić, M.D., Duraković, K.G. and Vukić, V.R.: Textural characteristics оf fermented milk beverages produced by kombucha. APTEFF 40 (2009) 63-69.
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16. Jovanović, S., Maćej, O. and Barać, M.: Influence of various coagulation factors on
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49, 2 (2004) 219-232.
17. Tamime, A.Y.: Fermented milks. Blackwell Science Ltd, a Blackwell Publishing
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ФИЗИЧКЕ И ТЕКСТУРАЛНЕ КАРАКТЕРИСТИКЕ ФЕРМЕНТИСАНИХ
МЛЕЧНИХ ПРОИЗВОДА ДОБИЈЕНИХ ПОМОЋУ КОМБУХЕ ГАЈЕНЕ НА
БИЉНИМ ЧАЈЕВИМА
Радомир В. Малбаша, Јасмина С. Витас, Ева С. Лончар и Спасенија Д. Милановић
Циљ овог рада је било истраживање физичких и текстуралних карактеристика
ферментисаних млечних производа добијених помоћу комбухе култивисане на
биљним чајевима.
У овом раду, ферментисани млечни производи добијени помоћу комбухе су
произведени коришћењем млека са 1,6% млечне масти додатком 10% (v/v)
инокулума комбухе гајене на екстрактима нане и коприве.
Процес ферментације је изведен на температурама од 37, 40 и 43°C. Ферментација је заустављена након што је достигнута вредност pH од 4,5. Са порастом
температуре процес ферментације је био краћи.
Физичке карактеристике су одређене применом стандардних метода анализе.
Текстуралне карактеристике су одређене анализом текстуралног профила. Добијени производи су показали добре физичке и текстуралне карактеристике, типичне
за јогурту сличне производе.
Кључне речи: ферментисани млечни производи, комбуха, биљни чај, физичке
карактеристике, текстура
59
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STARCH PROPERTIES OF VARIOUS ZP MAIZE GENOTYPES
Marija S. Milašinović-Šeremešićа*, Milica M. Radosavljevićа and Ljubica P. Dokićb
а
б
Maize Research Institute, S. Bajića 1, 11185 Belgrade, Serbia
The objective of this study was to investigate molecular and functional properties of
starches isolated from ZP maize genotypes of different genetic background. The protein,
fat, ash and resistant starch contents were very low. The amylose content in the isolated
starches of 10 ZP maize genotypes was characteristic for both types of maize starches,
normal and waxy. The waxy type had the highest average molecular weight of amylopectin (4.84 x 108 Da). The onset temperature of gelatinisation values of starches of 10 ZP
maize genotypes ranged from 62.1ºC to 65.0ºC. The waxy maize starch displayed a
significantly higher enthalpy change for gelatinised starch (ΔH=18.1 J/g) than normal
maize starches did (ΔH=13.6-15.6 J/g). Rapid Visco Analyser (RVA) profiles of starches
of ZP maize genotypes were typical for both types of maize starches, normal and waxy.
KEY WORDS: starch, maize, molecular characteristics, functional properties
INTRODUCTION
Starch is the second largest biomass produced on earth, next to cellulose. It is the
most important plant reserve material accumulating in fruits, seeds, roots and tubers in
the form of starch granules that provide 70-80% energy consumed by the world population. Commercial starches, used in the food-processing industry, are most often produced
from grain of common, waxy and high-amylose maize, then wheat, and different varieties
of rice, as well as, from potato, sweet potato and cassava (tapioca starch). Depending on
their botanical origin, starches differ in their chemical structure, size and shape of their
granules, and consequently in their functional and sensory properties. Compositions of
maize (Zea mays L.) starch vary depending on genotypes. Normal maize starch contains
about 25-30% amylose and 70-75% amylopectin; waxy starch consists mainly of amylopectin and 0-8% amylose; high-amylose starch consists of 40-85% amylose (1). Many
starches, such as high-amylose maize starches (2, 3) and sugary-2 starches (4), also contain intermediate components that are branched molecules with smaller molecular
weights and longer branch chains than amylopectin. Normal maize starch also contains
minor components including lipids (free fatty acids and triglycerides) and little phosphorlipids.
* Corresponding author: Marija S. Milašinović-Šeremešić, Maize Research Institute, S. Bajića 1, 11185 Belgrade, Serbia, e-mail: [email protected]
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Amylose is primarily a linear glucan, consisting of α 1–4 linked D-glucopyranose
with a few branches (5). The molecular size of amylose varies from 500 anhydroglucose
units (AGU) or 500 degree of polymerisation (DP) of high-amylose maize starch (6) to
more than 6000 AGU or DP 6000 of potato starch (7). Amylopectin is a highly branched
molecule, consisting of α 1–4 linked D-glucopyranose chains that are connected by α 1–6
branch linkages. Furthermore, amylopectin has a very large molecular weight (7 × 107 to
5.7 × 109 Da) (8). Amylopectin in the granule is present in the semi-crystalline structure,
whereas amylose is amorphous.
Starch granules possess different types of crystallinity, displaying A-, B- and C-type
X-ray patterns, depending on their amylopectin branch chain-length (9). The type A
structure is very common in cereals, the type B in raw potato and banana, while the type
C is typical for peas and beans. Normal and waxy maize starch granules display A-type
X-ray patterns, while high amylose maize starches display B-type. Starches of different
polymorphisms are known to display different enzyme digestibility (4, 10, 11, 12). The
A-type polymorphic starch is easily digestible, but the B-type and some C-type starches
are very resistant to enzyme hydrolysis. Resistant starch (RS) is the portion of starch that
is not digested in small intestine, but is fermented by microflora in the colon (13). Four
classes of RS have been proposed on the basis of mechanisms of enzyme indigestibility,
including inaccessibility of starch to amylases due to physical entrapment (RS1), inherent
granular structure of raw starch (RS2), molecular association of amylose or retrogradetion (RS3) and chemical modification (RS4).
In this study, we investigated the starch composition, amylose content, amylopectin
branch chain-length distribution, gelatinisation and pasting properties of starches isolated
from ZP maize genotypes of different genetic background. In terms of technological
value this information will be useful to recognise significant differences in the properties
of isolated starches of the selected ZP maize genotypes.
EXPERIMENTAL
Starches of various ZP maize genotypes (ZP 360, ZP 434, ZP 578, ZP 633, ZP 684,
ZP 808, ZP 74b, ZP 611k, ZP 704wx, ZP Rumenka) developed at the Maize Research
Institute “Zemun Polje”, Belgrade, Serbia were used in this study. The starches were isolated by applying a 100-g laboratory maize wet-milling procedure (14).
The moisture, ash, crude protein and crude fat contents of starch were determined
using the oven method (15), AOAC Method (16), microKjeldahl method (16) and Soxhlet method (16), respectively. The amylose content was determined by a rapid colorimetric method (17). RS content was determined according to the enzymatic-gravimetric
method (18) and McCleary method (19).
The thermal (gelatinisation) characteristics of the isolated starches were studied by
using a differential scanning calorimeter (DSC 2920 modulated, TA Instruments, New
Castle, DE) (20). Starch (2 mg, dry matter basis (dmb)) was accurately weighed in an
aluminium pan, mixed with 6 mg of deionised water and sealed. The sample was allowed
to equilibrate for 2 h and scanned at a rate of 5 ºC/min over a temperature range of 25120ºC. An empty pan was used as the reference.
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The Rapid Visco Analyser (RVA-4, Foss North America, Eden Prairie, MN) was used
for studying pasting properties (20). Starch suspension (8%, w/w, dmb), in duplicate for
each starch sample, was prepared by weighing starch (2.24 g, dmb) into a RVA canister
and making up the total weight to 28 g with deionised water. Starch suspension was
equilibrated at 30ºC for 1 min, heated at a rate of 6.0ºC/min up to 95ºC, maintained at
95ºC for 5.5 min, and then cooled to 50ºC at a rate of 6.0ºC/min. A paddle rotating speed
(160 rpm) was used throughout the entire analysis, except for rapid stirring at 960 rpm for
the first 10 s to disperse the starch sample.
The molecular weight distribution of amylopectin was estimated by a high-performance size-exclusion chromatography, HPSEC e.g. HPSEC-MALLS-RI system (coupled
with a multi-angle laser-light scattering, MALLS and a refractive index, RI detector). The
HPSEC system consisted of a HP 1050 series isocratic pump (Hewlett–Packard, Valley
Forge, PA), a multi-angle laser-light scattering detector (Dawn DSP-F, Wyatt Tech. Co.,
Santa Barbara, CA) and a HP 1047A refractive index detector (Hewlett–Packard, Valley
Forge, PA). Shodex OH pak SB-G guard column and SB-806 and SB-804 analytical columns (Showa Denko K.K., JM Science, Grand Island, NY) were used to separate amylopectin from amylose (8).
Statistical analysis. Results were expressed by means of values ± standard error of
three separate determinations. Data reported for the starch composition and gelatinisation
and pasting properties was assessed by the analysis of variance (ANOVA) and Duncan's
multiple range test was used for any significant differences at the P<0.05 level between
the means. RVA profiles were presented as means of two separate determinations. All the
analyses were conducted using statistical software package STATISTICA 8.1. (StatSoft
Inc. USA).
The data for the amylose, protein, fat and ash content and the molecular weight of
amylopectin in the isolated starches are given in Table 1. The protein content in the
starches ranged from 0.11% to 0.29%, pointing out to high quality ("purity") of obtained
starches. The highest protein content was obtained in starch samples isolated from ZP
611k and ZP 633, which can be attributed to the high percentage of hard endosperm in
these genotypes and the difficulties in the starch extraction. Starch samples isolated from
ZP 360 and ZP 704wx had the lowest values for the protein content, as these genotypes
had the high proportion of the soft endosperm fraction (21, 22). Although protein, oil and
ash are mostly determined by genetic factors (source of starch), the method of isolation
(extraction technique) also has a major impact on the “purity” of starch.
The amylose content in the isolated starches of 10 ZP genotypes was characteristic for
normal and waxy maize starches. The highest content of amylose was obtained in the
starch isolated from ZP 434 (26.0%). The isolated waxy type starch had the lowest value
for the amylose content (1.0%). The results concerning the average molecular weight of
amylopectin (Mw) of different ZP maize genotypes (Table 1) show that the waxy type of
starch had the highest Mw (4.84 x 108), which is consistent with the research carried out
by Sang-Ho and Jane (8) who had found a significant difference between Mw of normal
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and waxy starch. This difference can be attributed to the fact that all ADP-glucose is used
as a substrate for the biosynthesis of amylopectin in waxy starch, whereas the ADP-glucose substrate is partitioning between amylopectin and amylose for normal starch biosynthesis. The lowest Mw was found in the starches of ZP 74b (1.52 x 108) and ZP 633
(1.66 x 108) genotypes. Significant differences in Mw of various ZP genotypes were
found (Table 1). The genotypes ZP 434 and ZP 684 were not significantly different in
this parameter as well as the genotypes ZP 360, ZP 578 and ZP Rumenka.
Table 1. Contents of amylose, protein, fat and ash, and Mw of the isolated starches
Starch source
Amylose (%)
Protein (%)
Fat (%)
Ash (%)
ZP 74b
25.0 ± 0.3b
0.20 ± 0.00c
0.30 ± 0.02a
0.10 ± 0.00b
ZP 360
23.8 ± 0.2c
0.11 ± 0.05e
0.22 ± 0.02b
0.09 ± 0.01b
ZP 434
26.0 ± 0.2a
0.26 ± 0.04ab
0.05 ± 0.02e
0.01 ± 0.00d
ZP 578
23.5 ± 0.5cd
0.23 ± 0.02b
0.10 ± 0.01d
0.06 ± 0.03bc
ZP 611k
23.2 ± 0.1d
0.29 ± 0.01a
0.08 ± 0.00de
0.15 ± 0.02a
ZP 633
24.0 ± 0.2c
0.28 ± 0.02a
0.09 ± 0.01d
0.04 ± 0.00c
ZP 684
23.3 ± 0.5cd
0.21 ± 0.05ab
0.07 ± 0.01e
0.10 ± 0.02b
ZP 704wx
1.0 ± 0.1e
0.13 ± 0.02e
0.10 ± 0.03d
0.04 ± 0.00c
ZP 808
24.0 ± 0.2c
0.18 ± 0.02cd
0.15 ± 0.02c
0.09 ± 0.02b
ZP Rumenka
23.5 ± 0.3cd
0.18 ± 0.04cd
0.25 ± 0.02b
0.10 ± 0.01b
* Mw-molecular weight of amylopectin
** Means within a column followed by different letters are significantly different (P<0.05)
Mw x 108
1.52 ± 0.05f
2.27 ± 0.07d
2.87 ± 0.10b
2.27 ± 0.05d
2.68 ± 0.09c
1.66 ± 0.11f
2.97 ± 0.11b
4.84 ± 0.15a
1.91 ± 0.03e
2.25 ± 0.02d
The RS content in native starch of different ZP maize hybrids (Table 2) was very low
and ranged from 0.62 to 1.61% and 0.00 to 0.85% depending on the applied methods.
The method developed by McCleary gave lower values for the RS content although this
method allows a greater deviation for low RS concentrations in samples (<2%).
Table 2. RS content in the isolated starches
Starch source
ZP 74 b
ZP 360
ZP 578
ZP 611 k
ZP 704 wx
ZP 808
ZP Rumenka
RS (%)
(AOAC Official Method 991.43)
1.14 ± 0.09
1.17 ± 0.04
1.26 ± 0.15
1.50 ± 0.21
0.62 ± 0.24
1.61 ± 0.11
1.41 ± 0.07
RS (%)
(McCleary Method, 2002)
0.55 ± 0.20
0.70 ± 0.03
0.71 ± 0.10
0.80 ± 0.01
0.00 ± 0.00
0.78 ± 0.10
0.85 ± 0.05
Gelatinisation parameters of the starch isolated from 10 ZP maize genotypes are
shown in Table 3. The onset temperature of gelatinisation values of starches of 10 ZP genotypes ranged from 62.1ºC (ZP 74b) to 65.0ºC (ZP 434). In this study there was no relationship between the amylose content and gelatinisation temperatures of the starches (data are not given). The obtained results of the DSC analysis (Table 3) show that the starch
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isolated from ZP 74b had significantly lower gelatinisation temperatures (T0, Tp, Tc) and
enthalpy change compared to other samples of native starch which can be explained by
the lowest Mw in this sample. It is believed that the amylopectin chain length affects the
crystalline structure of amylopectin, and thus the parameters of gelatinisation (20). With
the decreasing amylopectin chain length the crystalline structure of amylopectin changes
resulting in a lower gelatinisation temperature. The starch isolated from ZP 434 genotype
had significantly higher gelatinisation temperatures. The waxy starch isolated from ZP
704wx had very similar gelatinisation temperatures to the normal starches isolated from
ZP 360, ZP 633, ZP 684 and ZP Rumenka. The waxy maize starch displayed a signifycantly higher enthalpy change for gelatinised starch (ΔH=18.1 J/g) than normal maize
starches did (ΔH=13.6-15.6 J/g). The highest value of enthalpy change for gelatinised
waxy maize starch (ZP 704wx) can be explained by the lowest amylose content and the
highest Mw of the starch.
Table 3. Gelatinisation properties of the starch isolated from various ZP maize
genotypes*
Starch sources
ZP 74b
ZP 360
ZP 434
ZP 578
ZP 611k
ZP 633
ZP 684
ZP 704wx
ZP 808
ZP Rumenka
T0 (°C)
62.1 ± 0.2d
63.3 ± 0.0bc
65.0 ± 0.5a
62.7 ± 0.2c
64.5 ± 0.5ab
63.3 ± 0.0bc
63.2 ± 0.3bc
63.7 ± 0.1b
62.9 ± 0.3c
63.7 ± 0.1b
Tp (°C)
66.6 ± 0.1e
68.3 ± 0.1bc
69.5 ± 0.2a
67.4 ± 0.1d
67.9 ± 0.4c
67.8 ± 0.0c
67.7 ± 0.5cd
68.8 ± 0.1b
67.4 ± 0.1d
68.3 ± 0.2bc
Tc (°C)
70.6 ± 0.5e
73.4 ± 0.0b
74.2 ± 0.4a
72.2 ± 0.1c
71.6 ± 0.6d
71.9 ± 0.3c
71.7 ± 0.6cd
74.7 ± 0.0a
71.6 ± 0.2d
73.1 ± 0.4b
ΔH (J/g)
13.63 ± 0.17d
15.01 ± 0.78bc
15.55 ± 0.16b
15.51 ± 0.39b
14.20 ± 0.15c
15.18 ± 0.58bc
14.53 ± 0.29c
18.10 ± 1.21a
14.30 ± 0.06c
14.84 ± 0.35c
* T0, Tp, Tc and ΔH are onset, peak and conclusion temperature and enthalpy changes of gelatinisation,
respectively.
** Means within a column followed by different letters are significantly different (P<0.05)
The RVA is considered to simulate food processing and is used to relate functionality
to structural properties (23, 24). RVA profiles of the isolated starches of normal ZP 434,
popping ZP 611k and waxy ZP 704wx genotypes are given in Figure 1. The profiles of
remaining normal starches from seven ZP genotypes were similar in behaviour to the
starch of ZP 434 genotype and therefore they are not presented in this paper.
It is observed that RVA profiles of the starches of ZP genotypes were typical for both
types of maize starch, normal and waxy (25, 26). Particularly, normal maize starch isolated from the selected ZP genotypes had a temperate peak viscosity (approximately about
2200 cP) and a high tendency of increasing viscosity during paste cooling (“setback”)
(approximately about 1300 cP) that is a result of a very high retrogradation rate of a linear fraction.
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3000
Viscosity (cP)
2500
ZP 434
2000
1500
ZP 611k
1000
500
ZP 704wx
0
0
500
1000
1500
‐500
Time (s)
Figure 1. RVA profiles of the selected starches
Starch isolated from ZP 611k genotype (popping maize) had a slightly lower “setback” viscosity (viscosity at 50°C), which corresponded to a slightly lower amylose
content in the sample (23.2%). Waxy maize starch (ZP 704wx) had the peak viscosity of
2822.4 cP and due to a small amount of a linear amylose fraction it had a very low
tendency of increasing viscosity during paste cooling (ΔV=429.6 cP).
CONCLUSION
The protein content in the starches was very low (<0.30%), pointing out to high quality (“purity”) of obtained starches. The amylose content in the isolated starches of various
ZP genotypes was characteristic for normal and waxy maize starches. The waxy type of
starch had the highest Mw (4.84 x 108 Da). Mw of the normal starch ranged from 1.52 x
108 to 2.97 x 108 Da. The RS content in native starch of various ZP genotypes was very
low (<1.61%). The onset temperature of gelatinisation values of starches of 10 ZP genotypes ranged from 62.1ºC to 65.0ºC. The waxy maize starch displayed a significantly
higher enthalpy change for gelatinised starch than normal maize starches did. RVA profiles of the isolated starches were typical for both types of maize starches, normal and
waxy. Some starches of specialty hybrids (ZP 74b, ZP 611k and ZP Rumenka) were
similar in behaviour to normal starch.
Despite significant differences in the content of amylose, Mw and DSC parameters of
some samples there were no significant differences in pasting properties of native starches isolated from the selected ZP maize genotypes with the exception of two specialty
genotypes (waxy and popping maize). On the basis of the results it can be concluded the
selected dent maize genotypes can be used in wet-milling for production of normal maize
starch which may be further modified physically, chemically or enzymatically to meet
specific needs and applications.
Acknowledgment
This study was financially supported by the Ministry of Education and Science of the
Republic of Serbia, Project TR31069.
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the dominant mutant amylose-extender (Ae1-5180) maize starch. Cereal Chem. 72
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4. Perera, C., Lu, Z., Sell, J., Jane, J.: Comparison of physicochemical properties and
structures of sugary-2 cornstarch with normal and waxy cultivars. Cereal Chem. 78
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5. Takeda, Y., Shirasaka, K., Hizukuri, S.: Examination of the purity and structure of
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6. Jane, J., Chen, J.F.: Effect of amylose molecular size and amylopectin branch chain
length on paste properties of starch. Cereal Chem. 69 (1992) 60-65.
7. Hizukuri, S., Takeda, Y., Yasuda, M.: Multi-branched nature of amylose and the
action of debranching enzymes. Carbohydr. Res. 94 (1981) 205-213.
8. Sang-Ho, Y., Jane, J.: Molecular weights and gyration radii of amylopectins determined by high-performance size-exclusion chromatography equipped with multi-angle
laser-light scattering and refractive index detectors. Carbohydr. Polym. 49 (2002)
307-314.
9. Hizukuri, S., Kaneko, T., Takeda, Y.: Measurement of the chain length of amylopectin and its relevance to the origin of crystalline polymorphism of starch granules.
Biochim. Biophys. Acta 760 (1983) 188-191.
10. Jane, J.: Current understanding on starch granule structures. J. Appl. Glycosci. 53
(2006) 205-213.
11. Jane, J.: Structure of starch granules. J. Appl. Glycosci. 54 (2007) 31-36.
12. Copeland, L., Blazek, J., Salman, H., Chiming Tang, M.: Form and functionality of
starch. Food Hydrocol. 23 (2009) 1527-1534.
13. Sharma, A., Yadav, B.S., Yada, R.B.: Resistant starch: Physiological roles and food
applications. Food Rev. Int. 24 (2008) 193-234.
14. Eckhoff, S.R., Singh, S.K., Zehr, B.E., Rausch, K.D., Fox, E.J., Mistry, A.K., Haken,
A.E., Niu, Y.X., Zou, S.H., Buriak, P., Tumbleson, M.E., Keeling, P.L.: A 100-g
Laboratory Corn Wet-Milling Procedure. Cereal Chem. 73 (1996) 54-57.
15. Kirk, R.S., Sawyer, R.: Pearson’s composition and analysis of foods. 9th Edition,
Longman, UK (1991) p.708.
16. Official method of analysis (17th ed.). Washington, D.C.: Association of Official
Analytical Chemists (1990).
17. McGrance, S.J., Cornell, H.J., Rix, C.J.: A simple and rapid colorimetric method for
the determination of amylose in starch products. Starch 50 (1998) 158-163.
18. Official method of analysis (17th ed., rev. 2.ed.). W. Horwithz. Gaithersburg, Maryland, AOAC International (2003), method 991.43.
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19. McCleary, B.V., Monaghan, D.A.: Measurement of resistant starch. J. AOAC Int. 85
(2002) 665-675.
20. Jane, J., Chen, Y.Y., Lee, L.F., McPherson, A.E., Wong, K.S., Radosavljević, M.: Effects of amylopectin branch chain length and amylose content on the gelatinization
and pasting properties of starch. Cereal Chem. 76 (1999) 629-637.
21. Milasinovic M., Radosavljevic M., Dokic Lj., Jakovljevic J.: Wet-milling properties
of ZP maize hybrids. Maydica 52 (2007) 289-292.
22. Radosavljevic M., Bekric V., Milasinovic M., Pajic Z., Filipovic M., Todorovic G.:
Genetic variability as background for the achievements and prospects of the maize
utilisation development. Genetika-Belgrade 42 (2010) 119-136.
23. Crosbie, G.B., Ross, A.S.: The RVA Handbook. St Paul: Eagan Press (2007) p.152.
24. Eliasson, A.-C.: Starch in Food: Structure, Function and Applications. Cambridge/
Boca Raton: Woodhead Publishing Limited/CRC Press LLC (2004) pp. 156-179.
25. Thomas, D.J., Atwell, W.A.: Starches. AACC, St. Paul, Minnesota, USA (1999).
26. Blaszczak, W.: Effects of High Pressure, Time of Treatment and Polysaccharide
Composition on the Physico-chemical Properties of Hylon VII and Waxy Maize
Starch, in Starch: Achievements in Understanding Structure and Functionality. Eds.
Yuryev, et al., New York, Nova Science Publishers (2007) pp. 179-228.
ОСОБИНЕ СКРОБА РАЗЛИЧИТИХ ЗП ГЕНОТИПОВА КУКУРУЗА
Марија С. Милашиновић-Шеремешићa, Милица М. Радосављевића
и Љубица П. Докићб
а
б
Институт за кукуруз „Земун Поље“, С. Бајића 1, 11185 Београд, Србија
Циљ овог рада био је да се испитају молекуларне и функционалне карактеристике скроба изолованих из ЗП генотипова кукуруза различите генетичке основе.
Садржај протеина, уља, пепела, као и резистентног скроба био је веома низак. Садржај aмилозe у изолованим скробовима био карактеристичан за нормалне односно
воштане кукурузне скробове. Воштани тип скроба (ЗП 704wx) имао је највећу просечну молекулску масу амилопектина (4,84 x 108). Почетна температура желатинизације скроба 10 ЗП генотипова кретала се у распону од 62,1°C до 65,0°C. Воштани
кукурузни скроб имао је значајно већу промену енталпије желатинизације (ΔH=
18,1 J/g) у односу на нормалне скробове (ΔH=13,6-15,6 J/g). РВА профили скроба
ЗП генотипова били су типични за нормалне, односно за воштани скроб кукуруза.
Кључне речи: скроб, кукуруз, молекуларне карактеристике, функционална својства.
Received: 30 July 2012
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MODIFICATION OF THE METHOD FOR DETERMINING PROTEIN
SOLUBILITY OF HEAT TREATED FULL-FAT SOYBEANS USING
EXTRACTION IN POTASSIUM HYDROXIDE: INTER-LABORATORY STUDY
Dragan V. Palića*, Kedibone Y. Modikab, Andre Oelofsec
and Marijana B. Sakača
a
University of Novi Sad, Institute of Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
b
Agricultural Research Council, Private Bag X2, Irene, 0062, South Africa
c
University of Pretoria, Faculty of Natural Sciences, Lynnwood Road, Hatfield, South Africa
Among a number of laboratory methods used to estimate the adequacy of heat treatment applied to full-fat soybean (FFSB) protein solubility in potassium hydroxide
(PSKOH) is an unofficial method, but it is used by many laboratories due to its simplicity. It has been reported that the results of PSKOH analysis of the same sample of FFSB
on the degree of heat treatment vary widely between laboratories. In this study, the
original PSKOH method has been modified and subjected to an inter-laboratory test,
with participation of 9 laboratories. Eight FFBS samples were processed by dry extrusion at temperatures ranging from 110 to 164C and analysed on PSKOH. Processed
FFSB samples were also assessed in a growth trial with broilers. Analysis of FFSB by the
PSKOH method generated for adequately-processed FFSB values between 67.1 and
76.5%. The values above 76.5% corresponded to under-processed and below 67.1% to
over-processed FFSB. The PSKOH method generated good precision, i.e. the repeatability and reproducibility limits of 3.48 and 10.86 %, respectively. The modified PSKOH
method can be recommended as a reliable indicator for quality control of heat processed
FFSB for the use in routine laboratory practice. This study could pave the way for establishing PSKOH as an official method for determining the nutritional value of FFSB following heat treatment.
KEY WORDS: broilers, protein solubility in potassium hydroxide, inter-laboratory
analysis
INTRODUCTION
The soybean is by far the most important oilseed crop in the world. Soybeans contain
highly valuable proteins and oils (crude protein ranging from 39-41% and oil from 1821%), which make them good feed alternatives to animal proteins and oils. As an animal
feed, it is used as a high-protein source (1). Soybean prior to oil extraction is referred to
* Correspoding author: Dragan V. Palić, University of Novi Sad, Institute of Food Technology, Bulevar cara
Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]
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as full-fat soybean (FFSB). Raw FFSB contains anti-nutritional factors (ANFs) (2),
which limits its use in diets for monogastric animals. The ANFs contained in soybeans
may cause unfavourable physiological effects (3, 4), and may decrease weight gain in
animals (5).
It has been shown (6, 7) that ANFs can be destroyed by moderate heating, which
leads to denaturation of tertiary and quaternary protein structures, allowing more effecttive penetration of digestion enzymes and increasing digestibility of soybean protein in
non-ruminants.
The problem relating to the availability of the amino acids in the heat-treated soybeans arises due to the fact that only an optimum level of heat treatment will produce
maximal availability of the amino acids to the animal. Under-processing of the FFSB limits amino acid availability due to only partial destruction of the ANFs. Over-processing,
on the other hand, decreases amino acid availability as a result of the Maillard reaction
that occurs between the aldehyde groups of sugars and free amino groups of amino acids
(mostly epsilon-group of lysine) (8). The main objective of heat processing of FFSB is to
achieve an optimum balance between degradation of ANFs and maintenance of amino
acid availability.
There are a number of laboratory methods that can be used to estimate the adequacy
of FFSB heat treatment. Commonly used methods for assessing the processed FFSB quality are those for the determination of urease activity index (UAI), trypsin inhibitor activity (TIA), nitrogen solubility index (NSI), protein dispensability index (PDI), and protein
solubility in potassium hydroxide (PSKOH) (9).
It has been demonstrated (10, 11) that the results of analysis of the same sample of
FFSB on the degree of heat treatment obtained by currently available analytical techniques vary widely between laboratories, causing uncertainty among soya processors, feed
manufacturers and end-users.
The provision of amino acids, either free or as protein, contributes a substantial amount to the cost of animal feedstuffs. The objective of any nutritionist is therefore to formulate diets that will provide the correct amounts of nutrients required by the animal at
the lowest possible cost (12). This implies that dietary formulations need to be done on an
available amino acid basis in order to optimise the dietary amino acid levels, thereby minimizing the cost. As a result of the uncertainties regarding the effect of processing on the
amino acid availability of full-fat soybeans (9), some nutritionists using FFSB in diets
have been forced to compensate for the possibility of reduced amino acid availability by
over-formulating diets on a total amino acid level. It is therefore recognised that, if the
full utilisation of FFSB used in diets is to be realised, reliable analytical procedures need
to be available for quality control of processed FFSB, which would enable feed manufacturers to determine the exact degree of processing of the soybeans (10). The results of this
would in turn provide an estimate of the availability of the amino acids contained in the
FFSB for the use in feed formulations and enable nutritionists to lower the cost of feed
formulations.
There are reports (13, 14) that protein solubility might be the most reliable indicator
for FFSB quality control and that therefore NSI, PDI and PSKOH would be the preferred
methods. Although being an unofficial method, PSKOH has been widely used by many
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laboratories, due to its simplicity. The preference in this study has been therefore given to
PSKOH method.
The aim of this study was to standardise, through an inter-laboratory study, the
PSKOH method, to establish its ranges for describing under-, adequate- and over-processed FFSB and its precision, i.e. the values for its repeatability and reproducibility limits.
EXPERIMENTAL
Raw soybeans, with moisture of 10-11%, were processed by dry extrusion, using industrial „Insta-Pro 2000R“ single screw extruder at 7 temperatures: 110, 120, 127, 136,
140, 145, 151 and 164°C.
In vivo trial. A total of 336 male Ross broilers were randomly allocated to 42 pens,
each containing 8 birds. On arrival, all broilers were sorted into equal weight groups, and
assigned at random to the different treatment pens, such that initial average weight and
weight distribution were similar for all pens. They were allocated to one of eight dietary
treatments containing the heat treated FFSB, with six replicates per treatment. The average body weight gain (ADWG) in the period from day 0 to day 14 and feed conversion
ratio (FCR) on day 14 were monitored as production parameters. The protocol was approved by the Animal Ethics Committee no. APIEC 04/01 (ARC-Irene, South Africa) to
ensure that it complies with South African and international standards for the care and
use of animals for experimental purposes.
Laboratory procedure. The procedure of the original PSKOH method of Araba and
Dale (15) was as follows.
An amount of 1.5 g of a FFSB sample, ground in a mill so it would pass through a 0.5
mm screen, was mixed with 75 ml of 0.2 % (0.036 N, pH 12.5) potassium hydroxide, and
then stirred for 20 min on a magnetic stir plate. The mixture was centrifuged at 2700 rpm
for 15 min. The supernatant was decanted, and filtered through glass wool. Fifteen ml, in
duplicate from a single filtrate, were transferred to digestion tubes. Total nitrogen was determined by the Kjeldahl method and the protein content was calculated. For the original
FFSB samples, the crude-protein content was also determined. Protein solubility was expressed as a percentage of the total protein soluble in a 0.2 % solution of potassium
hydroxide.
A review of this original PSKOH method revealed the following possible sources of
differences in results of analysis of the same FFBS sample obtained by different laboratories.
1. Milling through a 0.5 mm screen.
(It was very difficult to obtain a representative sample of FFSB when this is milled
through a 0.5 mm screen. This has been shown to be especially true for samples processed above 130C).
2. Mixing of FFSB sample with 75 ml of 0.2 % KOH.
(The volume and shape of the beaker, which both affect the protein solubility, was not
specified).
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3. Stirring of the sample on a magnetic stirring plate.
(The speed of stirring and the size of magnetic stir bar, have an effect on protein solubility, were not specified).
4. Centrifugation of mixture at 2700 rpm.
(Specification of “rpm” value means little in practice, where different types of centrifuges are used).
The modification of the original PSKOH method was then made, which resulted in
the following procedure.
1. The FFSB sample was milled using a laboratory hammer or cyclone mill equipped
with a 1.0 mm screen.
2. Milled sample was sieved using 1.000 mm and 0.600 mm sieves. The fraction that
passed through a 1.000 mm sieve and was retained on a 0.600 mm sieve was taken for
analysis. Thus, improved sample homogeneity was obtained.
3. 1.5 g (± 0.001 g) of the sieved sample was placed in a 250 ml Erlenmeyer flask
(bottom diameter = 85 mm and height = 145 mm).
4. 75.0 ml of 0.2 % KOH solution was added to the sample using a calibrated pipette
and the flask was sealed with a stopper.
5. The flask was placed on a horizontal shaker immediately after addition of the KOH
solution and was shaken at 100 cycles per minute for exactly 20 minutes at room
temperature.
6. The flask was left for exactly 2 minutes for its content to settle.
7. Without delay, the content of the flask was filtered through a wide-pore filter paper
(Whatman no. 1).
8. 15.0 ml of the supernatant was placed into a 250 ml flask and total nitrogen was
determined by Kjeldahl method. Crude protein content (% nitrogen x 6.25), i.e. soluble protein of the sample, was calculated. The crude protein content of the original
FFSB sample was also determined.
9. Protein solubility was expressed as a percentage of the total protein soluble in a 0.2 %
solution of potassium hydroxide.
Eight samples of FFSB used in the in vivo trial with chickens were analysed by 9 laboratories in duplicate using the improved PSKOH method. The method has therefore
been subjected to an inter-laboratory study, conducted according to the AOCS Official
Methods (16).
Data were analysed using the statistical programme SAS/STAT (17). Analysis of variance (ANOVA) was used to test for differences between treatments. Treatment means
were separated using Fishers' protected t-test least significant difference (LSD) at the 5%
level of significance.
Precision of the improved PSKOH method was determined according to the AOCS
Official Methods (18).
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In vivo trial
The results of broiler trial are shown in Table 1.
Table 1. Mean average daily weight gain (ADWG) during the period from day 0 to day
14 and feed conversion ratio (FCR) on day 14 of broiler chickens fed FFSB processed by
dry extrusion at different temperatures
Treatment (°C)
110
120
127
136
140
145
151
164
a,b,c,d
ADWG (g)
87.8bc
96.0bc
108.0bc
138.3a
132.0a
123.0a
97.2b
79.8c
FCR
2.081d
1.893cd
1.768c
1.382a
1.466a
1.529a
1.679c
1.891cd
Values in the same column with different superscript differ significantly (P<0.05).
The results showed that the best performance was achieved by chickens that were fed
the FFSB processed at 136, 140 and 145C and that there was no significant difference
between them (P>0.05). However, the difference between the groups that received the
FFSB processed at 127 and 136C, as well as at 145 and 151C, were significant (P
<0.05). Based on these parameters, a relation between the temperature of extruding and
the in vivo assessment of the degree of FFSB processing has been derived and is shown in
Table 2.
Table 2. Relation between the temperature of extruding and the in vivo assessment of the
degree of FFSB processing
Degree of FFSB processing
Under-processed
Adequately-processed
Over-processed
Temperature of extrusion (C)
< 136
136 – 145
> 145
It was concluded that the temperatures between 136 and 145 C define the range for
adequately-processed FFSB.
Inter-laboratory results
Average values of PSKOH in FFSB samples analysed by 9 laboratories in duplicate
are shown in Table 3.
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Table 3. Results of the determination of protein solubility in potassium hydroxide
(PSKOH) in FFSB samples processed by dry extrusion at different temperatures
1
Lab No
1
2
3
4
5
6
7
8
9
Average
SD
110ºC
89.80
87.26
94.51
95.79
88.55
84.65
88.57
95.21
89.69
90.45
3.83
120ºC
85.77
88.37
88.43
91.58
90.05
91.85
83.00
95.16
88.74
89.21
3.76
1
127ºC
85.03
88.13
85.86
88.18
87.18
88.04
82.33
90.40
86.69
86.87
2.45
PSKOH (%)
136ºC 140ºC
77.14 74.73
76.55 68.80
77.97 72.06
76.76 79.04
72.79 67.84
76.02 76.64
74.19 73.45
78.05 75.64
79.11 76.68
76.51 73.87
2.07
3.70
145ºC
67.23
66.17
70.44
67.92
57.68
71.59
65.36
68.39
69.51
67.14
4.07
151ºC
67.14
65.07
70.19
68.50
59.25
70.51
73.36
58.07
68.45
66.62
4.97
164ºC
58.66
57.02
61.10
63.33
51.06
65.32
60.62
69.31
63.00
61.05
5.12
Average of two replicates; SD = Standard deviation
The influence of temperature of processing on PSKOH values is shown in Fig. 1. A
high correlation (R2 = 0.94) was established between PSKOH values and treatment
temperature.
100
PSKOH (%)
80
60
y = -0.6218x + 161.59
R² = 0.9404
RSD = 2.30
40
20
0
110
120
130
140
150
160
170
Temperature (0C)
Figure 1. The influence of processing temperature on PSKOH values
The FFBS samples processed in this study at temperatures between 136 and 145C
represented adequately-processed FFSB (Table 1). The PSKOH values for these samples
obtained in the inter-laboratory study, shown in Table 3, were 76.5 and 67.1, respectively. Consequently, the PSKOH values for describing the degree of FFSB processing
have been established and are shown in Table 4.
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Table 4. PSKOH values for describing the degree of FFSB processing
Degree of FFSB processing
Under-processed
Adequately-processed
Over-processed
PSKOH (%)
> 76.5
67.1 – 76.5
< 67.1
The PSKOH range for adequately-processed FFSB was between 67.1 and 76.5%.
PSKOH values above 76.5 % corresponded to under-processed, whereas below 67.1%
corresponded to over-processed FFSB.
Precision parameters of the PSKOH method are shown in Table 5. The results obtained under the conditions of this study showed that the repeatability limit (r), i.e. absolute
difference between two single results of analysis of the same sample obtained in one laboratory, should not exceed 3.48 %. It was found that the reproducibility limit (R) of the
PSKOH method, i.e. absolute difference between single results of analysis of the same
sample obtained in different laboratories, should not exceed 10.86 %.
Table 5. Precision parameters of the PSKOH method
Parameter
Number of
laboratories
Number of laboratories
retained after eliminating
outliers
PSKOH values (%), average
of 9 laboratories
Repeatability standard
deviation (sr), %
Repeatability relative standard
deviation (RSDr), %
Repeatability limit (r)
[r = 2,8 x sr], %
Reproducibility standard
deviation (sR), %
Reproducibility relative
standard deviation (RSDR), %
Reproducibility limit (R)
[R = 2,8 x sR] , %
Sample
136ºC
140ºC
110ºC
120ºC
127ºC
9
9
9
9
9
9
9
90.45
89.21
1.073
Average
values
145ºC
151ºC
164ºC
9
9
9
9
9
9
9
9
9
86.87
76.51
73.87
67.14
66.62
61.05
2.075
1.407
1.085
1.022
1.380
0.782
1.107
1.242
1.187
2.326
1.620
1.418
1.384
2.056
1.151
1.814
1.621
3.005
5.811
3.941
3.037
2.862
3.864
2.190
3.100
3.478
3.941
3.839
2.504
2.118
3.807
4.178
5.373
5.270
3.878
4.357
4.303
2.883
2.768
5.153
6.223
7.903
8.633
5.061
11.033
10.750
7.012
5.929
10.659
11.700
15.044
14.756
10.858
According to the in vivo trial with broilers (Table 1), the samples which represent
adequately-processed FFSB were those processed between 136 and 145°C. The PSKOH
values for those temperatures were between 67.1 and 76.5%. These results are lower than
those established by Arabe and Dale (15). According to their results, a range of protein
solubility between 80 and 85 % appeared to be consistent with optimum soybean processing for broiler chickens. Values below 75 % indicated excessive heating. Globally accepted PSKOH values for adequately processed soybean are between 75 and 85 % (9). It
is to be noted that the above-mentioned authors obtained their values in single laboratory
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studies, contrary to the present study, which might be the explanation for the differences
in the results.
There is a good correlation (R2=0.94) between the PSKOH values and extrusion temperatures. A reduction in the protein solubility in 0.2 % KOH was found to be associated
with an increased heat treatment in soybeans and a decreased growth performance in chickens (19).
The PSKOH changed consistently as soybeans were heated at higher temperatures.
The only slightly inconsistent response of PSKOH was observed at 151°C, where the value was 66.62%, thus being on the border of the range of adequately- and over-processed
FFSB. The same FFSB sample was assessed as over-processed in the in vivo trial. Previous studies by Parsons et al. (20), Anderson-Haferman et al. (21) and Batal et al. (22) reported that PSKOH is a good indicator of FFSB over-processing.
Regarding results of the present study on precision of the PSKOH method, two single
determinations performed in one laboratory should not differ by more than 3.48 %, whereas two single determinations performed in different laboratories should not differ by
more than 10.86 %. Attempts to find in accessible literature the results on precision parameters for the PSKOH method obtained elsewhere were unsuccessful.
The optimum temperature for dry extrusion of full-fat soybean for use in poultry
feeding obtained in this study (between 136 and 145 °C) is in line with the results of Ruiz
et al. (6) who established, in a trial with broilers fed extruded FFSB, that body weight
gain and feed conversion ratio were best at treatment temperatures between 126 and 140
°C. Nelson et al. (23) stated that temperatures most commonly used commercially for
extruding raw soybeans were between 135 and 140°C.
CONCLUSION
Inter-laboratory analysis of FFSB by the improved PSKOH method generated for
adequately-processed FFSB values between 67.1 and 76.5 %. Values above 76.5 % corresponded to under-processed and below 67.1 % to over-processed FFSB. Improved
PSKOH method generated good precision, i.e. repeatability and reproducibility limits of
3.48 and 10.86 %, respectively. Based on the results of this inter-laboratory study, the
PSKOH method can be recommended as a reliable indicator for quality control of heat
processed FFSB, for the use in routine laboratory practice. This study could pave the way
for establishing PSKOH as an official method for determining the nutritional value of
FFSB following heat treatment.
Acknowledgement
This study was supported by the Protein Research Foundation of South Africa.
REFERENCES
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full fat soya, 12 March 1998, Irene, South Africa, Proceedings pp.10-13.
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C.: The binding of soybean agglutinin (SBA) to the intestinal epithelium of Atlantic
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Melo, V.M.M., Carlini, C.R. and Castelar, L.I.M.: Nutritional study of two Brazilian
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proteins. J. Nutr. Biochem. 12 (2001) 1-8.
5. Palacios, M.F., Easter, R.A., Soltwedel, K.T., Parsons, C.M., Douglas, T., Hymowitz,
M.W. and Pettigrew, J.E.: Effect of soybean variety and processing on growth performance of young chicks and pigs. J. Anim. Sci. 82 (2004) 1108-1114.
6. Ruiz, N., De Belalcazar, F., Diaz, G.J.: Quality control parameters for commercial
full-fat soybeans processed by two different methods and fed to broilers. J. Appl.
Poult. Res. 13 (2004) 443-450.
7. Zarkadas, L.N. and Wiseman, J.: Influence of processing of full fat soya beans included in diets for piglets. I. Performance. Anim. Feed. Sci. Tech. 118 (2005) 109-119.
8. Vohra, F. and Kratzer, F.K.: Evaluation of soybean meal determines adequacy of heat
treatment. Feedstuffs 63 (1991) 22-25.
9. Monary, S., Fullfat Soya Handbook, 2nd ed., American Soybean Assosiation, Brussels
(1996) pp. 22-28.
10. Davies, H.: Manufacturing of full-fat soybeans. Workshop: Processing of full-fat
soya, 12 March 1998, Irene, South Africa, Proceedings, pp. 21-23.
11. Palić, D., Morey, L., Modika, K.Y., Kokić, B., Djuragić, O., and Spasevski, N.: Precision of laboratory methods based on protein solubility in quality control of heat treated feedstuffs. Chem. Industry 66, 1 (2012) 53-57.
12. Palic, D., Moloto, K., Coetzee, S.E. and Djuragic, O.: Critical assessment of laboratory methods for full -fat soybean quality control. 1st International Congress on Food
Technology, Quality and Safety, 13-15. November 2007, Novi Sad, Serbia, Proceedings, pp. 197-202.
13. Peisker, M. and Dersjan-Li, Y.: Best use of soy proteins. Feed-mix 12 (2004) 18-22.
14. Caprita, R., Caprita, A. and Cretescu, I.: Protein solubility as quality index for processed soybean. Anim. Sci. Biotech. 43 (2010) 375-378.
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processing of soybean meal. Poultry Sci. 69 (1990) 76-83.
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19. Wiriyaumpaiwong, S., Soponronnarit, S. and Prachayawarakorn, S.: Comparative
study of heating processes for full-fat soybeans. J. Food Engineer. 65 (2004) 371-382.
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20. Parsons, C.M., Hashimoto, K., Wedekind, K.J. and Baker, D.H.: Soybean protein solubility in potassium hydroxide: An in-vitro test of in-vivo protein quality. J. Anim.
Sci. 69 (1991) 2918-2924.
21. Anderson-Haferman, J.C., Ahang, Y., Parsons, C.M. and Hymowitz, T.: Effect of
heating on the nutritional quality of Kunitz-trypsin-inhibitor-free and conventional
soybeans for chicks. Poultry Sci. 71, 10 (1992) 1700-1709.
22. Batal, A.B., Douglas, M.W., Engram, A.E. and Parsons, C.M.: Protein dispersibility
index as an indicator of adequately processed soybean meal. Poultry Sci. 79 (2000)
1592-1596.
23. Nelson, A.I., Wijeratne, W.B., Weh, S.W., Wei, T.M. and Wei, L.S.: Dry extrusion as
an aid to mechanical expelling of oil from soybeans. J. Am. Oil Chem. Soc. 64 (1987)
1341-1347.
МОДИФИКАЦИЈА МЕТОДЕ ЗА ОДРЕЂИВАЊЕ РАСТВОРЉИВОСТИ
ПРОТЕИНА У ТЕРМИЧКИ ТРЕТИРАНОЈ ПУНОМАСНОЈ СОЈИ
КОРИШЋЕЊЕМ ЕКСТРАКЦИЈЕ У КАЛИЈУМ ХИДРОКСИДУ: МЕЂУЛАБОРАТОРИЈСКО ИСПИТИВАЊЕ
Драган В. Палићa, Кедибоне Модикаб, Андре Оелофсев и Маријана Б. Сакачa
a
Универзитет у Новом Саду, Институт за прехрамбене технологије, Булевар цара Лазара 1,
21000 Нови Сад, Србија
б
Agricultural Research Council, Јужно-афричка република
в
Универзитет у Преторији, Јужно-афричка република
Међу методама за процену адекватности термичког третмана пуномасне соје је
и растворљивост у калијум хидроксиду (PSKOH), која је неофицијелна метода, али
је многе лабораторије користе због њене једноставности. У овом раду PSKOH метода је модификована и примењена у 9 лабораторија. Осам узорака пуномасне соје
је екструдирано на температурама од 110 до 164C и анализирано модификованом
PSKOH методом. Исти узорци су испитани и у in vivo огледима на пилићима.
Анализом пуномасне соје модификованом PSKOH методом су добијене вредности
од 67,1 до 76,5% за адекватно третирану соју. Вредности растворљивости изнад
76,5% одговарају недовољно термички третираној соји, а испод 67,1% сувише третираној. Модификована PSKOH метода је генерисала добру прецизност: репетабилност од 3,48% и репродуктивност од 10,86%. Модификована PSKOH метода се може препоручити као поуздан индикатор за контролу квалитета термички третиране
соје.
Кључне речи: бројлери, растворљивост протеина у калијум хидроксиду, интерлабораторијска анализа
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Review
THE NUTRITIVE VALUE OF POULTRY DIETS CONTAINING SUNFLOWER
MEAL SUPPLEMENTED BY ENZYMES
*Slavica A. Sredanovića, Jovanka D. Levića, Rade D. Jovanovićb
and Olivera M. Đuragića
a
b
University of Novi Sad, Institute of Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
Institute for Science Application in Agriculture, Bulevar despota Stefana 68b, 11000 Belgrade, Serbia
The international limitations imposed on the utilization of meat and bone meals in
animal diets, together with the increasing demand for soybean meal, create a necessity to
search for other protein sources to economically balance compound feeds. In this regard
it is important to note that sunflower is the best adapted high-protein crop available in
some European regions and that is useful to use it in poultry farming as the replacement
of other protein sources. Protein and many other nutrients are “imprisoned” to variable
degrees, inside sunflower meal fibrous structures, and remain less available for digestion
by the poultry’s own proteases and other endogenous enzymes. Added exogenous enzymes (phytase, hemicellulase, cellulase, carbohydrase, protease, etc.) offer a number of
creative possibilities for breakdown and “liberation” of these nutrients, their easier digestion and absorption, and thus development of new nutritional standards and new diets
formulation. Supplementation of poultry diets containing sunflower meal by different enzymes increasingly contribute to sustainable poultry farming by enhancing production
efficiency, increasing the effectiveness of nutrient utilization and upgrading in environmental protection.
KEY WORDS: sunflower meal, enzymes, poultry nutrition
INTRODUCTION
Sunflower meal (SFM), the important by-product obtained after the extraction of oil
from sunflower seeds, is used as a protein source in animal nutrition. The amount and
chemical composition of SFM depends on the variety of the seed, the processing method,
efficiency of oil extraction and the degree of dehulling or decortication. In our country,
the crude protein (CP) content of conventional SFM usually varies between 33% and
37%. The corresponding crude fiber (CF) contents are in the range between 18% and
23%. Thus, an inverse relation is seen between the CP and CF contents of SFM. These
meals are mixtures of protein containing kernel and hulls in the approximate ratio of
about 60:40% (1, 2). In other regions, CP content of SFM is very often lower than 28%,
and CF content is higher than 30%. Due to high hull levels, these meals are mainly used
* Corresponding author: Slavica A. Sredanović, University of Novi Sad, Institute of Food Technology, Bulevar
cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]
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Review
for feeding cattle and sheep. But, using SFM in this way is wasting of valuable proteins.
Therefore, some of the characteristics of SFM must be considered so that the maximum
benefit may be gained from this feed ingredient (3-8).
An apparent disadvantage of SFM is that it contains a relatively high level of fiber
compared to soybean meal (SBM). This characteristic of SFM may lead to bulky diets
which may be a problem for young chicks, in particular, because their digestive system
has a limited capacity. If SFM is incorporated at high inclusion rates, the nutrient and
energy densities of the resulting diet may be significantly diluted and growth stagnant.
The density of the diet is of prime concern in terms of nutrient intake and resultant
growth rate (9, 10). Inclusion of high fiber ingredients is also limited because of the poor
metabolizable energy contents. True metabolizable energy contents of sunflower meal is
negativly correlated with CF and hull content (11). Separation of the hull, as the main
source of fiber, from the kernel is the processing solution for improving the nutritional
and commercial values of SP. The CF content should be decreased to a minimum by
dehulling during processing of the sunflower seed for oil extraction or after oil extraction
by different fractionation procedures of SFM based on diametrically opposed physical
characteristics of the kernel and the hull. Several efficient fractionation procedures and
complex systems for separating hulls from kernels and meals, rendering high yields of
attractive protein fractions that contain 42-48 % of CP and 8-14% of CF have been
developed at the Institute of Feed Technology – now renamed Research Centre Feed-toFood at the University of Novi Sad, Serbia (2, 12, 13, 14). Technological solutions for the
production of SFM with 44% of CP, have been introduced and implemented in several
edible oil plants, based on some of these results. In the industrial conditions, from the
initial SFM containing 37-38% of CP, 35-40% of sunflower meal with 44% of CP
(protein fraction-through) and 65-60% sunflower meal with 33% CP (cellulosic fraction overs) may be obtained (1, 2, 13). The separation of sunflower hulls from kernels with
centrifugal separator is rather heavy due to the presence of hard conglomerates made of
kernels and hulls adhered to them. The remarkable amount of these conglomerates flow
over the sieve holes and directly reduced PF yield (13). The preliminary treating may be
applied in order to crush the existing agglomerates and enable the subsequent separation
of the kernels from the hulls adhered. It is very important that the hulls are not crushed
into too small particles, so that it could be separated later by mechanical fractionation.
Continuous investigations at our research Centre Feed to Food are focused on identifying
solutions to increase yield and enhance the quality of decellulosed high protein SFM and
we have made substantil progress in improving the technological process (14). However,
hull removal has not been totally successful, probably because of the tight binding of the
hull to the kernel, and it is useful to explore other options to upgrade SFM, so that the
maximum benefit may be gained from this feed ingredient in monogastric animals diets
(2, 9).
Based on the premise that SFM may contribute a significant portion of poultry diets
and that it contain high levels of non-starch polysacharides (NSP) and phytates it would
be useful to investigate the effect of enzyme supplementation on diets containing these
ingredients. Dietary supplementation with enzyme preparations is not a new concept, but
it becomes more fine-tuned with the production of specific enzyme preparations. The
addition of exogenous enzymes (phytase, hemicellulase, cellulase, pectinase, carbohydra80
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Review
se, protease, lipase, β-glucanase, etc.) offers a number of creative possibilities for breakdown and “liberation” of these nutrients, their easier digestion and absorption, and thus
development of new nutritional standards and new diets formulation (15). The objective
of the present study is to review some of research results and posible solutions obtained
by using of enzymes as additives for upgrading the nutritive value of poultry diets containing sunflower meal.
Sunflower meal as the substrate for enzyme’s actions
Enzymes are powerful but strictly specific catalysts that act on one or, at most, a limited group of compounds known as substrates. Careful focus should be directed to the
physicochemical characteristics of target feed ingredients before applying enzymes to
feed. Physicoohemical properties like major ingredients, target substrate and its amount,
and physical structure have influenced the eficacy and onset of the enzymatic hydrolysis.
By far, the varied enzyme efficacy due to ingredients cannot be simply explained. Amounts and characteristics of NSP, anti-nutritional factors, oligosaccharides and/or other
components, physical structure of mainly starch and protein and the degree of feed processing have been compounded to represent the efficacy of supplemental enzymes (16).
Thus, detailed information about chemical composition and nutritional properties of SFM
is a prerequisite for successful use of enzymes as additives for upgrading the nutritive
value of poultry diets containing this feedstuff. To describe the quality of SFM, chemical
composition of two conventional and two high-protein SFM with 42 and 44% CP in comparation to SBM, as the »standard« that feed and animal producers want to achieve with
other vegetable protein sources, is shown in Table 1.
Table 1. Chemical composition of soybean and sunflower meals*
Nutrient
Soybean meal
Sunflower meals
Dry matter [%]
90
90
90
90
Crude protein [%]
44
34
37
42
Crude fibre [%]
5
23
18
16
Crude fat [%]
1.5
1.5
1.6
1.7
ME for poultry [MJ kg-1]
9.25
5.44
6.70
7.20
Amino acids contents [%]
Lysine
2.74
1.18
1.28
1.40
Methionine
0.60
0.72
0.79
0.92
Cystine
0.63
0.55
0.60
0.68
Threonine
1.72
1.21
1.32
1.44
Tryptophane
0.59
0.45
0.47
0.48
Arginine
3.28
2.68
2.97
3.27
Glycine
1.86
1.92
2.04
2.12
Serine
2.25
1.40
1.61
1.71
Histidine
1.17
0.82
0.89
0.96
Isoleucine
2.13
1.47
1.60
1.64
Leucine
3.40
2.12
2.33
2.64
Phenylalanine
2.22
1.50
1.64
1.95
Tyrosine
1.62
0.81
0.88
1.01
Valine
2.19
1.78
1.93
1.96
*Compiled from Tables (17, 18) and our own research (1); ME = Metabolizable energy;
90
44
12
1.7
7.95
1.70
1.10
0.85
1.70
0.61
4.10
2.46
1.86
1.10
2.15
2.90
2.10
1.10
2.20
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If properly processed, SFM with 44% of protein can be fully compared to SBM by its
crude protein content (440 vs.440 g/kg). It contains considerably smaller amounts of lysine (17 vs. 27 g/kg), but significantly higher amounts of methionine (11 vs. 6) compared
to SBM. The strong lysine deficiency in SFM has to be balanced by adding the lacking
amount of lysine to obtain the utilisation of rich amino acid potential contained in
sunflower protein (1, 13).
A large number of investigations have found a direct dependence between the energy
value of SFM and the contents of dietary fiber (11, 19). As a result of these investigations, various formulas have been set, on which the base energy value of SFM can be
predicted trough nutrient content of individual fractions of fibers. Janssen and Care (20)
also pointed that the content of dietary fiber might be a good assumption of feed's nutritive value due to the impact of sunflower shell and core content of cell walls, and the
strong negative correlation between crude fiber content and digestibility of CP and fat.
The values of the metabolic energy of SFM were ranked from 4.94 to 9.39 MJ kg-10,
where the lower values are associated with higher levels of hemicellulose, and crude fiber
(9). The low level of metabolic energy for poultry can be overcome by adding fat to the
diets with SFM (9).
Unlike most other oilseed meals, SFM is not known to have harmful anti-nutritional
factors. Namely, SFM contains the polyphenolic compounds, chlorogenic and a cafeic
acid, but the concentrations of these antinutritional factors is not toxic in poultry diets
(21, 22). However, phytic acid is considered as anti-nutritional factor in poultry because
it binds phosphorous and other important nutrients and decreases their availability. Phosphorous is an essential nutrient in poultry diets, and its efficient use is essential for economic poultry production. Unavailable phosphorous is simply excreted and the result is a
serious phosphorous pollution problem. In order to become available to broiler chicks,
phosphorous from vegetable sources must be hydrolyzed with phytase as a catalyst, to
inositols and inorganic phosphates which are readily absorbed in the digestive tract (23).
By releasing phosphorous from phytate molecule, phosphorous supplementation in diets
may be considerably reduced or even cancelled, thus leading to the reduction of phosphorus excretion, with beneficial effects on the environmental issues (24). Furthermore, phytate has also the potential to form indigestible complexes with cations (Mg, Ca, Fe) and
bind with protein (25).
SFMs are used in animal nutrition mainly as protein sources, but they also contain
significant amount of dietary fibre (DF), which is defined as the sum of lignin and polysaccharides that are not digested by endogeneous secretion of the digestive tract of nonruminant animal species. In this nutrition context, the term DF includes any polysacharide reaching the hindgut and so includes resistant starch and NSP (26). Polysaccharides
are macromolecular polymers of simple sugars or monosacharides linked together by glycosidic bonds. NSPs have glycosidic bonds other than the bonds of starch which in some
case cause their resistance to starch degrading enzymes (27). The NSPs found in feedstuffs are primarily components of plant cell walls and they represent a group of heterogeneous compounds differing considerably in chemical composition and physical properties (28).
The types and levels of carbohydrates in SFM depend to a great extent on the technology of seed processing and the degree of dehulling or decortication and, hence, the
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data available in the literature differ greatly. According to the literature data, SFM with
33% crude protein contains 1-4% starch, 26-41% DF including 9-11% arabinoxylans, 1823% cellulose, 9-10% lignins, 2-5% pectins (2, 28, 29). In order to develop techniques to
counteract the antinutritive effects of soluble NSP, and understanding their chemistry,
physical properties and behavior on ingestion by monogastric is crucial. Further work is
required to characterise the type, levels and nutritive activity of the NSP and other DF
component found SFM.
It is convenient to classify five major classes of fibers, according to their chemical
structure and to their properties: four classes of water-insoluble polymers (lignins, cellulose, hemicelluloses, pectic substances) and one class of various water-soluble non-starch
polysacharides and oligosaccharides (water soluble pectins, -glucans, arabinoxilans)
(27). Solubility of fiber components is linked to their effects in the digestive tract of animals. NSPs are generally defined as water-soluble or insoluble. Plants generally contain a
mixture of both soluble and insoluble NSPs in a ratio that varies according to the type and
stage of maturity. The types and levels of carbohydrate polymers and monomers in sunflower seed are shown in Table 2. (28).
Table 2. Carbohydrates in sunflower seed (% dry matter) (28)
Carbohydrate
Starch
Total NSP
Cellulose
Rhamnose
Fucose
Arabinose
Xylose
Mannose
Galaktose
Glucose
Uronic acids
Soluble
Insoluble
4.5
23.1
8.7
0.3
0.1
3.0
5.3
1.1
0.9
0.4
3.4
0.2
0.1
0.6
0.1
0.3
3.2
Total
1.4
27.6
8.7
0.5
0.2
3.6
5.3
1.2
1.2
0.4
6.6
The NSP content of feedstuffs influences various aspects of animal performance. The
high NSP content in SFM limit their energy value and even more their protein value.
Their nutritional effects in monogastric animals are diverse and, in some cases, extreme.
It is, however, generally conceded that the major detrimental effects of NSP are associated with the viscous nature of these polysaccharides, their physiological and morphological effects on the digestive tract, and the interaction with the microflora of the gut.
Soluble fibers increase intestinal transit time, delay gastric emptying, delay glucose
absorption, increase pancreatic secretion, and slow down absorption, whereas insoluble
fibers decrease transit time, enhance water holding capacity and assist faecal bulking in
non-ruminant animals (3, 28). These include the effects on voluntary feed intake, supply
of available energy to the animal, including the digestibility and utilization of nutrients
other than NSPs and gut and animal health. These effects can be attributed to the effects
of NSPs on gut microorganisms, viscosity and water-holding capacity of the digesta (27).
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Some investigations have suggested that the negative effect of NSP can be overcome by
dietary modifications, including supplementation of diets with suitable exogenous enzyme preparations (30).
Enzymes and sunflower meal in poultry nutrition
Regardless of the goal of animal production, enzymes will aid performance through
removal of antinutritive factors (ANF), breaking open cell walls, releasing phytate phosphorus, improving protein digestibility and provision of substrates for beneficial microflora. Exogenous enzymes supplemented to feed are theoretically capable for improving
digestibility of feed by hydrolyzing the substrates that hinder digestion and specific antinutritional factors. However, in reality the practices do not exert the theoretically expected benefits.
Type and age of animal, quality of feed ingredients, including presence of target substrate and optimization of multi-enzyme combination are factors that are to be taken into
account upon using feed enzymes. Therefore, it needs a very careful approach to decide
the type of enzymes and their additional levels. (31).
Enzymes make it possible to upgrade the nutritional value of a feedstuff and can be
added to feeds as "multi-enzyme" products that contain a variety of different activities or
"specific-enzyme" products which are responsible for single-type of enzymatic activity
based on a particular dietary substrate, such as NSP (15). Not only the total fiber content,
but also the physical and chemical structure of fibrous polysaccharides and their anatomical arrangement within each specific ingredient, affect the accessibility of enzymes for
digestion of nutrients (27). Undoubtedly, a total depolymerisation of the NSP require
extremely complex enzyme activities. There are various types of fiber-degrading or NSPbreaking enzymes.
The efficacy of feed enzymes depends on their substrate specificity, activity and stability. Therefore, it is often very difficult to select potentially useful enzymes available in
the market. Selection of effective enzyme products for use in sunflower-containing diets
requires detailed knowledge of the substrates and their breakdown patterns in the gastrointestinal tract of the target species (31). Many commercially available enzyme formulations differ in their composition with respect to the number of individual enzymes and
their activities. Selected microbial enzymes must degrade NSPs to an extent that can
lower the viscosity in the intestine and improve feed utilization. Extensive research has
revealed that enzyme usage increases the efficiency of utilization of the feed. It is now
well documented that enzymes supplementation breaks NSP polymeric chains into smalller pieces, reduces the gut viscosity, and hence improves the nutritive value of fibrous
feedstuffs.
Poultry do not produce enzymes for the hydrolysis of NSP present in the cell wall of
SFM and they remain unhydrolized. This results in low feed efficiency. Most of the research work on NSP enzyme application in poultry feeds has focused on cereal grains,
such as wheat, barley, rye and triticale, or, alternatively, on diets based on corn and SBM,
with very little targeted to alternative protein meals like sunflower meal. Inconsistent results have been reported by several authors regarding the use SFM with enzyme supplementation in poultry diets (16).
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It has been recognized that the disruption of the cell wall matrix of SFM by exogenous microbial enzymes can lead to easy access of the endogenous proteolytic enzymes
to digest the entrapped proteins (32). Results obtained by Raza et al. (33) showed significant differences (p<0.05) among different experimental diets (varying in the level of
SFM and CF) for weight gain and feed conversion. The highest weight gain was observed
in chicks fed on the diet containing 10% SFM (6% CF in diet) with enzyme Grindazym
GP 5000, (produced by DuPontTM Danisco® Company and containing xylanase, P-glucanase and pectinase), while lowest gain was observed in chicks fed on the same diet
without enzyme supplementation. Francesch et al. (34) reported the results of dietary supplementation of an enzyme preparation (Grindazym GP 5000) containing xylanase, Pglucanase and pectinase activity included for four months in a barley : SFM-based (60 :
20%) layer diet. There was no significant effect of enzyme supplementation on the rate of
lay, daily food intake or body weight gain. However, a significantly positive effect was
observed during the first four weeks on egg weight and egg size, and there was also a reduction in the percentage of dirty eggs. High doses of enzyme also improved excreta
quality by reducing its water content. Sorensen (35) has reported that supplementation of
SFM-based diets with the same enzyme increased the nutrient utilisation of this product,
both in layers and broilers. Improvement in the performance of broilers with the addition
of multienzyme in high sunflower diets were also noticed by Meussen, (36), Raj et al.
(37), and Kocher et al., (32). Mushtaq et al. (38) reported that enzyme supplementation
had a pronounced effect in low nutrient concentration and high SFM diets for broilers
According to the results of Tavernari (39) the enzyme complex consisting of cellulase, β-glucanase, xylanase and phytase had a significant effect on the weight gain only
during the starter phase with the diets contained 20% SFM, which is possibly explained
by the immature digestive system of broilers at this age. Oliveira et al. (40) evaluated two
sunflower meal inclusion levels (0 and 15%) with or without an enzyme complex (cellulase, protease and amylase) in the diet of 21 to 42-day-old broilers, and did not find any
significant interaction between SFM and the enzyme complex. These authors concluded
that the dietary inclusion of 15% SFM improves live performance, but does not affect
carcass yield. El Sherif (41) found that SFM concentrated with energy and supplemented
with lysine and methionine was effectively utilized in grower and finisher broiler diets in
place of SBM without adverse effect on the production, and there were no beneficial effects of enzyme supplementation with this SFM. The results of Kocher et al. (32) clearly
indicate that comercial enzyme products have some effects in diets containing high
concentrations of SFM. However, these effects could not be seen after detailed analyses
of feed and digesta, and did not result in a significant improvement in the growth performance of broilers. Meng and Slominski (42) and Tabook et al. (43) have indicated that
the addition of commercially available multiactivity enzyme products did not result in an
improved broiler performance in diets, especially having increased concentration of SFM.
These controversial results reported regarding the nutritive value of SFM were attributed
to the differences in the variety, method of processing, age of birds and feed formulation
techniques employed in these studies (9).
It is clear from the literature that phytate levels in poultry feed are variable, and have
a negative effect on the overall efficiency of nutrient utilization by decreasing mineral
and protein solubility and digestibility, and increasing endogenous secretions (23). First
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research works regarding the usage of phytase in animal feed were conducted some thirty
years ago, but without wider application in field conditions. As a result of the growing
environmental concerns and more stringent environmental regulations, however, research
related to the production and application of phytase in animal feed has been intensified in
the recent years. The use of phytase has become standard practice to reduce phosphorus
levels in the environment and to compensate for the drastic increase of the cost of inorganic phosphates.
In order to be made available to broiler chicks, phosphorous from vegetable sources
must be hydrolyzed with phytase as a catalyst, to inositol and inorganic phosphates,
which are readily absorbed in the digestive tract (44). Results of numerous experiments
have shown that degradation of phytate by phytase has a twofold positive effect – release
of phosphorous and release of minerals, proteins and digestive enzymes. By releasing
phosphorous from phytate molecule, phosphorous supplementation in diets may be considerably reduced or even cancelled, thus leading to the reduction of phosphorus excretion (45), with beneficial effects on the environment. Phytase increases the digestibility of
phytate from around 25% to 50-70% in poultry, and its use has been on the increase since
banning the use of animal protein sources, such as meat and bone meal, in the EU. It is
also understood that phytase can improve the digestibility of other nutrients as well as
energy (46, 47).
All obtained results about the use of phytase in poultry feeding indicate that with the
addition of various phytase preparations in differently formulated diets, this enzyme improves availability of phytic phosphorous and other nutrients (44, 48, 49, 50) and reduces
phosphorous excretion (45,51).
Supplementation of poultry diets with exogenous enzymes as additives for upgrading
the nutritive value of poultry diets containing SFM has been investigated in our Institute.
The higher body weight by 8.36% and better feed conversion ratio by 2.84 % was observed in chicks fed on a diet containing 15% SFM (with 44% CP) supplemented with
enzymes (protease, hemicellulase, pectinase, β-glucanase) with respect to the trial diets
without enzyme supplementation (29). With the diet containing 10% SFM with 33% CF
supplemented by enzymes (cellulase, protease, lipase, α-amylase, β-glucanase) higher body weight by 10.23% and better feed conversion ratio by 8.15 % were obtained in comparasion with the diets without enzymes (52 ).
In one of our latest investigation (53), experimental diets were formulated on the basis
of corn, high-quality decellulosed SFM containing 44% of protein (20% in diets) and
SBM. Five broiler diets were tested. The first was a commercial diet based on corn and
SBM and in the others 20% of SBM was replaced with SFM containing 44% of crude
protein. The treatments were as follows: A – SBM without supplement; B – SFM without
supplement; C – SFM supplemented with l-lysine HCl; D – SFM supplemented with llysine HCl and enzyme complex containing protease, hemicellulase, pectinase and β-glukanase; E – SFM supplemented with l-lysine HCl and phytase and 30% lower phosphorous content. Both enzymatic supplements positively influenced live weight and feed
conversion ratio (FCR) in treatments D and E. There were no significant differences between these two treatments, but significant differences (P<0.01) were found for live
weight and FCR between treatments B, D and E. Significantly positive (P<0.01) effect of
added enzymes was found in treatments D and E in comparison with treatment A. It was
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also found that the treatment E with reduced phosphorous level and added l-lysine HCl
and phytase decreased the level of phosphorous in broiler feces up to 20%. The decrease
of available phosphorous level in diet E did not have any adverse effect on broiler feeding performances. From this experiment, it can be concluded that the supplementation of
poultry diets with 20% SFM containing 44% of crude protein by l-lysine HCl and enzyme complex (protease, hemicellulase, pectinase and β-glukanase) or phytase increasingly
contribute to the sustainable poultry farming by enhancing the production efficiency, increasing the effectiveness of nutrient utilization and upgrading environmental protection.
CONCLUSION
In spite of some conflicting results, in most studies SFM has been found to be a promising source of protein for poultry. Most of our research work suggested that the negative effect of SFM can be overcome by processing technologies and dietary modifications including supplementation of diets with suitable exogenous enzyme preparations. In
this context, research efforts have been directed to identify novel, alternative and economically viable SFM + enzyme combinations for a succesful replacement of other protein
sources in poultry diets.
Acknowledgement
This work is a part of Integrated and Interdisciplinary Research Project No. III 46012,
funded by the Ministry of Education and Science of the Republic of Serbia.
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45. Kornsbak A.: Korišćenje fitaze u hrani za životinje, Efikasnost i stabilnost. VIII Simpozijum Tehnologije Stočne Hrane, Petrovac na Moru, 02-06 juni 1998, Zbornik radova, p. 190-202.
46. Ravindran, V., Cabahung, S., Ravindran G., Selle, P.H. and Bryden, W.L.: Influence
of Microbial Phytase on Apparent Ileal Amino Acid Digestibility of Feedstuff For
Broilers. Poult. Sci. 78 (1999) 699-706.
47. Kornegay, E.T.: Dygestion of Phosphorus and Other Nutrients: The Role of Phytases
and Factors Influencing Their Activity. (In: Bedford, M.R., and Partridge, G.G., eds.
Enzymes in Farm Animal Nutrition). CABI Publishing, Wallingford UK (2001) pp.
237-271.
48. Mitchell R., Nir I., Nitsan Z. and Larbier M.: Effect of Phytase and 1,25-Dihydroxycholecalciferol of Phytate Utilisation and the Quantitative Requirement for Calcium
and Phosphorous in Young Broiler Chickens, Poult. Sci. 75, 1(1993) 95-110.
49. Cantor A. H.: Using Enzymes to Increase Phosphorous Availability in Poultry Diets.
Alltec’h 11th Annual Symposium, Kentacky USA, 17-19 May 1995, Proceedings, p.
349-353.
50. Sebastian S., Touchburn S., Chavez, E. And Lague, P.: The Effects of Supplementation Microbial Phytase on the Performance and Utilisation of Dietary Calcium, Phosphorous, Cooper and Zinc in Broiler Chickens Fed Corn-Soybean Diets. Poult. Sci.
75, 8 (1996) 729-763.
51. Vetesi M., Mezes M., Baskay Gy. And Gelencser E.: Effects of Phytase Suplementation on Performance, Calcium and Phosphor Output and Mechanical Stability of Tibia
in Broiler Chicken. 10th European Poultry Conference, Jerusalem, 21-26 June 1998,
Proceedings, p. 99.
52. Sredanović, S., Lević, J., Šanta, Š., Rac, M. and Harting, E.: "Dijaprot 33 F" i "Dijaprot 33 E" u ishrani pilića. 42. Savetovanje industrije ulja “Proizvodnja i prerada
uljarica”, Herceg Novi, 03.-08. juna (2001), Zbornik radova, p.107-111.
53. Lević, J., Sredanović, S., Đuragić, O., & Džinić, N. (2009). Formulation of New Diets
for Sustainable Poultry Farming. PTEP – J. Process. Ener. Agric. 13, 3 (2009) 244246.
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Review
ХРАНЉИВА ВРЕДНОСТ ОБРОКА ЗА ЖИВИНУ КОЈИ САДРЖЕ
СУНЦОКРЕТОВУ САЧМУ ДОПУЊЕНУ ЕНЗИМИМА
*Славица А.Средановића, Јованка Д. Левића, Раде Д. Јовановићб и
Оливера М. Ђурагића
а
Универзитет у Новом Саду, Институт за прехрамбене технологије, Булевар цара Лазара 1,
21000 Нови Сад, Србија
б
Институт за примену науке у пољопривреди, Булевар деспота Стефана 68б, 11000 Београд, Србија
Међународна ограничења наметнута за коришћењу месно-коштаног брашна у
оброцима животиња, заједно са повећањем потражње за сојином сачмом, стварају
потребу за проналажењем других извора протеина да би се смеше за животиње избалансирале на економичнан начин. У том смислу важно је напоменути да је сунцокрет најбоље прилагођен високо-протеински усев доступан у појединим европским регионима и да га је корисно употребљавати у живинарству као замену за
друге изворе протеина. Протеини и многе друге хранљиве материје су "затворени"
до променљивог степена, унутар влакнасте структуре сунцокретове сачме што смањује њихову доступност за варење сопственим протеазама и додатим ендогеним
ензимима. Додати ендогени ензими (фитазе, хемицелулазе, целулазе, карбохидразе,
протеазе...) нуде низ креативних могућности за разлагање и "ослобађање" тих хранљивих материја, њихово лакше варење и апсорпцију, а тиме и за развој нових прехрамбених стандарда и нове формулације оброка. Допуњавање оброка живине који
садрже сунцокретову сачму различитим ензимима све више доприноси одрживом
узгоју живине, унапређењем ефикасности производње, повећањем ефикасности коришћења хранљивих материја и унапређењем у заштити животне средине.
Кључне речи: сунцокретова сачма, ензими, исхрана живине
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CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF BERRY
FRUITS
Slađana M. Stajčić*, Aleksandra N. Tepić, Sonja M. Djilas, Zdravko M. Šumić,
Jasna M. Čanadanović-Brunet, Gordana S. Ćetković, Jelena J. Vulić
and Vesna T.Tumbas
University of Novi Sad, Faculty of Technology, Bul. Cara Lazara 1, 21000 Novi Sad, Serbia
The main chemical composition, contents of total phenolic (TPh), total flavonoid
(TF), and total monomeric anthocyianin (TMA), as well as the antioxidant activity of two
raspberry cultivars (Meeker and Willamette), two blackberry cultivars (Čačanska bestrna
and Thornfree) and wild bilberry were studied. The raspberry cultivars had the highest
total solids among fruits investigated. Bilberry fruits had the highest sugar-to-acid ratio.
Blackberry fruits were richer in crude fibers (cellulose) in comparison to raspberry and
bilberry fruits. The content of pectic substances was highest in the bilberry. Also, bilberry
had a highest content of TPh (808.12 mg GAE/100 g FW), TF (716.31 mg RE/100 g FW)
and TMA (447.83 mg CGE/100 g FW). The antioxidant activity was evaluated spectrophotometrically, using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
assay. The DPPH free radical scavenging activity, expressed as the EC50 value (in mg of
fresh weight of berry fruit per ml of the reaction mixture), of bilberry (0.3157  0.0145
mg/ml) was the highest. These results also showed that the antioxidant value of 100 g FW
bilberry, raspberry - Willamette, raspberry - Meeker, blackberry - Čačanska bestrna and
blackberry - Thornfree is equivalent to 576.50 mg, 282.74 mg, 191.58 mg, 222.28 mg and
272.01 mg of vitamin C, respectively. There was a significant positive correlation between the antioxidant activities and content of total phenolics (RTPh2=0.9627), flavonoids
(RTF2=0.9598) and anthocyanins (RTMA2=0.9496) in berry fruits.
KEY WORDS: berry fruits, main chemical composition, phenolic compounds,
antioxidant activity
INTRODUCTION
Free radicals and other reactive species can cause oxidation and biomolecular damages when the oxidative species exceed the antioxidative defense of the organism, resulting in oxidative stress. This is associated to aging and to the development of pathologies
such as cancer, cardiovascular disease, neurodegenerative disorders, diabetes, and inflam* Corresponding author: Slađana M. Stajčić, University of Novi Sad, Faculty of Technology, Bulevar cara
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mation (1-3). To prevent or slow down the oxidative stress induced by free radicals,
sufficient amounts of antioxidants need to be consumed. Fruit and vegetables contain a
wide variety of antioxidant compounds (phytochemicals) such as phenolics that may help
protect cellular systems from oxidative damage and lower the risk of chronic diseases (4).
Recently, an increasing number of studies have investigated the diverse protective
effects of berry fruits (5-7). Protective effects of berry fruits have been attributed to various classes of phenolic compounds, mostly flavonoids and anthocyanins (8-10).
The raspberry fruit is rich in phenolic compounds such as phenolic acids (ellagic acid
and hydrolysable tannins - ellagic acid derivatives), flavonoids (flavan-3-ols and their
oligomers - mainly dimmers, quercetin) and anthocyanins (cyanidin-3-sophoroside, cyanidin-3-(2-glucosylrutinoside), cyanidin-3-glucoside, pelargonidin-3-sophoroside, cyanidin-3-rutinoside, pelargonidin-3-(2-glucosylrutinoside), pelargonidin-3-glucoside, pelargonidin-3-rutinoside) (11-15). Anthocyanins constitute the main group of phenolic compounds in raspberry. In this fruit, the content of ellagic acid is reported to be high, but not
higher than of antocyanins (14).
Bilberries contain high quantities of anthocyanins (in which five antocyanidins – delphinidin, cyanidin, petunidin, peonidin, and malvidin are combined with three types of
sugars - galactose, glucose, arabinose), flavanols (catechin, epicatechin), flavonols (quercetin, myricetin, rutin), phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, p-coumaric acid, ellagic acid, gallic acid) and stilbene (trans-resveratrol) (16, 17).
It has been reported that ellagitannins and cyanidin-3-glucoside are the major phenolic compounds in blackberries. The anthocyanins (cyanidin-3-rutinoside and cyanidin3-malonyl glucoside), flavonols (quercetin and kaempferol glycosides) and flavan-3-ol
(epicatechin) were also identified in blackberries. Hydroxycinnamic acids are minor compounds, and they are found as ferulic, caffeic and p-coumaric acid esters (18).
The berries are not only available fresh, but are generally consumable frozen and
processed into juice, wine, jam, syrup, soft spreads and tea. Also, berries are interesting
as ingredients for use in ice cream and cake icing. Besides, they can be used in the development of functional foods with the objective of enhancing health conditions (19).
The berries have special significance for our country because they represent an important export product. According to raspberry and blackberry production, Serbia is
among the leading world countries, whereas bilberries are traditionally collected in
woods. Between 90 and 95% of cultivated raspberries in our country are North American
Willamette cultivar, which is characterized by the excellent taste and a dark red colour.
Besides the Willamette cultivar, in raspberry commercial plantings, Meeker is also cultivated to a minor extent. The predominant cultivar of blackberries in Serbia is Čačanska
bestrna (50% of total production), followed by Thornfree and Black saten (20-22).
The phenolic compounds in berries have been reported to have antioxidant, anticancer, antiinflammatory, and antineurodegenerative biological properties (23-24). Because
of the biological properties associated with berry fruits, the identification of their antioxidant activity is necessary for the evaluation of berry consumption on human health.
The data on chemical composition and antioxidant activity of berry fruits, examined in
this work, are poor concerning the growing region (25, 26). For these reasons, the aim of
this study was to investigate the main chemical composition, total phenolics, flavonoid
and anthocyianin content, as well as the antioxidant activity of two raspberry cultivars
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(Meeker and Willamette), two blackberry cultivars (Čačanka and Thornfree) and wild bilberry. Also, the correlation between the total phenolics/flavonoids/anthocyanins and antioxidant activity of berry fruits was investigated.
EXPERIMENTAL
Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, ascorbic acid, gallic
acid and rutin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). These
chemicals were of analytical reagent grade. The other chemicals and solvents used were
of the highest analytical grade, obtained from “Zorka” Šabac (Serbia).
Plant material
Two raspberry (Willamette and Meeker) and two blackberry (Čačanska bestrna and
Thornfree) cultivars were purchased from Alfa RS, Lipolist, Serbia. Wild bilberry from
the region of Kopaonik mountain, were purchased from ITN, Belgrade, Serbia. Fresh undamaged berries were freozen and stored at -20°C for two months. The fruits were defrosted and mashed before chemical analyses.
Main chemical composition
Contents of total solids, total ash, sugar (total sugars, reducing sugars, sucrose) were
assessed according to the Regulation on methods of sampling and chemical and physical
analyses of fruit and vegetable producst, 29/83 (27) Total solids were determined by
drying the samples at 105 °C to constant weight total ash was measured gravimetrically
by incenerating the samples at 525  25 °C to constant weight; cellulose (as crude fibers)
was determined by the Kirschner-Ganakova method; sugar content was assessed by the
method of Luff-Schoorl; pectin content was measured colorimetrically by carbasole
method; acidity was determined by titration with NaOH standard solution, and protein
content was evaluated by Kjeldahl's method (28).
Total monomeric anthocyanin content
Total monomeric anthocyanins (TMA) in raspberry, blackberry and bilberry fruits
was determined according to Giusti and Wrolstad method (29) based on the pH-differential method previously described by Fuleki and Francis (30). Anthocyanin content was
expressed as mg of cyanidin 3-glucoside equivalents per 100 g of fresh weight of berry
fruit (mg CGE/100 g FW).
Extraction for measurement of phenolics, flavonoids and antioxidant activity
The weighed sample of berry fruit (20 g) was extracted at room temperature using an
homogenizer, Ultraturax DIAX 900 (Heidolph Instruments GmbH, Kelheim, Germany).
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The extraction was performed using 80% of methanol aqueous solution with 0.05%
acetic acid with two portions of the solvent: 160 ml for 60 min, and 80 ml for 30 min.
The obtained extracts were combined and evaporated to dryness under reduced pressure.
The yields of the bilberry (B), raspberry - Meeker (M), raspberry - Willamette (W),
blackberry - Čačanska bestrna (Č) and blackberry - Thornfree (T) extracts were: YB =
15.74  0.71%, YM = 12.59  0.48 %, YW = 12.18  0.52%, YČ = 7.52  0.33% and YT =
9.65  0.41%, respectively.
Total phenolic content
The amount of total phenolics (TPh) in the berry fruit extracts was determined spectrophotometrically (UV-1800 spectrophotometer, Shimadzu, Kyoto, Japan) by the FolinCiocalteu method (31). The total phenolic content was determined from the regression
equation of the gallic acid calibration curve, and expressed as mg of gallic acid equivalents per 100 g fresh weight of berry fruit (mg GAE/100 g FW).
Total flavonoid content
Total flavonoids (TF) in the berry fruit extracts were measured by the aluminum
chloride spectrophotometric assay (32). Total flavonoid content was determined from the
regression equation of the rutin calibration curve, and expressed as mg of rutin equivalents per 100 g fresh weight of berry fruit (mg RE/100 g FW).
DPPH radical scavenging activity
The DPPH radical scavenging activity (SA) of berry fruits was determined spectrophotometrically using the modified DPPH method of Chen et al. (33). Briefly, 1 ml of
extract solution in distilled water or 1 ml of distilled water (blank) was mixed with 2 ml
of DPPH solution (2 mg of DPPH was dissolved in 50 ml of methanol). The range of the
investigated extract concentrations was 0.002 - 0.5 mg/ml. The mixture was shaken vigorously and left at room temperature for 30 min, then the absorbance was read at 517 nm
using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The capability to scavenge the DPPH radicals (DPPH radical scavenging activity) was calculated using the following equation:
SA (%) = 100 × (Ablank – Asample)/Ablank
where Ablank is the absorbance of the blank, and Asample is the absorbance of the sample.
Ascorbic acid was used as a reference compound.
Statistical analysis
All measurements were carried out in triplicate, and presented as mean ± SD. The
correlation and linear regression analyses were performed using Microsoft Office Excel
2003.
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Results of the determination of the main chemical composition of raspberry, blackberry and bilberry are shown in Table 1.
Table 1. Main chemical composition of raspberry, blackberry and bilberry
Raspberry
Compound
(g/100 g FW*)
Meeker
Willamette
Total solids
Ash
Cellulose
Pectin
Pectic acid
Protopectin
Acidity
Total sugars
Reducing sugars
Sucrose
Proteins
19.36  0.19
0.54  0.03
1.64  0.01
0.18  0.04
0.14  0.02
0.35  0.03
1.28  0.04
10.64  0.12
9.81  0.10
0.79
1.58  0.07
16.56  0.2
0.44  0.005
1.50  0.04
0.16  0.03
0.070  0.01
0.18  0.05
1.65  0.00
7.85  0.15
7.19  0.07
0.63
1.44  0.06
Blackberry
Čačanska
Thornfree
bestrna
11.96  0.38
15.57  0.52
0.29  0.01
0.41  0.01
2.2  0.10
2.97  0.03
0.29  0.01
0.30  0.01
0.1  0.00
0.10  0.00
0.15  0.00
0.17  0.00
1.36  0.02
1.39  0.02
5.36  0.03
5.98  0.08
1.46  0.16
1.32  0.03
3.71
4.43
1.4  0.05
1.49  0.03
Bilberry
14.75  0.29
0.25  0.004
0.91  0.02
0.1  0.00
0.38  0.01
0.25  0.01
0.52  0.05
7.84  0.08
6.69  0.11
0.94
1.01  0.09
*FW – fresh weight of berry fruits
Numerous parameters, like variety, growing conditions, harvesting, maturity stage,
transport and handling conditions affect the chemical composition of fruit. Besides, the
methods of sample preparation (freezing, storage temperature, time of storage, etc.), and
chemical analyses also influence the obtained results. For these reasons, it can be a quite
difficult job to interprete and compare the results obtained by different researchers. From
the results presented in Table 1, it is obvious that raspberry cultivars had the highest total
solids amnog the investigated fruits. The highest the total solids, the more convenient and
desirable is the fruit for processing. Generally, the highest share in total solids of fruit is
contributed by carbohydrates, i.e. sugars. The content of sugar depends on all above mentioned factors. Organic acids, together with sugars, play an important role in the sensory
characteristics of fruit. The dominant acid in berry fruit is citric acid. The results show
that among the investigated fruits a highest sugar-to-acid ratio had bilberry.
From the results shown in Table 1 it can be seen that blackberry fruits were richer in
crude fibers (cellulose) compared to raspberries and blueberries. On the other hand, bilberry had a highest content of pectic substances. Raspberry - Meeker had the highest share of water insoluble fraction (protopectin), which could be related to the fruit firmness,
as softening of the fruit is accompanied with solubilization of pectins due to the action of
different enzymes (34).
The feature that makes berry fruit very popular among consumers is their contents of
anthocyanins, phenolics and flavonoids, as they exhibit antioxidant activity (35-38) in
biological systems. It is observed that the contents of total phenolics, flavonoids and
anthocyanins in bilberry are higher than in raspberry cultivars and bilberry (Table 2).
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Table 2. Contents of total phenolics, flavonoids and anthocyanins in berry fruits
Berry fruit
Bilberry
Raspberry - W
Raspberry - M
Blackberry - Č
Blackberry - T
TPh
(mg GAE/100 g FW)
808.12  32.56
303.90  12.40
265.38  10.75
235.09  10.72
270.22  12.45
TF
(mg RE/100 g FW)
716.31  30.82
169.51  6.24
152.31  5.35
143.33  5.38
172.95  6.86
TMA
(mg CGE/100 g FW)
447.83  4.16
43,29  2.05
47,34  1.85
50.95  3.41
102.31  4.04
Our results of phenolic content in examined Meeker (3.04 mg/g) and Willamette (2.65
mg/g) raspberry were higher than those in the study of Milivojević et al. (26), who
reported for Meeker and Willamette raspberry phenolic content of 2.22 and 1.02 mg/g,
respectively. Lugasi et al. (39) reported phenolic content of 244 mg/100 g for red raspberry. In the study of De Ancos et al. (40) it was shown that the amount of total phenolics
in the fresh raspberries depends on the seasonal period of harvesting; late cultivars, Zeva
(1776.02 mg of GAE kg-1) and Rubi (1556.67 mg of GAE kg-1), showed the greater
phenolic content, and the early cultivars, Autumn Bliss (1212.42 mg of GAE kg-1) and
Heritage (1137.25 mg of GAE kg-1), the lowest ones.
Jovančević et al. (41) reported for wild bilberry (Vaccinium myrtillus L.) collected in
the summer of 2009, from 11 different localities in the mountain region of Montenegro
that the total phenolic content in all analyzed samples ranged from 3.92 to 5.24 mg
GAE/g FW, while the amounts of total anthocyanins varied between 0.27 to 0.46%.
Može et al. (16) showed that the total phenolic contents of bilberries (Vaccinium myrtillus
L.) sampled from seven different locations in Slovenia ranged from 1027 to 1629 mg/100
g FW. In the samples, 15 anthocyanins were identified by LC-MS/MS. Their contents
were 1210.3 ± 111.5 mg CGE/100 g FW.
Sellappan et al. (42) showed that the average total anthocyanin and polyphenolic
contents in blackberries were 116.59 ± 8.58 mg/100 g berry and 486.53 ± 97.13 mg/100 g
berry. Šamec and Piljac-Žegarac (43) reported for blackberries the total phenol, flavonoid
and anthocyanin content of 364.24 ± 9.09 mg GAE/100 g FW, 66.13 ± 3.76 mg CE/100 g
FW and 121.82 ± 2.30 mg CGE/100 g FW, respectively.
The differences in the reported results could be due to the environmental conditions,
period of harvesting, cultivar variability, or fruit maturity (40).
Several methods have been developed to determine the antioxidant activity of fruits, vegetables as well as herbs. Two major mechanisms, namely hydrogen atom transfer (HAT)
and single electron transfer (SET), are well known in the evaluation of the antioxidant
activity against free radicals. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay is one of the
methods that utilizes both the HAT and SET mechanism. It is considered to be predominantly based on the electron transfer reaction, whereas hydrogen-atom transfer reaction
is only a marginal pathway (44). The antioxidant molecules can quench DPPH free radicals (i.e. by providing hydrogen atoms or by electron donation, conceivably via a freeradical attack on the DPPH molecule) and convert them to a colorless/bleached product
(i.e. 2,2-diphenyl-1-hydrazine, or a substituted analogous hydrazine), resulting in a decre98
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ase in the absorbance at 517 nm (45, 46). Fig. 1 shows the dose response for the DPPH
radical scavenging activity of raspberry, blackberry and bilberry extracts. The DPPH free
radical scavenging activity of the extracts increased with increasing concentration.
100
80
SA (%)
60
Raspberry - Willamette
40
Raspberry - Meeker
Bilberry
20
Blackberry - Čačanska bestrna
Blackberry - Thornfree
0
0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration (mg/ml)
Figure 1. DPPH radical scavenging activity (SA) of the berry fruit extracts
The EC50 value, defined as the concentration of sample required for 50% scavenging
of DPPH radicals under experimental conditions employed, is a parameter widely used to
measure the free radical scavenging activity (47); a smaller EC50 value corresponds to a
higher antioxidant activity. The EC50 values of berry fruits (in mg of extracts or fresh
weight of berry fruit per ml of the reaction mixture) and ascorbic acid are shown in Table
3. Bilberry showed a higher DPPH free radical scavenging activity, expressed as EC50
value (0.3157  0.0145 mg FW/ml), than the raspberry - Willamette (0.6437  0.0290 mg
FW/ml), raspberry - Meeker (0.9500  0.0425 mg FW/ml), blackberry - Čačanska bestrna
(0.8188  0.0385 mg FW/ml) and blackberry - Thornfree (0.6691  0.0324 mg FW/ml).
Ascorbic acid (vitamin C), because of its antioxidant activity (EC50 = 1.82  0.07 µg/ml)
was used as a reference compound. These results show that the antioxidant value of 100 g
bilberry, raspberry - Willamette, raspberry - Meeker, blackberry - Čačanska bestrna and
blackberry - Thornfree is equivalent to 576.50 mg, 282.74 mg, 191.58 mg, 222.28 mg
and 272.01 mg of vitamin C, respectively.
Table 3. EC50 values of different berry fruit extracts/berry fruits (FW) and ascorbic acid
Berry fruit /
reference compound
Bilberry
Raspberry - W
Raspberry - M
Blackberry - Č
Blackberry - T
Ascorbic acid
EC50
(mg extract or ascorbic acid/ml)
0.0497  0.0023
0.0784  0.0035
0.1196  0.0054
0.0616  0.0029
0.0646  0.0031
0.0018  0.0001
EC50
(mg FW/ml)
0.3157  0.0145
0.6437  0.0290
0.9500  0.0425
0.8188  0.0385
0.6691  0.0324
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It is interesting to consider the correlation between the content of total phenolics/ flavonoids/anthocyanins and antioxidant activity of berry fruits, as phenolic compounds
contribute directly to the antioxidant activity (48). The 1/EC50 is representative of the
antioxidant activity because the higher is this value, the more efficient is the berry fruit.
The high correlation coefficients (R2>0.90), calculated from the regression analysis, indicates that there is a significant positive correlation between the content of total phenolics/flavonoids/anthocyanins in berry fruits and DPPH radical scavenging activity (Figure
2).
Content (mg/100 g FW)
1000
Phenolics
Flavonoids
Anthocyanins
y = 281.1x - 100.74
RTPh² = 0.9627
y = 284.35x - 201.21
RTF² = 0.9598
500
y = 201.1x - 203.1
RTMA² = 0.9496
0
0
0.5
1
1.5
2
1/EC50
2.5
3
3.5
Figure 2. Correlation between content of total phenolics/ flavonoids/anthocyanins
in berry fruits and 1/EC50 value
The linear relations shown in Fig. 2 demonstrate a high positive correlation between
the antioxidant activities of berry fruits, determined by DPPH method and the content of
total phenolics (RTPh2=0.9627), flavonoids (RTF2=0.9598) and anthocyanins (RTMA2 =
0.9496). This conclusion is in agreement with previous findings, obtained on blueberries
(9, 10, 49-51). Also, these results suggest that some other phenolic compounds (probably
some phenolic acids), in addition to anthocyanins and other flavonoids, are also responsible for the antioxidant activity of berry fruits.
CONCLUSION
In this work, the main chemical composition of the wild bilberry, two raspberry
cultivars (Willamette and Meeker), two blackberry cultivars (Čačanska bestrna and
Thornfree) was determined. The raspberry cultivars had the highest total solids among
fruits investigated. The highest sugar-to-acid ratio was found in bilberry. The blackberry
fruits were richer in crude fibers (cellulose) in comparison to the raspberry and bilberry
fruits. The content of pectic substances were highest in the bilberry. The contents of total
phenolics, flavonoids and anthocyanins in bilberry were higher than in the raspberry and
blackberry cultivars. A highest DPPH free radical scavenging activity showed the bilberry. The antioxidant value of 100 g FW bilberry, raspberry-Willamette, raspberry-Meeker,
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blackberry - Čačanska bestrna and blackberry - Thornfree is equivalent to 576.50 mg,
282.74 mg, 191.58 mg, 222.28 mg and 272.01 mg of vitamin C, respectively. A high positive correlation between the antioxidant activities of berry fruits and the content of total
phenolics, flavonoids and anthocyanins (R2>0.90) indicates that some other phenolic
compounds (probably some phenolic acids), in addition to anthocyanins and other flavonoids, were also responsible for antioxidant activity of berry fruits. The high antioxidant activity and significant positive correlation between the concentration of phenolics/flavonoids/anthocyanins and DPPH radical scavenging activity indicate that all investigated berry fruits can be considered as a good source of natural antioxidants that
may have potential health effects.
Acknowledgement
This research is part of the Project TR 31044 is financially supported by the Ministry
of Education Science and Technological Development of the Republic of Serbia.
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ХЕМИЈСКИ САСТАВ И АНТИОКСИДАТИВНА АКТИВНОСТ
БОБИЧАСТОГ ВОЋА
Слађана М. Стајчић, Александра Н. Тепић, Соња М. Ђилас, Здравко М. Шумић,
Јасна М. Чанадановић-Брунет, Гордана С. Ћетковић, Јелена Ј. Вулић и
Весна Т. Тумбас
Универзитет у Новом Саду, Технолошки факултет, Булeвар Цара Лазара 1, 21000 Нови Сад, Србија
У овом раду испитани су основни хемијски састав, садржај укупних полифенолних једињења, флавоноида и антоцијана, као и антиоксидативна активност малине (сорти Meeker и Willamette), купине (сорти Чачанска бестрна и Thornfree) и
дивље боровнице. Од испитаног воћа највећи садржај суве материје утвређен је код
обе сорте малине. Највећи однос шећера и киселина одређен је за боровницу. У односу на малине и боровницу, у купинама је одређен већи садржај целулозе. Највећи
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садржај пектинских материја одређен је за боровницу. Боровница је имала највећи
садржај укупних фенолних једињења, флавоноида и антоцијана (808,12 mg
GAE/100 g FW, 716,31 mg RE/100 g FW, односно 447,83 mg CGE/100 g FW). Антиоксидативна активност на стабилне 2,2-дифенил-1-пикрилхидразил (DPPH) радикале испитана је спектрофотометријском методом. Од испитаног бобичастог воћа највећу скевинџер активност на DPPH радикале, изражену као EC50 вредност (mg свежег бобочастог воћа/ml реакционе смеше), показала је боровница (0,3157 mg/ml).
Такође, утврђено је да је антиоксидативна вредност 100 g свеже боровнице, малине
- Willamette, малине - Meeker, купине - Чачанска бестрна и купине - Thornfree
једнака антиоксидативној вредности 576,50 mg, 282,74 mg, 191,58 mg, 222,28 mg,
односно 272,01 mg витамина Ц. Утврђена је значајна позитивна корелација између
антиоксидативне активности и садржаја укупних фенолних једињења (RTPh2=
0,9627), флавоноида (RTF2=0,9598) и антоцијана (RTMA2=0,9496) у бобичастом воћу.
Кључне речи: бобичасто воће, основни хемијски састав, антиоксидативна активност, фенолна једињења
Accepted: 22 october 2012
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TRADITIONAL MANUFACTURING OF WHITE CHEESES IN BRINE IN
SERBIA AND MONTENEGRO - SIMILARITIES AND DIFFERENCES
Slavica M. Vesković Moračanina*, Slavko Mireckib, Dejana K. Trbovića,
Lazar R. Turubatovića, Vladimir S. Kurćubićc and Pavle Z. Maškovićc
a
b
Institute for Meat Hygiene and Technology, Kaćanskog 13, 11000 Belgrade, Serbia
University of Montenegro, Biotechnical Faculty, Bulevar Mihaila Lalića 1, 81000 Podgorica, Montenegro
c
University of Kragujevac, Faculty of Agronomy, Cara Dušana 34, 32000 Čačak, Serbia
This paper presents the results of a study dealing with the processes in the production
of white cheese in brine, which are based on old, traditional technologies and are produced in Serbia (near Nova Varoš - Cheese from Zlatar) and Montenegro (Podgorica and
the surrounding of Danilovgrad). In both cases, fresh cow's milk without heat treatment
is used as a raw material. The paper presents the most important chemical quality parameters with the description of sensory properties. The autochthonous cheese in brine
from both area, show distinct and characteristic sensory properties of the product, and
also a high level of quality with the presence of certain individual differences. This research was aimed at a comparison of the autochthonous technologies, to save them from
oblivion, and also to show the quality parameters of cheese which are similar according
to the technological process, but are also very authentic.
KEY WORDS: white cheese in brine, traditional production, Serbia, Montenegro
INTRODUCTION
Autochtonous cheeses are products made from milk in specific geographical area as a
result of many years of development in traditional production. The awareness of the characteristics in such production is aided by the growing demand for organic and high quality food with labels of origin, whose market price, in comparison with conventional products, significantly increases from day to day. Today, native cheeses, are characteristic of
nations, states and regions, i.e. wealth and material part of the heritage of each country.
They are products of various flavors and consistency in relation to the industrially produced cheese, where the technology is strictly defined and controlled (1). Their specificity
is mostly related to the climate, geography, soil conditions, water, botanical composition
of natural meadows and pastures, breeds and breeding dairy cattle, as well as traditional
habits and customs of local people (2).
* Corresponding author: Dr Slavica M. Vesković Moračanin, Institute for Meat Hygiene and Technology, Kaćanskog 13, 11000 Belgrade, Serbia, e-mail: [email protected]
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Autochtonous cheeses are mostly produced from raw milk, without the use of starter
cultures. Their authenticity, which distincts them from other cheeses of the same type but
other region is based on, among the other things, characteristics and diversity of indigenous microorganisms, primarly lactic acid bacteria (LAB), which represent a significant
potential in the selection of technological and protective bacterial species (3, 4). The addition of commercial culture in autchthonous cheese production would lead to the loss of
the authenticity (5).
One of the most important representatives of autchthonous white cheese in brine in
Serbia is cheese of Zlatar. It is made from uncooked whole cow’s milk near Nova Varoš,
at the foothills and mountain slopes of Zlatar. Cheese production is taking place in rural
households, frequently during the summer breeding cattle on the mountains in summer
cottages. This method of processing milk was used in past more often.
In Montenegro, production of white cheese in brine is dominant, according to the diffusion and volume of production. In their household production, no procedures of standardization and pasteurization of milk and clean, starter organisms are used (1). It is usually produced in northern and north-eastern region and in the far south of Montenegro.
Enclosed in a number of sites, the indigenous production is retained and now have the names of the areas in which are produced. In the area of Podgorica and Danilovgrad, the
production of salt soused white cheese is developed and it defers, despite the mutual
proximity of production sites, characterized by differences in technology.
This paper presents the results of a research into the manufacturing process of the
indigenous white cheese in brine, which is produced in Serbia (near Nova Varos - Zlatar
cheese) and Montenegro (Podgorica and Danilovgrad surroundings). The paper describes
the most important chemical parameters of quality of the cheese along with a description
of their primary sensory characteristics.
EXPERIMENTAL
Recording indigenous technology of Zlatar cheese was performed by interviewing the
individual producers in the villages near Nova Varoš. Each of the interviewed manufacturers (6 of them) had a conditional and registered facility for the production of cheese, and
in this area represents a good host and producer. The laboratory study was carried out on
samples of ripened Zlatar cheese which were ready for consumption and the designed
tests were performed in three replicates. Part of the activities which includes a terrain part
in Montenegro was carried out in 10 households in the municipalities of Podgorica and
Danilovgrad. Each household is also traditionally engaged in the production of white
cheese in brine. The laboratory tests were performed on mature cheese samples, after 3-4
weeks of ripening.
The basic chemical quality parameters of Zlatar cheese (water content, total solids, fat
content, sodium chloride) were determined in the laboratory of the Institute of Meat Hygiene and Technology, Belgrade. The analyses were performed by accredited and regulated testing methods (6), and the evaluation of the results was carried out in accordance
with legal regulated standards of the quality of milk products (7, 8).
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Chemical analyses of cheese from Montenegro were performed at the Dairy Laboratory of the Biotechnology Faculty in Podgorica. The analyses (dry matter content, fat
content, salt content, protein content) were performed using FTIR spectrophotometry on
MilkoScan FT 120 FT. Milk fat content in dry matter of cheese and the water content of
fat-free cheese was determined in both cases by computation.
RESULTS AND DISCUSION
Characteristics of the autochtonous Zlatar - Serbian cheese
A survey conducted in order to collect valid data about the indigenous production of
Zlatar cheese showed that for this purpose use is made of full-fat cow's milk that was not
heat treated. Immediately after milking, the milk is squeezed through cheesecloth and
treated with rennet produced from stomach of calves (rennet one tablespoon per 10 liters
of milk). Making of the curd takes about 2 hours. For better separation of whey, curd is
cut into larger or smaller cubes, usually of the size 10x10 cm. After that, the formed curd
is transferred to the cotton gauze and hung on wooden hooks or specifically on tables to
achieve the necessary straining and self-pressing. This phase lasts up to 1 hour. In the
next phase of pressing the curd is transferred to a surface (the lump “rearrange”), the ends
of rag is set so as not to leave a big hole in the center of the cheese, and it is placed a
wooden board (“Circle”) which is loaded with stone. This phase lasts about 1.5 to 3 hours. The pressed clump, thickness up to 2 cm, is cut into regular slices in a square, measuring about ten inches, to fit into the appropriate wooden container. However, at this type of packing the price of production is higher, so the most cheese producers use the
plastic containers of 5 and 10 kg. Sometimes, during the new stacking slices of cheese,
old brine is changed with new one. Manufacturers of Zlatar cheese determine the amount
of salt used based on many years of experience (one closed fist of salt per pound of cheese). After each series of products, or when the container is loaded, the cheese is pressured
with a stone. The process of indigenous cheese ripening takes about 20-60 days, depending on time of year, i.e. the temperature of the ambient in which the cheese is ripening.
In the summer production of cheese, more cautions should be taken. These elements of
Zlatar cheese production are consistent with earlier defined terms, reported by some of
the authors of the present study (9, 10).
The slices of Zlatibor cheese are white-yellow to white color, regular shape and uniform thickness of about 1.5 cm. The smell is pleasant, distinct and lactic acid which is the
characteristic for this type of cheese. On the sections, cheese dough is tight, monolithic,
porcelain look with a small number of small cavities arranged properly. The taste is full,
distinctive, characteristic acid and moderately saline.
Characteristics of the indigenous production of cheese in brine – Montenegro
Producers from Podgorica and Danilovgrad, for the preparation of white cheese in
brine use, exclusively fresh cow's milk. Since no thermal treatments in the preparation of
cheese is applied, it is important that the milk comes from healthy animals, and also that
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1
V
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6.2(497.11+497.16)
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(
43, 107-113
Origin
the milk
m is with propeer hygiene, with the hygiene conttrolled processes. After milking,
depen
nding on the needd, milk is heated in pots up to 20 to 30°C. Rennet is added to the
heateed milk to the amoount that will coaggulate milk for 1.55 to 6 hours. The formed curd is
cut crrosswise, than intoo cubes, sized 5-66 cm. Green and clear
c
whey from th
he curd that comes out
o is a sign that the milk was clottted. The cheese curd
c
is transferred
d to the canvas
and than
t
hang on the hooks, to allow easy drying, withh occasional man
nual shaking to
helpss out as more wheey. When the wheey no longer goes out, the squeezed
d curd is placed
on th
he cheese-makingg table and presssed by wooden plank and stonee to make the
appro
opriate pressure or
o using the courtt, buckets, etc. Prressure should be around 1-2 kg
per kg
k of cheese curd. Squeezing and ppressing if the curdd takes 2 and 24 hours,
h
i.e. until
the ch
heese curd is draiined well. After squeezing, the currd is taken out of canvas and cut
into slices
s
that are thiccker in the winterr, and thinner in thhe summer. Each slice is treated
by saalt, and when the salt is being abssorbed, the slices are stacked in th
he container for
ripen
ning (wooden drums). Ripening takkes 2- 3 weeks. Good hygiene is necessary and
then cheese can be storred for weeks (11,, 12).
White
W
cured cheesse slices are characterized by mildd-sour odor and porcelain-white
p
colorr soft consistency, and compact struucture. On the sections, smaller and
d larger number
of ho
oles is seen, and they
t
are filled wiith the solution, which
w
matures an
nd in which the
ripen
ning process is com
mpleted.
Comparing
C
these autochtonous technnologies in cheesee production in Seerbia and Monteneg
gro, certian similarrities in their prepparation can be notticed (Figure 1 - a,
a b, c, d, e).
a)
b)
c)
d)
e)
Figu
ure 1. The proceddures in the preparration of autochthoonous cheese, inveestigated from
Serb
bia (Zlatar cheese)) and Montenegro: a) Filtration of fresh
f
heated milk, b) Adding the
rennet to thee milk, c) Cheese ccurd, d) and e) Preessing the cheese curd
Chemical
C
compossition of the tradiitional white cheese with brine prroduced in
Serbia and Montenegroo
Chemical
C
composittion of the traditioonal white cheese in brine, originatiing from Serbia
(Zlataar cheese) and Montenegro
M
is shhown in Table 1 and 2. On thee basis of the
determined values off water content iin fat-free dry matter
m
of cheese (Zlatar cheese
31±0.38; white cheese from Montenegroo 68.3±3.63), and on the basis of th
heir consistency
and appearance,
a
all of the samples belonng to the categoryy of cheese in brinee. Based on the
perceentage of milk fatt in dry matter of cheese (Zlatar cheese
c
54.24±5.90%, and white
cheesse from Monteneggro, 53.11±4.45), tthe samples belonng to category of full-fat
f
cheeses.
110
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Table 1. Results of the chemical analyses of the cheese – Serbia
Chemical quality parameter
Dry matter, %
Fat, %
Salt, %
Water on cheese fat-free dry matter, %
Milk fat on cheese dry matter,%
The determined values of Zlatar cheese SERBIA
Xmin.
Xmax.
X av.± SD
41.99
46.95
44.46 ± 2.49
20.50
28.00
24.25 ± 3.75
2.25
3.68
3.15 ± 3.90
72.88
73.68
73.31 ± 0.38
48.74
59.64
54.24 ± 5.90
SD - standard deviation; Xmin - minimum value; Xmax - maximum value; Xav - average value
Table 2. Results of the chemical composition of the cheese – Montenegro
Chemical quality parameter
Dry matter, %
Fat,%
Salt, %
Protein, %
Water on cheese fat-free dry matter,
%
Milk fat on cheese dry matter,%
The determined values of cheese MONTENEGRO
Xmin.
Xmax.
X av± SD
46.17
63.55
50.41 ± 4.02
22.49
39.36
26.88 ± 4.08
1.88
3.69
2.99 ± 0.49
14.37
21.78
18.82 ± 2.06
60.11
73.91
68.30 ± 3.63
47.37
62.40
53.11 ± 4.45
The results obtained in both types of autochthonous show some deviation, depending
of the sample. This could be expected (10, 13-16), since the samples were taken from
different households. During production processes, the households were using raw milk
with different content of milk fat and proteins. The technology itself is based on roughly
similar principles, but some production phases have their own characteristics.
CONCLUSIONS
The differences in the quality of autochthonous cheeses in Serbia and Montenegro are
the result of тхе differences in production practices and creative producers, as well as
other factors суцх ас climate, vegetation, geographical factors, habits and tastes of local
consumers and customers. Also, significant is the impact of earlier modes of production
which have left traces in the traditional dairy industry of both countries. Recording technology of autochthonous cheese production and the creation of records about them are
the necessity for saving the traditional technology from oblivion. Тhe data obtained may
serve as the basis for creating standardized production procedures, leading to the uniform
111
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quality of these products. Therefore, traditional dairy should not be seen as a return to the
past, but as an effort to preserve the indigenous technology, to gain their organized form,
the ethnographic richness of a given region so distinctive, a time stamp to the development of a nation. The affirmation of indigenous dairy products directly influences the
development of livestock on the one hand, and the identification revival of the pertaining
areas, on the other.
Acknowledgment
This investigation is a part of the Project No III 46009 financially supported by the
Ministry of Education and Science of Serbia.
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kvalitetu proizvoda od mleka i starter kultura, 2010.
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maj 2005. Zbornik radova, pp. 84-86.
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i senzorske karakteristike. Prehrambena industrija - Mleko i mlečni proizvodi 19, 1-2
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12. Mirecki, S: Mikrobiološki i hemijski kvalitet njeguškog sira. Poljoprivreda i šumarstvo 43, 4 (1997) 45-57.
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13. Maćej, O., Dozet, N. i Jovanović, S.: Karakteristike autohtone proizvodnje sjeničkog,
homoljskog, zlatarskog i svrljiškog sira. Poglavlje u monografiji "Autohtoni beli
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specifičnih originalnih mliječnih prerađevina u savremenim uslovima. Biotech.
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ТРАДИЦИОНАЛНА ПРОИЗВОДЊА БЕЛИХ СИРЕВА У САЛАМУРИ
У СРБИЈИ И ЦРНОЈ ГОРИ – СЛИЧНОСТИ И РАЗЛИКЕ
Славица М. Весковић Морачанина, Славко Мирецкиб, Дејана К. Трбовића,
Лазар Р. Турубатовића, Владимир С. Курћубићв и Павле З. Машковићв
a
б
Институт за хигијену и технологију меса, Каћанског 13, 11000 Београд, Србија
Универзитет у Црној Гори, Биотехнички факултет, Булевар Михаила Лалића 1, 81000 Подгорица,
Црна Гора
в
Универзитет у Крагујевцу, Агрономски факултет у Чачку, Цара Душана 34, 32000 Чачак,
У овом раду представљена су истраживања која су имала за циљ да опишу поступке производње меких белих сирева који су засновани на старим, традиционалним технологијама, а који се производе у Србији (околина Нове Вароши – златарски сир) и у Црној Гори (околина Подгорице и Даниловграда). У оба случаја, као
сировина, користи се свеже кравље млеко, без претходне термичке обраде. У раду
су дати најважнији хемијски параметри квалитета уз опис и оцену сензорских својстава. Сензорска својства аутохтоних меких белих сирева у саламури, са оба локалитета, показују особена и за производ карактеристична сензорска својства, као и
висок ниво квалитета уз постојање одређених индивидуалних оступања.
Наведена истраживања имала су за циљ да се аутохтона производња белих сирева у саламури опише, као и да се прикажу параметри квалитета сирева који су
слични по технологији припреме, али и веома аутентични.
Кључне речи: бели сиреви у саламури, традиционална производња, Србија, Црна
Гора
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Origina scientific paper
SUNFLOWER SEED FOR HUMAN CONSUMPTION AS A SUBSTRATE FOR
THE GROWTH OF MYCOPOPULATIONS
Marija M. Škrinjar*, Žarko M. Petrović, Nevena T. Blagojev and Vladislava M. Šošo
These mycological investigations are implicating samples of protein sunflower seed
from regular cultivation in the Vojvodina Province. Samples are examined in different
stages of production: reception in the silo, separation of massive fraction on peeler and
then peeling, kernel after peeling, hull, final product, i.e. kernels separated from visible
impurities on conveyor bel, that are later manually divided in two fractions – a) seemingly whole, undamaged kernels, without change of colour, and b) seemingly damaged kernels, broken, with change of colour. For the determination of viable count of moulds and
their isolation, two different media are used in parallel: Sabouraud maltose agar (SMA)
and malt/yeast extract with 50% of glucose (MY50G), favourable for growth of xerophilic
moulds.
All samples tested were contaminated with fungi. Total viable mould count per seed
varied from 1.6 (SMA) respecting 1.3 (MY50G) on reception, to 5.6 (SMA) and 7.5
(MY50G) cfu/seed in visually damaged sunflower kernels (final product). From seeds,
kernels and hull, numerous moulds were isolated, belonging to 8 genera and 13 species
(Alternaria alternata, Arthrinium phaeospermum, Aspergillus candidus, A. flavus, A. niger, A. ochraceus, A. versicolor, A. wentii, Cladosporium cladosporioides, Eurotium herbariorum, Penicillium aurantiogriseum, Rhizopus stolonifer and Trichoderma harzianum). Alternaria alternata, Aspergillus flavus, A.ochraceus, A. versicolor and Eurotium
herbariorum were isolated on both media. Aspergillus candidus, A. versicolor, C. Cladosporioides, P. aurantiogriseum and T. harzianum were isolated only on SMA, while A.
niger, A. wentii and R. stolonifer were exclusively isolated on MY50G. Most ubiquitous
species is A. alternata, which is isolated from all tested samples, while A. candidus, C.
cladosporioides and T. harzianum were isolated from sunflower seed on reception in silo,
using SMA medium.
KEY WORDS: sunflower, mycopopulations, xerophilic moulds, SMA, MYG50
* Corresponding author: Marija Škrinjar, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
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INTRODUCTION
Sunflower (Helianthus annus L.) is an annual plant native to the North America,
transferred to Europe in the 16th century. In the Vojvodina Province, its cultivation started during the World War I, but more intensive production dates from 1930. The highest
dispersion of sunflower in this area reached when new Soviet sorts were imported after
the World War II, and later, by implementation of domestic hybrids with high oil content
(1).
Based on the content of the main parts of sunflower seed (kernel/hull), oil and
proteins, two main types of sunflower can be distinguished: oil type and protein type. Oil
type is aimed for industrial production of oil and usually contains 20-30% of hull and
more than 40% of oil (referring on seeds with moisture content of 10%), so this type of
sunflower belongs to the oil seeds with high oil content. Considering the kernel size, this
sunflower type belongs to the group of small-size seeds. Protein type of sunflower is used
for industrial production of protein-based sunflower products (hulled kernels, protein
flour, „butter“ – sunflower kernel cream, etc.), or it can be used directly for consumption,
which is why it has been called confection sunflower. This type contains 30% of oil and
40-45% of hull, while the protein content in novel sorts is about 29%, and in hybrid sorts
more than 32%. Considering the kernel size, this type belongs to large-size seeds.
Crop plants, including oil seeds, are constantly, although at different extents, very
susceptible to contamination by different kinds of moulds (2-7). The level of mould contamination depends on diverse factors, such as: species, sort, hybrid, cultural practices
during the growing and maturation phases, conditions during harvest, transport and storage, as well as climatic conditions during growing, maturation ad harvest.
Sunflower is attacked by a number of diseases caused by various microorganisms,
including fungi. Of the fungal foliar diseases, leaf spot caused by Alternaria helianthi,
Septoria helianthi and other fungal pathogens are relatively important (8, 9). Fusarium
wilt is caused by different species of the genus Fusarium (F. solani, F. oxysporum, F.
helianthi, F. moniliforme and others). Sclerotinia wilt and head rot of sunflower are
caused by Sclerotinia sclerotiorum (10, 11). According to many data (3, 12, 13), sunflower seeds are highly contaminated with fungi which attack the plants at different stages
of development, harvesting and storage. In this respect, important role play Aspergillus
and Fusarium species, zigomycete and species from the group Dematiaceous Hyphomycetes (Alternaria spp., Cladosporium spp.). A lot of fungal species that contaminate
sunflower seeds are toxigenic and under proper conditions produce toxic metabolites –
mycotoxins, harmful for human and animal health.
Considering the aforementioned, the aim of this work was to investigate the mycological properties of the protein type of sunflower seeds aimed for the human consumption,
during storage and further processing to the final product, with the special focus on the
level of the fungal contamination, presence of the specific species and their moiety in
isolated mycopopulation.
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MATERIALS AND METHODS
The sunflower samples used for mycological investigation were protein hybrids, randomly sampled from one small enterprise for raw hulled sunflower seeds production in
the Vojvodina Province.
The following samples were used: seeds after the reception in the silo, massive seed
fraction before the peeling, kernels after the peeling, hull, and final product – kernels
cleaned from the impurities on the conveyer, and than manually divided into two fractions: a) seemingly whole undamaged kernels without discoloration and b) seemingly damaged kernels, broken or with present discoloration.
Determination of the total mould count per kernel/g, their isolation and identification. For the determination of the total mould count per kernel, the following method
was used: 10 g of the sample was treated with 100 ml of 4% sodium-hypochlorite, in
order to remove all moulds possibly present on the seed surface. Erlenmeyer flasks (300
ml) were shaken on the rotary shaker for 3 minutes, and then all samples were rinsed with
sterile distilled water (2 x 100 ml). On the surface of the solidified media containing antibiotics (1 ml of chloramphenicol and 1 ml of oxytetracyclin per 100 ml of the medium),
8 kernels were aseptically placed. Inoculated Petri dishes were incubated for 7 days at
25°C. For the determination of the total mould count per 1g of hull, the dilution method
was used, under the same incubating conditions.
For the both methods, two different media were used: Sabouraud-maltose agar (SMA)
and the medium containing yeast and malt extracts and 50% of glycose (MYG50: malt
extract – 10g; yeast extract – 2.5g; glycose – 500g; agar – 10g; distilled water – 1000ml),
which has been suggested by Pitt and Hocking (14) for isolation of xerophilic moulds.
All tests were conducted in triplicates. The mould growth was observed each day during 7 days, and the results were expressed as the average per kernel or gram.
Identification of isolated species was done according to Ellis and Samson et al.
(15,16).
As it was mentioned above, for total mould count determination, two different media
were used: SMA, general mycological medium, and MYG50, named for isolation of the
xerophilic species. The moisture content of sunflower samples used in this investigation
varied from 7.43% in kernel, 9.55% in seed, to 13.93% in hull, which is the reason why
the medium suitable for xerophilic moulds was used. After the incubation period, it was
observed that all tested samples were contaminated with moulds. In all samples, except in
hull, the higher contamination was observed on SMA, comparing to MYG50 (Table 1).
On the SMA medium, total mould count ranged from 1.5/g (hull) to 5.6/kernel (seemingly damaged kernels). On the MYG50 medium, total mould count varied from 1.2/kernel
(massive seeds before the peeling) to 7.5/kernel (seemingly damaged kernels).
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Table 1. Total viable count of moulds per sunflower seed/kernel and 1g of hull
determined by using SMA and MYG50 media
Sample
Seed after the reception in the silo
Massive seed fraction before the peeling
Kernels after the peeling
Hull
Seemingly whole undamaged kernels
Seemingly damaged broken kernels
Total viable count of moulds
SMA
MYG50
1.6
1.3
2.1
1.2
4.8
3.6
1.5
1.6
4.1
2.2
5.6
7.5
The moulds isolated from tested samples belong to 8 genera and 13 species (Table 2),
viz. Alternaria alternata, Arthrinium phaeospermum, Aspergillus candidus, A. flavus, A.
niger, A. ochraceus, A. versicolor, A. wentii, Cladosporium cladosporioides, Eurotium
herbariorum, Penicillium aurantiogriseum, Rhizopus stolonifer and Trichoderma harzianum. From Table 2 it could be seen that the genus Aspergillus was presented with 6 different species (A. candidus, A. flavus, A. niger, A. ochraceus, A. versicolor and A. wentii),
while all other genera were presented with the one species each.
Table 2. Fungal species isolated from sunflower seeds, kernels and hull by SMA and
MYG50 media
Species
Alternaria alternata (Fr.) Keissler
Arthrinium phaeospermum (Corda) M.B. Ellis
Aspergillus candidus Link
flavus Link
niger van Tieghem
ochraceus Wilhelm
versicolor (Vuill.) Tiraboschi
wentii Wehmer
Cladosporium cladosporioides (Fres.) de Vries
Eurotium herbariorum (Wiggers) Link
Penicillium aurantiogriseum Direckx
Rhizopus stolonifer (Ehrenb.) Lind.
Trichoderma harzianum Rifai
SMA
+
+
+
+
+
MYG50
+
+
+
+
+
+
+
+
+
+
+
+
+
It should be mentioned that some species, such as Alternaria alternata, Aspergillus
flavus, A.ochraceus, A. versicolor and Eurotium herbariorum, were isolated from specific
samples by using both media, while the others were isolated by using either one of them
(Table 3).
The highest frequency among isolated mycopopulations had the species Alternaria alternata and Eurotium herbariorum. Alternaria alternata was the most ubiquitously found
species, it was isolated from all tested samples and on both media, except from the seeds
from silo, where it was isolated only on SMA. On the other hand, Eurotium herbariorum
was mostly isolated by using MYG50 medium, which was expectable, since it is classified as typical xerophilic species. The species A. candidus, A. versicolor, C. cladosporium
and T. harzianum were found in just one of the tested samples.
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Table 3. Occurrence of fungal species on investigated samples determined by SMA and
MYG50
From the total of 13 isolated species, even 10 (A. alternata, A. candidus, A. flavus, A.
niger, A. ochraceus, A. versicolor, A. wentii, E. herbariorum, P. aurantiogriseum and R.
stolonifer) have been reported to produce some of the toxic metabolites (16). Table 4 lists
the toxic metabolites that this species produces under specific conditions.
Table 4. The most important toxic metabolites produced by moulds isolated from
sunflower seeds/kernels/hull
Fungal species
Alternaria alternata
Aspergillus candidus
A.flavus
A. niger
A. ochraceus
A. versicolor
A. wentii
Eurotium herbariorum
Penicillium
aurantiogriseum
Rhizopus stolonifer
Toxins
Alternariol, alterotoxin, tenuazonic acid
Candidulin, terphenyllin, xanthoascin
Aflatoxins, aflatrem, aflavinin, aspergillic acid, cyclopiazonic
acid, 3-nitropropionic acid, paspalinin
Malformin, naphthoquinones, nigragillin
Ochratoxin
Sterigmatocystin, nidulotoxin
Emodin, kojic acid, 3-nitropropionic acid, wentilacton, physicon
Sterigmatocystin
Ochratoxin A, penicillic acid, xanthomegnin, viomellein,
viridicatin (terrestric acid, penitrem A)
Toxic cyclic peptide
CONCLUSION
The protein type of sunflower was tested on its mycological properties, at various
processing stages. The obtained results showed that all samples were significantly contaminated with moulds. The total of 13 species, belonging to 8 genera, were isolated. Two
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different media were used for the mould isolation, one general medium for mould
growth, and other aimed particularly for the growth of xerophilic moulds. The mould
growth was observed on the both media, but the higher total mould count was detected
using general medium – SMA. Also, the higher number of different species were isolated
on SMA comparing to MYG50. About 76% of all isolated species are reported to produce different toxic metabolites.
REFERENCE
1. Vrebalov, T. and Škorić, D.: Površine, prinosi i privredni značaj suncokreta u svetu i
u našoj zemlji, in Suncokret. Ed. Milošević S., Nolit, Beograd (1988) 40-52.
2. Tabuc, C. and Stefan, G.: Assessment of mycologic and mycotoxicologic contamination of soybean, sunflower and rape seeds and meals during 2002-2004. Archiva
Zootechnica 8 (2005) 51-56.
3. Škrinjar M., Bandu, M., Dimić, E., Bjelobaba, K. and Romanić, R.: Infekcija semena
suncokreta žetve 2006 aflatoksigenim gljivama. Uljarstvo 38, 1-2 (2007) 41-47.
4. Deabes M. and Al-Habib, R.: Toxigenic fungi and aflatoxin associated to nuts in
Saudi Arabia. Journal of American Science 7, 8 (2011) 658-665.
5. Krnjaja V., Lević, J., Stanković, S. and Stepanić, A.: Fusarium species and their mycotoxins in wheat grain. Proc. Nat. Sci, Matica Srpska, Novi Sad 120 (2011) 41-48.
6. Kungulovski Dz., Avramovski, O., Atanasova, N., Pancevska, I. and Kungulovski, I.:
Mycotoxigenic molds in spices from macedonian stores. Proc. Nat. Sci, Matica
Srpska, Novi Sad 120 (2011) 155-164.
7. Luttfullah G. and Hussain, A.: Studies on contamination level of aflatoxins in some
dried fruits and nuts of Pakistan. Food Control 22 (2011) 426-429.
8. Achbani, E.H., Lamrhari, A., Laamaraf, N., Bahsine M.H., Serrhini M.N., Douira A.
and de Labrouche, D.T.: Downy mildew (Plasmopara halstedii): Importance and geographical distribution on sunflower in Marocco. Phytopath. Medit. 39, 2 (2000) 283288.
9. Maširević S. and Jasnić S.: Leaf and stem spot of sunflower. Biljni lekar 34, 4-5
(2006) 326-333.
10. El-Deeb A.A., Abdallah, S.M., Mosa, A.A. and Ibrahim, M.M.: Sclerotinia diseases
of sunflower in Egypt, Arab Universities. J. Agric. Sci. 8 (2000) 779-798.
11. Afzal R., Mughal, S.M., Munir, M., Sultana, K., Qureshi, R., Arshad, M. and Laghari,
M.K.: Mycoflora associated with seeds of different sunflower cultivars and its management. Pak. J. Bot. 42, 1 (2010) 435-445.
12. Vaidehi, B.K.: Seed mycoflora of sunflower – a perspective. Frontiers in Micro Biotec. Plant pathol. 1 (2002) 25-40.
13. Morar M.V., Dancea, Z., Bele, C., Salegean, D., Beke, A. and Baonca, I.: An approach upon the qualities of the raw material and raw oil from sunflower seeds resulting
in process of low capacities. Buletinul-Universitatii-de-Stiinte-Agricole-si-MedicinaVeterinara-Cluj-Napoca-Seria-Agricultura 60 (2004) 381-384.
14. Pitt, J.I. and Hocking, A.D.: Fungi and food spoilage, CSIRO Division of Food
Research, Sydney (1985) 398-400.
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15. Ellis, M.B.: Dematiaceous Hyphomycetes, Commonwealth Mycological Institute,
Kew, Surrey, England (1971) 25-58.
16. Samson, R.A., Hoekstra, E.S. and Frisvad, J.C.: Introduction to food- and airborne
fungi. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (2004) 64103.
СЕМЕ КОНЗУМНОГ СУНЦОКРЕТА КАО СУПСТРАТ ЗА РАСТ
МИКОПОПУЛАЦИЈА
Марија М. Шкрињар, Жарко M. Петровић, Невена Т. Благојев
и Владислава М. Шошо
Универзитет у Новом Саду, Технолошки факултет, Нови Сад, Србија
Миколошка испитивања у овом раду баве се узорцима сунцокрета из редовног
гајења на подручју Војводине. Узорци су испитивани у различитим фазама производње: полазно семе из силоса, крупнија фракција одвојена на љуштилици и која
иде на љуштење, језгро које излази након љуштења са љуштилице, љуска, финални
производ, тј. језгра пречишћена од видљивих нечистоћа на конвејерској траци и та
зрна су на основу визуелног прегледа ручно одвојена у две фракције: а) наизглед
цела, неоштећена зрна без промене боје, и б) наизглед оштећена зрна, поломљена
или са променом боје. За испитивање укупног броја плесни и њихово изоловање,
коришћене су паралелно две подлоге: Sabouraud-малтозни агар (SMA) и сладни/квасни екстракт са 50% глукозе (MYG50).
Сви тестирани узорци су били контаминирани плеснима. Укупан број плесни по
зрну је варирао од 1.6 (SMA) и 1.3 (MYG50) – на пријему, до 5.6 (SMA) и 7.5
(MYG50) cfu/зрно у узорцима видљиво оштећеног језгра (финални производ). Из
зрна, језгра и љуске изолован је велики број плесни, које су сврстане у 8 родова и
13 врста (Alternaria alternata, Arthrinium phaeospermum, Aspergillus candidus, A. Flavus, A. niger, A. ochraceus, A. versicolor, A. wentii, Cladosporium cladosporioides, Eurotium herbariorum, Penicillium aurantiogriseum, Rhizopus stolonifer and Trichoderma
harzianum). Врсте Alternaria alternata, Aspergillus flavus, A.ochraceus, A. versicolor и
Eurotium herbariorum су изоловане са обе подлоге. Aspergillus candidus, A. Versicolor, C. cladosporioides, P. aurantiogriseum и T. harzianum су изоловане само на SMA
подлози, док су А. niger, A. Wentii и R. stolonifer изоловане само са MY50G подлоге. Најраспрострањенија врста је била Alternaria alternata, која је изолована из свих
испитиваних узорака, док су A. candidus, C. Cladosporioides и T. Harzianum изоловане само из зрна сунцокрета на пријему у силос, и то само на SMA подлози.
Кључне речи: сунцокрет, микопопулације, ксерофилне плесни, SMA, MY50G
Received: 17 August 2012
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INFLUENCE OF ECOPHYSIOLOGICAL FACTORS ON THE PRESENCE OF
OCHRATOXIN A IN DRIED VINE FRUITS: A REVIEW
Vladislava M. Šošo*, Marija M. Škrinjar and Nevena T. Blagojev
Grapes derived products, especially dried vine fruits (raisins, sultanas, currants), are
very often used in human nutrition, and along with wine, are of significant economic importance in Mediterranean countries, especially in Spain, Greece, Turkey and Italy. The
diversity of climate in the areas where grape is grown, indicate that moulds, potential
producers of ochratoxin A (OTA), are ubiquitously distributed. Considering this fact,
OTA itself is commonly isolated from these products. Great efforts are taken to eliminate
ochratoxigenic moulds and preform detoxification of the products, but efficient methods
have not been found so far. Because of that, the inhibition of mould growth is of great importance.
It is well known that the ecophysiological factors highly influence the presence of
toxigenic moulds in different food products, including production of OTA. The aim of this
work was therefore to summarize results obtained so far on the presence of OTA in dried
vine fruit, and to outline the influence of ecophysiological factors on mould and OTA
presence in this commodity.
KEY WORDS: dried vine fruits, ochratoxin A occurrence, ecophysiological factors,
INTRODUCTION
Grapes and dried vine fruit are one of the most important agricultural products and,
consequently, are of major commercial interest. Grape is served as fresh, dried vine fruit,
preserved and processed in jellies or jams, crushed for juice or wine making (1).
In view of the importance of the production scale, dried vine fruit presents second
product that is made from grapes, after wine. Today, dried vine fruits are usually commercially classified in three groups: raisins (dried white grapes), sultanas (dried white
grapes from seedless varieties) and currants (dried black seedless grapes) (2).
With grape drying, as one of methods of preservation, great profit is made in several
countries that are famous for vine growing. Countries that are recording significant dried
vine fruit production are Greece, Turkey, Mexico, Chile, Australia, South Africa, Iran
and India (1). In the Mediterranean region, vine is one of the most important agricultural
crops. In support of that, two of the biggest dried vine fruits producers in world, are in
* Corresponding author: Vladislava M. Šošo, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected],
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this region, Turkey and Greece (3). Taking in consideration the fact that Serbia is not producing dried vine fruit, great share of imported dried vine fruit originate from these
countries.
In order to obtain dried vine fruit of good quality, both physical (berry size, berry
colour, the nature of waxy cuticles) and chemical (moisture content, sugar content and
acidity) fruit properties at harvest affect dried vine fruit quality. These properties are influenced by several factors, and some of them cannot be manipulated by the grower (variety, the age of vine, soil and climate conditions), while some others, such as soil improvement, irrigation management, nitrogen and potassium nutrition, etc., which can be improved by the grower. All these parameters together are of essential significance in fine
quality vine production (1).
Grape ripening is accompanied by increase in the sugar content, decrease in acidity
and formation of specific colour, texture, aroma and taste, and creration of favourable
conditions for growth of specific microorganisms, such as moulds.
Grape harvest can be done manually for the purposes of table grapes production or
mechanically for dried vine fruit, grape juice or wine production.
Drying is probably one of the oldest and the most cost effective methods for fruit preservation. Even though, new preservation methods and ability to supply market with fresh
fruits during the whole year are developed, drying stayed one of the most used technological operations worldwide, especially in the countries of extensive growth. Drying
also keeps diversity of the product on the market and responds to consumers demands.
Grape drying can be performed in several ways: a) „dry-on-vine” method, where grapes
is dried on vine directly, b) drying in open sun (traditional method), where grape bunches
are spread over either the ground or on a platform in thin layer directly exposed to the sun
(this method can be done also in shadow), c) drying under controlled conditions in drying
chambers. In Mediterranean countries, grapes are often dried by exposing to the solar
radiation, or in drying chambers performing cold or hot procedure (4).
In order to improve drying process, pre-treatments are done to enhance water diffusion through the berry waxy cuticle and improve generally quality of dried vine fruit. Commonly used pre-treatments are dipping in the hot water or application of the chemicals
(sulphur, soda, ethyl or methyl oleate emulsions). When grape is dried in open sun, rapid
quality drop might be observed, which can be expressed in colour change and presence of
foreign matter.
Dried vine fruit is a nutritionally worthy food. It is rich in potassium, sugar, dietary
fiber, iron and vitamin A. Grape and its products are rich in phenolic compounds which
demonstrate a wide range of biochemical, and pharmacological effects, including anticancerogenic, antiatherogenic, anti-inflammatory, antimicrobial, and antioxidant activities. It
is eaten as a snack food without further processing and is used for cooking, baking and
brewing (1, 3).
Among technological parameters of quality (size of the berries, uniformity, conditions of the berry surface, moisture content, and chemical composition) for creating healthy safe product, microbiological quality (absence of the decay, moulds, yeasts and foreign matter, insect infestation) is of essential importance (1).
Because of its frequent use, dried vine fruit is extensively investigated, including
frequent contaminants such as toxigenic moulds, whereas mycotoxins are extremely ha124
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zardous to human health (1, 2). In veiw of this, the aim of this work was to summarize results obtained so far on the presence of ochratoxin A (OTA) in dried vine fruit, and to
outline the influence of ecophysiological factors on mould and OTA presence in this
commodity.
OCHRATOXIN A – PROPERTIES AND OCHRATOXIN A PRODUCING
MOULDS
Mycotoxins are extracellular toxic secondary metabolites from a large number of filamentous fungi. They are not essential products of metabolism, so their production is takes
place only in certain circumstances. Alimentary intake of mould toxins provokes intoxications, so called mycotoxicoses (5).
Because they cause rot and can have pathogenic effects, moulds play an important
role in fruit and vegetable spoilage. They can be found in nature in every region where
suitable conditions for their development are present.
In the largest group of toxigenic moulds that are infesting fruits, species form the
genera Alternaria, Penicillium and Aspergillus are included, and the toxins that can be
found in fruit tissue are aflatoxins, ochratoxin A, patulin and Alternaria toxins. Some of
these mycotoxins are cancerous and most of them have stable chemical structure during
the processing and because of that they are present in final product, and can easily reach
the consumer. Consumers will notice evident fruit spoilage, and skip intake, but with processed products situation is different - mycotoxins remain in the food due to their stability
and present significant source of these toxins. A common characteristic of all mycotoxins
is that their production and amount depend on the substrate on which they are growing,
water activity of the substrate (aw), temperature, and interaction with other mycopopulation on/at the substrate (6).
OTA (Figure 1) was first discovered as a metabolite of Aspergillus ochraceus in
1965, during a large investigation made on fungal metabolites, which was performed in
order to find new mycotoxins. Soon after that, OTA was found in corn sample in the
USA. International Agency for Research on Cancer (IARC) defined OTA as potential
cancerous agent for people from group B2 (7). OTA exhibits nephrotoxic, carcinogenic,
immunotoxic, teratogenic and mutagenic effects (2), and also disturbs physiological state
of cell in many ways, primarily by inhibiting the enzymes responsible for synthesis of
phenylalanine tRNA complex. Besides, it inhibits mitochondrial ATP production and
promotes lipid peroxidation (6).
There is an opinion that OTA is involved in the human disease called Balkan Endemic Nephropathy (BEN) and can promote cancer of the urinary tract. BEN is a chronic
nephritis that often occurs in the populations living in areas bordering with the Danube
and Sava River in parts of Romania, Bulgaria, Serbia, and Croatia (5). Because of its chemical structure, phenylalanine-dihydroxycoumarin derivatove, OTA is very resistant on
heating and hydrolysis (6).
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Figure 1. Structural formula of ochratoxin A
Ochratoxin A is an extracellular metabolite produced by some species from the genera Aspergillus and Penicillium. Ochratoxigenic species from these genera are presented in
Table 1 (2).
Table 1. Ochratoxigenic species from the genera Aspergillus and Penicillium
Genus
Section
Circumndati
Aspergillus
Flavi
Nigri
Penicillium
Viridicata
Species
ochraceus
melleus
ostianus
sclerotiorum
elegans
stenynii
westerdijakiae
alliaceus
glaucis
carbonarius
niger „agregate“
nordicum
verucosum
A. niger “aggregate” presents the most complicated taxonomic subgroup, which includes eight morphologically very similar taxons: A. niger, A. tubingensis, A. acidus, A.
Brasiliensis, A. costaricaensis, A. lacticoffeatus, A. piperis and A. vadensis (8). With respect to A.niger usage for enzyme and citric acid production intended for human consumption, it is very important that industrial isolates do not produce OTA (9, 10). Apart from
the mentioned ochratoxigenic moulds, some species from Aspergillus genus (A. Albertensis, A. auricomus i A. wentii) show the ability for OTA biosynthesis only in special conditions (11). Further investigations revealed that, regardless from the substrate, A. Carbonarius (section Nigri) is the main ochratoxigenic fungus, where its isolates are in 75 to
100% cases ochratoxigenic (investigation was done on 48 isolates from dried grapes in
Argentina) (12).
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The Aspergillus section Circumdati has some of the most significant OTA producers
(Table 1). Recent investigations have shown that A. steynii has a different ochratoxigenic
ability depending on the origin of isolation (13).
The genus Penicillium has two ochratoxigenic species – P. nordicum and P. verrucosum (2, 14). According to the literature data, some other Penicillium species (P. aurantiogriseum, P. chrysogenum etc.) are also having ability to produce OTA (15).
According to the data (16), at least 77 countries have definite regulations for mycotoxins, while 13 countries are known to have no regulation; whereas there are no available data for about 50 countries, most of which are in Africa (17, 18). On the basis of the
opinion adopted by the European Food Safety Authority (EFSA) it was considered necessary and appropriate for the protection of public health to establish maximum levels for
ochratoxin A in those foodstuffs that are significant contributors to the exposure of OTA
or for those foodstuffs that are not necessarily a significant contributor to the exposure of
OTA, but there is evidence that there can be found a very high level of OTA in these
commodities. Those commodities are: cereals, dried vine fruit (currants, raisins and sultanas), coffee, wine, grape juice, foods for infants, green coffee, spices and liquorice (19,
20). The European Union legislation authorities have set up a maximum level for OTA in
dried vine fruit 10 μg/kg.
MYCOPOPULATIONS AND OTA IN DRIED VINE FRUIT
A large number of investigations have been carried out in order to determine mycopopulations that are forming on grapes during ripening process (21, 22), but in spite of that,
there is not enough data regarding ochratoxigenic mycopopulations during the drying
process. After discovering OTA in dried vine fruit in the late 90’s, focus was directed on
the representation and importance of contaminants from genus Aspergillus section Nigri
on this commodity (23, 24). In the MAFF (Ministry of Agriculture, Fisheries and Food,
U.K.) investigations, 301 dried vine sample from the market was tested on OTA presence
and 286 were contaminated with this mycotoxin (9% had concentration of OTA higher
than the European limit). MacDonald et al. (23) examined 60 samples of dried vine fruit
from UK market, and in 88% cases OTA was found. These samples were imported from
Greece and Turkey. Average contamination was around 9.0 μg/kg in currants, 4.6 μg/kg
in sultanas and 7.5 μg/kg in raisins from Greece, and around 5.7 μg/kg for sultanas from
Turkey. After these investigations the European Committee set a maximal concentration
of OTA in dried vine fruit at 10 μg/kg. Studies carried out after the adoption of regulations have revealed that contamination of dried vine fruit with OTA is worldwide,
although mean concentration level is low and under the European limit (3, 25-28). Table
2 shows the results of numerous investigations on OTA in dried vine fruit (29).
A. carbonarius is contributing mostly to dried vine contamination with OTA (30-36).
A. niger also participate in OTA production, because it is often isolated, but its strains do
nott always have ochratoxigenic properties (30, 32-37). Iamanaka et al. (37) isolated
ochratoxigenic species A. ochraceus from Brazilian dried vine fruit. OTA contamination
in dried vine fruit can be prevented by controlling black Aspergillus species right before
harvest, with fast drying after the harvest, and eliminating mouldy berries during process
(32-34).
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Table 2. OTA concentration in dried vine fruit from different countries
Country
Sweden
Germany
United Kindom
Morocco
Turkey
Greece
Brasil
Canada
a
Dried vine fruit
type
raisins and currants
raisins and currants
raisins
sultanas
raisins
sultanas
sultanas
sultanas
raisins
sultanas
currants
Incidence of
contamination
with OTA
96/118a
101/106
17/20
17/20
6/20
179/264
17/27
29/43
67/85
36/66
2/2
OTA level
(mean) μg/kg
<0.1-34.6
≤ 21.4
0.3 – 19.5
0.5-20
0.05-4.95
0.026-54
≤ 13.2
0.1-33.9
max. 26.6 (2.29)
max. 26.0 (3.11)
max. 4.85 (2.81)
– number of OTA contaminated samples / total number of samples
Australian dried vine fruit, dried in open sun, that is not preserved with SO2, was in
100% contaminated with A. niger and allied species, which grew during the drying process and showed a high degree of resistance against strong solar radiation in continental
parts of Australia (32). In addition to the above species, much rarely isolated are species
from the genera Penicillium, Alternaria, Epicoccum, Trichoderma, Rhizopus and Cladosporium (32). From currants, species Xeromyces bisporus was isolated (38).
In comparison with other grape products, dried vine fruits belong to the group of
products that are usually the most contaminated with OTA, where concentration of this
toxin reaches the level of 50-70 μg/kg (39).
The investigations showed that OTA level in dried vine fruit of the Mediterranean
area is much higher than of non-Mediterranean areas (Chile, USA and Australia). In favour of these results, researchers from Greece, Stefanaki et al. (40), published a study on
OTA in Greece’s dried vine fruit - sultanas, raisins and currants. Sampling was done during storage and production. Sultanas appeared to be less contaminated (mean 2.1 μg/kg)
than currants (mean 2.8 μg/kg). However, 7.5% of tested samples had OTA level higher
than 10 μg/kg, exceeding the limit prescribed by the European regulatory EC No.1881/
2006.
The altitude of vine growing and drying have a significant influence on the OTA concentration. Dried vine fruit from vineyards at the sea level of cc 500 m was less contaminated than from vineyards from 1000 m a.s.l. (41-43).
In Turkey, Meyvaci et al. (3) have investigated sultanas in the period from 1998 to
2000, and tested 264 samples on OTA presence (Table 2). They showed that 9.8% of
samples had OTA level higher than the European limit (10 μg/kg); the average OTA concentration was 3.4 μg/kg, and maximum 54 μg/kg.
Another author (44), investigated 53 samples of Turkish sultanas intended for European market and found that 4% of them had a higher level of OTA than it is prescribed by
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the European regulations, while contamination of all samples were in the range from 0.5
to 58 μg/kg.
In Morocco, of 20 samples of raisins tested on OTA presence 30% were contaminated, but the level of OTA did not exceed the limit prescribed by the European regulations (10 μg/kg) (45).
In Californian dried vine fruit OTA level was lower than in the Mediterranean, while
in the Argentinian one the level of contamination was similar (31).
On Serbian markets, commonly dried vine fruit is imported from Turkey, Greece and
Iran. Investigations have revealed that all tested samples were contaminated with moulds.
The isolated species belonged to 4 genera: Aspergillus (60%), Eurotium (40%), Penicillium (40%) and Mucor (20%), and the the most ubiquitously isolated species was A. niger
(in 33% of samples). Ochratoxin A was isolated from dried vine fruit, and ranged between 40-56 μg/kg. These values exceed the limit prescribed by the European regulatory
(10 μg/kg) (46, 47).
INFLUENCE OF ECOPHYSIOLOGICAL FACTORS ON OTA BIOSYNTHESIS
Fungal growth and OTA production are determined by a wide range of different factors. They can be roughly defined as physical, biological and chemical, and they are interacting between themselves and influence further fungal growth and mycotoxin production (48).
The OTA synthesis can be conditioned due to the effects of intrinsic (water activity,
pH, nature of substrate) and extrinsic (climatic conditions, temperature and humidity)
factors. Species from the genera Aspergillus and others have the ability to grow on various substrates, also in different climatic regions, and because of that they are distributed
worldwide. Growth of species from the genus Aspergillus is often related with regions of
warm climate (48).
It is very hard to predict the level of contamination of agricultural products and food
with mycotoxins, because it depends on numerous factors, such as: moisture content,
temperature, type of product, presence of endogenous fungal species, storage conditions,
time of storage, type and time of transportation (49,50). Control of certain parameters
could reduce production of mycotoxins (11).
Water activity (aw)
Fungal growth and mycotoxin production ars determined by numerous abiotic and
biotic parameters and their interaction. Water activity is perhaps the most critical factor
that influencesthe germination, growth and establishment of moulds on nutrient worthy
substrates (51). Esteban et al. (52) were investigated the effect of different water activity
values on OTA production using twelve A.niger isolates, cultured on Czapek Yeast Agar
(CYA) and on Yeast Extract Sucrose agar (YES) where aw ranged from 0.82 to 0.99. For
A. niger, it is known that minimal aw needed for fungal growth is 0.77 (53), but for OTA
production it is found that optimal aw is 0.90-0.99, depending on the strain and culture
medium.
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Romero et al. (54) investigated a mixture of four A. carbonarius strains (isolated from
dried vine fruits) on OTA production at 30°C on CYA using different aw values (0.83,
0.85, 0.87, 0.89, 0.90, 0.93 and 0.95). They find out that limiting water activity of mixed
inoculum for OTA production is 0.87 aw, indicating xerotolerant behaviour of A. carbonarius isolates. Pitt and Hocking (38) reported that the lower aw limit for ochratoxin A
production is near 0.92 aw. A. ochraceus grows optimally at 0.95 - 0.99 aw, but minimal
aw needed for fungal growth is 0.77.
Regarding discussions of the mentioned authors, it can be suggested that lowering on
0.85 aw can inhibit OTA production. This fact can be used to control and prevent OTA
production in dried vine fruit (11).
Temperature
After water activity, second limiting factor for OTA production is temperature. Optimal temperature for OTA production is between 25-30°C for A. ochraceus (55), 10-20°C
for A. carbonarius (56) and for A. niger “aggregate” 20-25°C (57). Because of their ability to grow in a wide range of temperatures, OTA can be constantly formed in field. This
fact is very important for some products, such as grapes and dried vine fruit, because
main contaminants are the species of A. carbonarius and a few species from A. niger “aggregate”, also main producers of OTA.
Ouselati et al. (58) examined the temperature effect and light exposure on the fungal
growth and mycotoxin production. They simulated average Tunisia’s temperatures (night
temp./daily temp. - 20/30, 30/37 and 25/42°C), where night temperature lasted 11h and
daily 13h. The tested isolates were six strains of A. carbonarius isolated from Tunisian
vineyards. The influence of these temperatures was examined on the OTA production of
A. carbonarius using synthetic nutrient medium (SNM) similar to grape composition
between the onset of ripening and ripeness (0.99aw). The highest concentration of OTA
was observed in the temperature regime of 20/30°C. Also, the sunlight influence was
tested using the same strains, at constant temperature of 25°C with 11h of darkness and
13h of light. Control sample was inoculated - medium incubated only in darkness and in
light for 24h. The highest concentration of OTA was found in the samples that were
incubated for 24h in light. Between the samples incubated in the regime 11/13h - darkness/light and 24h - darkness, the differences were not significant. Concentrations of
OTA produced under different conditions of temperature and day/night regime, dod not
appear to be significant, but, on the other hand, light enforced fungal growth, directly implying higher OTA level in the samples (59).
A. carbonarius produces OTA optimally at cooler temperatures: 15°C and 0.95 - 0.97
aw or 20°C and 0.98 - 0.99 aw. Little or no OTA was formed at temperatures above 35°C.
A. ochraceus grows strongly at 37°C, indicating a maximum for growth of at least 40°C.
The growth was reported down to 0.79 aw on glucose/fructose media and down to 0.81 aw
on media based on NaCl (38). A. ochraceus grows at moderate temperatures, and on
commodities with aw higher than 0.80, so it can be frequently isolated in storage. Because
of the ability to grow at high temperatures, A. carbonarius is often related with fruit
ripening, especially with grapes (11).
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Pardo et al. (60) indicated that the germination occurred down to 0.80 aw at 20 or
30°C and 0.85 aw at 10°C. A. westerdijkiae and A. steynii are closely related to A. ochraceus with perhaps slightly faster growth rates on CYA at 25°C, but the growth does not
occur at 37°C . The growth temperatures for A. niger are: minimum 6-8°C, maximum 4547°C, and optimum 35-37°C (33, 38).
Investigations of ochratoxigenic moulds showed that P. verrucosum can grow on some commodities only under temperatures of 30°C and at 0.80 aw (11).
In any case, storage conditions are of great importance, so keeping the temperature
and aw away from optimums, can reduce mould growth and OTA production.
Effect of the substrate
Among the many different parameters that can determine expression of the ochratoxigenic character of moulds, substrate has an important role (66). Same isolates of one species grown in the same conditions (temperature, humidity) produced different amounts of
OTA, depending of the medium (67). Medina et al. (66) tested ochratoxigenic capability
of A. ochraceus in the same liquid media that had different natural supplements, such as
corn extract, peptone, coconut extract. They also optimized and designed semi-synthetic
media that could stimulate OTA production by ochratoxigenic strains of A. ochraceus.
The amount of free carbon (C), pH value, presence of some metals and chemical composition of the medium, are directly implicated in the biosynthesis of OTA by some species from the Aspergillus and Penicillium genera (66, 67). According to Muhlercoert et
al. (60), A. ochraceus produces OTA at a pH in the range from 5.5 to 8.5. The presence of
some metals directly influences the pH value and thereby the biosynthesis of OTA. For
example, addition of 0.12 mg/l of Zn, at the pH 6.5 increased by 50% colony growth of
A. ochraceus and also OTA production. On the other hand, addition of Fe in the same
amount, decreased the colony growth by 40% and as well as the OTA production. In same investigations, the concentration of OTA in the substrate was not in correlation with
biomass, and increased with the decrease of glucose in the substrate. Lactose does not
stimulate fungal growth and along with that biomass, but has a positive effect on OTA
production.
Pitt and Hocking (38) reported that A. ochraceus grew well between the pH 3 and 10,
and slowly at the pH 2.2, A.niger on pH 4.0-6.5 and A. carbonarius grew over a wide pH
range (2–10).
Effects of climate conditions
The influence of different climate conditions and regions on the growth of toxigenic
moulds and occurrence ochratoxigenic capability, and OTA concentration in the final
product are known in the agricultural industry (61). In order to determine closely the influence of climate and regions on grapevine contamination, many researchers have investigated small-scale field plots. However, the obtained results were not always in accordance with the expectations. A few investigations have shown a certain correlation between toxin concentration and the area of vine growth in the Mediterranean region, i.e.
Greece and Italy. Results have revealed that in southern regions grape is contaminated
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with moulds that are having higher predilection for OTA production (40, 62). For example, OTA is found more frequently in wines originating from warmer regions (South Europe) than in the ones from colder regions (63). In Greece, higher concentrations of OTA
are found in wines from islands with wet than from islands with more continental climate
(64). Also, Battilani et al. (65) reported strong effect of rainfall on OTA concentrations in
grape bunches, but on the other hand Belli et al. (56) did not find correlation between
these factors.
Leong et al. (32) have reported that A. carbonarius is main OTA producer in Australia, which characterized only a few isolates of A. niger species. From tested strains, ochratoxigenic ability of A. carbonarius isolated from grapes was the highest and outreached
37.5 μg/kg.
Interaction of the main ecophysiological factors
Estimation of the interaction of the main ecophysiological factors and different fungus
species is very important in designing a model in order to minimise the risk of OTA in
foodstuffs.
Valero et al. (68) have investigated in vitro the effect of biotic factors on A. carbonarius growth and OTA production, using SNM. The tested strains were isolated from
dried vine fruit and grapes. They were cultured on the same SNM agar plate with one positive OTA A. carbonarius strain, at two different temperatures (20 and 30°C) and two aw
values (0.92 and 0.97).
The differences in the OTA biosynthesis at both temperatures and at 0.92 aw were not
significant. At 0.97 aw and temperature of 30°C, the production of OTA was reduced
when A. carbonarius was grown in paired cultures (comparing that grown alone). At 0.97
aw and 20°C, there was no clear interaction between moulds, and the level of synthetisis
of OTA remained the same.
The reduction in the OTA production by toxigenic moulds that are cultured together
on the same medium at 30°C and at 0.97 aw can be explained on various ways: a) limited
space for A. carbonarius growth because of the growth of the other cultured species,
which normally leads to lower OTA production; b) antagonistic fungi consume specific
nutrients needed for OTA production; c) OTA decomposition by other moulds, and d)
interaction between moulds can provoke excretion of the substances that diffuse towards
A. carbonarius colonies and blocking the OTA production. It was suggested (68) to keep
the drying temperatures above 30°C, to prevent potential OTA biosynthesis.
In Australia, Leong et al. (33) revealed that 25°C is an optimal temperature to prevent
the synergistic effect between A. carbonarius (5 strains) and A. niger (2 strains) for OTA
production. They found that the optimal mould growth was at 15°C, at an optimal aw. in
the range of 0.95-0.98.
CONTROL MEASURES TO REDUCE THE OTA LEVELS IN DRIED VINE
FRUIT
Contamination with OTA can be significantly reduced by using appropriate agrotechnical measures and fungicides in the vineyards, in order to prevent growth of some
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species from the Aspergillus genera (2). At harvested fruit, SO2 can be used as efficient
reagent to control mould growth (69) and the presence of residual SO2 (that is usually
added to prevent browning) can help to control the development of moulds. Besides, the
rapid drying of grapes at a temperature above 30°C reduces aw to safe levels that are not
favourable for mould growth and OTA production. Elimination of the discoloured berries
and the ones that are dark coloured after drying, can reduce the final OTA concentration
in the products, because OTA contamination is related with this changes (32-34). Occasionally, frequent turning over of grapes during the drying process is important, because
moisture accumulation and increased sugar level in grapes can stimulate growth of Aspergillus section Nigri and biosynthesis of OTA (42).
Drying methods can influence on final concentration of OTA in dried vine fruit (70).
Drying in controlled conditions (temperature and humidity) is favourable comparing drying in open air under solar radiation. Drying in chambers at 50°C prevents OTA production because that temperature inhibits the growth of A. carbonarius, and partly degrades
already formed toxin, due to the elevated temperature. Dipping of dried vine fruit in olive
oil or ethyl-oleate can enhance water permeability thought the berry membrane and also
eliminate some OTA. However, these procedures are very expensive and they are performed only in cases when severe mould contaminations are observed. Investigations (71,
72) have revealed that in the case of drying under controlled conditions, in climate chambers with monitored temperature and humidity, 24% less contamination occurred than in
the cases when grapes were dried in open sun.
CONCLUSION
On analysing the investigations that have been done worldwide, it clearly comes out
that great effort should be put in the optimisation of fruit drying process, as well as in the
application of adequate agricultural measures in vineyards, because the only way to prevent OTA production is to avoid microbiological contamination. Microbiological contamination, especially with moulds can incur not only big economic losses, but also favourable conditions for the production of OTA, which is very toxic and hazardous to human
health. OTA has been frequently isolated from dried vine fruit, and in many cases, its entration was above the pean limit, which implicates that this commodity can be a significant source of this mycotoxin. It has been shon that the drying process has an important
influence on the OTA level. Drying in open sun is frequently applied, but it brings the
risk of secondary contamination with foreign matter, insects and microorganisms. The
mycotoxicological quality of dried vine fruit is influenced by the region of origin and climatic conditions of area where grapes are harvested and dried.
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61. Lopez de Cerain, A., Gónzalez-Peñas, E., Jiménez, A.M. and Bello, J.: Contribution
to the study of ochratoxin A in Spanish wines. Food. Addit. Contam. 19 (2002) 10581064.
62. Pietri, A., Bertuzzi, T., Pallaroni, L. and Piva, G.: Occurrence of ochratoxin A in Italian wines. Food. Addit. Contam. 18 (2001) 647-654.
63. Belli, N., Marin, S., Sanchis, V. and Ramos, A.J.: Ochratoxin A (OTA) in wines,
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УТИЦАЈ ЕКОФИЗИОЛОШКИХ ФАКТОРА НА ПРИСУСТВО
ОХРАТОКСИНА А У СУВОМ ГРОЖЂУ
Владислава М. Шошо, Марија M. Шкрињар и Невена Т. Благојев
Универзитет у Новом Саду, Техолошки факултет, Булевар цара Лазара 1, 21000 Нови Сад, Србија
Производи од грожђа, пре свега сушени производи, веома често се користе у
људској исхрани и, заједно са вином, имају веома велики економски значај у земљама Медитерана, посебно у Шпанији, Грчкој, Турској и Италији. Разноликост у
клими и регионима на којима се узгаја грожђе, указује на то да плесни, потенцијални произвођачи охратоксина А (ОТА), имају велико распрострањење. Самим тим,
овај токсин се често изолује из ових производа. Велики напори се улажу у циљу
сузбијања раста охратоксигених плесни и детоксификације већ контаминираних
производа. Међутим, значајно ефикасних метода за детоксификацију и елиминацију ОТА из намирница још увек нема. Из тог разлога спречавање раста токсигених
пленси је од непобитног значаја.
Данас је веома добро познато да екофизиолошки фактори имају јак утицај на
присуство охратоксигених плесни на различитим прехрамбеним производима, као
и на производњу ОТА. Циљ овог рада је, стога, да сумира резултате који су до сада
добијени на пољу испитивања присуства ОТА у сувим производима од грожђа и да
истакне утицај екофизиолошких фактора на присутност плесни и ОТА у овој врсти
производа.
Кључне речи: суво грожђе, појава охратоксина А, екофизиолошки фактори
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THE IMPACT OF THE MANUFACTURING PROCESS ON THE HARDNESS
AND SENSORY PROPERTIES OF MILK CHOCOLATE
Danica B. Zarića, Biljana S. Pajinb, Ivana S. Lončarevićb*, Dragana M. Šoronja Simovićb

and Zita I. Šerešb
a
b
Ihis Tehno Experts d.o.o., Research Development Center, 11000 Belgrade, Serbia
The aim of this paper was to examine the impact of the manufacturing process on the
textural characteristics and sensory properties of milk chocolate. The research was conducted on the samples of chocolate produced in a ball mill during 30, 60 and 90 minutes
of refining, each of them being pre-crystallized at 26, 28 and 30°C. A chocolate mass of
identical ingredient composition was also produced using a standard manufacturing process at the same pre-crystallization temperatures. Chocolate hardness was examined
using a piece of equipment called Texture Analyser, measuring the stress intensity which
leads to chocolate crushing. Sensory analysis was performed using the point scoring
method. The new manufacturing process, i.e. the manufacturing of chocolate in a ball
mill improves sensory properties and hardness of milk chocolate.
KEY WORDS: milk chocolate, ball mill, hardness, sensory evaluation
INTRODUCTION
Chocolate mass can be produced in two ways: using a standard or traditional manufacturing process (1, 2) and using the unconventional one in a ball mill (3,4). The traditional process includes: mixing of raw materials, refining in a five-roller mill, conching,
tempering, moulding and final crystallization, and it has been well known and used in
unaltered form for the last 150 years (5). Present-day trend of manufacturing rationalization has resulted in introducing a new method which combines the first two phases of the
traditional process: conching and refining. A ball mill consists of a vertical or a horizontal double-wall cylinder with hot water flowing between them (6). In the central part of
the cylinder there is a paddle mixer that operates at the speed of 50-70 rpm. The balls, i.e.
the refining medium fill up to 60-80% of the cylinder. The balls are usually made of
stainless steel or any other material used in the food industry. The mill must be provided
with the mass recirculation system. The chocolate mass with constant recirculation goes
through a thick layer of rotating balls, in the process of which the particles are constantly
* Corresponding author: Ivana S. Lončarević, University of Novi Sad, Faculty of Technology, Bulevar cara
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refined and subjected to shear force and friction. The speed of mass recirculation is 3-6
kg/min. The time of refining and processing of one loading is 90-120 minutes. The advantages of manufacturing chocolate in a ball mill in comparison with the conventional
manufacturing process are reduced costs of maintenance, workforce, production and initial costs.
Ball mills are constantly improved, so that the chocolate could have suitable sensory
and rheological properties. Mazzeti company has created a ball mill with a container for
storing chocolate mass that is equipped with a thin layer evaporator, which in addition to
refining also enables the correction of chocolate mass viscosity and taste (7). Duyvis
Wiener company has created “Taste Changer” for eliminating unwanted moisture and volatile acids. According to this procedure, heated dry air is added to the chocolate mass
under pressure and controlled flow rate. Dry air takes over moisture and volatile acids,
while the increased temperature causes volume expansion of cocoa butter which in this
way coats solid particles more easily and contributes to the improvement of the chocolate
mass rheological properties (8, 9).
After being manufactured in a ball mill, the chocolate mass is tempered, moulded and
hardened through final crystallization. Tempering or pre-crystallization is favourable for
the creation of crystallization centres within a stable crystal form, which results in a product having good physical and sensory properties (10).
Chocolate hardness is one of the most important factors in defining physical properties of this product and it is determined by the measuring intensity of the force required
for chocolate crushing. Chocolate hardness depends on the refinement of solid particles
as well as on their distribution by size (11). Chocolate sensory properties are also an unavoidable factor in determining the consumers’ acceptance of this product (12).
EXPERIMENTAL
Material
The raw materials used for chocolate production included: cocoa butter (Theobroma,
Amsterdam), cocoa liquor (Cargill), medium-grain sugar (Crvenka AD, Serbia), whole
milk powder (total fat 25%, proteins 28% and carbohydrates 37% ) - Imlek, Serbia, skimmed milk powder (total fat 1%, proteins 35% and carbohydrates 51 %) - Imlek, Serbia,
hazelnut paste (total fat 25%, proteins 28% and carbohydrates 37% ) - Arslanturk, Turkey, Ethylvanilin (FCC, Norway), soya lecithin with a minimum insoluble content of
65% in acetone (Soyaprotein AD, Serbia), polyglycerol polyricinoleate or PGPR (Danisco, Malaysia) and SM-chocolate mass produced by standard manufacturing process
(Jaffa, Srbija).
Methods
Production of chocolate mass in a ball mill. The chocolate was manufactured in a laboratory ball mill with a homogenizer (capacity 5 kg), of a domestic manufacturer. All
raw materials were added to the homogenizer exept for the 10% of cacao butter. The
mixing time was 20 minutes. After mixing, the mass was transfered into the ball mill. The
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milling time was 90 minutes and the remaining quantity of the cacao butter was added in
the 80th minute. The diametar of the balls in the mill is 9.1 mm and the rotation speed of
mixer is 50 rpm.The ball mill is equipped with the mass-circulation system, with a speed
of 10 kg/hour. The internal diameter of the ball mill is 0.250 m, and the height is 0.31 m.
The volume of the space provided for balls and 5 kg of chocolate mass is 0.0152 m3.
Chemical analyses. The basic chemical composition is determined using standard
AOACC methods (13), as shown in Table 1.
Table 1. Chemical methods for basic chemical composition of milk chocolate mass
determination
Quality factor
Moisture [%]
Total fat [% d.m.]
Proteins [% d.m.]
Carbohydrates [% d.m.]
Cocoa solids [% d.m.]
No fat cocoa components [% d.m.]
Saccharose [% d.m.]
Lactose [% d.m.]
Energy value [kcal]
Energy value [kJ]
Method/Principle
Thermogravimetric
Determination of the petrol ether extract
Kjeldahl method
Polarimetric
Spectrophotometric
Spectrophotometric
Polarimetric
Iodine metric titration
Calculation
Calculation
Production of the chocolate mass using standard manufacturing process. The chocolate mass manufactured in a standard way is mixed in a melanger for about 20 minutes
(with one half amount of the cocoa butter and emulsifier) and then refined in a five-roller
mill and conched. The remaining amount of cocoa butter is added while conching. Conching lasted for 12 hours, after which the remaining amount of emulsifier is added and
the process is continued for another 6 hours.
Pre-crystallization of the chocolate mass. Pre-crystallization of the chocolate mass is
performed in the laboratory precrystallizer – a modified Brabender farinograph (14). The
process of pre-crystallization is controlled indirectly by the changes of the mass resistance on the occasion of mixing, which is registered on a force/time diagram – the thermoreogram. The following pre-crystallization temperatures were applied: 26oC, 28oC and
30oC for both chocolate masses.
Symbols used in the work. Symbols of the chocolate masses used in work are listed in
Table 2.
Determination of chocolate hardness. The determination of the chocolate textural
properties was performed using Texture Analyser following the original method 3-Point
Bending Rig HDP/3PB. Working conditions were: measuring cell 5 kg; temperature
20oC; speed of cylindrical sonde before the analysis: 1.0 mm/s; speed of cylindrical sonde
during the analysis: 3.0 mm/s; speed of cylindrical sonde after the analysis: 10.0 mm/s;
distance: 40 mm; texture measuring was performed on 3 repeated occasions, after seven
days of stabilization of the manufactured chocolate. The subject of the measurement was
the intensity of force used to crush the chocolate.
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Table 2. Symbols of the chocolate masses
Symbol of the chocolate mass
R1-90-26
R1-90-28
R1-90-30
R1-60-26
R1-60-28
R1-60-30
R1-30-26
R1-30-28
R1-30-30
SM -26
SM -28
SM -30
Refining time (min)
90
90
90
60
60
60
30
30
30
in five-roller mill
In five-roller mill
In five-roller mill
Pre-crystallization temperature (ºC)
26
28
30
26
28
30
26
28
30
26
28
30
Sensory analysis. A committee of 6 members scored from 1 to 5 the following quality
parameters: appearance, structure, chewing, taste and smell. The obtained scores of these
parameters were multiplied by a defined coefficient of importance (14), and the category
of quality was defined on the basis of the total number of points. The chocolate samples
were analysed seven days after their stabilization.
Statistical analyses. The results of measuring hardness and the total number of pondered points were processed by statistical testing for significant difference between two
means using t-test at the significance threshold of 95%, α = 0.05 (program packages Statistica 8.0 and Origin 6.1). The influence of independent variables (x and y) on the above
dependent variables (z) was mathematically defined by means of regression analysis of
experimental values. The response function z was defined by the regression equation
(mathematical model) of the following form:
z = b0 + b1x + b2y + b11x2 + b12xy + b22y2,
in which: b0, b1, b2, b11, b12 and b22 are regression coefficients; x is pre-crystallization
temperature (tp); y is refining time (τ); z is response function of characteristic parameter
value: the chocolate hardness, the total number of points for sensory quality). The regression coefficients b1 and b2 indicate linear effect of the independent variables x and y on
the dependent variable z, b11, and b22 indicate square effect, while b12 indicates linear interaction of independent variables.
On the basis of obtained experimental - real (ze) and theoretical - expected values (zt)
the following statistical parameters were calculated: the standard error of the regression
σ, p and t-values, the coefficient of determination and analysis of variance for the selected regression expression.
The standard error of the regression is defined by the following relation:
σ
Σ(z e  z t ) 2
n2
The calculation of the coefficient of determination (r2) solved to determine the discrepancy between the experimental and the theoretical values.
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Origin
Sttatistical processiing of the results included only thhe examination off the chocolate
mass manufactured in the ball mill.
LTS AND DISCU
USSION
RESUL
Chemical analyssis
Both
B
chocolate maasses had identicaal chemical compoosition, as they were
w
made from
the same
s
raw materiaals, but in differeent manufacturingg processes, whicch is shown in
Tablee 3.
Table 3.
3 Chemical compposition of the millk chocolate mass
Quality factor
Q
M
Moisture
[% d.m.]
T
Total
fat [% d.m.]
P
Proteins
[% d.m.]
C
Carbohydrates
[% d.m.]
C
Cocoa
componentss [% d.m.]
N fat cocoa compponents [% d.m.]
No
S
Saccharose
[% d.m
m.]
L
Lactose
[% d.m.]
E
Emulsifiers
[% d.m
m.]
E
Energy
value [kcall]
E
Energy
value [kJ]
1.10
32.41
8.76
52.98
30.14
4.74
42.67
10.31
0.50
538.64
2251.53
Hardness oof the chocolate mass
m
The graph showingg experimental reesults of measurinng the hardness of chocolate R1
and SM
S using Texturre Analyser follow
wing the originall method 3- Poin
nt Bending Rig
HDP//3PB is presentedd in Fig. 1.
Figu
ure 1. Comparativee illustration of thhe hardness of the chocolate mass manufactured
m
in
the ball
b mill (R1) and the chocolate mass made by standaard manufacturing
g process (SM)
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Origin
In
n the chocolate mass
m
produced by standard manufaccturing process it was noted that
an increase in the pre-ccrystallization tem
mperature leads too a decrease in thee force required
for ch
hocolate crushing. An increase in thhe pre-crystallizattion temperature in
n the chocolate
mass produced in the ball mill, irrespecctive of the refiniing time, leads to
o an increase in
the fo
orce required for chocolate crushinng (increased harddness values), whiich is most clearly seen
s
in the most of
o the homogeneous systems – the one
o in which the chocolate mass
was refined
r
during 90 minutes. The incrrease in hardness as a result of the increase
i
in precrystaallization temperaature was also connfirmed by the stattistical data processsing.
The results of statistical data processing are shown inn the 3D and conttour diagram in
Fig. 2.
2 The increase inn the pre-crystalliization temperaturre had a significan
ntly greater influen
nce on the increasee in the hardness of the chocolate mass
m
R1, than on the refining time. By
B testing statisticcal significance oof certain parametters in the regresssion equation it
has been
b
noted that thhe regression coeffficients b1, b0 andd b11 indicate certtain changes of
the dependent variablee, i.e. hardness, byy changing the inddependent variablee x, i.e. the precrystaallization temperaature.
a)
b)
Z1 = 3518.727 – 261.002 tp + 11.213 τ + 4.942 tp2 – 0.118 tp τ + 0.0
017 τ 2
Fiigure 2. The impaact of the refining time and pre-crysstallization temperrature on the
hardness of
o the chocolate m
mass manufacturedd in the ball mill R1:
R
a) 3D diagraam, b) contour diagram
The calculated valuue of standard erroor of the regressioon (σ = 13.864) co
onfirms that the
selectted mathematical model gives a biit larger dispersioon of experimentaal values, while
the value of coefficiennt of hardness deteermination (r2= 0.894) indicates thaat physical propertiees of chocolate R11 are determined bby variations of inndependent variablles by 89.4%.
The analysis of varriance in regressioon equation confirrms that at the 95
5% significance
level (α = 0.05) and byy applying the sellected regression equation,
e
it is posssible to predict
the behaviour
b
of the chocolate
c
hardnesss under varying refining time and pre-crystallization temperature
t
(calcuulated F = 12.874 > tabular F0.05;6;3 = 8.94).
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Origin
Sensory analysis of chocolate
The graph showingg comparative expperimental results of the milk choco
olate mass produced
d in the ball mill (R1)
(
and of that pproduced by standdard manufacturing
g process (SM)
is sho
own in Fig. 3.
Figu
ure 3. Comparativve illustration of seensory marks of thhe chocolate masss manufactured
in th
he ball mill (R1) and
a the chocolate mass manufactureed by traditional manufacturing
m
prrocess (SM)
The chocolate masss produced by staandard manufactuuring process, pree-crystallized at
30°C
C provides a chocoolate of suitable, ii.e. very good sennsory quality (hig
ghly glossy surface, tiny grainy structture, shelly fractuure and suitable taaste and smell). Itts external propertiees are significantlyy better in compaarison with the choocolates pre-crysttallized at 26°C
and 28°C.
2
These very good sensory prooperties result from
m the optimal viscose properties
and suitable
s
pre-crystaallization of cocoaa butter. The choccolate SM-28 had the worst fracture and
a the lowest totaal of pondered poiints.
The chocolate masss produced in thee ball mill refinedd during 90 minutees and pre-crystallizzed at higher tem
mperatures providdes a chocolate off suitable, i.e. exccellent sensory
quality (highly glossy surface, tiny grainny structure, shellly fracture, and su
uitable taste and
smelll). Such good sennsory properties reesult from the opttimal viscose prop
perties and suitable crystallization off cocoa butter. Thee chocolate R1-600 had a very good sensory quality
as weell as better markss for external propperties, structure and
a chewing in co
omparison with
the ch
hocolate R1-90. Refining
R
time of 330 minutes providdes a chocolate of good sensory
quality only, i.e. the obbtained chocolate form had weakerr deformation, parrtially damaged
surface, coarse grain fracture,
f
uneven sstructure and evident properties of slower melting
in thee mouth.
The graph showingg statistically proccessed data on deependence of the chocolate
c
mass
R1 seensory properties on the pre-crystaallization temperatture and refining time is seen in
the 3D
D and contour diaagram in Fig. 4. Inn the regression eqquation which deffines the dependencee of the total of seensory analysis poondered points on the refining time and pre-crystallization temperature, the coefficient off square dependennce of the refinin
ng time and the
coeffficient of linear innteraction betweenn the refining timee and the pre-crysttallization temperatu
ure have the greattest influence.
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Origin
a)
b)
Z2 = 8.476667 – 0.365833
0
tp- 0.0244056 τ + 0.0062500 tp2 + 0.00100 tpτ + 0.000117 τ 2
Figure 4. Impactt of the refining tim
me and precrystalllization temperatu
ure on the
sensory scorees of the chocolatee mass manufactuured in the ball milll R1:
a) 3D diagraam b) contour diaggram
The value of standdard error of the rregression shows a slight dispersio
on between the
experrimental values annd the theoretical curve (σ = 0.0955). The selected reegression equation is
i representative, as the variations oof independent vaariables are signifficantly responsible for the sensory quality
q
of the choocolate mass (r2 = 0.9824). The vaariance analysis
confiirms that the regreession equation is statistically signiificant as a wholee, as it has been
calcu
ulated that F > F0.005;6;3= 8.94.
NCLUSIONS
CON
Milk
M chocolate prooduced in a ball m
mill is harder than the chocolate pro
oduced by standard manufacturing prrocess, irrespectivve of the applied pre-crystallizatio
on temperature.
The force
f
required forr crushing the choocolate produced in a ball mill is in
i average 2.25
timess greater in compaarison with the chhocolate producedd by standard manufacturing process. By introducing the
t new manufactturing process, i.ee. the ball mill, th
he sensory propertiees of milk chocolaate have been improved.
nowledgement
Ackn
This work is fundded by the Ministtry of Science annd Technological Development,
Repu
ublic of Serbia (Project TR 31014).
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7. Mazzeti Renato S.A.: Catalogo Generale 2009. www.mazzetirenato.it (accessed 20th
October 2011).
8. Duyvis W.: Brochures about W95/W100 Ball Mill 2009. www.duyviswiener.com.
(accessed 11th October 2011).
9. Kennedy's Confection: The new approach to chocolate processing, Duyvis Wiener
(2009) 18-21.
10. Afoakwa E. O.: Chocolate Science and Technology, 1st Edition, While-Blackwell,
Oxford (2010).
11. Afoakwa, E.O., Paterson, A., Fowler, M., Vieira, J.: Relationship between rheological, textural and melting properties of dark chocolate as influenced by particle size
distribution and composition. European Food Research and Technology 227, 4 (2008)
1215-1223.
12. Popov-Raljić, J. V., Laličić-Petronijević, J. G.: Sensory properties and Colour Measurements of Dietary Chocolate with Different Compositions During Storage for Up to
360 Days. Sensors 9 (2009) 1996-2016.
13. AOAC, Official Methods of Analysis, 17th ed., Maryland, USA: Association of Official Analytical Chemists (2000).
14. Pajin. B.: Praktikum iz tehnologije konditorskih proizvoda, Faculty of Technology,
University of Novi Sad (2009) 36-39.
УТИЦАЈ ПОСТУПКА ПРОИЗВОДЊЕ НА ЧВРСТОЋУ И СЕНЗОРНЕ
КАРАКТЕРИСТИКЕ МЛЕЧНЕ ЧОКОЛАДЕ
Даница Б. Зарића, Биљана С. Пајинб, Ивана С. Лончаревићб, Драганa M. Шороња
Симовићб и Зита И. Шерешб
a
b
Ihis Tehno Experts д.o.o., Истраживачко-развојни центар, 11000 Београд, Србија
Циљ овог рада био је да се испита утицај поступка производње на текстуру и
сензорне карактеристике млечне чоколаде. Испитивани су узорци чоколаде која је
произведена у кугличном млину уситњавањем 30, 60, 90 минута и свака чоколада је
преткристалисана на 26, 28 и 30°C. Такође је произведена чоколадна маса идентич147
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ног сировинског састава стандардним поступком, која је преткристалисана на исте
температуре. Чврстоћа чоколада је испитана на уређају Тexture Analyser, мерећи јачину силе која доводи до лома чоколаде. Нови поступак производње чоколаде у
кугличном млину побољшава сензорне карактеристике и чврстоћу млечне чоколаде.
Кључне речи: млечна чоколада, куглични млин, чврстоћа, сензорна анализа
Received: 10 February 2012
Accepted: 12 April 2012
148
CHEMICAL TECHNOLOGY AND PROCESS ENGINEERING
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PARTITIONING OF CELLULOLYTIC ACTIVITY IN THE POLYETHYLENE
GLYCOL/DEXTRAN TWO-PHASE SYSTEMS
Mirjana G. Antova,*, Branimir Z. Jugovićb, Milica M. Gvozdenovićc
and Zorica D. Knežević Jugovićc
a
b
University of Novi Sad, Faculty of Technology, Novi Sad, Serbia
Serbian Academy of Science and Arts, Institute of Technical Science, Belgrade, Serbia
c
University of Belgrade, Faculty of Technology and Metallurgy, Belgrade, Serbia
This study is concerned with the partitioning of cellulolytic activity in the polyethylene glycol/dextran two-phase systems. In the system of 10% (w/w) polyethylene glycol
1500/5% (w/w) dextran 500,000/80% (w/w) crude enzyme at the pH 5, 100%, yield of
cellulolytic activity from Penicillium sp. in the top phase was achieved in a single extraction step. Addition of KH2PO4 to this system at a concentration of 15 mmol/L improved
the purification factor in the top phase for cellulolytic activity from crude preparation to
a value of 2.6, although it had an adverse effect on the yield in the same phase.
KEY WORDS: aqueous two-phase system, cellulolytic activity, partitioning, purification
INTRODUCTION
An aqueous two-phase system (ATPS) is the medium that enables selective partitioning of biomaterials such as proteins, nucleic acids, organelles and whole viable cells,
from complex mixtures (1). This system is formed by mixing the solutions of two
mutually incompatible polymers or polymer and salt above critical concentrations. The
basis of separation is the uneven distribution of biomaterials between two phases, both
having high water content. This high water content combined with the low interfacial
tension of the system allows non-destructive partitioning of sensitive biomaterials and is
often referred as biocompatibility. Even more, the biocompatibility of the phases allows
preservation of biomolecules’ native structure while the presence of polymer can even
improve their stability (1). Partitioning is governed by numerous factors that can be manipulated to achieve desired separation and purification results, which makes ATPS very
flexible for the application (1, 2).
Being the medium that is very well suited for the partitioning of biomaterials, ATPS
has found wide and advantageous application in bioseparation of enzymes as well. There
are numerous examples of extraction of enzymes in ATPS in downstream processing
with the aim of their isolation and purification (1,3-5).
* Corresponding author: Mirjana G. Antov, University of Novi Sad, Faculty of Technology, Bulevar cara
Lazara 1, 21000 Novi Sad, Serbia, e-mail address: [email protected]
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Cellulases, enzymes belonging to the family of glycosyl hydrolases, play a key role in
organic carbon turnover and have important and wide application in industry. At present,
cellulases are used in the food, brewery and wine, animal feed, textile and laundry, pulp
and paper industries, as well as in agriculture (6). In addition, cellulases have recently
gained additional attention because of their application in the production of biofuel from
lignocellulosic substrates (7). So, the demand for these enzymes is growing rapidly, becoming a driving force for the research on cellulases production and downstream processing.
In this study, partitioning of cellulolytic activity in polymer/polymer two-phase systems was studied with the aim to establish the conditions in which the highest possible
yield and purification factor in the top phase can be achieved. Several factors were investigated in polyethylene glycol/dextran ATPS - molecular weight of polyethylene glycol (PEG) and its concentration, as well as addition of different salts. Partitioning parameters of cellulolytic activity from commercial enzyme preparation and crude enzyme
from Penicillium sp. were determined and compared.
EXPERIMENTAL
Preparation of ATPS
Polyethylene glycols having molecular weights 1500 g/mol (PEG 1500), 4000 g/mol
(PEG 4000) or 6000 g/mol (PEG 6000) (Merck, Germany) and fractionated dextran with
moleculat weight ~500,000 g/mol (Fluka, Switzerland) were used for the preparation of
ATPS. Ten-gram phase systems were prepared by adding adequate quantities of PEG,
dextran, enzyme solution and 10 mmol/L acetate buffer pH 5.0, to achieve desired concentrations (%, w/w). The mixtures were vortexed for 5 minutes and the phases were allowed to separate in graduated tubes for 12 hours. Then, the top phase was carefully removed with a pipette, leaving a small amount at the interface, and the bottom phase was
then sampled through the interface. Samples of each phase were analysed for enzyme activity and protein.
Commercial enzyme
Commercial preparation Celluclast 1.5 L™ (Novozyme) was prepared for partitioning
experiments by dilution in the 10 mmol/L acetate buffer pH 5.0 to make basal enzyme
solution.
Crude enzyme from Penicillium sp.
Crude enzyme preparation was obtained by submerged cultivation of Penicillium sp. 300 mL Erlenmeyer flasks, containing 100 mL medium with 1.5 g sugar beet extraction
waste (particle size 400 m) and 0.5 g (NH4)2SO4 in 0.15 mol/L KH2PO4, pH 4.5, were
inoculated with 106 spore/mL and incubated at 28oC and 200 rpm. After 4 days, the cultivation was stopped and content of flasks was filtered to obtain crude enzyme (CE).
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Enzyme assay
Cellulolytic activity was determined according to König et al. (8) - 1.5 mL of 4%
(w/v) carboxymethylcellulose in 0.1 mol/L acetate buffer, pH 5, and 0.5 mL enzyme
solution were kept in a water bath at 40oC for 30 minutes. The reaction was stopped by
addition of DNS reagents, followed by boiling at 100oC for 5 minutes and absorbance
was read at 540 nm. One unit was determined as the amount of the enzyme catalysing the
formation of 1 mol of glucose per minute at 40 oC and pH 5.0. Protein concentration
was determined by Bradford method (9) with bovine serum albumin as standard.
Partition parameters
The partition coefficient for cellulolytic activity in the ATPS systems was defined as
activitytop phase
K
[1]
activitybottom phase
and the yield in the top and the bottom phase, respectively, as
100  Vt  K
Yt (%) 
Vt  K  Vb
[2]
100  Vb
[3]
Vt  K  Vb
where Vt and Vb are the volumes of the top and bottom phase, respectively.
The purification factor of crude enzyme in the top phase was defined as
specific activitytop phase
PFt 
[4]
specific activityCE
where specific activity represents the ratio between the enzyme activity and protein concentration in the sample.
The results are the mean value of at least three measurements of activity (the accuracy
is considered to be 5%) on a minimum of three replicas for every partition experimental
point.
Yb (%) 
The influence of molecular weight of PEG on the partitioning of cellulolytic activity
The selection of molecular weight of polymer is usually the first step in the partitioning experiments with the aim of finding a suitable phase system where selective separation of target material is achieved. Results of the distribution of cellulolytic activity
from commercial preparation and crude enzyme between two phases of polyethylene
glycol/dextran two-phase systems obtained at different molecular weights of the top
phase polymer are given in Figure 1.
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a)
b)
Yb
100
Yt
80
Yield (%)
Yield (%)
80
60
40
20
0
100
60
40
20
PEG 1500
PEG 4000
PEG 6000
PEG molecular weight (g/mol)
0
PEG 1500
PEG 4000
PEG 6000
PEG molecular weight (g/mol)
Figure 1. Influence of the molecular weight of PEG on the partitioning of cellulolytic
activity between the top and the bottom phases from a) commercial enzyme preparation
in 10% (w/w) PEG/5% (w/w) dextran/35% (w/w) basal enzyme solution ATPS and b)
crude enzyme from Penicillium sp in 10% (w/w) PEG/5% (w/w) dextran/80% (w/w)
crude enzyme ATPS
It is known that the phase polymer molecular weight influences the material partitioning both by altering the phase diagram, i.e. by influencing the composition of the
phases, and by changing the number of polymer-enzyme interactions in general. Usually,
the partition coefficient of enzyme and consequently top phase yield decrease as the PEG
chain length increases (1), but in some cases, the partition parameters show just opposite
dependence (10, 11). This was also the case with the results obtained with commercial
enzyme preparation – although the the top phase yield did not change very much with the
change of the moleculat weight of PEG (from approx. 71 to 79%), still the highest
amount of cellulolytic activity was partitioned to the top phase of the system containing
the longest investigated PEG molecule (Figure 1a). On the other hand, the decrease of the
molecular weight of PEG was followed by an increase in the yield of cellulolytic activity
from crude enzyme from Penicillium sp. and in the system containing PEG 1500 enzyme
activity was completely partitioned to the top phase (Figure 1b). In addition, the top
phase yields of the enzyme activity from crude preparation were in average higher in
comparison to those from commercial preparation.
The influence of concentration of PEG 1500 on the partitioning of cellulolytic
activity
Further investigations were carried out with PEG 1500 to establish the influence of
the concentration of the top phase polymer on the partitioning of cellulolytic activity into
the phases of ATPS (Figure 2).
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a)
b)
Yb
100
Yt
80
Yield (%)
Yield (%)
80
60
40
20
0
100
60
40
20
10
12
14
PEG concentration (%, w/w)
0
10
12
14
16
PEG concentration (%, w/w)
Figure 2. Influence of the concentration of PEG on the partitioning of cellulolytic
activity between the top and the bottom phases from a) commercial enzyme preparation
in PEG 1500/5% (w/w) dextran/35% (w/w) basal enzyme solution ATPS and b) crude
enzyme from Penicillium sp. in PEG 1500/5% (w/w) dextran/80% (w/w) crude enzyme
ATPS.
The most favourable conditions for cellulolytic activity from commercial enzyme to
be partitioned in the top phase were in the system containing 12% PEG 1500 (Figure 2a).
As for the enzyme activity from crude preparation, the highest obtained top phase yield
of cellulolytic activity was achieved in the system with the lowest investigated PEG
concentration, while at the other two concentrations small portions of the enzyme were
partitioned to the bottom phase (Figure 2b).
The influence of concentration of added salt on the partitioning of cellulolytic
activity
It is known that the addition of salt to a polymer-polymer two-phase system can influence the partition behaviour of the material (1) and that it may be a powerfull tool for
the improvement of partitioning parameters. So, to the systems with highest cellulolytic
activity from commercial and crude preparation partitioned in the top phase, observed in
the previous experiments, three salts were added at concentrations that do not change
equilibrium in ATPS (12).
The presence of the three tested salts in ATPS influenced only slightly the ratio
between the top and bottom phase yields during the partitioning of cellulolytic activity
from commersial preparation (Figure 3a). Contrary to that, the addition of the salts dramatically influenced distribution of cellulolytic activity from the crude enzyme between
the phases in way that favoured its partitioning to the bottom phase of the system (Figure
3b). However, the system with 15 mmol/L KH2PO4 provided the most favourable conditions for the selective distribution of cellulolytic activity from crude preparation in the
top phase, and hence the highest purification factor was obtained (Table 1).
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a)
b)
Yb
100
Yt
80
Yield (%)
Yield (%)
80
60
40
20
0
100
60
40
20
no salt
(NH4)2SO4
Na2SO4
Added salt (15 mmol/L)
KH2PO4
0
no salt
(NH4)2SO4
Na2SO4
KH2PO4
Added salt (15 mmol/L)
Figure 3. Influence of added salt on the partitioning of cellulolytic activity between the
top and the bottom phases from a) commercial enzyme preparation of 12% (w/w)
polyethylene glycol 1500/5% (w/w) dextran/35% (w/w) basal enyzme solution ATPS and
b) crude enzyme from Penicillium sp. in 10% (w/w) polyethylene glycol 1500/5% (w/w)
dextran/80% (w/w) crude enzyme ATPS
Purification of crude enzyme in ATPS
Since the aim of downstream processing of enzymes is not only to achieve high yield
but also their separation from contaminants, purification factor of cellulolytic activity
produced by cultivation of Penicillium sp. was also determined throughout all partitioning experiments. Its highest obtained values in the top phase along with corresponding
compositions of ATPS are presented in Table 1.
Table 1. Purification factor of cellulolytic activity from crude enzyme from Penicillium
sp. in the top phase of ATPS
Composition of ATPS
10% (w/w) PEG 4000/5% (w/w) dextran/80% (w/w) crude enzyme
12% (w/w) PEG 1500/5% (w/w) dextran/80% (w/w) crude enzyme
10% (w/w) PEG 1500/5% (w/w) dextran/80% (w/w) crude enzyme in
15 mmol/L KH2PO4
PFt
2.33
2.45
2.60
By comparing the results presented in Figures 1b, 2b and 3b with those in Table 1, it
can be noticed that the compositions of ATPS that enabled the highest partitioning into
the top phase were not the same as those that provided the most appropriate conditions
for the selective distribution of cellulolytic activity in the same phase. So, in the downstream processing of enzymes in ATPS, the factors influencing the partitioning have to be
carefully selected to enable good balance between both bioseparation parameters.
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CONCLUSION
The polyethylene glycol/dextran 500,000 two-phase system appeared to be a suitable
medium for the partitioning of cellulolytic activity to the top phase. It was shown that the
extraction of cellulolytic activity from crude enzyme in this system can be useful technique in the downstream processing for both isolation and purification. Appropriate conditions for the favourable and selective partitioning of enzyme activity to the top phase were created by selection of polyethylene glycol molecular weight and concentration, and
by addition of salt to the system. The observed differences in responses to the changes of
the factors influencing partitioning between cellulolytic activities originated from two
sources can be explained by the differences in the complexity of the matrix in commercial, partially purified, enzyme preparation and, on the other side, in crude unpurified enzyme. It might be that the partitioning of contaminants also creates such environment in
the phases which in turn may additionally influence partitioning behaviour of the enzyme
activity.
Acknowledgement
The financial support from the Ministry of Education and Science of the Republic of
Serbia (Grant No. 46010) is gratefully acknowledged.
REFERENCES
1. Albertsson, P-Å.: Partition of Cell Particles and Macromolecules. John Wiley & Sons,
New York, (1986) pp.12-17.
2. Hatti-Kaul, R.: Aqueous Two-phase Systems: A General Overview. Appl. Biochem.
Biotech. 19 (2001) 269-277.
3. Ruiz-Ruiz, F., Benavides, J., Aguilar, O. and Rito-Palomares, M.: Aqueous Twophase Affinity Partitioning Systems: Current Applications and Trends. J. Chromatogr.
A 1244 (2012) 1-13.
4. Walter, H., Brooks, D.E. and Fisher, D.: Partition in Aqueous Two-Phase systems Theory, Methods, Uses and Applications in Biotechnology, Academic Press, Orlando
(1985) pp.121-153.
5. Antov, M.: Aqueous Two-phase Systems – Principles of Partitioning and Application
(in Serbian), Faculty of Technology, Novi Sad (2006) pp.71-95.
6. Bhat, M.K.: Cellulases and Related Enzymes in Biotechnology. Biotechnol. Adv. 18
(2000) 355-383.
7. Wilson, D.B.: Cellulases and Biofuels. Cur. Oppin. Biotechnol. 20 (2009) 295-299.
8. König. J., Grasser, R. Pikor, H. and Vogel, K.: Determination of Xylanase, βGlucanase, and Cellulase activity. Anal. Bioanal. Chem. 374 (2002) 80-87.
9. Bradford, M. M.: A Rapid and Sensitive Method for the Quantitation of Microgram
Quantities of Protein Utilizing the Principle of Protein-dye Binding. Anal. Biochem.
72 (1976) 248-254.
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10. Bim, M.A. and Franco T.T.: Extraction in Aqueous Two-phase System of Alkaline
Xylanase Produced by Bacillus pumilus and its Application in Kraft Pulp Bleaching.
J. Chromatogr. B 743 (2000) 349-356.
11. Antov, M., Peričin, D. and Dašić, M.: Aqueous Two-phase Partitioning of Xylanase
Produced by Solid-state Cultivation of Polyporus squamosus. Process Biochem. 41
(2006) 232-235.
12. Freire, M.G., Claudio, A.F.M., Araujo, J.M.M., Coutinho, J.A.P., Marrucho, I.M.,
Lopes, J.N.C. and Rebelo, L.P.N.: Aqueous Biphasic Systems: A Boost Brought
About Using Ionic Liquids. Chem. Soc. Rev. 41 (2012) 4966-4995.
РАСПОДЕЛА ЦЕЛУЛОЛИТИЧКЕ АКТИВНОСТИ У ДВОФАЗНИМ
СИСТЕМИМА ПОЛИЕТИЛЕНГЛИКОЛ/ДЕКСТРАН
Мирјана Г. Антова, Бранимир З. Југовићб, Милица М. Гвозденовићв
и Зорица Д. Кнежевић Југовићв
а
Универзитет у Новом Саду, Технолошки факултет, Бул. Цара Лазара 1, Нови Сад
б
Српска Академија наука и уметности, Факултет техничких наука, Београд
в
Универзитет у Београду, Технолошко металуршки факултет, Београд
У раду је испитана расподела целулолитичке активности у воденим двофазним
системима полиетиленгликол/декстран. Максимално могућ 100% принос целулолитичке активности добијене култивацијом Penicillium sp. постигнут је у двофазном
систему састава 10% (m/m) полиетиленгликол 1500/5% (m/m) декстран 500000/
80% (m/m) сирови ензим на pH 5 у само једном кораку екстракције. Додатак
KH2PO4 у концентрацији 15 mmol/l у овај систем, иако је смањио расподелу целулолитичке активности из сировог ензимског препарата у горњу фазу система, побољшао је фактор пречишћавања у тој фази на вредност 2,6. Разлике у одзиву
између целулолитичких активности из два испитивана извора на промене фактора
који утичу на расподелу могу се објаснити различиом комплексошћу њихових матрикса – комерцијалног, делимично пречишћеног, и сировог непречишћеног препарата добијеног култивацијом. Наиме, и присуство самих контаминената може додатно утицати на расподелу ензимске активности.
Кључне речи: водени двофазни систем; целулолитичка активност; расподела; пречишћавање.
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COMPARISON OF LIFE CYCLE ASSESSMENT FOR DIFFERENT VOLUME
POLYPROPYLENE JARS
Nevena M. Krkić*, Vera L. Lazić and Danijela Z. Šuput 
When deciding what packaging is the most appropriate for a product there are many
factors to be considered. One of them is the impact of the packaging on environment. In
this work, life cycle inventory and life cycle assessment of two different volume packagings were compared. The data were collected on the types and amounts of materials and
energy consumption in the process of packaging and distribution of hand cream packed
in polypropylene jars of 200 and 350 mL. Life cycle inventory (LCI) and life cycle impact
assessment (LCA) were calculated. It was found that the total mass flow was higher for
the jars of 350 mL. After analyzing individual flows, it was found that in both cycles
(polypropylene jars of 200 and 350 mL),the consumption of fresh water was a dominant
flow. This fresh water flow is mostly (95%) consumed in the injection molding process of
manufacturing jars from polypropylene granules. The LCA analysis showed no significant difference in global warming potential between different volume jars. The process
that mostly affected global warming was the production of polypropylene jars from polypropylene granules by injection molding for both jar volumes. Judging by the global
warming potential, there is no difference of the environmental impact between investigated jars, but considering the mass flow and water consumption, more environmental
friendly were the 200 mL jars.
KEY WORDS: Life cycle assessment, polypropylene, jars, packaging
INTRODUCTION
Through its entire life cycle the packaging significantly affects the environment. The
environmental impact begins with the exploitation of the raw materials for packaging
production, continues through the packaging process, and ends when the packaging
appears as the packaging waste, after the utilization of the packaged product. Depending
on the type of packaging, raw materials production and production of the packaging itself
have a major impact on the environment, as they affect the ratio of natural resources. In
addition, these processes consume some amount of energy, which also has effect on the
environment. The process of packing, depending on the type of product and type of
* Corresponding author: Nevena M. Krkić, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
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packaging, can also have a significant impact on the environment, and this aspect should
be taken into account when choosing packaging. The packaging process should be managed properly to ensure the percentage of scrap is minimal (1, 2).
After using the product, its packaging emerges as packaging waste in landfills where
it shows a negative effect on the waterways, land and pollutes the air. Industrialization,
high technology and consumer demand for safe products lead to the problem of large
quantities of used, discarded packaging (3). The life cycle of packaging includes all of
these phases, starting from the raw materials production and delivery, packaging material
production, packaging preform formation and processing, delivery of the packaging
material or packaging, packaging formation, filling and closing, storage, distribution and
delivery to retail and in the end of the cycle, processes of separation, recycling and
disposal (4).
Environmental friendly or eco-friendly is the packaging that uses less energy and makes less pollution during the production, application and removal than other materials
with the same purpose. For the purpose of easier separation of packaging materials for
recycling, universal labels have been introduced to indicate the consumer that the packaging material is recyclable, and should be separated for recycling (5). In order to consider
the life cycle impacts on the environment, a relatively new method, called the product
(packaging) life cycle assessment (LCA), was developed. It is the only standardized
method that is currently used to assess the product (packaging) life cycle. The goal, the
motivation for the research, must be clearly defined from the very beginning, because it
may later affect the certain phases of the life cycle. In the case of packaging, the analysis
begins with the process of raw materials extraction from the environment, continues in
the production, product consumption and ends when the packaging or its derivatives enter
the waste streams. Operations such as transport, recycling, maintenance must be considered in the analysis (6). Assessing the product life cycle includes life cycle inventory forming and assessment.
Evaluation of packaging life cycle includes the analysis of the packaging along with
the evaluation of life cycle inventory. Life cycle assessment is equivalent to ecological
balance and environmental profiles (7).
Apart from the possibility of packaging life cycle assessment, this method allows a
comparison of similar packagings, which makes it even more important. This comparison
is possible between the systems of similar packaging, such as the original packaging and
reduced weight packaging. In such cases, life-cycle assessment is used to confirm intuition, but also to quantify the effects of such changes. Sometimes, however, the same product can be packaged in containers that can be drastically different. Life cycle assessment
can be used to compare the overall environmental burden, despite the many differences
between the packaging systems, although it is necessary to take particular care when interpreting such comparisons (8).
The aim of this paper was to assess and compare the life cycle impact of two containers of different volume made of polypropylene (PP), which are used for packaging of
pharmaceutical products.
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EXPERIMENTAL
Packaging process scheme forming
The process of packaging chamomile and olive oil hand cream in PP jars of 200 mL
and 350 mL was recorded at the factory for production of cosmetics and chemical products („Yuco-hemija“ DOO, Bački Jarak, Serbia). The process of packaging was followed through the liner filler, which is composed of volumetric filler, aluminum foil sealing equipment, labeling and closing machine.
The supplier of the PP jars of 200 mL was the company from Subotica, Serbia, while
the supplier of PP jars of 350 mL was from Nova Pazova, Serbia. The empty jars are delivered in bags, 300 pieces per bag of 200 mL and 200 pieces per bag of 350 mL. The
user stores the empty jars for a period of 2 to 3 months.
The PP jars were disinfected with alcohol before printing, then placed on the conveyor belt, which is connected to the printer, where the date and expiry date are set to be
printed.
Cream filling in the PP jars is made on the linear filler. Filling the PP jars, previously
placed on the conveyor belt, is done with two dozers that allow precise cream dosage.
The weight of the cream to be filled into the jars is adjusted. Based on the known volume
and density of the cream, the mass is determined, which may deviate ±2%, in accordance
with the specification. The speed of the PP jars filling for 350 mL is 10 pieces/min and
for the 200 mL jars, 16 pieces/min.
After completing the filling process, sealing with aluminum foil (Al foil) is carried
out. The supplier of the aluminum foils is from Subotica, Serbia. Sealing is done on the
part of the linear filler, sealer. The heater is used to adjust the operating temperature and
sealing time, which is defined based on packaging formats. The operating temperature
and time are set via the appropriate clip. The sealer can operate manually or automatically, which is regulated with the aid of a button on the device itself.
After sealing, the PP jars are labeled. The device for labeling consists of the conveyer
belt, a wheel that sets the width and height of the containers that are labeled and label
carrier. A wheel is used to set the width and height of the jars and the jars are then manually placed on the conveyor belt and the carrier sticks the label to the jar. The labeling
machine is automatic, with manual placing of the jars on the conveyor belt.
In the end of the filling process, the PP jars are closed. The closing unit consists of the
clips for setting jar width, according to which appropriate closure and closing tool is selected. A photocell regulates the unit operation. For small amount of packed products, the
closing process can be performed manually.
Individual packaging is put into the transport boxes made of corrugated cardboard.
The supplier of transport boxes is from Novi Sad, Serbia. The transport boxes are formed
by gluing. A package contains 24 pieces of 350 mL jars, or 48 pieces of 200 mL jars. The
transport boxes dimensions are: 380x290x235 mm, for 200 mL jars and 420x320x140
mm, for 350 mL jars. The transport boxes are placed on wooden pallets that are stored in
the warehouse.
The factory delivers products up to 1,000 km distance. The greatest distance is up to
Nis, where during the final product delivery raw materials and packaging is supplied. The
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product transport is carried out by trucks and vans, which consume Euro diesel. The vans
(“Kango” and “Cleo”) are used for short distance transport for economical reasons.
Collecting relevant data (inputs and outputs)
The software system used, GABI 4, requires information about most important machine characteristics and preprocessing of the collected data.
Further, the software used requires defining of the following parameters:
 Functional unit - represents the amount of cream sales per year for the two largest
consumers („Mercator group“ and „Delta Maxi“), from Belgrade, Serbia. Functional unit was calculated based on the sales in February and December 2010.
 Reference flow - number of PP jars that will be required for the amount of cream
defined by functional unit to be delivered to the customer. For the 350 mL jar, this
number is 1824 pieces, and for the 200 mL jar, 3192 pieces.
 Product system - product system with the system boundaries (Fig. 1).
Figure 1. Life cycle scheme for PP jars of 350 mL (with asterisk) and 200 mL
(unmarked)
* In Fig. 1, the numbers marked with an asterisk represent data regarding all life cycle stages for
350 mL jars and unmarked numbers for 200 mL jars.
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Origin
All
A necessary data for lifecycle steps: printing, fillingg, sealing, closing, labeling, over
packiing, transport, disstribution, sale and cream consumpption for PP jars of
o 200 mL and
350 mL
m was collectedd at the factory ("Y
Yuco hemija" froom Bački Jarak, Serbia)
S
(Fig. 1).
For liife cycle steps: PP
P jars production by injection moldding, transport box
xes production,
Al fo
oil production and disposal of used jjars the data were used from the sofftware program
GAB
BI 4. A 100% of laandfill disposal waas assumed for useed PP jars.
RESULTS AND DISCUSSIION
Life ccycle inventory
After
A
entering all the
t data shown inn Fig. 1, using sofftware GABI 4, life cycle inventory for
f PP jars was calculated
c
(total m
mass of all input and
a output flows for the system,
i.e. in
nput and output) and
a it is shown in F
Fig. 2.
Figure 2. Life cycle inventory for PP jjars with differentt volume (350 mL
L and 200 mL)
The sum of all inpuut and output flow
ws is lower for 2000 mL PP jars (PP
P 200): 8067.00
kg, compared to 350 mL
m PP jars (PP 3550): 8467.44 kg. After
A
analyzing th
he material and
energ
gy flows within thhe system (Fig. 1), it can be seen thhat there is different material and
energ
gy consumption foor the cycles of PP
P jars of different volumes. This leads to different
total mass inventory. One
O of the reasonss is significantly loower mass of the PP 200: 31.3 ±
2 g, compared
c
to PP 350:
3
58.8 ± 2 g. Although it requiires fewer PP 350
0 pieces: 1824,
comp
pared to 3192 piecces of PP 200 to ddeliver the same am
mount of cream to
o the consumer,
the PP
P material usage is still higher forr PP 350 (107.25 kg) than for PP 200
2 (99.91 kg).
Thereefore, the amountt of waste PP thaat gets to the landdfill after cream consumption
c
is
higheer for PP 350. Thee mass of aluminuum foil for sealingg jars after filling is lower for PP
350 (2.49
(
kg), comparred to the PP 200 (3.24 kg). The coorrugated cardboard masses used
for trransport packagingg were: 22.68 kg ffor PP 350 and 9.005 kg for PP 200 (Fig.
(
1).
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Origin
Analyzing
A
the weaak points of the syystem („weak poinnt analysis“ functiion of GABI 4)
and breaking
b
down thhe total inventoryy to individual floows, it has been found that the
domiinant flow is freshh water consumpttion, followed by the emissions intto the air: CO2,
steam
m and exhaust gasees, as shown in Fiig. 3.
Figu
ure 3. Life cycle inventory
i
of PP 350 and PP 200 mL
L, individual flow
ws of materials
aand energy
The system weak point analysis shhowed that the innjection molding process of the
produ
uction of PP jars from PP granulees has the highesst degree of conttribution to the
overaall life cycle invenntory (Figs. 3 and 4). The two mostt important items in
i the life cycle
inven
ntory of PP jars, fresh
fr
water consum
mption and emissiions to air originaate mainly from
the prrocess of PP jars injection
i
molding (Fig. 4).
Figure 4. Inventory of thhe PP jars injectionn molding processs
The total water coonsumption in thee life cycle of PP
P 350 was 6167.0
0 kg, of which
5856.7 kg or 95% was spent in the proceess of injection molding.
m
Similarly,, the total water
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consumption in the life cycle of PP 200 was 5668.4 kg, and for the production of jars by
injection molding 5455.8 kg or 96%.
Total emissions in the PP 350 life cycle is 968.7 kg and during the molding process
419.7 kg or 43% is emitted. In the PP 200 life cycle, of a total 1036.8 kg emitted, the
molding process makes up about 391.0 kg or 38%.
PP jars life cycle assessment (LCA)
Appropriate tools were used to calculate the life cycle assessment (LCA) on global
warming, calculated as equivalent kg of CO2. It was observed that the impact of PP 350
life cycle is slightly higher (Fig. 5). For the PP 350 global warming potential was 563.1
kg CO2equiv and for PP 200, 555.5 kg CO2equiv.
600
500
LCA PP
Injection moulding
kg CO2 equiv.
400
300
200
100
0
PP 350
PP 200
Figure 5. Life cycle assessment on global warming potential (kg CO2 equiv) and
contribution of injection molding (kg CO2 equiv) process for different volume PP jars
The LCA and system weak point analysis allowed us to single out process that mostly
affected global warming. This process is the production of PP jars from PP granules by
injection molding (Fig. 5). Of the total PP 350 LCA on global warming, only the injection molding process produces 475.9 kg or 85% of CO2eqiv. For the PP 200, the injectionmolding process makes up 443.3 kg CO2eqiv or 80% of the total life cycle CO2eqiv production.
The relevant data for the PP jars injection molding, manufacturing of cardboard transport packaging and production of aluminum foil were taken from the software. This data
refer to the characteristics of these processes in the EU (especially Germany). It would be
useful to continue the research and collect data for these processes in our country. With
these data collected, a more comprehensive analysis of the LCA could be given, as well
as possible improvement measures at the most sensitive points of the system. For example, the PP jars lightweightning (material saving), recycling of discarded jars, injection
molding process optimization.
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CONCLUSION
Comparing environmental impacts of cream jars of different volume, it was found that
the usage of the larger volume packaging did not bring about a reduction in life cycle
inventory, as well as it did not reduce packaging impact on the environment. The life cycle assessment of global warming potential showed only a slight difference between different volume jars, but the life cycle inventory was considerably lower for the 200 mL jars.
This is partly due to the fact that the mass of a 350 mL PP jar is two times greater than
that of 200 mL jar, requiring thus a larger amount of the material to distribute the same
amount of cream to the consumer. Highest contribution to the life cycle inventories (70 %
or more) showed the production process of PP jars by injection molding.
Acknowledgement
This paper is based on the life cycle inventory and life cycle assessment calculation
done in the GABI 4 LCA software. The authors would like to thank Mr. Damjan Korda
(general manager at Yuco - Hemija DOO), as well as all employees in Yuco - Hemija
who helped us immensely in the phase of collecting data for this paper.
REFERENCES
1. Dean, D.A.: Plastics-an Introduction, in Pharmaceutical Packaging Tehnology. Eds.
Dean, D.A., Evans, E.R. and Hall, I.H., Taylor and Francis, London (2000) pp. 264326.
2. Lazić, V. and Novaković, D.: Ambalaža i životna sredina, Faculty of Technology,
Novi Sad (2010) p. 122.
3. Lazić, V., Krkić, N., Gvozdenović, J. and Novaković, D.: Packaging Life Cycle
Assessment. Journal on Processing and Energy in Agriculture. 14, 1 (2010) 61-64.
4. Kirwan, M.J. and Strawbridge, J.W.: Plastics in Food Packaging, in Food Packaging
Technology. Eds. Coles, R., McDowell, D. and Kirwan, M.J., Blackwell Publishing,
San Francisco (2003) pp. 233-237.
5. Lazić, V., Gvozdenović, J. and Popović, S.: Packing Between the Need and Environmental Problems. Journal on Processing and Energy in Agriculture. 13, 3 (2009) 226228.
6. Robertson, L.G.: Food Packaging Principles and Practice, Massey University, Palmerton North (2006) p. 518.
7. Huang, C.C. and Ma, H.W.: A Multidimensional Environmental Evaluation of Packaging Materials. Sci. Total Environ. 324, 1-2 (2004) 161-172.
8. Sonnemann, G., Castells, F. and Schuhmacher, M.: Integrated Life Cycle and Risk
Assessment for Industrial Processes, Lewis Publishers, Boca Raton (2004) p. 40.
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ПОРЕЂЕЊЕ УТИЦАЈА ЖИВОТНИХ ЦИКЛУСА ПОЛИПРОПИЛЕНСКИХ
КУТИЈИЦА РАЗЛИЧИТИХ ЗАПРЕМИНА НА ЖИВОТНУ СРЕДИНУ
Невена М. Кркић, Вера Л. Лазић и Данијела З. Шупут
Универзитет у Новом Саду, Технолошки факултет, Булeвар цара Лазара 1, 21000 Нови Сад, Србија
При одабиру амбалаже балансира се између технолошког, економског, маркетиншког и еколошког оптимума. Све ове захтеве потребно је задовољити у што већој мери. Чест је случај да се економски и еколошки најоправданија решења поклапају. У овом раду је, са еколошког становишта, анализирана оправданост употребе
амбалаже веће запремине, уз примену софтвера за процену утицаја животног циклуса.
Сакупљени су подаци о врстама и потрошњи материјала и енергије у процесу
паковања и дистрибуције једног типа креме за руке, који су унесени у софтвер за
израчунавање инвентара животног циклуса и процену утицаја животног циклуса на
околину. Уочено је да је укупни инвентар маса већи за циклус полипропиленских
кутијица од 350 mL, у односу на кутијице од 200 mL. Разлагањем инвентара на поједине токове и након анализе слабих тачака система уочено је да у оба циклуса
(полипропиленске кутијице од 200 и 350 mL) доминира потрошња свеже воде, која
се највећим делом (око 95%) троши у поступку производње кутијица бризгањем из
гранулата полипропилена. Поређењем поступка бризгања, уочено је да је инвентар
за овај процес, као и потрошња свеже воде, мања код бризгања кутијица од 200 mL.
Аналогно инвентару животног циклуса, утицај животног циклуса амбалаже за паковање креме за руке на глобално загревање већи је за циклус кутијица од 350 mL.
Резултати овог рада су показали да је у анализираном случају еколошки прихватљивија амбалажа мање запремине. Да би се даље утицало на смањење утицаја
амбалаже на животну средину потребно је радити на уштеди материјала и производњи полипропиленских кутијица мање масе, увести рециклирање материјала одбачених кутијица и радити на оптимизацији процеса производње кутијица методом
бризгања.
Кључне речи: животни циклус, утицај, полипропилен, кутијице
Received: 3 February 2012
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USING THE ANSYS FLUENT FOR SIMULATION OF TWO-SIDED
LID-DRIVEN FLOW IN A STAGGERED CAVITY
Jelena Đ. Marković*, Nataša Lj. Lukić, Jelena D. Ilić, Branislava G. Nikolovski,
Milan N. Sovilj and Ivana M. Šijački
University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
This paper is concerned with numerical study of the two-sided lid-driven fluid flow
in a staggered cavity. The ANSYS FLUENT commercial software was used for the
simulation, In one of the simulated cases the lids are moving in opposite directions
(antiparallel motion) and in the other they move in the same direction (parallel motion).
Calculation results for various Re numbers are presented in the form of flow patterns and
velocity profiles along the central lines of the cavity. The results are compared with the
existing data from the literature. In general, a good agreement is found, especially in the
antiparallel motion, while in the parallel motion the same flow pattern is found, but the
velocity profiles are slightly different.
KEY WORDS: cavity benchmark; fluid flow; two-sided lid driven cavity; parallel motion; antiparallel motion
INTRODUCTION
In the past decades, flow in a lid-driven cavity has been studied extensively as one of
the most popular fluid problems in the computational fluid dynamics (CFD). This
classical problem has attracted considerable attention because the flow configuration is
relevant to a number of industrial applications. ANSYS FLUENT uses conventional
algorithms for calculation of macroscopic variables. Computational advantages of this
commercial software are simplicity of the problem setup, parallel computing and higher
precision.
Two-sided lid-driven staggered cavity appears to be a synthesis of two benchmark
problems: a lid-driven cavity and backward facing step. Furthermore, it has all the main
features of a complex geometry. Nonrectangular two-sided lid-driven cavities have been
recently introduced and investigated as a potential benchmark problem by Zhou et al. (1),
Nithiearasu and Liu (2) and Tekic et al. (3). Zhou et al. Presented a solution for the flow
in a staggered cavity obtained by using wavelet-based discrete singular convolution.
Nithiarasu and Liu solved the same problem using the artificial compressibility-based

* Corresponding author: Jelena Marković , University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]
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characteristic-based split scheme. Tekic et al. solved this problem by using the latticeBoltzmann method. The aim of this work was to study two-sided lid-driven staggered
cavity utilizing the commercial software package FLUENT. Solutions are presented in
the parallel and antiparallel motion of the lid and the flow pattern which develops under
these conditions.
Figure 1. Schematic diagram of two-sided lid-driven staggered cavity: (a) antiparallel;
(b) parallel motion.
MATHEMATICAL FORMULATION
General Scalar Transport Equation: Discretization and Solution - ANSYS FLUENT uses a control-volume-based technique to convert a general scalar transport equation to an algebraic equation that can be solved numerically. This control volume technique consists of the integration of the transport equation about each control volume, yielding a discrete equation that expresses the conservation law on a control-volume basis.
Discretization of the governing equations can be illustrated most easily by considering
the unsteady conservation equation for transport of a scalar quantity Φ. This is demonstrated by the following equation written in integral form for an arbitrary control volume
V as follows:

[1]
V t dV    v  d A      d A  V S dV
where ρ is the density, v - velocity vector; A - surface area vector;  - diffusion
coefficient for Φ, S source of Φ per unit volume. Equation [1] is applied to each control
volume, or cell, in the computational domain. The two-dimensional, triangular cell shown
in Figure 1 is an example of such a control volume. Discretization of Equation [1] on a
given cell yield
N
N

V   f v f  f  A f    f  A f  SV
[2]
t
f
f

faces
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where Nfaces represents the number of faces enclosing the cell, Φf is the value of convected through the face f, A f is the area of the face f and V is the cell volume.
The equations solved by ANSYS FLUENT take the same general form as the one
given above and apply readily to multi-dimensional, unstructured meshes composed of
arbitrary polyhedra.
Figure 2. Control volume used to illustrate discretization of a scalar transport equation.
For relatively uncomplicated problems (laminar flows with no additional models activated) in which convergence is limited by the pressure-velocity coupling, a converged solution can often be obtained more quickly using SIMPLEC. With SIMPLEC, the pressure-correction under-relaxation factor is generally set to 1.0, which aids in convergence
speedup. In the present study, a slightly more conservative under-relaxation value was
used, and it is equal to 0.7 .Special practices related to the discretization of the momentum and continuity equations and their solution by means of the pressure-based solver is
most easily described by considering the steady-state continuity and momentum equations in the integral form:
 v  d A  0
[3]
  vv  d A   pI  d A    d A   FdV
[4]
V
where I is the identity matrix,  is the stress tensor, and F is the force vector.
Discretization of the Momentum Equation - previously described a discretization
scheme for a scalar transport equation is also used to discretize the momentum equations.
For example, the x-momentum equation can be obtained by setting   u :
aP u 
a
nb
nb
u nb 
p
^
f
Ai  S
[5]
If the pressure field and face mass fluxes are known, Equation [5] can be solved in the
previously outlined manner, and a velocity field can be obtained. However, the pressure
field and face mass fluxes are not known a priori and have to be obtained as a part of the
solution. There are important issues with respect to the storage of pressure and the
discretization of the pressure gradient term. ANSYS FLUENT uses a co-located scheme,
whereby pressure and velocity are both stored at cell centers. However, Equation [5]
requires the value of the pressure at the face between cells c0 and c1, shown in Figure 2.
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Therefore, an interpolation scheme is required to compute the face values of pressure
from the cell values.
Discretization of continuity equation- Equation [1] may be integrated over the control
volume to yield the following discrete equation
N faces
J
f
Af  0
[6]
f
where Jf is the mass flux through the face vn . In order to proceed further, it is necessary
to relate the face values of the velocity, vn , to the stored values of velocity at the cell
centers. Linear interpolation of cell-centered velocities to the face results in an unphysical
checker-boarding of pressure. ANSYS FLUENT uses a procedure similar to that outlined
by Rhie and Chow (4) to prevent checkerboarding. The face value of velocity is not averaged linearly; instead, momentum-weighted averaging, using weighting factors based on
the aP coefficient from the equation [5], is performed. Using this procedure, the face flux,
Jf, may be written as:
^
a p ,c vn ,c  a p ,c vn ,c
Jf  f
 d f (( pc  (p) c  r0 )  ( pc  (p) c  r1 ))  J f  d f ( pc  pc1 ) [7]
a p ,c  a p ,c
0
0
1
0
1
1
0
0
1
1
0
where pc , pc and vn ,c , vn ,c , are the pressures and normal velocities, respectively,
0
1
0
1
^
within the two cells on either side of the face, and J f contains the influence of velocities
in these cells (Figure 2). The term d f is a function of a P , the average of the momentum
equation of the a P coefficients for the cells on either side of the face f.
Spatial Discretization - By default, FLUENT stores discrete values of the scalar  at
the cell centers (c0 and c1 in Figure 2). However, the face values  f are required for the
convection terms in Equation [2] and they have to be interpolated from the cell center
values. This is accomplished using an upwind scheme. Upwinding means that the face
value f is derived from quantities in the cell upstream, or „upwind“, relative to the direction of the normal velocity vn in Equation [2]. The diffusion terms are centraldifferenced and are always second-order accurate.
When second-order accuracy is desired, the quantities at cell faces are computed
using a multidimensional linear reconstruction approach (5,6). In this approach, higherorder accuracy is achieved at cell faces through a Taylor series expansion of the cellcentered solution about the cell centroid. Thus, when second-order upwinding is selected,
the face value f is computed using the following expression:
 f ,SOU      r
[8]
where  and  are the cell-centered value and its gradient in the upstream cell, and r
is the displacement vector from the upstream cell centroid to the face centroid. This formulation requires the determination of the gradient  in each cell. Finally, the gradient
 is limited so that no new maxima or minima are introduced.
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Origin
Siimulation setup - Mesh was createdd with 140x140 number
n
of elemen
nts with grid refinem
ment adjacent to thhe walls. Densityy of the fluid was set to 1 kg/m3, and
a viscosity to
0.001
1 Pas. Reynolds number
n
was calculated as Re = uL
L/. where ρ repreesents the fluid
densiity; μ is dynamic visocity
v
of the fluuid; L is the characcteristical length of
o cavity, and u
lid veelocity in the x dirrection. The veloccity of the movingg lid was calculateed based on desired Re number. Bounndary conditions w
were set as no-sliip for the left and right wall, and
for th
he upper and bottoom moving lid as moving walls witth defined velocitty and direction
of moving
m
depending on the case (parrallel or antiparalllel). Starting con
nditions for the
first-o
order upwind scheeme were taken as 0.5 velocity of the
t moving lids, and results were
used as starting conditiions for the seconnd-order upwind sccheme.
Validatioon or results of oone-sided lid –driiven square cavitty
In
n order to validatee the simulation m
mehod, a popular benchmark
b
probleem of one-sided
lid drriven square caviity is simulated ffor different Re numbers
n
and com
mpared with the
resulsst in the available literature. Figure 3 shows the u- annd v-velocity proffile, through the
geom
metric center of thhe cavity. The oobtained results are
a in good agreeement with the
resultts of Chen et al. (66) and Ghia et al ((7).
Figu
ure 3. Velocity proofiles u – and v- aalong the vertical and
a horizontal cen
nterlines of the
sqquare cavity.
Antiparalllel motion of the lids
l
The results for anttiparallel motion oof lids are listed in
i Table 1. Stream
mfunction contours at various Re nuumbers are presentted in Figure 4, while
w
the results obtained
o
for the
veloccity u – and v-proofiles through the mid-section of thhe staggered caviity are given in
Figurre 5. For comparisson sake, the resullts obtained by Teekic et al. (3) are also
a presented.
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Origin
Itt is evident that with
w the increase iin the Re numberr, extreme values of the velocity
comp
ponents also increease in magnitudde. Furthermore, the
t inertial forcess are dominant
comp
pared to the viscoous ones. As a reesult, the gradiennts close to the moving
m
lids are
stronger for higher Re..
As
A previously menntioned, three studdies on staggered cavity, (1)-(3) sh
howed unsteady
behav
vior for Re numbbers above 1000. In the present stuudy, symmetric and
a asymmetric
patterrns are achieved even
e
at Re numbeers lower than 10000. Multiple vorticces are formed,
moree precisely there arre three primary vvortices, although in Table 1 the thiird vortex is referred
d to as secondary for easier compaarison of the resultts. Primary vortices are all vertically aligned along thee mid-section of thhe cavity. Opposeed to this, secondaary vortices are
locateed in the left and right
r
bottom corneer of the cavity.
Figure 4. Streamffunction contours at various Re num
mbers – antiparallel motion.
With
W the increase of
o Re, the primaryy vortex located inn the left bottom corner
c
grows at
the ex
xpense of the prim
mary vortex locatted in the upper right
r
corner. With
h the further increasse of the Re numbber, the bottom lefft corner vortex disappears, and theere are two primary
y vortices along the long diagonall of the cavity, seecondary vorticess appear in the
corneers next to the movving lid.
Figu
ure 5. Velocity proofiles u – and v- aalong the vertical and
a horizontal cen
nterlines of the
staggered cavity
c
–antiparalleel motion (Rea – Tekic
T
et al. results (3))
Velocity
V
profiles allong the vertical ccenterline of the cavity
c
differ for so
ome Re values.
The most
m
notable diffference is for Re =100. While the results of Tekic et al. (3) show
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Origin
moree flattened profiless, ANSYS FLUEN
NT results show thhe existence of a sine-like
s
curve,
moree similar to the profiles
p
which T
Tekic et al. (3) showed
s
for higheer Re numbers
(Re=1000). The veloccity profiles along the horizontal centerline are almost identical.
Conssidering that there is a very good aggreement betweenn the present study
y and the work
of Gh
hia et al. (6) andd Chen et al. (7), and also betweenn results of Tekicc et al. (3) and
previiously mentioned authors, the reasoons for disagreements with present study could be
found
d in different Re number definitionn and different booundary condition
ns implementations caused by the diffferent numerical aapproach.
To summarize the results, the locaations of the centers of the vorticees are listed in
Tablee 1 and comparedd with (1) and (33). It can be noticed that the results for primary
vorticces are in good aggreement with the results in the avaiilable literature.
Tablle 1. Locations annd secondary vortiices – antiparallel motion, aZhou et al. (1), b Tekic
et al.(33), c present study
(xc1,yc1)
(0.9781, 1.1600)
(0.4219, 0.2518)
(0.9637, 1.1551)
(0.4494, 0.2543)
(1.10383,1.17135)
(0.41911,0.343648)
(1.0172, 1.1091)
(0.3828, 0.2889)
First secondary
vortex
(xc2,yc2)
(1.3556, 0.4405)
(0.0444, 0.9595)
(1.3484, 0.4476)
(0.0460, 0.9543)
(1.27995, 0.69013)
(0.032408, 0.8355)
(1.3556, 0.4486)
(0.0444, 0.9514)
100b
(1.0031, 1.1382)
(0.4082, 0.2693)
100c
(1.20251, 1.25965)
(0.452649,0.360417)
Re
50a
50b
50c
100a
Primary vortex
R
Re
Primary vortex
(xc1,yc1)
First secondary
vortex
(xc2,yc2)
(1.3500, 0.4656)
(0.0500, 0.9344)
(1.3522, 0.4607)
(0.0554, 0.9506)
(1.25479, 0.60078)
(0.05631, 0.60016)
(1.3250, 0.4844)
(0.0750, 0.9063)
Second secondary
vortex
(xc3,yc1)
(0.4703, 1.625)
(0.9219, 0.2375)
(0.4382, 1.1345)
(0.9824, 0.2862)
40
00a
(0.7000, 0.7000)
40
00b
(0.6822, 0.6859)
40
00c
(0.472214,0.445062))
000a
10
(0.7000, 0.7000)
(1.3502, 0.4457)
(0.0460, 0.9543)
10
000b
(0.7000, 0.7000)
(1.3250, 0.4844)
(0.0750, 0.9063)
(0.8811,0.2167)
(0.5301, 1.1962)
(0.90263, 0.89427)
(0.04669, 0.84673)
000c
10
(0.6934, 0.6972)
(1.3371, 0.4851)
(0.0722, 0.9280)
(0.82157, 0.13123)
(0.61360, 0.96692)
(0.42548, 1.07007)
(0.7256,0.2000)
(0.5339, 1.1907)
Parrallel motion
As
A expected, paralllel motion of thee opposite lids deevelops a differen
nt flow pattern
comp
pared to the antipparallel motion. F
Figure 6 shows the
t streamfunctio
on contours for
differrent Re numbers.
mfunction contouurs at various Re numbers
n
– parallel motion.
Figure 6. Stream
Itt can be noticed thhat two primary coounter-rotating voortices are presentt and that a free
shearr layer forms betw
ween them. Compaared to the previoous studies of flow
w inside rectangularr cavities, where the
t free shear layeer is formed alongg a horizontal cen
nterline (6), (8),
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Origin
in thee staggered cavityy the free shear layyer is formed alonng the shorter diag
gonal. The flow
is no longer symmetriccal due to the uppper lid moving froom the offset, whille the lower lid
movees towards the offfset. At low Re nuumbers, a secondaary vortex is preseent close to the
corneer of the right waall and offset. As the Re numberr increases, this vortex
v
gains in
streng
gth at the cost of the upper primaryy vortex. At higееr Re numbers, secondary vortex
causees splitting of thee primary vortex and formation of a second secon
ndary vortex as
show
wn in Figure 6. Booth primary vorticces have become more
m
prominent and
a larger in size, so
o that viscous efffects are confined to the thin bounddary layers close to the walls of
the caavity (9). As menntioned by Sahin aand Owens (10), fluid
f
begins to rottate like a rigid
body with a constant angular
a
velocity aat high Re numbers. Figure 7 show
ws the u- and vveloccity profiles alongg mid sections of the cavity. As diiscussed in the prrevious section,
with the increase in thhe Re number, exttreme velocity vaalues also increasee in magnitude.
Furth
her, the free shearr layer formed beetween the two prrimary vortices sh
hrinks with the
increase in the Re nuumber due to turbbulence. The proffiles confirm asym
mmetrical flow
aboutt the horizontal ceenterline of the cavvity, as previouslyy mentioned. Com
mpared with the
resultts of Tekic et al., it
i can be seen thatt the obtained proffiles are quite sim
milar.
Figu
ure 7. Velocity prrofiles u – and v- aalong the vertical an horizontal cen
nterlines of the
staggered cavity – parallel m
motion (Rea – Tekkic et al. results (3
3)).
As
A in the case of anntiparallel motionn, the results of Teekic et al. (3) give more flattened
profilles, while the pressent study shows tthe existence of a minimum velociity pitch. These
differrences occur at lower values of Re number (50 and 100). Velocity pro
ofiles along the
horizzontal centerline shhow relatively good agreement for the Re values 50,, 400 and 1000,
whilee for the Re=100 there
t
is a more siggnificant differencce. In general, thee velocity profiles ob
btained by simulaation in the presennt study are more symmetrical
s
and have
h
more pronounced minimum andd maximum velocity pitch. These diifferences, as prev
viously mentioned, could be a result of the different R
Re calculation prrocedure, and imp
plementation of
the diifferent boundary conditions.
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CONCLUSION
Results of the ANSYS FLUENT commercial software simulation of two-sided liddriven flow inside a staggered cavity are presented in this article. Both antiparallel and
parallel motions of two facing lids are investigated. The benchmark results obtained with
ANSYS FLUENT are in good agreement with the results available in the literature.
For antiparallel motion of lids in a staggered cavity results show symmetrical and asymmetrical flow patterns. Velocity profiles along the horizontal centerline are in a good
agreement with existing data from the literature, while the profiles along the vertical centerline are slightly different from those used for comparison, especially for Re=50 and
Re=100. These differences could be explained by the different Re calculation procedures
and different boundary conditions implementation methods, considering the different
numerical approach. The situation is quite similar in case of parallel motion of lids. Unlike for antiparallel motion, steady-state asymmetric patterns are obtained for all investigated Re numbers. It can be noticed that a free shear layer is formed along the short diagonal of the staggered cavity. All the main features of the flow are shown, streamline
contours, horizontal and vertical velocity components along the mid sections of the cavity
are visually presented, while the location of vortices is presented in Table 1.
Acknowledgement
This research was financially supported by the Ministry of Science and Technological
Development of the Republic of Serbia (Project No. 46010)
REFERENCES
1. Zhou, Y.C., Patnaik, B.S.V., Wan, D.C., and Wei, G.W.: Dsc Solution for Fow in a
Staggered Double Lid Driven Cavity. Int. J. Num. Meth. Eng. 57 (2003) 211-234.
2. Nithiarasu, P. and Liu, C.-B.: Steady and Unsteady Incompressible Flow in a Double
Driven Cavity Using the Artificial Compressibility (Ac)-Based Characteristic-Based
Split (Cbs) Scheme. Int. J. Num. Meth. Eng. 63 (2005) 380-397.
3. Tekić, P., Rađenović, J., Lukić, N., and Popovic, S.: Lattice Boltzmann Simulation of
Two-Sided Lid-Driven Flow in a Staggered Cavity. Int. J. Comp. Fluid Dyn. 24
(2010) 383-390.
4. Rhie, C.M. and Chow, W.L.: Numerical Study of the Turbulent Flow Past an Airfoil
with Trailing Edge Separation, AIAA 21 (1983) 1525-1532.
5. Barth, J. and Jespersen, D.: The Design and Application of Upwind Schemes on Unstructured Meshes, AIAA-89-0366, AIAA 27th Aerospace Sciences Meeting, Reno,
Nevada, (1989) 10-13
6. Chen, S., Tolke, J., and Krafczyk, M.: A New Method for the Numerical Solution of
Vorticity-Streamfunction Formulations. Comp. Meth. Applied Mech. Eng., 198
(2008) 367-376.
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7. Ghia, U., Ghia, K.N., and Shin, C.T.: High-Re Solutions for Incompressible Flow
Using the Navier–Stokes Equations and a Multigrid Method. J. Comput. Phys. 48
(1982) 387-411.
8. Perumal, D.A. and Dass, A.K.: Simulation of Flow in Two-Sided Lid-Driven Square
Cavities by the Lattice Boltzmann Method, Advances in fluid mechanics VII. Boston,
MA: WIT Press (2008) 45-54.
9. Patil, D.V., Lakshmisha, K.N., and Rogg, B.: Lattice Boltzmann Simulation of LidDriven Flow in Deep Cavities, Comput. Fluids 35 (2006) 1116-1125.
10. Sahin, M. and Owens, R.G.: A Novel Fully Implicit Finite Volume Method Applied
to the Lid-Driven Cavity Problem – Part I: High Reynolds Number Flow Calculations., Int. J. Numer. Methods Fluids 42 (2003) 57-77.
СИМУЛАЦИЈА ТОКА У ДВОСТРАНО ВОЂЕНОМ ПОКРЕТНОМ КАНАЛУ
ПОМОЋУ ANSYS FLUENT ПРОГРАМСКОГ ПАКЕТА
Јелена Ђ. Марковић, Наташа Љ. Лукић, Јелена Д. Илић, Бранислава Г. Николовски,
Милан Н.Совиљ и Ивана М. Шијачки
Рад се бави проблематиком нумеричке анализе струјања флуида у каналима у
којима струјање флуида настаје услед кретања горње и доње странице канала. Комерцијални софтвер ANSYS FLUENT је коришћен за симулацију двострано вођеног струјања флуида. Симулација је урађена за два случаја, први – када се горња и
доња страна крећу у супротним смеровима (антипаралелно струјање) и други –
када се горња и доња страна крећу у истом смеру. Резултати прорачуна за низ
вредности Рејнолдсовог броја приказани су у виду путања струјања флуида и
профила брзина дуж хоризонталне и вертикалне централне линије канала. Добијени
резултати су упоређени са потојећим подацима у литератури. Генерално уочено је
добро слагање са резултатим претходних истраживања, нарочито када се ради о
антипаралелном струјању. У случају паралелног струјања, визуелно ток флуида је
исти, али потоји мала разлика у профилима брзина.
Кључне речи: симулација, Ansys Fluent, струјање флуида, двострано вођени
покретни канали
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FINITE ELEMENT SIMULATION OF SINK PASS ROUND TUBES
USING ANSYS
M.P. Nagarkara, R.N. Zawareb and S.G. Ghalmea 
a
b
SCSM College of Engineering, Ahmednagar (M.S.) - 414005 India
PDVVP College of Engineering, Ahmednagar (M.S.) - 414111 India
Modeling and simulation of metal forming processes are increasingly in demand from
the industry as the resulting models are found to be valuable tools considering the optimization оf the existing and development of new processes. By the application of modeling and simulation techniques, it is possible to reduce the number of time-consuming experiments such as prototyping. Seamless tubes of various sizes and shapes are manufactured by various processes like sinking, fixed plug, floating plug, moving mandrel, cold
working and hot working.
The present work deals with the simulation of round tubes while passing through the
sink pass, using ANSYS software. The simulation results are the displacement and von
Mises stresses. The procedure can be used to improve the product quality and to study
the effect of various parameters like die angle on the product quality.
KEY WORDS: Sink pass, Finite Element Analysis (FEA), Finite Element Method
(FEM), Seamless tubes
INTRODUCTION
In most of the industries, cold working process is used for the production of various
components. Cold working of metal and alloys is done below their recrystallization temperature. (1, 3, 4)
There a five tube drawing methods: sinking, rod drawing, floating plug drawing, tethered plug drawing, and fixed plug drawing. Choosing the right method or a combination of methods for a particular application requires understanding of the characteristics
of each of them.
In the tube drawing, the die angle and bearing length are of essential importance for
the finished tube’s appearance. The die angle influences the tubing’s surface finish-a
gentle angle results in a smooth finish, whereas a steep angle results in a rough finish.
The bearing length must be long enough to ensure the correct diameter and roundness,
but not so long as to increase and mar the surface finish.
* Corresponding author: Mahesh P. Nagarkar, SCSM College of Engineering, Ahmednagar (M.S.)-414005,
India, e-mail: [email protected]
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The die, most commonly used in tube drawing, is a sintered tungsten carbide insert
encased in steel, with a cobalt content of approximately 10 %. Higher cobalt content provides more shock resistance, whereas a lower content provides better wear resistance.
The basic tube drawing processes are sinking, rod (mandrel) drawing, and several types
of plug drawing. (1, 4, 5)
Kim et al. investigated the process parameters related to the tool configuration. They
proposed a conventional straight type and terraced type of mandrels to obtain successfully formed shaft without any defect. For both types of mandrel, finite element (FE)
analyses of drawing processes were carried out, using the ductile fracture criterion to
predict a forming failure. The results are further used to design advanced mandrel shapes
(6).
An analytical procedure based on the upper bound method for the investigation of
formation features of the workpieces in dies for extrusion and/or drawing of fin-tubes and
fin-bars has been developed by Kiuchi and Jima. Through a series of analyses, the effects
of working conditions on shapes and dimensions of manufactured fin-tubes and fin-bars
have been consistently clarified. The procedure offers a systematic approach to the design
of dies and the pertaining processes (7).
In cold lubricated extrusion of round tubes, the material properties and surface quality
of the extruded products are influenced by the die profile. Streamlined dies induce less
redundant work and render desirable distribution of strength. Experiments have been
carried employing A12024 as working material. The effects of the process parameters
such as area reduction, die length, tubular shape ratio (thickness to outer diameter) and
friction condition, etc. on the extrusion power are used to study the metal flow. The theoretical predictions concerning both the extrusion load and metal flow appeared to be in
good agreement with the experimental results (8).
This paper discusses the modeling and simulation of the sink pass using ANSYS.
Quarter models of the die and the tube are modeled. The model is meshed using Solid 93
3-D element. Then, it is solved by applying quarter symmetry and boundary conditions.
Sinking
In the tube sinking process, the tube is drawn through a die to reduce the outside and
inside diameters. Sinking does not use an internal support, such as mandrel. Theoretically, the wall thickness does not change. However, it may increase or decrease, depending on the die angle and diameter-to-thickness ratio. Multiple sinking operations are
generally used for commodity tubing for theе applications such as low-cost lawn furniture (1, 2, 3, 9, 10).
In a sinking process, the typical die angle is 24°, with a relatively long bearing. Wall
thickening is caused by a lower angle, whereas higher angles result in the wall thinning.
Sinking uses a long bearing to achieve the correct size and optimal roundness, making
this process suitable for a final sizing operation.
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Figure 1. Sinking operation
Figure 2. Sinking operation – Die and tube showing various zones
FE Simulation using ANSYS
The finite element method is the numerical analysis technique for approximate solutions to the varieties of engineering problems. The finite method is originated as a method
of stress analysis. The finite element procedure produces many simultaneous algebraic
equations which are generated and solved by using computer. It transforms a physical
system having an infinite number of unknown into a finite number of unknowns. ANSYS
is a computer software for solving FEM and partial differential equations.
ANSYS can also be used to solve problems involving fluid flow, heat transfer,
electromagnetic field, diffusion, and many other phenomena. In this paper, we have used
it to predict the deformation and stress fields within solid bodies subjected to external
forces. (4, 11)
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Origin
F Modeling of D
FE
Die and Tube for Sink Pass
To construct the geeometric model inn ANSYS, 2-D model
m
of die and tu
ube is modeled
using
g key points, liness and areas as shown in Fig. 3. Thee c/s is then revolv
ved about the x
axis to
t obtain the quartter model (see Figg. 4).
Figure 3. 2-D
D Model of die andd tube
Figure 4. Soliid model of die annd tube
Meshing of Diee and Tube for Siink Pass
Meshing
M
is done by
b using 20-node solid95 since thee material has nonlinear characteristics. Refer Fig. 6.
Element Descripttion
SOLID95 E
SOLID95 element can tolerate irreguular shapes withouut as much loss off accuracy. SOLID9
95 elements have compatible displaacement shapes. It
I is well suited to
o model curved
boundaries.
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Origin
The element is deefined by 20 noddes having three degrees of freed
dom per node:
transllations in the nodaal x, y, and z directions. The elemeent may have any spatial orientation. SOLID95 has pllasticity, creep, sttress stiffening, laarge deflection, and
a large strain
capab
bilities. (11)
Figure 5. SOLID95 - 3-D
D 20-node structuural solid element.
Figure 6. Meshhed model of die and
a tube
FE Contact Pair
The analysis of thee sink pass is conccerned with the coontacts taking plaace between the
innerr surface of the diee and outer surfacee of the tube. A coontact pair is geneerated using the
contaact wizard (Fig. 7)). Here, a frictionnless contact (i.e. the
t coefficient of friction = 0) is
modeeled. Various lubbricants can also be incorporatedd in the modeling
g by inputting
vario
ous values of the coefficient of frictiion.
Figgure 7. Contact paair between the diee and the tube
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Origin
FE Boun
ndary Condition
ns
In
n the simulation, the
t die is fixed w
while the tube is moved
m
past the diie along the Xdirection. Boundary coonditions are appllied on the nodes.. As the number of
o unknowns in
the global
g
force vectoor–there are moree global displacem
ment vector matriices than equations (see Fig. 8). Affter applying the boundary condittions the numberr of unknowns
becom
mes equal to or less than the numbeer of the equationss.
Figure 8. Bounddary conditions diee and tube
Analysis
A
is carried out in the ANSYS
S 10 environmentt.
The material used for the tube in thhe sink pass is EN
N-31, with the modulus of elasticity of
o 210,000 N/mm22 and Poisson’s raatio 0.3. This is a non-linear
n
type off contact analysis, contact
c
between thhe die inner and thhe tube outer surffaces. Thus it is necessary
n
to introdu
uce the non-linear nature of the maaterial in the analyysis. Fig. 9 showss the non-linear
behav
vior of the tube material
m
as a stress-strain diagram. In the analysis, the part under
study
y is the tube and not
n the die. Hencee the die materiall is assumed to haave a very high
valuee of the modulus of
o elasticity of 2.1 x 109 N/mm2. Thus
T
one can observe the stresses
and displacement
d
plotss only for the tubee.
The materials used is having the following mechanicaal properties:
Material
M
for the diee:
Ex = 2.1 * 109 N/m
mm2
ν = 0.3
184
Material for tube:
t
EN-31
Ex = 210,000
0 N/mm2
ν = 0.3
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Origin
Fiigure 9. Non-lineaar material model for the tube
Nodal
N
Solution
Figurre 10. Displacemeent in the X-directtion Figure 11. Displacement in the
t Y-direction
Fiig. 10 shows the displacement of the tube after com
mpletion of the siinking process.
This is the total displaacement of the tubbe. Figs. 11 and 12
1 represent the reeduction of the
diameter by 7.996 mm
m in the Y-directioon and 8.129 mm in
i the Z-direction. Fig. 13 shows
the von-Mises
v
stress plot
p for the sinkinng process. The maximum value of the induced
stresss in the sinking prrocess is 64.299 N
N/mm2. This valuee is well below thee material yield
limit..
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Figure 12. Displacement in the Z-direction
Elemental Solution
Figure 13. The von Mises stress plot
CONCLUSION
From Figs. 10, 11 and 12, it can be seen that the sink pass of round tubes is simulated
successfully using ANSYS software. Fig. 11 shows the total displacement of the tube
during the sink pass, while Figs. 11 and 12 show the reduction in the diameter during the
sinking process. The von Mises stress plot (Fig. 13) shows that the induced stresses are
well below the permissible stresses of the steel. Thus the sink pass operation is successfully carried out without material failure. Standard ANSYS has a lot of advanced nonlinear features, solution methods, and convergence tools. It serves as a virtual prototyping
tool for such processes. Simulation reduces the modification, redesign and re-analysis
time, thus giving greater flexibility to the designers.
In future, the effect of the increase in the land and die angle can also be studied and
optimum land and die angle can be obtained using ANSYS. Also, this model can help to
simulate sinking process of various grades of steel and with various lubricants.
186
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REFERENCES
1. Bayoumi, L. S.: “Cold drawing of regular polygonal tubular sections from round tubes” International Journal of Mechanical Sciences, 43, 11 (2001) 2541-2553.
2. Technology Report, “Tube Shapes Up”, www.eastonbike.com/downloadable_files
_unprotected/r&d_files/R&D-11%20Tube%20Shapes.pdf (accessed on Jan 30, 2012).
3. Lamprecht, L.P. and Bevan M.: “The safe use of shock-tube and detonating cord systems in shaft sinking - a global trend” The Journal of The South African Institute of
Mining and Metallurgy, 99, 01 (1999) 1-4.
4. Pelletier, H., Krier, J., Cornet, A. and Mille, P.: “Limits of using bilinear stress–strain
curve for finite element modeling of nanoindentation response on bulk materials”
Thin Solid Films, 379, 1-2 (2000) 147-155.
5. http://www.powerultrasonics.com/content/chapter-1-introduction, 5 March, 2008. (accessed on Jan 30, 2012).
6. Kim, S.W., Kwon, Y.N., Lee, Y.S. , Lee, J.H.: „Design of mandrel in tube drawing
process for automotive steering input shaft”, Journal of Materials Processing Technology, 187–188 (2007) 182-186.
7. Kiuchi, M., I-I-Jima, S.: “Computer-Aided Simulation of Extrusion and/or drawing of
Fin-Tubes and Fin-Bars”, CIRP Annals - Manufacturing Technology, 42, 1 (1993)
261-264.
8. Yang, D.Y., Kim, H.S., Lee, C.M.: “Investigation into Lubricated Extrusion of Round
Tubes through Streamlined Dies Considering the Plastic Flow”, CIRP Annals - Manufacturing Technology, 36, 1 (1987) 169-172.
9. Kalpakjian, S. and Schmid, S.R.: “Manufacturing Processes for Engineering Materials”, Fourth Edition, Pearson Education, India (2009) pp. 260-326.
10. ASM Handbook, Vol. 14A, Metalworking: Bulk Forming, ASM International, (2005)
pp. 448-458.
11. ANSYS – Help.
СИМУЛАЦИЈА ПРОЦЕСА ИЗРАДЕ ОКРУГЛИХ ЦЕВИ ПОМОЋУ
КОНАЧНИХ ЕЛЕМЕНАТА ПРИМЕНОМ ANSYS СОФТВЕРА
M. P. Nagarkara, R.N. Zawareb и S.G. Ghalmea
a
b
SCSM Инжењерски колеџ, Ахмеднагар (M.С.)-414005, Индија
PDVVP Инжењерски колеџ, Ахмеднагар (М.С.)-414 111, Индија
Моделовање и симулација процеса обликовања метала се све више примењују у
индустрији, имајући у виду да добиjени модели представљају корисне алате у оптимизацији постојећих и развоју нових процеса. Применом техника моделовања и
симулације се смањује број дуготрајних експеримената као што је израда прототипова. Бешавне цеви различитих величина и облика се производе различитим процесима као што су пропуштање кроз отвор, обрада уз помоћ непокретног клина, об187
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рада уз помоћ покретног клина, обрада уз помоћ покретног вретена, хладно ваљање
и топло ваљање.
Овај рад се односи на симулацију процеса обликовања округлих цеви техником
пропуштања кроз отвор, уз примену ANSYS софтвера. Резултати симулације су
померање и von Mises-ова напрезања. Поступак се може применити за повећање
квалитета производа и за испитивање утицаја различитих параметара као што је
угао алата за обликовање на квалитета производа.
Kључне речи: Обликовање пропуштањем кроз отвор, анализа коначних елемената
(FEA), метода коначних елемената (FEM), бешавне цеви.
Received: 03 May 2012
Accepted: 05 August 2012
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TENSION MECHANICAL PROPERTIES OF
RECYCLED GLASS-EPOXY COMPOSITE MATERIAL
Jelena M. Petrović, Darko M. Ljubić, Marina R. Stamenović, Ivana D. Dimić,
and Slaviša S. Putić
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia
The significance of composite materials and their applications are mainly due to their
good properties. This imposes the need for their recycling, thus extending their lifetime.
Once used composite material will be disposed as a waste at the end of it service life.
After recycling, this kind of waste can be used as raw materials for the production of
same material, which raises their applicability. This indicates a great importance of
recycling as a method of the renowal of composite materials. This study represents a
contribution to the field of mechanical properties of the recycled composite materials.
The tension mechanical properties (tensile strength and modulus of elasticity) of once
used and disposed glass-epoxy composite material were compared before and after the
recycling. The obtained results from mechanical tests confirmed that the applied recycling method was suitable for glass–epoxy composite materials. In respect to the tensile
strength and modulus of elasticity it can be further assessed the possibility of use of
recycled glass-epoxy composite materials.
KEY WORDS: recycling, glass-epoxy composite materials, tension mechanical
properties
INTRODUCTION
Modern constructions require materials with special properties and forms that can respond to difficult working conditions (increased load, pressure, speed, impacts, vibration).
These conditions are the field for the applications of composite materials (CMs), and the
last thirty years has been a period of their intensive development. The former is not only
due to their good mechanical properties and light weight of produced components, but
also due to the following factors [1, 2]: easy tailoring of desired properties such as high
strength and modulus of elasticity, low density, relatively good impact strength, good
dynamic strength and cracks growth resistance, good oxidative and corrosion resistance,
and freedom in design and shaping and forming that facilitate easy integration of parts,
reducing the consumption of materials and tools, along with the favorable total cost of
*Corresponding author: Jelena Petrović, University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia, e-mail: [email protected]
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production. The use of CMs is growing every day and for that reason their adequate disposal and subsequent recycling must be carried out after the completion of their service
life. Otherwise, these materials will end up on a landfill in the form of waste, which further pollutes and distorts the environment [3].
The industrial CM waste is usually used as a raw material for the same CM production. The quantity of obtained waste is low compared to the production volume. If the
CM waste recycling is necessary, additional processing can be required, such as gradual
warming up before grinding. The fiber reinforced thermoplastic polymers can be recycled
by melting and casting. This is not case with the fiber reinforced thermoset polymers
which are dominant in the market. One of the possibilities for their recycling is grinding
and the use as fillers in a new material. A second method is the treatment with suitable
chemicals which abstract the reinforcing fibers from the thermoset matrix by dissolution
of the polymer matrix. Thermal treatment at high temperatures of both components can
be a third method for recycling of fibers reinforced thermoset composites, where the fibers are separated from the polymer matrix [4, 5].
The primary method for the recycling of composites is grinding to the desired particle
size and further use as filler in a new composite material production. The better strength
and thermal properties with ground glass-epoxy composite as filler in the epoxy-resinbased composites can be achieved in comparison with the same epoxy resin composites
with common fillers. Also, many pyrolytic methods have been developed for recycling of
composites. Combustion of composite materials gives energy and other useful byproducts. The solvent method for glass fibers (GFs) recycling from polymer matrix was also
developed [6].
There are many possibilities for application of recycled components from composite
materials. Recycled components from composite materials can be used as the reinforcing
for lumber (reinforced thermoplastics substituting even wood). Recycled fibers can be
used as reinforcing for asphalt (i.e. asphalt for bridges), as interlayer between two pure
glass layers in special cast boards and in the process of stirring of volume cast mixtures
which provide increased reinforcing due to the remaining recycled fibers [7].
The significance of recycling, based on wide spectrum of applications of recycled
components from CMs is undeniable. In this study, glass-epoxy composite material
(GECM) reinforced with non-andrecycled glass mats (from the lab-scale performed recycling) was firstly molded by handcrafted mold and mechanical properties were tested.
Тhe aim was to investigate the mechanical properties of recycled glass-epoxy composite
materials (RGECM), compare their mechanical properties with those of GECM, and to
validate the applied recycling method.
EXPERIMENTAL
Molding and Composition of GECM with non-recycled GFs
GECM with non-recycled GFs was molded by handcrafted mold. The mold consisted
of two metal plates screwed with screw bolts to ensure adequate pressure force [8]. Once
placed in a mold, CM was left 24 h at room temperature to cure and harden. After 24 h
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the mold was opened and hardened CM without any significant defects was taken out of
the mold and left to cure completely in air during 7 days at room temperature. The specimens for mechanical testing were cut from the prepared CM.
The reinforcing for CM preparation were 20 mm long “E”-glass-fibers based on lowalkali (wt<1%) silicate glass with surface density 550 g/m2 and volume fraction 60%. Eglass-fibers have good mechanical, hydro-thermal and electrical properties (Tables 1 and
2).
Table 1. Composition of „Е“-glass
Structural component
SiO2
Al2O3
B2O3
Na2O and K2O
MgO
CaO
TiO2
Fe2O3
Fe
Fraction (wt%)
52 – 56
12 – 16
5 – 10
0–2
0–5
16 – 25
0 – 1.5
0 – 0.8
0–1
Table 2. Physical properties of "Е"-glass
Property
Specific weight, g/m3
Tensile strength, МPа
Modulus of elasticity, GPа
Elongation at break, %
Thermal elongation, 10-6 К-1
Thermal conductivity, W/mК
Dielectric constant, 
Electrical resistivity, cm
Moisture absorption, at 20C  65% wt
Value
2.6
2400
73
3.3
5
1
6.7
1014
0.1
The polymer matrix used in this study was epoxy resin. The properties of used epoxy
resin are given in Table 3.
The CM with non-recycled GFs (Tables 1 and 2) and epoxy resin polymer matrix
(Table 3) was prepared by previously described method. The GFs as structural components in a form of glass mat were obtained by cutting into 2 cm long continual fibers
(Figure 1). The polymer matrix was synthesized from 2,2-bis(4-hydroxyphenyl)propane,
bisphenol A and epichlorohydrin. 3-Aminomethyl-3,5,5-trimethylcyclohexylamine (modified cycloaliphatic amine) was used as hardener in the epoxy resin system. The molded
GECM contained 47 wt% of GFs regularly dispersed (in the form of a glass mat) in the
epoxy matrix.
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Origin
Table 3. Prooperties of epoxy resin
r
Unit
Propertyy
App
pearance
Epo
oxy number
Epo
oxy equivalent
Den
nsity
Visccosity at 25C
Color (Gardner colorr scale)
Non
n-volatile componnents content
Org
ganic chlorine content
n/100 g
g/cm3
mPа·s
% min
% max
R
Reference
Yelllow viscous
liquid
0.51 – 0.54
196 – 185
10000 – 15000
3
99
0.3
Analysis
results
Yeellow viscous
liquid
0.52
192
1.26
13700
Less
L than 3
99.5
0.17
Figure 1. Appeearance of cut glasss fibers
Recyccling of GECM
The obtained and tested CMs weree recycled and thiis resulted in the recycled GFs.
Oncee obtained recycledd GFs were used ffor GECM preparration with recycleed GFs.
In
n order to obtain GFs
G from CM, epooxy resin must bee completely remo
oved with a suitable reagent. The 50-g CM samples weere immersed firsstly in concentrateed sulfuric acid
(98 wt%),
w
where small amount of epoxy resin was rem
moved. To removee the remained
epoxy
y resin, the samplles were further kkept in 200 cm3 soolution of nitric acid (68.5 wt%)
at 90°C during 5 h. Affter that, the epoxyy resin was complletely removed and recycled GFs
(RGF
Fs) were obtainedd. The RGFs werre separated from
m nitric acid by fiiltration, rinsed
with distilled water annd neutralized wiith ammonium hyydroxide solution (25 wt%) and
again
n with distilled waater till pH 7 wass attained. The neeutralized and rinssed RGFs were
dried
d in an oven for 24 h at 110°C, cooled, and madde ready for the preparation of
RGECM. The loss of GFs
G during the reecycling process was
w 5.8 wt%, whicch is negligible
amou
unt since the proccess consists of seeveral phases. Figgure 2 compares th
he appearances
of no
on-recycled and reecycled GFs.
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Origin
Figure 2. Apppearance of non--recycled (left) annd recycled (right) GFs
Molding and Componen
nts of RGECM with
w recycled GFss
After
A
recycling annd obtaining RGF
Fs, the CM was molded
m
with RGF
Fs by the same
method as in the prepparation of GECM
M with non-recycled GFs. The stru
uctural componentss of RGECM withh RGFs were the glass mat (reinfoorcing) obtained from
f
RGFs and
epoxy
y resin as the mattrix. The standardd specimens for mechanical
m
testing
g were cut from
the prrepared RGECM.
Teensile testing
Fiive specimens foor mechanical tessting of GECM (N-1,
(
N-2, N-3, N-4,
N
N-5) and
RGECM (R-1, R-2, R-3, R-4, R-5) were prepared The
T specimen dim
mensions were
250x25x2.5 mm. Befoore testing, the speecimen's thicknesss and width were precisely measured
d (±1%). Further machine processiing of specimens was performed with
w a diamond
tool tip
t moving at a sppeed that reducess generation of heeat in the specimeen. Cutting was
carrieed with a not cheed cutter thickness of 1 m/min on the machine ALG
G-100. Testing
was performed
p
accordiing to the standardd test method AST
TM D3039 9.
The testing was caarried out on the ttensile tester SCH
HENCK TREBEL
L RM 100 with
u of hydraulic jaws, and the defo
formations (1) in the longitudinal direction were
the use
contin
nuously recorded..
The incorporated looad was registeredd by the measurinng cell (capacity of
o 100 kN). The
elong
gation was measuured by using duaal extensimeter Hottinger
H
DD1. There
T
were two
parallelly connected exxtensimeter relateed to the measuree elongationon on
n the both sides
of thee specimen, and the
t parallel connection to the extennsimeter facilitated
d the averaging
of thee measured values. The measuring range of the exteensimeter was  2.50
2
mm, and it
work
ked on the principlle of measuring taape with accuracy of 0.05.
The cross-sectionall dimension, the vvalues of tensile sttrength, and the modulus
m
of elasticity
y of the samples were
w calculated by using th eequationns (1-3) [10].
The cross-section
c
of thhe specimens was calculated with thhe following equattion:
A 0 bd
[1]
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Tensile strength was calculated with equation (2) as follows:
Rm,1
Pmax
bd
[2]
where: Rm,1- tensile strength in longitudinal direction, MPa; Pmax- maximal force at break,
N; A0 - cross-section of specimen, mm2; b - specimen wideness, mm; d - specimen, thickness, mm
The modulus of elasticity (Elong) was calculated from equation (3) where ratio P/1
was determined by linear regression method from the straight part of registered curve
stress - strain:
E uzd 
  P 1


   1 b  d
[3]
RESULTS AND DISSCUSION
The tensile test in longitudinal direction was performed on five specimens of each
prepared CM (GECM and RGECM), and the tensile strength and modulus of elasticity in
longitudinal direction were obtained. It may be noted that the test was successful because
in the all tested specimens the fracture occurred in the middle of the specimen (the
measurement part). The calculated values of the tensile strength in longitudinal direction
and the corresponding modulus of elasticity are given in Table 4. Figure 3 shows the
percentage deviation of the tensile strength and modulus of elasticity of the RGECM
specimens from the corresponding mean values of the GECM specimens.
Table 4. Results from tensile testings
Specimen
N-1
N-2
N-3
N-4
N-5
R-1
R-2
R-3
R-4
R-5
194
Type of
GFs
Nonrecycled
Recycled
Specimen
wide
b, mm
Specimen
thickness
d, mm
Cross
section
А0, mm2
Max force
at break
Рmаx, N
Tensile
strength
Rm1, МPа
Modulus of
elasticity
Е1, GPа
14.9
15.0
14.8
15.0
14.9
14.8
15.0
14.8
14.9
14.8
2.6
2.6
2.7
2.5
2.7
1.8
2.0
2.2
2.1
1.9
38.7
39.0
39.9
37.5
40.2
26.6
30.0
32.6
31.3
28.1
8500
8640
9600
9260
8970
5700
5950
5850
5990
5660
219.64
221.54
240.60
246.93
223.13
214.29
198.33
179.45
191.37
201.42
3.17
4.81
2.88
3.52
4.14
2.84
3.05
3.76
2.92
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-5
-10
increase
Deviation from GECM, %
0
+
decrease
5
-
-15
-20
Average values for GECM:
% ((Rm= 230,4 MPa))
-25
% ((E1 = 3,7 GPa))
-30
-35
R-1
R-2
R-3
R-4
R-5
Test samples
Figure 3. Deviation of tension test results
The relative uniformity of the obtained values of maximum force at break Pmax for
both GECM and RGECM can be noted. However, the values of Pmax are smaller for the
RGECM samples than for the samples from GECM.
Based on the results for the five tested specimens for each material the calculated
mean tensile strength of the two materials were 230.37 MPa for GECM and 196.97 MPa
for RGECM, and the mean values of the modulus of elasticity 3.70 GPa for GECM and
3.21 GPa for RGECM, respectively. Also, it was observed that the values for the tensile
strength and the modulus of elasticity for RGECM are lower compared to GECM. The
deviations from the mean value of measured (calculated) values both for the tensile
strength and modulus of elasticity are relatively small in this type of testing. The minimum of tensile strength deviation for GECM was 3.14% for sample N-5 and the maximum 7.2% for sample N. The minimum of tensile strength deviation for RGECM was
0.7% for sample R-2 and maximum 8.9% for sample R-3 for RGECM.
The explanation for the slightly higher dispersion of the results for the modulus of
elasticity of both materials can be the fact that it was relatively difficult to accurately determine the elasticity modulus because of the relatively small initial curvature in the
stress-strain curves ( – ). In regard of the tensile strength, it is well known that due to
different orientation of fibers in the glass mat as the reinforce, all the GFs are not under
the same stress. Different stresses can occur with short fibers, due to the different orientation of individual fibers, which cannot coincide in each sample, and therefore leads to
the different maximum force at break.
Figure 4 shows a schematic representation of a short fiber that is inserted into the
matrix exposed to the longitudinal tensile stress σa. It can be seen that there are areas
close to the ends of fiber that are not exposed to the entire load, and the mean stress in the
fibers of limited length is slightly smaller than that which would have an infinitely long
fiber exposed to the same external load.
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

Short fiber
l
Figure 4. Schematic representation of deformations around short fiber inserted in the
matrix exposed to the axial tension
Also, if we compare the deviations of the composites with non-recycled GFs and
RGFs (Figure 3), it can be observed that the tensile properties of the composite material
obtained by using RGFs as reinforcement are worse, the tensile strength is by 14.5%, and
the modulus of elasticity by 13.2% lower compared to the values of the materials formed
with non-recycled GFs. The differences in the values of the tensile properties of the two
composites tested were expected. An explanation follows from the fact that recycled fiber
surface layer was damaged during the recycling process (cooking, exposure to acids,
etc.), thus good bonding of GFs with the matrix (epoxy resin) is disturbed as compared to
the non-recycled fibers good interaction with the polymer matrix. By applying the same
type of loading, the breaking of the fiber-matrix bonds in the composite with RGFs occur
easier and at lower loadings than in the CM with non-recycled GFs, because of the poorer
fiber-matrix adhesion. A confirmation of the conclusions is certainly the SEM images
shown in Figures 5 and 6, where above phenomena are observed at higher magnifications.
Figure 5 The breaking of the fibermatrix bonds
Figure 6 Poorer fiber-matrix adhesion
CONLUSIONS
The aim of this study was to examine and compare the properties of the composites
prepared with RGFs and non-recycled GFs, as well as to present the possibility for recycling of CMs.
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The obtained values of tensile properties of the composites with RGFs are acceptable
and satisfactory, although they are lower than the corresponding values of the composite
with non-recycled GFs (tensile strength values were 14.5% and module of elasticity by
13.2% lower than the corresponding values CM with non-recycled GFs). It can be concluded that the RGECM retains its tensile properties with minimal fluctuation compared
to GECM, and as such it can be used for different purposes.
Also, on the basis of the obtained results it can be concluded that the method of
recycling GECM based on the exposure to nitric acid can be applied to recycle small
amounts of the material, and further research should be directed toward the improvement
of the applied method to solve the problem of recycling of the compounds from the decomposed epoxy resin from composite material obtained by boiling in nitric acid. The
method should be developed in the direction of the application of several different acids
to shorten the time of exposure of the composites to acid attack and increase the efficiency of the recycling process at lower temperatures 11,12. The recycling of composite
materials and recycling in general can significantly save the energy and the raw materials,
and certainly pollution would be drastically lowered.
REFERENCES
1. Lubin, G.: Handbook of Composites, Van Nostrand, New York (1982) pp.328-375.
2. Flueler, P. and Farshad M.: Arrest of rapid crack propagation in polymer pipes,
Materials and Structures 28, 2 (1995) pp. 108-110.
3. Ilić M., Miletić S.: Osnovi upravljanja čvrstim otpadom, Institut za ispitivanje materijala, Beograd (1998) pp. 59-79.
4. Yang Y., Boom R., Irion B., Van Heerden D.-J., Kuiper P., De Wit H.: Recycling
of composite materials, Chem. Eng. Process. (2011), doi:10.1016/j.cep.2011.09.007
5. Job S.: Composite recycling – summary of recent research and development, Materials KTN Report, September (2010) pp. 26. Available online from www.compositesuk.co.uk/LinkClick.aspx?fileticket=LXN-MfM0360%3D&tabid=111&mid=550.
6. Perović, G., Tanasković, M.: Mogućnosti primene savremenog pristupa upravljanja
komunalnim čvrstim otpadom u našoj zemlji, Zbornik radova, Međunarodna konferencija otpadne vode i komunalni čvrsti otpad i opasan otpad Budva 20-22. Septembar, Udruženje za tehnologiju vode i sanitarno inženjerstvo, Beograd (1999), pp. 367372.
7. Ilić, M., Stevanović, H., Mladenović, A.: Plan upravljanja komunalnim čvrstim otpadom, Regionalni centar za životnu sredinu za Centralnu i Istočnu Evropu, Beograd
(2003) pp. 43-46.
8. Krivokuća, M. P.: Uticaj staklenog ojačanja na statička i dinamička svojstva laminarnih kompozitnih materijala, magistarski rad, Univerzitet u Beogradu, Tehnološkometalurški fakultet, Beograd (1999).
9. Annual book of ASTM Standards, American Society for Testing and Materials, Philadelphia, PA, Vol.15.03 (1999).
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10. Pickering, S.J.: Thermal methods for recycling waste composites, Management,
Recycling and Reuse of Waste Composites, WP and CRC Press, Cambridge, UK
(2010) pp. 65–101.
11. Jovanović, O.: Monitoring i ublažavanje posledica zagađenosti životne sredine, VŠSS
Beogradska politehnika, Beograd (2004) pp. 129-143.
12. Tchobanglous, G., Theisen, H., Virgil, S. A.: Integrated Solid Waste Menangment,
McGraw Hill, New York (1993) chapter 8.
МЕХАНИЧКA СВОЈСТАВА
РЕЦИКЛИРАНОГ СТАКЛО-ЕПОКСИ КОМПОЗИТНОГ МАТЕРИЈАЛА
Јелена М. Петровић, Дарко M. Љубић, Марина Р. Стаменовић, Ивана Д. Димић,
и Славиша С. Путић
Примена композитних материјала, захваљујући својим добрим својствима, сваким даном постаје све већа што намеће питање могућности њиховог рециклирања и
тиме продужења њиховог животног века. Након једанпут коришћеног композитног
материјала у одређене сврхе врши се њихово одлагање у виду отпада. Овакав отпад
углавном представља сировину за производњу исте врсте композита поступком рециклаже, што повећава њихову примену. Имајући ту чињеницу у виду, овај рад
представља допринос у подручју истраживања механичких својстава рециклираних
композитних материјала. У раду су приказана затезна механичка својства стаклоепокси композитниог материјала који је био у експлоатацији, поступак његове рециклаже, као и затезна механичка своства стакло-епокси рециклираног композитног материјала. Поређењем резултата се дошло до података о исправности поступка рециклаже стакло-епокси композитног материјала као и процене о даљој могућности примене, узимајући у обзир добијене вредности за затезну чврстоћу и модул
еластичности.
Кључне речи: рециклажа, стакло-епокси композитни материјали, механичка
својства
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HYDRODYNAMICS OF GAS-AGITATED LIQUID-LIQUID
EXTRACTION COLUMNS
Milan N. Sovilj
Although the non-agitated extraction columns (spray column, packed column, perforated plate column, sieve plate column, etc) can handle high flow rates and are simple
and cheap, there have been relatively few applications of these columns because they
suffer from serious backmixing of the continuous phase. It was shown that the backmixing
is reduced when the spray column is operated with dense packing of drops. Another way
of increasing the efficiency of a non-agitated extraction column is to introduce an inert
gas (air, nitrogen, oxygen) as a mixing agent in the two-phase liquid-liquid (L-L) system.
This method of energy introduction increases the turbulence within the new three-phase
gas-liquid-liquid (G-L-L) system, which causes an improved dispersion of droplets, and,
consequently, a higher dispersed phase holdup and therefore a great mass transfer area.
The present study reports the hydrodynamics in the non-agitated extraction columns, as
well as the axial dispersion for the two- and three-phase systems.
KEY WORDS: extraction columns; gas-liquid-liquid system; hydrodynamics
INTRODUCTION
Because of their simplicity, low cost, and versatility, non-mechanically agitated columns are still extensively used in extraction processes. They are also a convenient and
inexpensive way to experimentally test theoretical models of mass transfer in simple
extraction systems. In regard with the dispersed phase holdup, spray extraction column,
as one of the simplest extraction columns, can operate in three modes of packing of the
dispersed phase drops: dispersed, restrained, and dense.
Although they can handle high flow rates and are simple and cheep, there have been
relatively few applications of these columns because they suffer from serious backmixing
of the continuous phase. It was shown hat the backmixing was reduced when the spray
column operated with dense packing of drops. One of the way to increase the efficiency
of a spray column is to introduce an inert gas as a mixing agent in the two-phase L-L
system. This method of energy introduction increases the turbulence within the threephase G-L-L system, which causes the increase of average dispersed phase holdup and a
* Corresponding author: Milan N. Sovilj, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
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larger mass transfer area. Mass-transfer or chemical reactions for G-L-L systems may be
also encountered in gas absorption, gas-liquid reactions, and fermentation, often with a
heterogeneous liquid catalyst, or liquid-liquid reactions with gas agitation. Some examples can be cited: absorption of SO2 into the aqueous emulsion of xylidine in water (1);
purification of crude naphthalene with H2S04 accompanied by air sparging (2); air
oxidation of hydrocarbon in aqueous emulsion, fermentation of hydrocarbons, in which
the substrate is dispersed in an aqueous culture medium with air bubbling; and extractive
fermentation of useful species, such as alcohols and steroids, which are produced in the
aqueous phase by the metabolism of the relevant microorganisms and are extracted in situ
into the coexisting organic phase of an extractant, shifting the reaction favorably. A few
examples of more complicated systems containing solid particles are air oxidation of
substituted benzyl alcohol catalyzed by palladium catalyst in the presence of an aqueous
phase, which gives rise to the favorable formation of aldehyde (3), and competitive
liquid-phase hydrogenation of cyclohexanone and cyclohexene catalyzed, by Ru catalyst
in the presence of water (4). Packed towers operated under gas-liquid countercurrent
conditions have found increased applications in distillation, absorption, and liquid-liquid
extraction processes. They are also becoming increasingly important environmental
protection technologies. The extraction of hydrogen peroxide by means of deionized
water from anthraquinone working solution via anthraquinone process was carried out in
a gas-agitated sieve plate extraction column (5). The effect of superficial velocity of air,
dispersed phase and continuous phase on the overall plate extraction efficiency has been
investigated in the mentioned paper. The correction for the prediction of the overall plate
extraction efficiency was also presented.
Gas-liquid-liquid columns have many advantages over any other conventional gasliquid or liquid-liquid contactors,. They are of simple configuration without moving parts
and require no seal, need little space and maintenance. In this columns it is easily and in
the widely intreval adjusts the resistance time of the liquid phases. They allow comparatively large liquid-phase volumetric mass-transfer coefficients or interfacial area to be
achieved with relatively low energy consumption. Namely, the efficiency of non-agitated
extraction columns (spray, packed, perforated, and sieve plate column) can be cosiderably increased by introducing an inert gas as a mixing agent in the two-phase L-L system.
The transition and steady state behavior of the gas agitated two-phase L-L dispersions is
well characterized for spray columns, where the gas is introduced into the continuous
liquid phase at the base of the column and the second liquid phase is dispersed at the top
of the column. Dispersions and emulsions can also arise as a consequence of liquid entrainment by bubbles as they pass through a liquid-liquid interface. This mode of dispersion or emulsion formation is pertinent also to batch type processes, where neither liquid
phase is dispersed initially, and the gas is again introduced from below. Such examples
are pyrometallurgical processes for the production of nickel and copper, processes for
electro-organic synthesis, and the dispersion or emulsification of oil slicks in breacking
waves (6). The introducing gas as a mixing agent in the two-phase L-L dispersion caused
the formation of large liquid-liquid interface area due to the presence of the smaller disperd phase drops. Coalescence times for drops at the liquid-liquid interface were found to
be rapid and appear to be unafected by the rate of bubble passage. The energy introduced
by the mixing agent increases the turbulence within the three-phase G-L-L system, which
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brings about an improved dispersion of the droplets and, consenquently, a higher dispersed phase holdups, and also higher backmixing in the continuous phase. Galkin et al. (7)
concluded that the extraction efficiency was nearly three times greater for conventional
columns when air was introduced into the sieve plate extraction column at the lower inlet,
and claimed that the process was more efficient than by the use of stirring or pulsation of
the column.
The aim of this paper is to give a critical review of the hydrodynamics characteristics
of non-mechanically agitated extraction columns, which use an inert gas (air, oxygen, nitrogen, etc.) as a mixing agent in the two-phase liquid-liquid system. The energy thus
introduced increases the turbulence within the now, three-phase G-L-L system, which
brings about an improved dispersion of the droplets and, consequently, a higher holdup
and larger mass transfer area.
HYDRODYNAMIC CHARACTERISTICS OF GAS-LIQUID-LIQUID SYSTEMS
The hydrodynamics of a system represents one of the main difficulties in the scale-up
of liquid-liquid extractors. As for the design, difficulties arise mainly because of the dispersion in radial and axial directions; however, in most cases, the radial dispersion has a
small influence. The main hydrodynamic characteristics in the non-mechanically agitated
extraction column are the slip velocity, dispersed phase holdup, gas phase holdup, drop
size distribution, and axial dispersion in the continuous phase. In the following text we
will discuss the effects of these characteristics on the operation of non-mechanically gasagitated liquid-liquid extraction columns.
Dispersed phase holdup and slip velocity
In the generalization of dispersed phase holdup data, for a specified value of the slip
velocity (us) for the countercurrent flow in an L-L system in the spray extraction column,
use can be made of the familiar Thornton-Pratt relationship (8):
us 
uc
u
 d
1   d   d
[1]
where: uc, ud - superficial velocity of the continuous and dispersed phase, respectively, d
– dispersed phase holdup. For a packed column voidage (e), the slip velocity has the next
form:
us 
uc
u
 d
e 1   d  e  d
[2]
Equation (1) was corrected by the additon of a new part, as follows (9):
uc
u
m
 d  u 0 1   d 
1   d   d
[3]
wherein the viscosity ratio of the phases (m) is defined by:
m  1.22  d /  c 
0.2
[4]
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where: u0 - velocity of a single drop, d, c – dynamic viscosity of the dispersed and continuous phase, respectively. If the static dispersed phase holdup is defined as:
 st  u d / u s , when uc = 0, еq. [1] can be rewritten in the form:
us 
ud
 st

uc
1   d


ud
d
[5]
On the basis of the amount of experimental data simple empirical equations for the
estimation of the slip velocity and dispersed phase holdup in the two-phase L-L columns
were derived by several authors (11-15). The equation presented by Kumar et al. (11)
predicts the slip velocity for the dispersed phase holdup (0.01 to 0.75) and Reynolds
number (7 to 2450). This equation gives the average absolute value of the relative errors
of 14.5% and 13.5% for the dispersed holdup and slip velocity, respectively. Kumar and
Hartland (15) derived an empirical expression for the prediction of the dispersed phase
holdup and slip velocity in droplet dispersions settled under gravity. This equation is
valid in a wide interval of the dispersed phase holdup (0.01 to 0.76) and Reynolds
number (0.61 to 3169), with the average absolute value of the relative error of 14.3% and
12.8% for the dispersed holdup and slip velocity, respectively. Sovilj (13) proposed an
empirical relationship for the prediction of the dispersed phase holdup and slip velocity in
the liquid-liquid spray extraction columns. A good agreement between the experimental
and predicted values of the slip velocity by this equation were obtained for the dispersed
phase holdup in the range (0.0097 to 0.362) and Reynolds number (58 to 1067). The
average deviations for the slip velocity and dispersed phase holdup were 9.6% and
14.0%, respectively. On the basis of a large bank of published experimental data for eigth
different types of extraction columns (rotation disc, asymetric rotation disc, Kühni, WirzII, pulsed perforated-plate, Karr reciprocatind-plate, packed, and spray columns), Kumar
and Hartland (15) presented a unified correlation for the prediction of dispersed phase
holdup in the two-phase L-L dispersion. The average error of predicted data on the entire
data sets using this equation was 18.1%, which is better than that achieved by most authors in attempting to correlate their own experimental results. The highest error was 22.7%
for the rotating disk and asymetric rotating disk columns, and a lowest 14.1% for the
spray extraction columns. The errors for the pulsed perforated-plate and packed columns
were 19.0% and 18.3%, respectively.
The Experimental procedure applied in the paper (14) was as follows: at the beggining of each run, the cylindrical part of the spray extraction column was filled to about
half its volume with water (continuous phase) throuh a water distributor at the top of the
column, and the level of continuous phase (Hc) was recorded. At that moment, the dispersed phase (toluene) was introduced at the bottom of the column with a chosen flow rate, and two-phase dispersion occurred. The position of the interface (Hb) corresponded to
the height of the two-phase dispersion above the toluene inlet. When the volume of the
two-phase dispersion (water-toluene) became constant, air (gas phase) was introduced at
the bottom of the column via a gas distributor, and the interface level in the column increased. At that moment, the continuous phase was introduced in the column at the chosen
flow rate. The new position of the interface (Ht) corresponds to the height of the threephase G-L-L dispersion above the toluene and air inlet (14).
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On the basis of the experimental procedure for the estimation of dispersed phase
holdup, explained in the previous text, the dispersed phase holdup (dt) in the three-phase
dispersion (air-water-toluene) was calculated from the following relationship (14):
 dt 
Hb  Hc
Ht
[6]
The mean value of the dispersed phase holdup in the three-phase dispersion was
determined with an uncertainty of  3%.
In the G-L-L system, the continuous phase holdup (ct) is defined by:
[7]
 ct  1   dt   g
where: dt – dispersed phase holdup in the G-L-L system, g - gas phase holdup. On the
other hand, the basic eq. [1] for the G-L-L system in the spray extraction column can be
expressed as (16):
uc
u
 d  uCH 1   dt   g 
1  dt   dt
[8]
where: uCH - characteristic velocity identified as the mean relative velocity of the droplets
extrapolated to the essentially zero flow rate, defined by Thornton (17).
The effective slip velocity of the dispersed liquid was also analyzed by using the longitudinal dispersion coefficients of the measured dispersed liquid (18,19). However, the
studies in these papers were limited only to the air-kerosene-water system. Wang et al.
(20) expressed the slip velocity in the G-L-L system air-anthraquinone-aqueous working
solution in the form:
us 
ud
d

uC
1  g  d
[9]
The average dispersed phase holdup in the spray extraction column increased with
increasing the dispersed and gasesous phase superficial velocities, at the constant value of
the continuous phase superficial velocity (5), fig. 1.
Figure 1. Effects of gas superficial velocity on the holdup of dispersed phase
[Source: Cheng et al., Ind. Eng. Chem. Res. 47 (2008) 741-7418]
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The average dispersed phase holdup exibits a relatively small increase with the increase in the gas phase superficial velocity. Moreover, Billet and Braun (21) concluded that
an initial sinking of dispersed phase holdup takes places at gas phase superficial velocities below 0.2 cm/s. They clamed that within that gas flow rate range, the energy input
is sufficient to produce an intensive turbulence and, consequently, to form a large number
of droplets (21). On the other hand, the increase in the dispersed phase holdup observed
by them was below 20% for the gas phase superficial velocity within the range from 0.2
to 0.6 cm/s at a constant dispersed phase superficial velocity of 0.4 cm/s.
Drop size and gas phase holdup
The average drop size in most of the investigated two-phase L-L systems can be expressed as a Sauter drop diameter, d32, in the form:
d 32 
n d
n d
i
3
i
i
2
i
[10]
where: ni – number of the drop diameters in the limited range, di – values of the drop
diameters in the given range.
Nishikawa et al. (22) measured the effects of the volume fraction of dispersed phase,
viscosity of liquids, impeller speed and impeller-to-vessel diameter on the average drop
size of a dispersion in a mixing vessel for the two-phase: water (continuous phase)-honey
bees` wax (dispersed phase). Hatate et al. (23) also measured the mean droplet size for
several systems using two columns and correlated it as a function of the superficial gas
velocity, interfacial tension, and column diameter. The same authors (24) measured the
average gas holdups, the longitudinal distribution of the volume fraction of a dispersed
liquid (droplet), and the longitudinal dispersion coefficients of a dispersed liquid, using
two bubble columns (0.066- and 0.122 m i.d.) with a perforated plate as a gas sparger.
Their columns were operated batchwise or continuously with respect to the liquids. They
found smaller average gas holdups for the air-kerosene (dispersed liquid)-water (continuous liquid) system than for the corresponding ones without kerosene, over a range of
superficial gas velocity: ug = 0.007-0.09 m3/(m s), analyzed the longitudinal distribution
of the volume fraction of the dispersed liquid, using a dispersion model allowing for the
slip velocity. Diaz et al. (25) examined the dependence of the mean droplet size on the
superficial gas and liquid velocities, and measured the dispersion coefficients of both
liquids, for the air-kerosene-water system. Priestly and Ellis (26) also found that the efficiency of non-mechanically agitated extraction column with different packings can be
considerably increased by the introduction of an inert gas as a mixing agent in two-phase
L-L systems. On the other side, Kato et al. (27) extended the studies of Hatate et al.
(23,24) from a single-stage to the multistage bubble columns of the same diameter. Their
study was also limited to the air-kerosene-water system with a few additional ones for the
measurement of average gas holdups. Using the organic liquids (kerosene, dibutyl phthalate, or groundnut oil) dispersed in water, Bandyopadhyay et al. (28) measured the average gas holdup of air in a bubble column (0.2 m in diameter) with a multiple nozzle
sparger plate, operated batchwise with respect to the liquids. They found that the fractio204
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nal holdup depends on the gas velocity, liquid properties, phase inversion in the liquid
mixture, as well as on the spreading coefficient of the organic liquid. In the presence of a
liquid with a negative spreading coefficient, the holdup is a minimum at the phase inversion point but the reverse is true for a liquid with a positive coefficient of spreading. The
model assumes that the particles rise or fall with the slip velocity caused by the density
difference between the dispersed and continuous phases and explains well the behavior of
the solid particles in the suspension bubble columns (16,17). The longitudinal distribution
of the fractional gas holdup was measured in the bubble columns with two immiscible
liquids (29). The columns were operated batchwise with respect to both liquids, over a
wide range of relevant physical properties and average volume fraction of the dispersed
liquid. The average gas holdups could be correlated by an empirical expression presented
in the literature for a single liquid phase, when it was applied to the individual liquid phases, allowing for their volume fraction. Doungdeethaveeratana and Sohn (30) investigated
a novel solvent extraction process without moving parts, in which the emulsion is generated the by bottom gas injection rather than by mechanical stirring. They found that this
process had a number of advantages over the mixer-settler unit or the spray extraction
column, which provideed a sufficiently large intefacial area for mass transfer. Yan (31)
studied the process of extracting hydrogen peroxide from an anthraquinone working
solution with the bottom air injection in a spray column. This result showed that the extraction efficiency was 2-3 times higher than that of conventional liquid-liquid extraction
without air introduction. Lü et al. (32) presented the results of the extraction of hydrogen
peroxide with deionized water from the anthraquinone solution via anthraquinone process, which was carried out in a gas-agitated sieve plate extraction column. Experimental
procedure for the estimation of dispersed phase holdup in the three phase G-L-L system
was described in the paper (13). The author presented the hydrodynamic characteristics
of the air-water-toluene three-phase G-L-L system in a countercurrent spray extraction
column. If the position of the interface was maintained constant in the cylindrical section
of the column by the adjustable overflow tube, the average dispersed phase holdup on the
three-phase G-L-L dispersion was calculated by the following relation (13):
g 
Ht  Hb
Ht
[11]
The uncertainty of the average gas holdup measurements in the phase system airwater-toluene was estimated to be  5%. The average gas phase holdup increased with increasing superficial velocity of the gas phase in the three-phase G-L-L systrem, whereas
the gas phase holdup determined at a constant ratio of the contiuous and gas phases
decreased with incresing the superficial velocity of the dispersed phase (12). The hydrodynamics of a spray extraction column operated with the liquid-liquid and gas-liquidliquid systems was intensively investigated (35-37).
Hikita et al. (38) derived an empirical correlation for the prediction of the average gas
phase holdup in a three-phase G-L-L system, as follows:
 g  0.672 Ca
0.578
Mo
 0.131
 g

 L



0.062
 G

 L



0.107
[12]
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where: Ca=(ucL)/ and Mo=  L4 g /  L  3 - characteristic parameters,  - interficial tension, g - acceleration due to gravity, L - density of the liquid phase, g - gas phase density, G, L - dynamic viscosity of the gas and liquid phase, respectively. The predictions
may be seen to be in reasonable agreement with their experimental data. Asai and Yoshizawa (29) showed the relation for the calculation of the average gas holdup in the three
phase air-water-kerosene system which was based on the relationship for the G-L system,
when the mean volume fraction of the dispersed phase was dt = 0.50:
[13]
 g  1   dt  gc   gd  dt
where: gc, gd - average gas phase holdup in continuous and dispersed phase of the GL-L dispersion. As a check of the presented static pressure measurements, the values of
(g) for the system air-water-kerosene were determined by measuring the difference in the
total liquid height between the sparged and unsparged conditions. Although this technique of the measurements of the liquid level under the sparged conditions was not so easy
because of the violent fluctuation, the average values of several measurements agreed
with the values obtained from the static pressure measurements within an error of about
10%. Asai and Yoshizawa (29) presented experimental values of the gas-phase holdup in
the two-phase air-water system and three-phase air-water-kerosene system. Both systems
gave the same observed values and were in good agreement with the predictions, eq. [13].
At the same time, figure 4. in the paper (29) shows the data for gas-phase holdup in the
three-phase system air-water-kerosene obtained from empirical relationship of Bandiotphayay et al. (28), whose form is:
0.011
g  0.65 u
0,496
g

0.25
dt
 M  


 g M 
0.20  dt < 1.0 [14]
where: ug - superficial velocity of the gaseous phase, M, M – density and dinamic viscosity of the liquid mixture, respectively. Asai and Yoshizawa (29) concluded that this
expression predict worse values for the air-kerosene-water system than those for the airwater system, but still it gives rise to larger values than their data for both systems. This
is not in line with the findings of Hatate et al. (23), Kato et al. (24) and Bandyopadhyay
et al. (28), who found lower average gas holdups for the air-kerosene-water system.
However, these experiments were performed with a perforated plate or a multiple nozzle,
in the region of lower superficial gas velocity, where the flow mechanism was known to
vary with the configuration of the gas sparger. Therefore, this different observation may
be possibly attributed to the different effect of the liquid physical properties on the fractional gas holdups in the different flow regimes. In fact, the data of Bandyopadhyay et al.
(28) reveal a reduction of the difference in the gas phase holdups between both systems
with an increase of the gas flow rate. Bandyopadhyay et al. (28) claimed that eq. [14] correlates their data for the air-kerosene-water system worse than those for the air-water
systems, but still it gives rise to larger values than for the experimental data of Asai and
Yoshizawa (29) for both systems. These authors presented graphically the relation between the average gas phase in the G-L (air-water) and G-L-L (air-kerosene-water) system, fig. 2.
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Figure 2. Average gas holdups for the air-kerosene-water and air-water systems:
DT = 0.064 m; t = 9.7  1.9oC
[Source: Asai and Yoshizawa, Ind. Eng. Chem. Res., 30 (1991) 745-751]
Wang et al. (20) presented an empirical expression for the calculation of the gas-phase holdup in a gas-agitated sieve plate extraction column, as follows:
3.104
0.131
0.062
0.107
  c4 g 
 u g c 
 g 
 g 

u 







 g  0.215 
exp   0.0071 d  [15]
3 
uc 

  
 c 
 c 
 c  
where: g, c – density of the gaseous and continous phase, respectively, g, c – dynamic
viscosity of gaseous and continuous phase, respectively.
The effects of the gaseous superficial velocity on the gas holdup in different liquid-liquid and gas-liquid-liquid systems have been widely investigated, which is presented, fig.
3.
Figure 3. Average gas holdups for the different gas-liquid-liquid and gas-liquid systems
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Drop size and interface area
In chemical engineering, the rate of mass transfer between two different phases often
directly determines the production rate of the process (e.g., the gas absorption rate in gasliquid systems). The mass transfer rate is directly proportional to both the mass transfer
coefficient and the specific interfacial area between the different phases. Both parameters
depend mainly on the (local) hydrodynamic situation inside the system. For the design
purposes as well as for the improvement of the existing production facilities, it is very
important to have a better insight into the phenomena that affect these parameters.
The knowledge of the dispersed phase drop size is of primary importance in the design of liquid-liquid non-mechanicall agitated extraction columns. It affects the dispersed
phase holdup, the residence time of the dispersed phase, and the free throughputs. Furthermore, together with the dispersed phase holdup, it determines the interfacial area disposable for mass transfer and affects both the continuous and dispersed phase mass transfer coefficients. It is therefore important to be able to predict the drop diameter as a function of the column geometry, physical properties of the liquid-liquid system, and direction of mass transfer (39).
Seibert and Fair (40) proposed a new equation for the prediction of the Sauter drop
diameter in the packed and spray extraction columns, as follows:
  
d32  1.15  

  g 
0.5
[16]
where  is a correction factor calculated from the experimental drop diameter data assumed from the literature,  - difference of density. Its values are  = 1.0 for no mass
transfer or transfer from the continuous phase to the dispersed phase and  = 1.0 – 1.8 for
mass transfer from the dispersed phase to the continuous phase. Kumar and Hartland
(39) presented a relationship for the limiting value of the drop size in the absence of agitation or at low levels of agitation in the liquid-liquid extraction columns in the following
form:
1/ 2
[17]
d32  C1  /   g
where the constant, C1, is a function of the column geometry, mass transfer, and the characteristics of the liquid-liquid system employed. Vedaiyan et al. (41) proposed an empirical correlation for the calculation of the Sauter drop diametar, given by:
  

d 32  1.59 
 g 
0.5
 u 02 


 2 g d0 
0.167
[18]
where: u0 – superficial velocity of the dispersed phase at the nozlle, d0 – diameter of the
nozlle of the distributor of dispersed phase. The gas-liquid interfacial area, which is determined by the gas holdup and the Sauter mean bubble diameter, determines the production rate in many industrial processes. The effect of additives on this interfacial area is
often not undrestood, especially in multiphase systems (gas-liquid-solid, gas-liquid-liquid). The addition of a third phase can cause the gas-liquid system to become completely opaque, which means that conventional techniques to study the interfacial area cannot
be used (41). The influence of different additives (1-octanol; dodecane, and toluene) on
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the interfacial area was studied in a stirred vessel and in a bubble column under coalescing and noncoalescing conditions (42). It was found that the addition of toluene to a
noncoalescing electrolyte system decreased the interfacial area to a large extent by turning it into a coalescing system, due to the interaction between gas bubbles and liquid
organic droplets. Furthermore, around the toluene solubility concentration, both the gas
holdup (measured using an electric conductivity technique) and the interfacial area increased to the values similar to those observed in noncoalescing systems. The cause of this
remarkable phenomenon lies probably in the presence of a small toluene layer around the
gas bubbles, which can be formed beyond the solubility point (43). This layer is absent at
the concentrations below the solubility limit and a large surface tension gradient exists
between these two situations, which can be responsible for the sharp change in the coalescence behavior.
A comparison of ultrasonic spectroscopy with a digital camera technique was performed in a flat (20x3x150 cm) bubble column using the ultrasonic technique in combination with the electrical conductivity method and a digital camera technique with digital
image analysis, simultaneously (43). The camera was placed 10 cm in front of the column, and the ultrasonic transducers were mounted into the wall of the column (the measurement path length was 20 cm). Measurement of the exact size distribution using the
ultrasonic technique was difficult, mainly due to the small attenuation and ultrasonic velocity differences. These differences were small due to the low gas holdups that were applied (1%), which was necessary for the digital camera technique to work optimally.
The interfacial area could, however, be determined accurately, and together with the measurement of the gas holdup using the electrical conductivity technique, the Sauter mean
bubble diameters were calculated. The value of the interfacial area (a) of the bubble size
distribution can be calculated from the Sauter mean diameter (d32) and the gas phase
holdup (g), according to the following relation:
a
6 g
d 32
[19]
The liquid-liquid interfacial area and liquid-phase mass-transfer coefficients in the
emulsion bubble columns were measured by Fernandes and Sharma (43), who took advantage of the alkaline hydrolysis reaction of several esters for their determination. For
the analysis they assumed complete mixing of both the continuous and dispersed liquids.
Yoshida et al. (44) measured the mean diameter of kerosene dispersed in the water phase
of bubble columns, operated batchwise with respect to both liquids. They studied the variation of the oxygen absorption into water with the addition of kerosene, liquid paraffin,
toluene, and oleic acid. They claimed that in the previous studies, the effects of physical
properties on the various characteristics of the bubble columns were not clarified. Assai
and Yoshizawa (29) presented the longitudinal distribution of volume fraction of the dispersed liquid over a wide range of relevant physical properties and average volume fraction of the dispersed liquid. The observed longitudinal distribution of the volume fraction
of the dispersed liquid was analyzed by means of the dispersion model, allowing for the
slip velocity caused by the density difference between both liquid phases. The observed
Peclet numbers based on the slip velocity were empirically correlated as a function of the
relevant system parameters.
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The hydrodynamic characteristics of air-anthraquinone working solution-water threephase system used for the production of hydrogen peroxide were determined in a gasagitated sieve plate extraction column (5). The effects of the superficial velocities of the
gaseous phase, organic dispersed phase and continuous phase on the organic dispersed
phase holdup were investigated. The organic dispersed phase holdup increased with the
increase of the superficial velocity of the gaseous phase and organic dispersed phase. The
effect of the superficial velocity of the continuous phase on the organic dispersed holdup
could be neglected. Based on the equation of the relative velocity between the organic
dispersed phase and continuous phase, a method used to calculate the organic dispersed
holdup was proposed (20). The organic dispersed holdup of gas-liquid-liquid systems (including liquid-liquid systems) in this study and in data from the literature (32) were calculated using the method proposed in that study (20). The calculation data were well consistent with the corresponding experimental data (20) and the literature data (33,34), and
the relative error were 4.9-15.5%.
Axial mixing
A theoretical and experimental study has been carried out on the dynamics of twophase countercurrent flow with interfacial transfer in a packed bed absorption column
(45). An eight-parameter model has been formulated consisting of axially dispersed plug
flow for the gas phase and a piston-diffusion exchange model for the liquid phase. In
addition, three limiting cases of this model have been analyzed. Solutions of the models
have been obtained in the Laplace domain with four possible transfer functions for each
model as a result. Only two of these transfer functions have been found useful for an experimental study of the absorption of a poorly soluble gas. Experimental measurements
of these two transfer functions, in the form of frequency characteristics, have been carried
out in a 0.105 m diameter column packed to a height of 2.1 m by glass spheres 0.01 m in
diameter. The absorption system studied was water-air-oxygen. Evaluation of the parameters of the formulated models was carried out in the frequency domain. The results
showed that the models with a stagnant liquid zone are considerably better than the axially dispersed models. For a more reliable assessment of the various models, however, a
combination of several independent measurements is recommended.
Axial mixing arises in the packed columns from the fact that the „packed“ fluid do not
all move through a packed bed at a constant and uniform velocity, either because of either
velocity gradients in the fluid, or eddy motion in the packed voids. Axial mixing tends to
reduce the concentration driving force for mass transfer that would exists for piston flow
(45). To achive a given separation, more transfer units are required for the axial-mixing
case owning to the reduced drivning force. Longitudinal dispersion coefficients of the
continuous phase were experimentally obtained in spray type liquid-liquid extraction columns (45). The method used was unsteady-state measurements of a KCl solution as the
tracer. It was concluded that the increase in the continuous phase velocity greatly increased the axial mixing coefficient in binary mixtures (Ecb), and the increase in the dispersed
phase velocity decreased the axial mixing coefficient (45). Small dispersion coefficients
were found for small tower lenghts and these coefficients increased as the tower lenght
increased. Also, at a long lenghts, where the end of the effects became negligible, Ecb was
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indipendent of the lenght. A decrese in the tower diameter from 35.8 mm to 27 mm caused a decrease in Ecb of approximately 20% for a range of the continuous phase velocities. A comparison of the Peclet and Reynolds numbers for spray towers with those for
packed beds gave comparable values. Using the data of the work (46) and the values from
the literature (47), the following realatioship was obtained using the method of least squares:
( uc  4.5 mm/s)
[20]
Ecb  3,43 x10 4 uc0.42
where Ecb – continuous phase axial dispersion cofficient of the two-phase system, uc was
used in mm/s. The correlation coefficient was 0.94, with an average deviation of  8%.
Diaz et al. (25) showed that high axial dispersion coefficients were deduced in both
the liquid phases for the three-phase air-water-kerosene system (Ect), in which water was
the continuous phase and kerosene the dispersed phase. They also found that the values
of the axial dispersion Peclet number of the water phase were 0.1 to 1.2, decreasing with
the increase in the flow rates of air or kerosene, or when the flow water rate was reduced.
Diaz et al. (25) concluded that the Peclet number for the kerosene phase decreased to the
values between 0.4 and 0.1 when the air flow rate increased. Kato et al. (27) investigated
the axial dispersion in the multistage bubble columns for the air-water-kerosene system.
They concluded that the dispersion-phase coefficient in the three-phase G-L-L system
(Edt) increased with increasing the gas phase superficial velocity and coluumn diameter,
and was independent of the the total liquid velocity in the range from 0.05 to 1.0 cm/s.
Kato et al. (27) derived also an empirical equation in which Edt depends on the gas phase
superficial velocity, column diameter and gravitattion acceleration.
Asai and Yoshizawa (29) measured the longitudinal dispersion coefficients of the
continuous (Ec) and dispersed phase (Ed) in bubble columns (with air as a gaseous phase)
operated batchwise with respect to two immiscible liquids (2-ethylhexanol, water or kerosene). They concluded that the longitudinal coefficients Ec and Ed of the continuous and
dispersed phase were independent of the clear liquid height. It was shown that Ec and Ed
increased with the increase in the volume fraction of disperesed liquid () in the system
air-2-ethylhexanol-water, which had a highly viscous dispersed liquid. For the air-kerosene-50% aqueous sucrose solution system with a highly viscous continuous liquid, Ec increased and Ed decreased with an increase in . All observed longitudinal dispersion coefficients of both liquids were correlated by empirical correlations (29). Figure 4 shows the
effect of the gaseous superficial velocity (ug) on the longitudinal coefficient of the continuous and dispersed phase of (Ec, Ed) in the system air-etylhexanol-water (29). These
authors concluded that the increase in the visosity of the dispersed liquid apears to rather
improve the dispersioin of both liquids, fig. 4.
The continuous phase axial dispersion coefficients of the three-phase G-L-L system in
a gas-agitated spray extraction column, described above, were examined by Sovilj (48).
The system used was water as a continuous, toluene as a dispersed, and air as a gaseous
phase. The experimental values of the continuous phase axial mixing coefficients were
obtained by unsteady-state measuring of the concentrations of a tracer solution (solution
of potassium chromate in water) in the continuous phase. The increase in the gas phase
superficial velocity increased the continuous phase axial mixing coefficient. A nonlinear
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dependence between the continuous phase axial mixing coefficient and continuous phase
superficial velocity was observed.
Figure 4. Effects of the gas phase superficial velocity on the longitudinal dispersion
coefficients Ec and Ed or the air-2-ethylhexanol-water system:
DT = 0.064 m; t = 14.0 ± 2.9oC
[Source: Asai and Yoshizawa, Ind. Eng. Chem. Res., 31 (1992) 587-592]
No correlation was found between the continuous phase axial dispersion coefficient
and the dispersed phase superficial velocity. The increase in the dispersed phase holdup
generated a growth of the continuous phase axial dispersion coefficient. The continuous
phase axial dispersion coefficients in the spray extraction column were higher for the
three-phase air-water-toluene system (48) than those obtained for the two-phase watertoluene system (49) under the same operating conditions. Regression analysis showed
that the mean increase in the continuous phase axial dispersion coefficient in the threephase system (Ect) was approximately 90%. In the paper (48), an equation for the prediction of the continuous phase axial mixing coefficient was developed, as is given below:
u 2 d  
u c d 32
 0.124  c 0 c 
E ct



0.77
 u c d0 c

 c



0.87
 dt0.24  g0.23
[20]
where: Ect- continuous phase axial dispersion coefficient of the three-phase system, d0 –
orifice diameter, dt - dispersed phase holdup in the G-L-L dispersion. The average deviation for eq. [16] was 17.7%. Seventy-one percent of the predicted continuous phase axial
dispersion coefficients lied within the  20% limits and 84% within the  30% limits.
These results are in accordance with the results for the two-phase system (48) and with
the conclusion of Horvath et al. (50) that an average deviation within 30% was sufficient
for the use with back mixing models.
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CONCLUSION
This review article deals with the hydrodynamic characteristics of the non-mechanically agitated extraction columns. An inert gas (air, nitrogen, oxygen) as a turbulence
agent was introduced in the two-phase liquid-liquid system. In the new, three-phase gasliquid-liquid system, the gaseous phase causes intensive turbulence, which caused improving of the average dispersed phase holdup and a larger mass transfer area. Mass-transfer
or chemical reactions for three-phase systems may be also encountered in the gas absorption, gas-liquid reactions, and fermentation, often with a heterogeneous liquid catalyst, or
liquid-liquid reactions with gas agitation. Different empirical equations which describe a
function of the dispersed phase holdup, gas phase holdup, and axial distribution coefficient were analyzed and compared.
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Review
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ХИДРОДИНАМИКА ЕКСТРАКЦИОНИХ КОЛОНА ТЕЧНО-ТЕЧНО
АГИТОВАНИХ ГАСОМ
Милан Н. Совиљ
Технолошки факултет, 21000 Нови Сад, Булевар цара Лазара 1, Република Србија
У овом раду дат је приказ и анализа хидродинамичких карактеристика екстракционих колона течно-течно код којих се користи инертан гас као агитатор. Увођењем гаса у колонски уређај са двофазним системом течно-течно формира се знатно
ефикаснији трофазни систем гас-течно-течно, пошто се ситњењем капи дисперговане фазе повећава специфична површина одговорна за пренос масе у систему. Дат
је и анализиран утицај средњег садржај дисперговане и гасне фазе на пренос масе у
трофазном систему гас-течно-течно. У исто време, приказане су и корелације за
сваку од хидродинамичких величина, као и њихова тачност у предвиђању ових величина у колонском уређају. Коначно, приказан је и утицај повратног мешања на
хидродинамику екстракционих колона течно-течно, као и гас-течно-течно. Анализиране су и одговарајуће емпиријске корелације које дају везу између коефицијента
повратног мешања и хидродинамичких карактеристика екстракционих колона.
Кључне речи: екстракционе колоне, систем гас-течно-течно, хидродинамика
Received: 4 April 2012
Accepted: 13 June 2012
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MICROFILTRATION OF DISTILLERY STILLAGE: INFLUENCE OF
MEMBRANE PORE SIZE
Vesna M. Vasić*, Marina B. Šćiban, Aleksandar I. Jokić, Jelena M. Prodanović
and Dragana V. Kukić 
Stillage is one of the most polluted waste products of the food industry. Beside large
volume, the stillage contains high amount of suspended solids, high values of chemical
oxygen demand and biological oxygen demand, so it should not be discharged in the
nature before previous purification. In this work, three ceramic membranes for microfiltration with different pore sizes were tested for stillage purification in order to find the
most suitable membrane for the filtration process. Ceramic membranes with a nominal
pore size of 200 nm, 450 nm and 800 nm were used for filtration. The influence of pore
size on permeate flux and removal efficiency was investigated. A membrane with the pore
size of 200 nm showed the best filtration performance so it was chosen for the microfiltration process.
KEY WORDS: Microfiltration, distillery stillage, wastewater
INTRODUCTION
Stillage (distillery wastewater) is the main waste product generated in the distilleries.
Its pollution potential is one of the most serious problems today, so distillery industries
are forced to develop new techniques for stillage purification and utilization. The amount
and composition of stillage are variable and depend on the feedstocks used for bioethanol
production, as well as various aspects of the production process.
It is characterized by a high content of organics and total solids, low pH and a very
large volume, increasing together with the ethanol production. To produce 1L of ethanol,
approximately 10 to 15 liters of distillery stillage are generated (1). Also, it has very high
biological oxygen demand (BOD), chemical oxygen demand (COD) and a high BOD/
COD ratio. The amounts of inorganic substances such as nitrogen, potassium, phosphates, calcium and sulfates are also very high (2). Distillery stillage contains some feedstock components and degraded yeast cells. Many of those components are characterized
by a high nutritive value. They contain vitamins (with large amounts of those classified
as group B), proteins rich in exogenous amino acids, and mineral components (3).
* Corresponding author: Vesna M. Vasić, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
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Compared to other distillery wastewaters (sugar-based stillage and stillage from cellulosic materials), the stillage from the fermentation of starch-based feedstocks contains
more suspended solids due to the presence of grains remaining after fermentation. An important characteristic of stillage obtained from molasses-based feedstocks include color
components. Phenolics from the feedstock, melanoidins from Millard reaction of sugars
with proteins, caramels from overheated sugars and furfurals from acid hydrolyses can
contribute to the color of the effluent (4). Chromium, copper, nickel and zinc were found
at levels significantly above detection limits in some distillery effluents, especially in the
stillages from cellulosic feedstocks. Some heavy metals are originating from feedstocks
used for bioethanol production. The processing equipment used in the pretreatment of
cellulosic feedstocks (acid hydrolysis) is often made of corrosion-resistant alloys. Heavy
metals contained in these alloys may leach into the feedstock during the hydrolysis,
resulting in detectable levels in the stillage(4). Also, corrosion of piping, reactors and
heat exchangers may contribute to heavy metal content in the stillage.
Considering high pollution potential of distillery wastewater, it should not be disposed in the nature without previous treatment. Stillage disposal in the environment can be
adverse. High COD and nutrient content may result in eutrofication of natural waters,
colored compounds block out sunlight penetration in rivers and lakes, reducing photosynthetic activity and dissolved oxygen concentration. Disposal of distillery wastewater
on land is also harmful, and can affect the vegetation and groundwater quality.
Different techniques for distillery stillage purification have been explored. Stillage is
usually treated first with a screw decanter to remove solids (1). Also, centrifugation can
be successfully used as a technique for solids separation (5). Further, stillage can be concentrated in the multi-effect evaporators with the co-production of condensate, which is
lower in organics and almost devoid of inorganic salts. However, significant energy required to evaporate the stillage can negatively impact the energy balance of ethanol production (4). Coagulation and flocculation are also commonly used methods to remove
particulates and organic matter from wastewaters. They are usually conducted by adding
chemicals such as salts of aluminium and iron and polyelectrolytes. The limitations of
coagulation and flocculation are: an increased salinity of the effluent, the storage and
handling of corrosive chemicals, need for pre- and post-dosing adjustment of pH and
sludge handling (6). Biological treatment processes such as anaerobic and aerobic digestion, as well as combination of these two methods have been successfully used for stillage treatment. Although the biological processes have several advantages such as the easy access and a large scale operation, the major drawbacks of these processes are high
energy consumption, high labor costs, and large variations of the treatment efficiency
with the change in feedstocks used for bioethanol production (1). However, it is hard and
sometimes impossible to meet the environmental standards with aforementioned kinds of
purification. Membrane separation techniques are widely used for distillery wastewater
treatment, offering a possibility to improve the value of stillage and to meet environmental standards. The most commonly used membrane processes for wastewater purification
are: microfiltration, ultrafiltration, nanofiltration and reverse osmosis.
The current trend in the membrane market has shown significant improvements in
technical efficiencies of membrane systems, which makes them a cost competitive alternative to conventional treatment systems. Permeate flux rate and throughput are critical
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measures of membrane performance and play important roles in determining the cost of
membrane filtration system (7).
The aim of this study is to evaluate the application of microfiltration for stillage treatment, and to find the most suitable membrane for the filtration process.
EXPERIMENTAL
Experimental material
The experiments were performed using distillery stillage from the ethanol factory
Reachem, Srbobran (Serbia), where it was obtained from starch feedstocks.
Microfiltration experiment
The experiments were carried out in a conventional cross-flow microfiltration (MF)
unit (Figure 1).
Figure 1. Schematic representation of the experimental setup for cross-flow
microfiltration (8)
The feed was circulated by a peristaltic pump (ISMATEC, Switzerland), under the
conditions of complete recirculation of the fluid. The feed suspension was concentrated
to a volume concentration factor (VCF) of 1.88. The permeate was constantly drained
away from the system, collected and analyzed. The transmembrane pressure difference
was adjusted by the regulation valve. The inlet and outlet pressures of the membrane
module were measured by two pressure gauges. The average of these two pressure values
gave the value of transmembrane pressure (TMP) as the outside of the membrane is
vented to the atmosphere. The experiments were performed under the TMP of 0.6 bar.
The membrane module used was a MembraloxTM 1T1-70 module (SCT, Bazet, France).
The single channel ceramic membranes used had nominal pore sizes of 200 nm, 450 nm
and 800 nm (TAMI Deutschland) with the length of 250 mm and inner/external diameter
of 6/10 mm. The useful membrane surface was 4.33×10−3 m2. The membranes were
cleaned according to the recommendation of the manufacturer аfter each experiment; the
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cleaning sequence was a classical acid-base one (alternate washing with 0.2% solution of
NaOH and 0.2% solution of HNO3, both with recirculation, and rinsing with distilled
water). After that the water flux of the membranes was measured; the measurement provided the reference to assess the effectiveness of the membrane cleaning. The permeate
flux was calculated from the time needed to collect 10 mL of permeate. All measurements were carried out in triplicate, and the results were averaged. All experiments were
carried out at the room temperature (25ºC).
Analitical methods
Feed and permeate samples obtained after the microfiltration of stillage were analyzed
for dry matter, ash, organic dry matter, suspended solids, BOD and COD using Standard
Methods (9).
Dry matter content was determined by gravimetric method, drying the sample at 103105ºC. Suspended solids were determined by centrifugation at 3000rpm during 10 minutes (centrifuge MLW T52.1); supernatant was poured off and the residue was determined
by gravimetric method, drying at 103-105ºC. COD was determined by dichromate reflux
method. BOD was measured using a VELP SCIENTIFICA BOD system according to the
manufacturer manual. Total nitrogen was determined by Kjeldahl method (10)
The stillage was analyzed immediately after its bringing from the factory. The results
of the analyses are presented in Table 1.
Table 1. Results of the analyses of stillage and permeates obtained after
microfiltration
220
Parameter
Stillage
Dry matter (mg/L)
Ash (mg/L)
Organic dry matter (mg/L)
% of Organic dry matter (%DM)
Suspended solids (mg/L)
Ash of suspended solids (mg/L)
Organic dry matter of
suspended solids (mg/L)
% of Organic dry matter of
suspended solids (mg/L)
COD (mgO2/L)
BOD (mgO2/L)
BOD/COD *100 (%)
Total Kjeldahl nitrogen (mg/L)
63700
9640
54060
85
18340
1165
Permeate from the membrane
with pore size of:
200 nm
450 nm
800 nm
50910
42590
40700
15625
4340
4150
35285
38250
36550
69
90
90
-
17175
-
-
-
94
-
-
-
102000
89000
87
2866
64500
48000
74
1480
64000
47750
74
1420
62600
46320
74
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As can be seen, the stillage had high values of COD and BOD, as well as the values
of dry matter and total nitrogen. About 29% of dry matter is in form of suspended solids.
According to these results it was confirmed that the stillage is highly polluted. Further,
the stillage was passed through the microfiltration membranes with different pore sizes,
applying the same conditions for all membranes (TMP = 0.6bar, feed flow rate (Q) = 100
L/h, pH = 3 and t = 25ºC). The permeates were collected and also analyzed (Table 1).
The filtration time for the membranes with pore sizes of 200, 450, 800 nm was 3 hours 36
minutes 28 seconds, 4 hours 11minutes 8 seconds, and 5 hours 14 minutes 18 seconds,
respectively.
Based on the presented results, it can be said that the values of COD, BOD, total nitrogen and dry matter decreased compared to the initial values of the stillage. The removal efficiency of COD for the membranes of 200, 450, 800nm was 36.7%, 37.3% and
38.7%, respectively. The total Kjeldahl nitrogen content was lower by 48.4%, 50.5% and
51.2% respectively, whereas the BOD/COD ratio was the same for all permeates, which
indicates that the same kind of organic matters that pass through examined membranes
are biodegradable. Suspended solids were completely removed from the stillage. The ash
content of the permeate obtained after filtration through the membrane with pore size of
200 nm was higher by about 60% compared to initial value in the stillage. Arora et al.
(11) reported similar results obtained after the ultrafiltration of thin stillage. Their results
showed that ash content of permeate was higher than in thin stillage. They concluded that
this may be attributed to the solubility of mineral components in the stillage stream,
which allowed them to pass through the membrane. However, ash content was reduced in
permeates for the membranes with pore sizes of 450 nm and 800 nm, which can be explained by particles accumulation within pores of the membrane. The sizes and the shapes of
particles in stillage are very variable and dependend of the feedstocks used for bioethanol
production. Therefore, pores can be blocked with components of large molecular weights.
Considering the pore size of the membranes for microfiltration, it cannot be expected
to remove all organic pollution from wastewater by their application, but it can be reduced considerably. That makes microfiltration suitable as a pretreatment for ultrafiltration
or reverse osmosis (6).
The effect of the membrane pore size on the permeate flux is shown in Figure 2.
40.00
200nm
450nm
800nm
35.00
30.00
Flux (L/h)
25.00
20.00
15.00
10.00
5.00
0.00
0
5000
10000
15000
20000
Time (s)
Figure 2. Time dependence of the permeate flux in the microfiltration of stillage
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Experimental results suggest that there is a fast initial flux decrease for all membranes, it takes place during the first ten minutes of filtration and does not decrease significantly later on. As can be seen from the figure, similar flux rate profiles were observed
for all membranes. The membrane with pore size of 800 nm had the lowest value of
permeate flux. The reason for this can be found in the fact that the membranes with a larger pore sizes may be blocked due to the presence of components with large molecular
weights. Numerous examples show that membrane fouling is more severe with increasing
pore size. There appears to be an optimum pore size, above which severe membrane fouling reduces the flux. Attia et al. (12,13), while processing skim milk on aluminium oxide
MF membranes, found higher permeate fluxes with a 0.2 μm membrane than a 0.8 μm
membrane. Stopka et al.(14) reported similar results obtained after the microfiltration of
model yeast suspension and beer. Their study showed that the permeate flux for the membrane with a pore size of 500 nm was lower compared to that observed for the membrane
with pore size of 200 nm. Arora et al. (11) presented results of ultrafiltration of thin
stillage, using membranes with pore sizes of 10 and 100 kDa. The results showed that the
membrane with larger pore size (100 kDa) had lower flux.
It is evident that the membrane with a pore size of 200 nm had the best performance,
in terms of permeate flux. The values of the flux for the membranes of 450 nm and 800
nm were lower compared to the membrane of 200 nm for 13.8 % and 31.2 %, respectively. This can be explained by the foulants accumulation within the larger pores of the
membrane, which leads to their clogging. In contrast, the difference in the removal efficiency of COD, BOD, total nitrogen, and dry matter (Table 1) for all membranes is insignificant.
CONCLUSIONS
Based on the obtained results it can be concluded that the membranes with the pore
size of 450 and 800 nm had lower permeate flux than the membrane with pores of 200
nm. The difference in the permeate quality for all membranes is insignificant. The membrane with the pore size of 200 nm showed the best results in terms of duration of the
filtration process. Hence, it was chosen for the microfiltration of investigated starch based
distillery stillage. Eventually, it can be concluded that the selection of the membrane is
very important for the successful implementation of the filtration process, both from the
economic standpoint and the standpoint of environmental protection.
Acknowledgement
This research was supported by the grant number TR 31002 from the Ministry of
Education and Science of the Republic of Serbia.
REFERENCES
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techniques for distillery stillage treatment. Czech J. Food Sci. 24 (2006) 261-267.
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technologies and potential applications. J. Hazard. Mater. 163 (2009) 12-25.
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6. Ryan, D., Gadd, A., Kavanagh, J. and Barton, G.W.: Integrated biorefinery wastewater design. Chem. Eng. Res. Des. 87 (2009) 1261-1268.
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Rausch, K.D.: Microfiltration of thin stillage: Process simulation and economic analyses. Biomass Bioenerg. 35 (2011) 113-120.
8. Jokić, A., Zavarago, Z., Šereš, Z. and Tekić, M.: The effect of turbulence promoter on
cross-flow microfiltration of yeast suspension: A response surface methodology
approach. J. Membr. Sci. 350 (2010) 269-278.
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12. Attia, H., Bennasar, M. and Tarodo de la Fuente B.: Study of the fouling of inorganic
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УТИЦАЈ ПРЕЧНИКА ПОРА НА МИКРОФИЛТРАЦИЈУ ЏИБРЕ
Весна М. Васић, Марина Б. Шћибан, Александар И. Јокић, Јелена М. Продановић
и Драгана В. Кукић
Универзитет у Новом Саду, Технолошки факултет, Булевар Цара Лазара 1, 21000 Нови Сад, Србија
Џибра представља један од најзагађенијих отпадних токова прехрамбене индустрије. Поред велике запремине, џибра садржи велику количину суспендованих чес223
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тица, има велику вредност хемијске потрошње кисеоника (ХПК) и биолошке потрошње кисеоника (БПК), па се не сме испуштати у околину без претходног пречишћавања. У циљу проналажења најпогодније мембране за процес филтрације џибре, испитане су три керамичке мембране за микрофилтрацију са различитим пречницима пора. За филтрацију су коришћене керамичке мембране са средњим пречником пора од 200 nm, 450 nm и 800 nm. Испитан је утицај пречника пора на флукс
пермеата и ефикасност пречишћавања. Мембрана са пречником пора од 200 nm показала је најбоље перформансе, па је стога изабрана за процес микрофилтрације испитиване џибре.
Кључне речи: микрофилтрација, џибра, отпадна вода
Received: 31 Маy 2012
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INFLUENCE OF THE OPERATING PARAMETERS ON THE FLUX DURING
MICROFILTRATION OF THE STEEPWATER IN THE STARCH INDUSTRY
Zita I. Šereša*, Ljubica P. Dokića, Biljana S. Pajina, Dragana M. Šoronja Simovića,
Drago Šubarićb, Jurislav Babićb and Aleksandar Z. Fišteša
a
b
Josip Juraj Strossmayer University, Faculty of Food Technology, Franje Kuhača 18, 31000 Osijek, Croatia
The subject of the work is the possibility of applying microfiltration through a
ceramic tubular membrane with 100 nm pore sizes to the steepwater obtained in the
production process of corn starch. The dry matter content should be reduced in the
steepwater permeate. Thus the consumption of the process water would be reduced, the
nutrients from the steepwater could be exploited as feed and the wastewater problem
would consequently be solved. The objective of the work was to examine the influence of
the operating parameters on the permeate flux during steepwater microfiltration. The
parameters that vary in the course of microfiltration, were the transmembrane pressur
and flow rate, while the permeate flux and dry matter content of the permeate and retentate were the dependent parameters, constantly monitored during the process. Another
objective of this study was to investigate the influence of static turbulence promoter on
the permeate flux during steepwater microfiltration. Static mixers enhance permeate flux,
thus the microfiltration can be performed longer. As a result of the statistical analysis,
the optimal conditions for steepwater microfiltration were determined. The maximum
value of the permeate flux without mixer (25 lm-2h-1) was achieved at a pressure of 2 bars
and a flow rate around 100 lh-1. With the use of static mixer the flux is 2,5 times higher
compared to the one obtained without the mixer. The dry matter content of the permeat
after 2.5 hours of mucrofiltration was lowered by 40%.
KEY WORDS: steepwater, microfiltration, ceramic membranre, permeate flux, dry
matter content
INTRODUCTION
Governments of the developed countries have tried to increase the pressure on the
largest waste producers in order to reduce the undesired environmental pollution. For
example, the Commission of the European Communities introduced the Integral Pollution
and Prevention Control Directive. The purpose of the directive is to achieve integrated
prevention and the control of pollution arising from the particular activities listed in its
Annex I. Among others, the directive defines the Best Available Techniques (BAT) as
* Corresponding author: Zita I. Šereš, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1,
21000 Novi Sad, Serbia, e-mail: [email protected]
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the most effective and advanced stage in the development of activities and their operation
methods which indicate the practical suitability of particular techniques for providing in
principle the basis for emission limit values designed to prevent and, where that is not
practicable, generally to reduce emissions and the impact on the environment (1, 2). One
of them is membrane technique.
Membrane separation is a filtration technique in which a feed stream is fractionized
with a porous membrane. Some of the dissolved solids are held back because their molecular size is too large to allow them to pass through. The size range depends upon the
pore sizes of the used membrane. Fractionation of the feed stream occurs, with some molecules being concentrated on the upstream side of the membrane, which is known as the
concentrate or retentate. The smaller molecules pass through the membrane into the permeate stream. There are few membrane processes where they can be characterized by driving forces that cause mass transfer of solutes (e.g. difference in concentration – dialysis), difference in electric potential - electro-dialysis), difference in pressure – microfiltration, ultrafiltration, nanofiltration, reverse osmosis) (3, 4, 5).
The main problem in the performance of microfiltration is concentration polarization
and fouling of the membrane. Concentration polarization causes deposition of retained
compounds on the membrane surface. A number of reviews have described the process in
detail (5, 6). The pure water flux of micro- and ultrafiltration membranes is usually high,
but when separation starts through the membrane, the permetae flux falls very quickly,
which is caused by the gel formation on the membrane surface. This gel layer forms a
secondary barrier to the flow through the membrane (5, 7). There is no possibility for
avoiding membrane fouling but it can be limited by applying a number of different techniques which enhance membrane flux. These techniques might be pre-treatment of feed
stream, backflushing, fluidized bed, fluid instability, application of electric, magnetic and
ultrasonic fields (5). Fluid instability can be more useful in overcoming concentration
polarization and membrane fouling, various possibilities have been tested: turbulence
promoters, pulsation and rotating membrane filter (Taylor vortex flow). Turbulence promoters as static mixers were applied for permeate flux enhancement during the separation
of non-sucrose compounds from sugar beet syrup (8). There are several papers dealing
with the application of membrane filtration for purification of wastewater from starch
processing industry or for filtration of the starch suspensions (9, 10). Membrane filtration
is used in order to achieve an increase in the quality of the finished sweetening and syrup
products. It has also found its application in the process of water elimination, i.e. dehydration in the course of the production. It is used to isolate proteins from diluted process
flows (11).
The aim of this work was to look into the possibility for steepwater microfiltration in
order to examine the influence of the oprating parameters on the permeate flux during
steepwater microfiltration. Another objective was to investigate the influence of static
turbulence promoter on permeate flux during steep water microfiltration, in order to
enhance permeate flux. Generally, the results and the optimization can serve for the determination of the suitable operating conditions for the steepwater concentration. The dry
matter content could be reduced in the steepwater permeate and the process water in the
starch industry could be reused. Thus, the consumption of the process water would be
reduced and the nutrients from the steepwater could be exploited as a feed .
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EXPERIMENTAL
Microfiltration experiments were conducted on the samples of steepwater, which were
obtained from the corn starch wet milling plant „Jabuka“, Pančevo (Serbia). The procedure of microfiltration on a single-channel ceramic membrane with 100 nm pore sizes on
the laboratory apparatus for microfiltration has already been published (12).
The central part of the apparatus is the module with the membrane inside. In this study, use was made of the ceramic membrane of GEA manufacturer (Germany). The membrane is single-channel, 250 mm lenght, with the inner diameter of 6.8 mm and outer diameter of 10 mm. The membrane is made of -Al2O3 with TiO2 layer. The active membrane surface equals 0.005 m2. The pore sizes of the membrane are 100 nm. This pore
size is twice smaller than that usually used for starch wastewater, e.g. ny Cancino-Madariaga and Aguirre (13). These authors used a 0.2 μm PVDF membrane of 7.5 m2. Their
experiment was carried out in a real production plant on wastewater solutions with and
without a prior sedimentation step. Šaranović et al. (12) investigated microfiltration of
wheat starch wastewater on ceramic membrane with 200 nm pore sizes, and achieved a
dry matter decrease of about 50–60%. For this investigation of steepwater microfiltration,
the membrane with 100 nm pore sizes could be used because it contains smaller particles
and no starch. Dry matter content was 6.5%, out of which proteins were 50%, lactic acid
26%, carbohydrates (as dextrose) 2.5%, and total ash 21.5%.
The static turbulence promoter used during experiments was the stainless steel Kenics
static mixer. The static turbulence promoter was inserted inside the whole membrane tube
and was fixed properly to avoid any movement due to the fluid flow (12).
The microfiltration experiments were planned based on a full 23 factorial designed
experiment (14). In this experiment, the factors, i.e. the independent parameters were the
transmembrane pressure (p) and flow rate (Q). Table 1 shows the values for the independent parameters which varied during the course of filtration.
Table 1. Varied values of independent variables
Independent variables
Varied values
Q [L/h]
50 / 150/ 200
P [bar]
1 / 2/ 3
Q - flow rate [L/h]
P - transmembrane pressure [bar]
The dependent parameters monitored during the process of microfiltration, permeate
flux and dry matter content of permeate and retentate were determined at the beginning,
during and at the end of microfiltration (4).
The determination of dry matter content in steepwater and of permeate and retentate
was based on the following: defined volume of steepwater, permeate or retentate weight
in the laboratory glass, with a known mass of the glass. The glass with the content of the
sample was put in the water bath. When the water evaporated, the glass with the content
was dried at 105oC to a constant weight.
The experimental data were processed with computer programmes Statistica for Windows 8.0 and Origin 6.1.
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The membrane was cleaned before each experiment with 0.5% solution of Ultrasil 11.
The effectiveness in membrane cleaning was assessed by examining the water flux recovery. The cleaning procedure was repeated until the 95% of original water flux was restored.
The influences of transmembrane pressure and flow rate on the permeate flux with the
time were analyzed by means of a statistical multifactorial analysis of the experimental
data (12).
Responses fitted with the polynomial model [1] of the second degree were: permeate
flux without static mixer - JNSM, and permeate flux with static mixer - JSM:
z = b0+b1·x+b2·y+b11·x·x+b22·y·y+b12·x·y
[1]
where z - JNSM or JSM [l/m2h], x - P [bar], y - Q [l/h] and b0, b1, b2, b11, b22, b12 - coefficients.
Fig. 1 compares the dependence of the fluxes of distilled water and steepwater on the
transmembrane pressure for the microfiltration on the ceramic membrane with pore sizes
of 100 nm at flow rate of 200 L/h, and at room temperature. It shows how many times are
the permeate flux smaller compared to the water flux. The water flux is the basic parameter for flux comparison with the permete steepwater flux. It is evident that the permeate
flux of steepwater is 5-10 times reduced at transmembrane pressures of 1-3 bars compared to the water flux. Fig. 1 shows just the preliminary experiments at the first few minutes of the microfiltration.
After this experiment, the main experiments were started based on a full 23 factorial
design. At each combination of pressure and flow rate, the microfiltration were stopped
after cca. 3 hours. Figures 2, 3, and 4 show the results of these experiments.
1600
1400
3200
distilled water
steepwater
Q=200 l/h
M 100 nm
NSM
2800
2
1800
Flux [L/m h]
2
Flux [L/m h]
2000
1200
2400
2000
1000
1600
800
1200
600
800
400
400
200
0
distilled water
steepwater
Q=200 l/h
M 100 nm
SM
0
1.0
1.5
2.0
2.5
3.0
P [bar]
1.0
1.5
2.0
2.5
3.0
P [bar]
Figure 1. Dependence of the fluxes of distilled water and steepwater permeate on the
transmembrane pressure in the microfiltration on the ceramic membrane with pore sizes
of 100 nm at flow rate of 200 L/h, at room temperature with (SM) and without (NSM) the
static mixer
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The results of fitting the experimental values of the permeate flux after 2.5 hours of
microfiltration of the second-order polynomial are shown in Table 2.
The second-order polynomials (flux as a function of the pressure and flow rate) [2]
and [3] stand for the case without and with the use of the static mixer, respectively:
JNSM= 18.74-23.1617·P+0.3838·Q+2.725·P2+0.0264·P·Q-0.0015·Q2
[2]
JSM= 199.0367-69.175·P-1.2737·Q+11.596·P2+0.0264·P·Q+0.0044·Q2
[3]
They approximate well the experimental results for the system without (R2 =0.85) and
with static mixer (R2 =0.98). The relatively high values of R2 obtained for all responses
indicate good fit of the experimental data to equation (12). The closer the value of R2 to
the unity, the better the empirical model fits the actual data (16). The significance of each
coefficient was determined through the t-values. The larger the magnitude of the t-value
the more significant is the corresponding coefficient. The polynomial model tested for the
selected responses were significant at the 95% confidence level (p-value; 0.05, Table 2).
Table 2. Results of fitting the experiemntal values of the permeate flux,
after 2.5 hours of microfiltration
Factor
b0
b1
b2
b11
b22
b12
R2
Without static mixer
value
t-value
18.7400
0.33969
-23.1617
-0.93401
0.3838
0.57054
2.7250
0.50156
-0.0015
-0.68931
0.0264
0.34360
0.85
With static mixer
value
t-value
199.0367
4.72421
-69.1750
-3.68837
-1.2737
-2.50324
11.5960
2.77319
0.0044
2.70383
0.0264
0.45431
0.98
In order to facilitate comparisons of the significance of individual coefficients, they
were expressed as a fraction of the largest t-values of the observed correlation (17). The
significance of individual coefficients of average permeate flux correlation with or without static mixers are shown in Fig. 2. The most important linear factor influencing permeate flux during the 2.5 hours microfiltration without turbulence promoter is the pressure,
as well as in the system with the turbulence promoter. Among the quadratic coefficients
the greatest impact on the microfiltration process in the system without turbulence promoter has the suspension flow rate, while the most significant is quadratic effect of transmembrane pressure in the system with static mixer. The interaction between mentioned
parameters is more important in the system without static mixer.
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100
100
80
80
60
60
40
40
20
20
0
0
b0
b1
b2
b11
b22
significance [%] NSM
b12
b0
b1
b2
b11
b22
b12
significance [%] SM
Figure 2. Significance of the individual coefficient of the average permeate flux
correlation with and without static mixer
Based on the obtained experimental values and using the program Statistica 8.0 a
regression equation was obtained, which best describes the dependence of the flux on the
transmembrane pressure and flow rate, and the graphs depicting two dependent variables
are shown in Fig. 3. In the case without static mixer, the most important linear factor
influencing the permeate flux without turbulence promoter is the pressure and the figure
shows that the highest flux values without static mixer caan be achieved (over 25 L/ m2h)
when the flow rate is held around 150 L/h and the transmembrane pressure under 2 bars.
Figure 3. Dependence of the steepwater permeate flux on the transmembrane pressure
and flow rate for the microfiltration without static mixer
It could be expected that the permeate flux would increase with the transmembrane
pressure. However, there is a negative effect of a higher transmembrane pressure: the
cake layer may become more compact as the transmembrane pressure increases, leading
to a greater flux reduction (16). At higher steepwater flow rates, with increasing transmembrane pressure, the permeate flux initially increases, eventually reaching a stationary
value (18). A higher steepwater flow rate results in a higher tangential shear stress and
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the particles on the membrane surface are more unstable (19). Consequently, less cake
mass can be formed under a higher flow rate, which leads to an increase in the average
permeate flux. It can be noticed that with increasing flow rate at all transmembrane
pressures, the average permeate flux in the system without static mixer increases.
Figure 4. Dependence of the steepwater permeate flux on the transmembrane pressure
and flow rate in the microfiltration with static mixer
The effects of the transmembrane pressure and flow rate on the permeate flux in the
system with static mixer are presented in Fig. 4. Evidently, the increases in the transmembrane pressure results in a decreased permeate flux at all flow rates. In the system
with the presence of static mixer a steady state value of the permeate flux is not achieved
with increasing transmembrane pressure, as it is the case in the system without static
mixers. The main reason is that the turbulence promoter allows the creation of the
secondary flow to improve mass transfer and mixing of fluids near the surface membrane,
which also reduces the deposition (7). On comparing the values of permeate flux in the
system with and without static mixers it can be seen that the increase in the permeate
flux, achieved by increasing the transmembrane pressure at low flow rates and pressures
is around 300%. The static mixer as turbulence promoter provides a high-speed flow and
better mixing, and allows slower deposition of particles on the membrane surface and
reduces the thickness of the cake (20). With the increase of steepwater flow rate, the
permeate flux increases at all transmembrane pressures, but the flux increase is more
evident increase at lower transmembrane pressures. At higher pressures, it may happen
that some particles from steepwater penetrate into the membrane pores, decreasing thus
the mixing effects.
Fig. 5 clearly illustrates the flux decline during the time of microfiltration with and
without the use of a static mixer under the same operating conditions. The flux decline
without static mixer is very fast, and can be described by the following equation [4]:
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[4]
J NSM  32.05  479.81  e -t 0.84 
where the R is 0.98. From the picture, it can be seen that after just 5 minutes the flux
declines from 352 to 50 l/m2h. After that, until 225 minute the declination is slower, but it
ends with 20 l/m2h. The use of a static mixer during the ultrafiltration is much better. The
flux decline is slower (at 129 minute the flux reaches 55 l/m2h). The flux decline with the
use of static mixer can be described by equation [5], where the R2 is 0,99:
2
J SM  28.06  371.75  e -t 44.49 
[5]
400
J (NSM, 2 bar, 150 l/h)
J (SM, 2 bar, 150 l/h)
350
2
J (l/m h)
300
250
200
150
100
50
0
0
50
100
150
200
250
300
350
t (min)
Figure 5. Time dependence of the steepwater permeate flux with (SM) and without
(NSM) the use of the static mixer under the same operating conditions
The dry matter content of the retentate increased in average by 10%, and the dry matter content of the permeate decreased by about 20–40%, depending on the microfiltration
mode (NSM or SM). This means that during the steepwater microfiltration permeate contains 40% less particles, and thus it can be considered for the use as recicled water in the
corn starch production.
CONCLUSIONS
On the basis of the study of the effects of the steepwater microfiltration conditions,
the following conclusions can be drawn:
 The permeate flux of steepwater is lower by 5-10 times compared to the water flux.
Such an effect can be ascribed to the increased adsorption and adhesion of particles and
solutes on the membrane, which leads to an effective decrease in the diameter of the
pores and a decline in the permeate flux. Such a change, i.e. flux decline, is explained
by the concentration polarization and the formation of a layer containing wastewater
compounds on the membrane surface.
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 After 2,5 hours of microfiltration without static mixer, the maximum value of the
permeate flux (25 lm-2h-1) was achieved at the pressure under 2 bars and the flow rate
around 150 lh-1.
 By using static mixer, the maximal flux is around 90 lm-2h-1, which is almost 4 times
higher than the flux value reached in the system without a static mixer.
 The dry matter content of the retentate increases in average by 10%, and the dry matter
content of the permeate is lowered by about 20–40%, depending on the microfiltration
mode (NSM or SM)
Acknowledgement
The authors acknowledge the financial support of the Secretariat for Science and
Technological Development of the Province of Vojvodina through the project „Cookies
and crackers with functional characteristics with special dietary needs“.
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УТИЦАЈ РАДНИХ ПАРАМЕТАРА НА ФЛУКС ПЕРМЕАТА ТОКОМ
МИКРОФИЛТРАЦИЈЕ ВОДЕ ОД МОЧЕЊА У ИНДУСТРИЈИ
СКРОБА
Зита И. Шереш a, Љубица П. Докић a, Биљана С. Пајин a, Драгана М. Шороња
Симовић a, Драго Шубарић б, Јурислав Бабић б и Александар З. Фиштеш a
a
б
Свеучилиште Јосипа Јурја Штросмајера, Факултет Прехрамбене технологије, Фрање Кухача 18, 31000
Осијек, Хрватска
У овом раду се испитује микорфилтрација воде од мочења при добијању кукурузног скроба. На овај начин би се смањила потрошња свеже воде током технолошког процеса, а нутријенти из воде од мочења би се могли користити као сточна
храна. За регенерацију нутријената из воде од мочења се користила керамичка мембрана са отворима пора од 100 nm. Током експеримената се пратио утицај трансмембранског притиска и протока на зависне параметре, који су флукс пермеата и
сува материја пермеата и ретентата. Други циљ рада је да се прати утицај статичког
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мешача на флукс пермеата током микрофилтрације воде од мочења. Статистичком
анализом експерименталних података су се утврдили оптимални услови микрофилтрације воде од мочења. Максимална вредност флукса пермеата која се добија без
употребе статичког мешача износи 25 l/m2h, и постиже се при трансмембранском
притиску од 2 бара и при протоку од 100 l/h. Уз употребу статичког мешача постиже се флукс пермеата за 2,5 пута већи од вредности флукса постигнутом без мешача. Након 2,5 сата микрофилтрације пермеат садржи за 40% мање суве материје у
односу на полазну сировину, воду од мочења.
Кључне речи: вода од мочења, микрофилтрација, керамичка мембрана
Received: 24 April 2012
Accepted: 25 Septemбer 2012
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ANTIBACTERIAL ACTIVITY OF LEMON, CARAWAY AND BASIL
EXTRACTS ON LISTERIA SPP.
Gordana R. Dimića*, Sunčica D. Kocić-Tanackova, Olivera O. Jovanovb, Dragoljub D.
Cvetkovića, Siniša L. Markova and Aleksandra S. Velićanskia 
a
b
Jugoinspekt, Dunavska 23, 21000 Novi Sad, Serbia
Commercial extracts of three spices (lemon, caraway and basil) against Listeria monocytogenes, L. innocua and L. welshimeri were investigated using disc diffusion method.
Lemon and basil extracts inhibited all the organisms at the level of ≥ 5 µl (lemon) and
20 µl (basil). These extracts produced inhibitory zones of 9-19 mm (lemon) and 8-11.5
mm (basil). The extract of caraway showed activity only against L. innocua at the highest
level (20 µl), producing an inhibitory zone of 14.7 mm. Generally, the lemon extract was
the most effective. This extract exhibited greater inhibitory activity against L. monocytogenes, while the basil extract had the strongest effect on L. welshimeri.
KEY WORDS: Antibacterial activity, essential oils of spices, Listeria spp., food
contamination
INTRODUCTION
Food contamination by microorganisms and their development and, hence, its decontamination represent a serious problem. Chemical agents to prevent microbial growth and
various additives that are used in food industries, are considered to be potentially harmful
to human health.
In seeking of possible alternatives antimicrobial activities of compounds of natural
origin that can be found in many plants are currently in progress worldwide. Spices are
aromatic plants that are widely used in the food industry and culinary food preparation
for flavouring. However, their essential oils and extracts can have another role, and that is
to controle the growth of harmful microorganisms. It is necessary the spice to be effective
enough to ensure that the product is safe and also have acceptable both odour and taste.
Numerous studies document the inhibitory effects of some essential oils and extracts
of spices, plants, or their major active constituents on the bacteria Escherichia coli, Aeromonas spp., Enterococcus faecalis, Salmonella enterica Typhimurium, Staphylococcus
aureus, Shigella spp., Bacillus spp., Listeria. monocytogenes, Micrococcus spp., Yersinia
enterocolitica, Pseudomonas aeruginosa, Proteus vulgaris, Streptococcus spp., Lacto* Corresponding author: Gordana R. Dimić, University of Novi Sad, Faculty of Technology, Bulevar cara
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bacillus spp., Enterobacter spp. (1-11). Gram-positive bacteria are generally more sensitive than Gram-negative bacteria (12, 13).
Species of the genus Listeria (gram-positive rod-shaped bacteria) are frequent in the
human environment and their growth at low temperatures and high salt concentrations, in
vacuum packed food and that packaged under modified atmospheres (MAP) could be a
problem, especially if the contamination with pathogenic L. monocytogenes is present.
Also, ready-to-eat (RTE) products are popular and can be consumed without further
cooking. Salads and sandwiches are at risk of contamination because they require much
manual manipulation during their preparation. Even the products that are held at refrigerator temperature during their manufacture, the storage and distribution provide the
opportunity for growth of psychrotolerant pathogens and spoilage bacteria. Hence, the
consumption of the food contaminated with L. monocytogenes can constitute a serious
health risk (listeriosis) to humans. Listeria spp. was isolated from homemade white cheeses from various public bazaars (L. monocytogenes, L. innocua, L. seeligeri, L. grayi, L.
ivanovii and L. welshimeri) (14), from fresh salad vegetables (L. monocytogenes, L.
welshimeri and L. murray) (15), liquid whole eggs ( L. innocua) (16). L. innocua was the
dominant species in fresh chicken meat and chicken products that are examined by
Kosek-Paszkowska et al. (17) and the highest prevalence in food products from supermarkets in Bangkok (18). The results of this study indicate the highest number of cases of
Listeria spp. of contamination in meat, meat products and raw vegetables. Previously, we
have reported that Listeria spp. were frequent in raw chicken, pork, and beef meat (19).
In this work we studied the antibacterial activity of some commercial spice extracts
against pathogenic and nonpathogenic of Listeria species that contaminate food.
EXPERIMENTAL
Spice extracts
Three different commercial extracts, of lemon, caraway and basil, were provided by
ETOL, Celje, Slovenia. The botanical name and main components as typical contents of
the spice samples are listed in Table 1. These extracts are intended for use in the food
industries. Test concentrations for antibacterial examination were 0, 5, 10 and 20 µl.
Table 1. List of spice extracts tested
Common name
Lemon
Caraway
Basil
Scientific name
Citrus limon
Carum carvi
Ocimum basilicum
Plant family
Rutaceae
Apiaceae
Lamiaceae
Main component
Limonene
Carvone
Estragole
Percentage (%)
65.0
70.0
86.7
Test bacteria
The foodborne and spoilage bacteria used as test organisms were L. monocytogenes,
L. innocua and L. welshimeri, all isolated from fresh meat. Until the experiment cultures
of the bacteria were kept on tryptone soy yeast extract (TSYEA) slants agar at 4oC.
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Preparation of the inoculum
Bacterial inoculums were prepared from overnight culture on the TSYEA slant. Cultures were directly suspended in saline and the optical density of the suspension was
measured by MacFarland standards and then the suspensions were diluted to the appropriate bacterial concentrations (106 cells/ml) needed in the experiments, using the same
solution.
Antibacterial assays
In these studies, the disc diffusion method was used. TSYEA was poured in the Petri
plates (9 mm diam) in a thickness of 4 mm. Each of test cultures was inoculated with a
sterile swab by rotating the plate three times for 1/3 rounds between each smear and left
to dry for 10 min at ambient temperature. Sterile discs (diameter of 6 mm), were placed at
the center of each plate, after which the spice extracts were added in the above amounts.
The plates were incubated at 37o C for 48 h. If the organism was more or less sensitive to
the extract, around the disc appeared a clear zone of the growth inhibition. The diameter
of the inhibitory zone was measured in mm. All tests were done in triplicate. The values
are presented as means ± SD of three measurements.
The results of the antibacterial disc diffusion assays are summarized in Table 2.
Table 2. Antibacterial activity of lemon, caraway and basil extracts on the growth of
Listeria spp.
Strain
L. monocytogenes
L. innocua
L. welshimeri
L. monocytogenes
L. innocua
L. welshimeri
L. monocytogenes
L. innocua
L. welshimeri
Quantity of spice extract (µl/disc)
5
10
20
Inhibition zone diameter (mm)
Lemon
9.5 ± 0.26
12.5 ± 0.35
19 ± 0.36
9 ± 0.21
16.9 ± 0.44
17.5 ± 0.26
9.5 ± 0.27
15.5 ± 0.35
18 ± 0.48
Caraway
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
14.7 ± 0.28
0 ± 0.00
0 ± 0.00
0 ± 0.00
Basil
0 ± 0.00
0 ± 0.00
10 ± 0.13
0 ± 0.00
0 ± 0.00
8 ± 0.11
0 ± 0.00
10 ± 0.22
11.5 ± 0.26
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The extracts of lemon, caraway and basil showed inhibitory potential against Listeria
spp., the lemon extract being the most efficient. The inhibitory effect of this extract at the
lowest volume for all three Listeria species was not practically different. The data
indicate that L. innocua was equally sensitive to the increase in the level of oil at 10 and
20 µl, with the inhibition zones of 16.9 and 17.5 mm, respectively. At the level of 20 µl,
L. monocytogenes was the most sensitive, with a slightly larger inhibitory zone (19 mm).
The volumes of the caraway extract less than 20 µl did not affect the growth of the
examined species. With 20 µl, it was effective only against L. innocua. The basil extract
appeared to be a weaker inhibitor of L. innocua, but it was effective against L. welshimeri
and L. monocytogenes.
The study showed that of the different spices tested for their antibacterial effect only
lemon extract inhibited the growth of all bacteria, with L. monocytogenes being the most
and L. innocua the least susceptible, while L. monocytogenes, L. innocua and L. welshimeri showed moderate sensitivity to basil extract. On the other hand, no inhibition zones
of caraway were observed on L. monocytogenes and L. welshimeri. However, the basil
extract was less potent than the caraway extract against L. innocua.
Smith-Palmer et al. (12) examined the inhibitory activity of essential oils of 23 species plant and two essences against some Gram-nagative and Gram-positive bacteria,
including also L. monocytogenes, and they found low activity of pure lemon oil and basil
against L. monocytogenes when they were used in quantities of 25 µl in agar well technique. Fernandez-Lopez et al. (20) showed that the lemon extract (from an industrial byproduct) having a high water content was not active against L. monocytogenes and L.
innocua, as well as against lactic acid bacteria (except for one strain of Lactobacillus
lactis, with an inhibitory zone of 8.5 mm). Friedly et al. (21) studied the sensitivity of L.
monocytogenes and L. innocua to commercial citrus essential oils and the results of the
disc diffusion assay showed inhibition zone diameters of 8.4 and 8.8 mm, for 10 µl of
lemon essential oil. Espina et al. (22) evaluated the antimicrobial effects of three essential
oils of lemon, orange, and mandarin against six microorganisms: L. monocytogenes,
Staphylococcus aureus, Enterococcus faecium, Salmonella Enteritidis, Escherichia coli
O157 : H7 and Pseudomonas aeruginosa. Lemon and orange oils (15 µl/disc) showed
weak/no inhibition zone (< 12 mm) against these microorganisms. The results that were
obtained using broth dilution method showed minimum inhibitory concentraton (MIC) of
lemon oil of 1.0 µl/ml for L. monocytogenes and minimum bactericidal (MBC) > 30
µl/ml. Shan et al. (23) tested the antimicrobial properties of 46 extracts from spices and
herbs and reported that caraway extract had no inhibitory activity against five food-borne
bacteria, L. monocytogenes, B. cereus, S. aureus, E. coli and S. anatum. L. monocytogenes was the most resistant of the tested Gram-positive organisms in this investigation.
Pure oil from caraway and suspensions in ethanol in the ratio of 2:1 and 1:1 showed
microbiostatic effect on L. monocytogenes ATCC 19115 (more sensitive) and L. monocytogenes ATCC 19112, with an inhibitory zone of ≤ 13 mm (24). In several report, basil
essential oil was less effective in inhibiting L. monocytogenes and Pseudomonas aeruginosa compared to the other Gram-positive and Gram-negative bacteria (25). However,
Hossain et al. (26) reported that essential oils and methanol extracts of basil displayed a
great antibacterial activity against L. monocytogenes and other tested bacteria. Nguefack
et al. (27) investigated the inhibitory effects of essential oils from five aromatic plants on
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the strains of L. monocytogenes, L. innocua and S. aureus. L. innocua and three strains L.
monocytogenes were more sensitive to oil isolated from basil Ocimum gratissimum than
Ocimum basilicum, while one strain of L. monocytogenes was resistant to O. basilicum.
Lemon, caraway and basil essential oils and extracts contain many biologically active
compounds that give rise to their antimicrobial actions. The most important active ingredients of lemon are monoterpenes such as limonene, γ-terpinene, β-pinene and the aldehydes geranial and neral (22, 28). Iacobellis et al. (29) determined carvone, limonene,
germacrene D and trans-dihydrocarvone as the main components of caraway. Linalool
was found as a main component in basil oil investigated by Hanif et al. (30). Kocić-Tanackov et al. (31) identified estragol as the major component present in basil extract.
Numerous publications have documented different data on antibacterial activity of the
spice essential oils and extracts. The data are based on different test methods, different
methods of obtaining and composition of oils and extracts and the caracteristics of the
test microorganisms. Although it is not always easy to compare different studies, there
are indications that the application of these natural antimicrobial agents can slow or
prevent the growth of microorganisms (32, 33), and the mechanisms of the action are
being intensively examined. The flow cytometry of L. innocua stained with carboxy fluorescein diacetate showed that essential oil permeabilized the cytoplasmic membrane (27).
CONCLUSION
The results of this study indicate that lemon, caraway and basil extracts have the potential as a natural antimicrobial agents to be used for the future practical application in
preservation of the food from microbiological spoilage and prevention of foodborne
diseases.
Acknowledgement
These results are part of the Project No. TR - 31017 financially supported by the Ministry of Science and Technological Development of the Republic of Serbia.
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АНТИБАКТЕРИЈСКА АКТИВНОСТ ЕКСТРАКАТА ЛИМУНА, КИМА И
БОСИЉКА НА LISTERIA SPP.
Гордана Р. Димића*, Сунчица Д. Коцић-Танацкова, Оливера О. Јовановб, Драгољуб
Д. Цветковића, Синиша Л. Маркова и Александра С. Велићанскиа
a
Универзитет у Новом Саду, Технолошки факултет,Булевар цара Лазара 1, 21000 Нови Сад, Србија
б
Југоинспект, Дунавска 23, 21000 Нови Сад, Србија
Три комерцијална екстракта зачина (лимун, ким и босиљак) против Listeria monocytogenes, L. innocua и L. welshimeri су били испитивани коришћењем диск
дифузионе методе. Екстракти лимуна и босиљка су инхибирали све организме на
нивоима од ≥ 5 µl (лимун) и 20 µl (босиљак). Ови екстракти производили су зоне
инхибиције од 9-19 mm (лимун) и 8-11,5 mm (босиљак). Екстракт кима је показао
активност само против L. innocua на највишем нивоу (20 µl), производећи зону
инхибиције од 14,7 mm. Генерално, екстракт лимуна је био најефективнији. Овај
екстракт је показивао већу инхибиторну активност против L. monocytogenes, док је
екстракт босиљка најјаче деловао на L. welshimeri.
Кључне речи: антибактеријска активност, етарска уља зачина, екстракти, Listeria
spp., контаминација хране
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ANTIFUNGAL ACTIVITY OF THE BASIL (Ocimmum basilicum L.) EXTRACT
ON Penicillium aurantiogriseum, P. glabrum, P. chrysogenum,
AND P. brevicompactum
Sunčica D. Kocić-Tanackova*, Gordana R. Dimića, Dušanka J. Pejin,
Ljiljana V. Mojovićb, Jelena D. Pejina and Ilija J. Tanackovc 
a
b
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000, Belgrade, Serbia
c
University of Novi Sad, Faculty of Technical Sciences, Trg Dositeja Obradovića 6,
21000 Novi Sad, Serbia
This study was aimed at investigating the antifungal potential of basil (Ocimmum
basilicum L.) extract against toxin-producing Penicillium spp. (P. aurantiogriseum, P.
glabrum, P. chrysogenum, and P. brevicompactum) isolated from food.
The basil extract composition was determined by the GC-MS method. The major component identified in the extract was estragole (86.72%).
The determination of the antifungal activity of basil extract on Penicillium spp. was
performed using the agar plate method. Basil extract reduced the growth of Penicillium
spp. at all applied concentration levels (0.16, 0.35, 0.70, and 1.50 mL/100mL) with the
colony growth inhibition from 3.6 (for P. glabrum) to 100% (for P. chrysogenum).
The highest sensitivity showed P. chrysogenum, where the growth was completely inhibited at the basil extract concentration of 1.50 mL/100mL. The growth of other Penicillium spp. was partially inhibited with the colony growth inhibition of 63.4 % (P. brevicompactum), 67.5% (P. aurantiogriseum), and 71.7% (P. glabrum).
Higher concentrations (0.70 and 1.50 mL/100mL) reduced the growth of the aerial
mycelium of all tested Penicillium species. In addition, at the same extract concentrations, the examination of microscopic preparation showed the deformation of hyphae with
the frequent occurrence of fragmentations and thickenings, occurrence of irregular vesicle, frequently without metulae and phialides, enlarged metulae.
The results obtained in this investigation point to the possibility of using basil extract
for the antifungal food protection.
KEY WORDS: Basil extract, antifungal activity, Penicillium spp., food
* Corresponding author: Sunčica D. Kocić-Tanackov, University of Novi Sad, Faculty of Technology, Bulevar
cara Lazara 1, 21 000 Novi Sad, Serbia, e-mail: [email protected]
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INTRODUCTION
Penicillium species are videly spread in the nature and are frequent contaminants of
food. Food products with the medium (0.75 - 0.9 aw) and low (0.75 aw) moisture content,
as well as the acid medium favour their development. These species are frequent contaminants of storage products such as fruits, vegetables, spices, sausages, cheese, grain and
grain products (flour, bread, cakes), etc. (1-3).
The metabolic activity of this species results in spoilage of food products and high
economic damage. On the other hand, the toxin-producing species (P. aurantiogriseum,
P. brevicimpactum, P. chrysogenum, P. glabrum, P. expansum, P. nordicum, P. rugulosum, P. solitum, P. verrucosum, etc.) biosynthesize toxic metabolites – mycotoxins (ochratoxin A, roquefortine C, patulin, citrinin, nephrotoxic glycopeptides, verrucosidin, citromycetin, botryodiploidin, mycophenolic acid, etc.). The consumption of food products
contaminated with these mycotoxins, both by human and animal, leads to the occurrence
of mycotoxicoses, which, further, have cytotoxic and cancerogenic effects on human cells
(liver cells in the first place) (1-5).
Extracts and essential oils extracted from spices and other herbs, as well as their biologically active components, have attracted of attention many authors to investigate their
antimicrobic activity (6-9). In food production and processing, the extracts and essential
oils obtained from spices and other herbs are important in the food prevention from microorganisms, especially of short shelf-life products, which are the most sensitive to microbiologic spoilage (bread, bakery products, cakes, salads, fresh fruit and vegetable).
Their use in the food industry decreases the use of synthetic preservers and additives,
and, at the same time, improves the freshness and sensory of the product quality.
The paper presents а study of the antifungal effect of basil extract on the growth of
toxin-producing Penicillium species isolated from food.
EXPERIMENTAL
Basil extract
For the testing of the antifungal activity, a commercially available, food grade basil
extract was provided from ETOL Tovarna arom in eteričnih olj d.d., Celje, Slovenia.
Determination of basil extract composition
The composition of the extracts was determined by Gas Chromatography – Mass
Spectrometry (GC-MS) analysis carried out on a Varian T2100 GC-MS instrument equipped with data processor. A fused silica capillary column VF-5MS (30 m x 0.25 mm i.d.,
0.25 m film thickness, Varian) was used for the separation of the sample components.
The carrier gas, ultra pure helium, was passed through moisture and oxygen traps at a
constant flow rate of 0.62 cm3/min. The following temperature program was used: injector temperature 230°C, initial temperature 40°C (held 5 min), temperature increase
5°C/min to 200 °C and held at this temperature for 25 min. The mass spectrometer was
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operated in the electron ionization mode. The data acquisition was carried out in the scan
mode (range 50-550 m/z). The injection volume was 1 μl. The compounds were identified by matching the mass spectra with NIST Mass Spectra Library stored in the GC-MS
database.
Fungal strains
The following fungal strains from the genus Penicillium were used as test microorganisms: P. aurantiogriseum Dierckx, P. glabrum (Wehmer) Westling, P. chrysogenum
Thom, and P. brevicompactum Dierckx. The fungal cultures were isolated from cakes
and fresh salads from different varieties of ready-for-use vegetables and maintained on
Potato Dextrose Agar (PDA) (Merck, Darmstadt) at 4 C as a part of the collection of the
Laboratory for Food Microbiology at the Faculty of Technology, University of Novi Sad,
Serbia.
Determination of the basil extract effect on the Penicillium spp. growth
The agar plate method was applied to test the antifungal activity of basil extracts. The
basic medium for the antifungal tests was PDA. The medium was divided into equal
volumes (150 mL), poured into Erlenmeyer flasks (volume 250 mL) and autoclaved at
121°C for 15 min, and cooled to 45C. The extracts were added to the PDA to achieve
the following concentrations: 0, 0.16, 0.35, 0.70, and 1.50 mL/100mL. The PDA containning different concentrations of basil extract was poured into sterile Petri dishes ( 9 cm),
12 mL per dish.
The seven-day fungal cultures grown on PDA were used to prepare the fungal spore
suspension tests. Suspensions of the fungi were prepared in a medium containing 0.5%
Tween 80 and 0.2% agar dissolved in distilled water and were adjusted to provide initial
spore count of 106 spores/mL by using a haemocytometer. For each extract dose and
fungal species, including the controls, the dishes were centrally inoculated by spreading 1
µL of a spore suspension (103 spores/mL), using an inoculation needle. After the inoculation, the Petri dishes were closed with parafilm.
The effect of the basil extract on fungal growth was evaluated by a daily measurement
of the diameter of the radial colony growth during 14 days of incubation at 252ºC. The
parafilm was removed from the Petri dishes in which no colony growth was observed
after 14 days, and the dishes were further incubated for 16 days (30 days in total) at
252°C. In the Petri dishes in which fungal growth was observed from 15th to 30th day,
the concentration of basil extract used was considered to be the minimal inhibitory concentration (MIC). If there was no visible fungal growth after 30 days, the fungal spores
were transferred using a wet cotton baton to the PDA in which no basil extract was added, and were incubated for 5 days at 252 ºC for the determination of fungicide effect
(MFC).
The inhibitory effect of the basil extract on fungal growth after 14 days was calculated from the following formula:
I (%) = (C-T)/C100
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where I is the inhibition (%), C is the colony diameter in the control dish (cm) and T is
the colony diameter in the test dish (cm) (10).
Each antifungal test was carried out in 3 series (2 replications in each series). Values
are presented as means±SD of six measurements.
The changes in the macroscopic and microscopic features of the fungi were also observed and compared to the controls. The macroscopic features of spores were observed
using a binocular, magnifying glass Technival 2, Carl Zeiss and the microscopic features
using a microscope Aristoplan, Leitz.
Colony diameter (cm)
4
0.00 m L/100m L
0.35 m L/100m L
1.50 m L/100m L
3
0.16 m L/100m L
0.70 m L/100m L
2
1
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Colony growth inhibition (%)
The basil extract at the investigated concentrations exhibited the capacity to reduce or
inhibit the growth of the Penicillium species. The effect of the basil extract on the Penicillium spp. growth is presented in Figures 1-4. Table 1 shows the inhibitory effect (%) of
the basil extract on the colony growth of Penicillium species on the 14th day of incubation.
The concentrations of basil extract, 0.7 and 1.5 mL/100mL resulted in the delay or no
growth of investigated moulds with different inhibitory effect on growth rate decline. The
extract concentration of 0.7 mL/100 mL delayed the growth of P. aurantiogriseum, P.
glabrum and P. brevicompactum for 1 day. The germination of P. chrysogenum at this
concentration was noticed on the first day. The highest applied concentration (1.5 mL/
100 mL) was minimal fungicidal concentration (MFC) for P. chrysogenum, while the
growth of other molds was delayed by 4 (P. aurantiogriseum), 7 (P. brevicompactum),
and 8 (P. glabrum) days (Figures 1-4). The growth rate decline with the increase of the
basil extract content in the PDA medium was more expressed in P. aurantiogriseum and
P. chrysogenum, compared to the other two molds, pointing to their higher sensitivity (Figures 1-4).
0.16 m L/100m L
0.70 m L/100m L
100
0.35 m L/100m L
1.50 m L/100m L
80
60
40
20
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Time (days)
Time (days)
(a)
(b)
Figure 1. The influence of basil extract on the growth rate (a) and colony growth
inhibition (b) of P. aurantiogriseum
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5
0.00 m L/100m L
0.16 m L/100m L
0.35 m L/100m L
0.70 m L/100m L
1.50 m L/100m L
0.16 m L/100m L
0.70 m L/100m L
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4
3
2
1
0
1
2
3
4
5
6
7
8
0.35 m L/100m L
1.50 m L/100m L
100
80
60
40
20
0
1
9 10 11 12 13 14
2
3
4
5
6
7
8
9 10 11 12 13 14
Time (days)
Time (days)
(a)
(b)
inhibition (b) of P. glabrum
0.00 m L/100m L
0.16 m L/100m L
0.35 m L/100m L
0.70 m L/100m L
1.50 m L/100m L
0.16 m L/100m L
0.70 m L/100m L
3
2
1
0
1
2
3
4
5
6
7
8
0.35 m L/100m L
1.50 m L/100m L
100
80
60
40
20
0
1
9 10 11 12 13 14
2
3
4
5
6
7
8
9 10 11 12 13 14
Time (days)
Time (days)
(a)
(b)
inhibition (b) of P. chrysogenum
0.00 m L/100m L
0.6 m L/100m L
0.35 m L/100m L
0.70 m L/100m L
3
1.50 m L/100m L
2
1
0
1
2
3
4
5 6 7 8
Time (days)
(a)
9 10 11 12 13 14
0.16 m L/100m L
0.70 m L/100m L
100
0.35 m L/100m L
1.50 m L/100m L
80
60
40
20
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Time (days)
(b)
inhibition (b) of P. brevicompactum
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The lowest applied basil extract concentration (0.16 mL/100mL) inhibited the growth
of all investigated fungi at a low inhibition level, from 3.6 to 9,8%. The weakest antifungal effect of the extract at this concentration was observed against P. glabrum, with the
inhibition rate of 3.6%, while the growth of other fungi was decreased by 4.3% (P. chrysogenum), 7.3% (P. aurantiogriseum), and 9.8% (P. brevicompactum). The increased extract concentration (0.35 mL/100mL) in agar medium affected the increase of the growth
inhibition of the investigated fungi from 9.5% (P. chrysogenum) to 18.7% (P. aurantiogriseum). The growth of P. aurantiogriseum was inhibited significantly (44.7%) at the
concentration of 0.7 mL/100mL, and the reaction of the other investigated fungi was similar, with the inhibition degree from 19.9% (P. glabrum) and 26.7% (P. chrysogenum)
(Table 1).
The strongest antifungal effect at the highest applied extract concentration (1.5 mL/
100mL) was observed for P. chrysogenum. The growth of this fungi was completely
stopped at this concentration, while in the other fungi, the growth was partially inhibited
with high inhibition degree of 63.4% (P. brevicompactum) and 71.7% (P. aurantiogriseum) (Table 1).
Table 1. Inhibitory effect (%) of basil extract on the colony growth of Penicillium
species after 14 days of incubation
Fungi
P. aurantiogriseum
P. glabrum
P. chrysogenum
P. brevicompactum
Basil extract concentration (mL/100mL)
0.16
0.35
0.70
1.50
7.3
18.7
44.7
67.5
3.6
13.8
19.9
71.7
4.3
9.5
26.7
100.0
9.8
13.4
20.5
63.4
These results are in accordance with our previous investigations, showing a strong antifungal activity of basil extract towards Fusarium spp. (F. oxysporum, F. proliferatum,
F. subglutinans, and F. verticillioides) isolated from cakes. Their growth was completely
inhibited at the extract concentration of 1.5 mL/100mL (11). The antifungal investigations performed by Adigüzel et al. (12) by disc-difusion method are in contrast to our findings. They reported the inefficiency of the ethanol, methanol and hexane basil extracts
on the growth of Alternaria aternata, Aspergillus flavus, F. oxysporum and Penicillium
spp. at the concentrations of 300 µg/disc.
However, a number of studies report on strong antifungal action of basil essential oil.
Doube et al. (13) using the agar plate method, showed that basil oil, in a concentration of
1.5 mL/L inhibited completely the growth of 22 species of molds, including the aflatoxigenic strains Aspergillus parasiticus and A. flavus.
Zollo et al. (14) reported that basil oil inhibited completely the growth of Candida
albicans and A. flavus at a concentration of 5000 ppm, during 7 days of incubation, using
microdilution method. Soliman and Badeaa (15) found that basil oil acts as a fungistatic
agent against F. verticillioides in a concentration of 2000 ppm, and as a fungicid agent in
concentration of 3000 ppm. The results presented by Fandohan et al. (16) showed a complete growth inhibition of F. verticillioides at concentrations higher than 2.7 µL/mL.
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The antifungal activity of tested basil extract depends on the content of major and minor components. The content of estragol (methyl chavicol) was the highest in the investigated basil extract (86.72%). Besides estragol the basil extract contained: trans-alfa-bergamotene (2.91%), eucalyptol (2.67%), trans-ocimene (1.04%), cadinol (0.74%), linalol
– syn. linalool (0.72%), methyl-eugenol (0.71%), δ-cadinene (0.49%), camphor (0.42%),
β-elemene (0.37%), δ-guaiene (0.29%), menthol (0.27%), β-pinene (0.23%), limonene
(0.22%), bornyl acetate (0.21%), α-pinene (0.16%), β-caryophyllene (0.15%), myrcene
(0.15%), sabinene (0.13%), fenchone (0.13%), γ-muurolene (0.12%), borneol (0.10%),
menthone (0.10%), β-sesquiphelladrene (0.10%), cubenol (0.11%), carvone (0.07%), βselinene (0.07%), cis-α-bergamotene (0.06%), camphene (0.05%), cis-ocimene (0.05%),
p-cymene (0.05%), α-humulene (0.04%), aromadendrene (0.03%), terpinolene (0.02%),
γ-terpinene (0.02%), α-guaiene (0.01%). The majority of authors address linalol, estragol,
eugenol and methyl cinnamate as the major antimicrobial components of basil extracts
and essential oil (17-23). The study performed by Lis-Balchin et al. (18) point to a strong
antifungal effect of oil which contained estragol as the main component on the growth of
Aspergillus niger, A. ochraceus, and Fusarium culmorum (inhibition growth of 71.0 to
94.76%). These results are in accordance with the results and investigations of Baratta et
al. (19), who found that estragol type oil inhibited the growth of A. niger by 93.1%. Reuveni et al. (17) investigated the effect of eteric basil oil components on the growth of Rhizopus nigricans and F. oxysporum. They found linalol and estragol to be more efficient
against R. nigricans (100% of inhibition), compared to eugenol (38.1% of inhibition). Eugenol exhibited stronger inhibition towards F. oxysporum (100% of inhibition), in contrast to linalol and estragol, where the inhibition values were 26.4 and 30.3%, respectively.
Besides the effect of growth reduction, the investigated extract at higher concentrations caused the macro- and micromorphologicl changes of the fungi. The extract concentrations of 0.70 and 1.5 mL/100mL reduced the growth of the aerial mycelium in all tested species. In addition, at the same extract concentrations, the examination of the microscopic preparation showed deformation of hyphae, with frequent occurrence of fragmentations and thickenings, occurrence of irregular vesicle, frequently without metulae and
phialides, enlarged metulae. These macro- and micromorphologic changes point to the
possible changes at cellular level (reduction in the cellular growth, decrease in the oxygen
uptake, inhibition of the synthesis of lipids, proteins and nucleic acids, changes in the lipid profile of the cell membrane and inhibition of the synthesis of the fungal cell wall)
due to the action of extract components with functional groups of cellular enzymes (2325).
CONCLUSIONS
This study proved that the tested basil extract can be used as a protective agent against
Penicillium spp, frequent contaminants of food.
Acknowledgement
The study is a part of the investigations realized within the scope of the Project No. TR31017, financially supported by the Ministry of Education, Science, and Technological
Development of the Republic of Serbia.
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АНТИФУНГАЛНА АКТИВНОСТ ЕКСТРАКТА БОСИЉКА (Ocimmum
basilicum L.) НА РАСТ Penicillium aurantiogriseum, P. glabrum, P. chrysogenum
И P. brevicompactum
Сунчица Д. Коцић-Танацкова*, Гордана Р. Димића, Душанка Ј. Пејина, Љиљана В.
Мојовићб, Јелена Д. Пејина и Илија Ј. Танацковв
а
в
Универзитет у Новом Саду, Факултет техничких наука, Трг Доситеја Обрадовића 6, 21000 Нови Сад,
Србија
б
Оваj рад приказује антифунгални потенцијал екстракта босиљка на раст токсинпродукујућих Penicillium врста (P. aurantiogriseum, P. glabrum, P. chrysogenum и P.
brevicompactum) изолованих из хране.
Састав екстракта босиљка одређен је GC-МS методом и у највећем проценту садржавао је естрагол (86,72%). Утицај екстракта на раст плесни испитан је методом
агар плоча. При свим примењеним концентрација екстракта (0,16; 0,35; 0,70 и 1,50
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mL/100mL) редукован је раст Penicillium spp. уз инхибицију раста колонија од 3,6
(P. glabrum) до 100% (P. chrysogenum).
Највећу осетљивост показала је плесан P. chrysogenum. Њен раст је био потпуно
спречен при концентрацији од 1,5 mL/100mL. Раст осталих Penicillium spp. је делимично инхибиран са високим процентом инхибиције раста колоније од 63,4 (P. brevicompactum), 67,5 (P. aurantiogriseum) и 71,7% (P. glabrum). Више концентрације
екстракта (0,70 и 1,5 mL/100mL) су редуковале мицеларни раст свих тестираних
врста. Такође, при овим концентрацијама су у микроскопском препарату уочене деформације хифа са честом фрагментацијом и задебљањима, везикуле неправилног
облика, појава проширених метула, али често без метула и фијалида.
Добијени резултати указују на могућност коришћења екстракта босиљка у антифунгалној заштити хране.
Кључне речи: Екстракт босиљка, антифунгална активност, Penicillium spp., храна
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CHARACTERIZATION OF ANTIOXIDANT AND ANTIMICROBIAL
ACTIVITIES OF NETTLE LEAVES (Urtica dioica L.)
Zoran Z. Kukrića*, Ljiljana N. Topalić-Trivunovića, Biljana M. Kukavicab,
Snježana B. Matoša, Svetlana S. Pavičića, Mirela M. Borojab and Aleksandar V. Savića 
a
University of Banja Luka, Faculty of Technology, Vojvode Stepe Stepanovića 75, 78000 Banja Luka,
Republic of Srpska, BiH
b
University of Banja Luka, Faculty of Natural Sciences and Mathematics, Mladena Stojanovića 2, 78000
Banja Luka. Republic of Srpska, BiH
Samples of stinging nettle or common nettle (Urtica dioica L.) were collected from
the area of Banja Luka. To measure and evaluate the content of chlorophyll (a and b),
carotenoids, and soluble proteins, as well as peroxidase activity (POD, EC 1.11.1.7.),
fresh nettle leaves of different developmental stages were used. Dried nettle leaves were
used to obtain ethanol extract. The dry residue of ethanol extract was dissolved in methanol and the obtained solution was used to determine the content of total phenols, flavonoids, flavonols, as well as non-enzymatic antioxidant activity and antimicrobial activity.
The non-enzymatic antioxidant activity was determined by different methods: FRAP,
DPPH, and ABTS. The results were compared to those of standard substances like
vitamin C, BHT, and BHA. Antimicrobial activity was screened by using macrodilution
method.
The obtained results showed insignificantly higher content of chlorophyll, carotenoids, and proteins in young nettle leaves as well as an increase in the soluble peroxidase
activities. Native electrophoresis of the soluble fraction showed the presence of two peroxidase isophorms in the soluble protein fraction of nettle leaves. The total phenolic
content in nettle extracts amounted to 208.37 mg GAE/gdw,, the content of total flavonoids
was 20.29 mg QE/gdw, and the content of total flavonols was 22.83 mg QE/gdw. The
antioxidant activity determined by FRAP method was 7.50 mM Fe(II)/gdw, whereas the
antioxidant activity measured by using DPPH and ABTS methods, with IC50 values, were
31.38 and 23.55 μg mL-1, respectively. These results showed the weak and moderate antioxidant capacity of stinging nettle.
Extract of Urtica dioica L. was tested for antibacterial acivity against various Grampositive and Gram-negative bacteria: Bacillus subtilis IP 5832, Lactobacillus plantarum
299v (Lp299v), Pseudomonas aeruginosa, and Escherichia coli isolated from food and
Escherichia coli isolated from urine samples. Ampicillin, erythromycin, ciprofloxacin,
and gentamicin were used as positive control. The results showed that minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract
ranged from 9.05 to more than 149.93 mg mL-1.
* Corresponing author: Zoran Z. Kukrić, University of Banja Luka, Faculty of Technology, Vojvode Stepe
Stepanovića 75, 78000 Banja Luka, Republika Srpska, BiH,
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KEY WORDS: Urtica dioica L., content of chlorophyll (a and b), carotenoids and soluble proteins, total phenols, flavonoids and flavonols, antioxidant and
antimicrobial activity
INTRODUCTION
Common nettle (Urtica dioica L), a herbaceous perennial flowering plant, is a member of the Urticaceae family. Traditional herbal medicine in the Balkan countries uses
stinging nettle leaves in the form of an herbal infusion as a remedy for the treatment of
diarrhea, vaginal discharge, internal/external bleeding (1). Being rich in chlorophyll, nettle leaves are used for the treatment of anemia as well as general well-being, and more
recently as natural food colorant. The nettle extract is a common ingredient in cosmetics,
e.g. in shampoos and hair growth lotions.
The stinging nettle leaf contains chlorophyll, vitamin C, vitamin K, panthotene acid,
carotenoids, B group vitamins (B1 and B2), tannins, essential oil, proteins, and minerals
(Fe, Cu, Mn and Ni) (2). Stinging nettle hairs contain acethylcholin and histamine (1),
while the stem and root contain flavonoids (3,4).
Animal studies proved that nettle leaf extract may inhibit blood clotting (platelet
aggregation), can decrease total cholesterol levels as well as enhance the overall liver
function (5,6,7). Water extract of stinging nettle makes significant inhibition of adenosine
deaminase activity in prostate tissue in the patients with prostate cancer (8). Adding dried
powder of nettle into laying hens diets significantly increases egg production, proves the
modulating effects of the immune parameters (9), and lowers the total cholesterol and
triglycerides concentration (10). It has also been reported that the stinging nettle extract
exibits antioxidant, antimicrobial, antiulcer, and analgesic activities. (11)
The aim of the present study is to evaluate the total, non-enzymatic and enzymatic,
antioxidant capacities of ethanol nettle extract as well as its antimicrobial activity.
EXPERIMENTAL
Materials and instruments
All reagents used in this research were of pro analysis grade. Folin-Ciocalteu; DPPH;
TPTZ (Sigma Chemical Co., St. Luis, USA); Gallic acid; BHT; BHA; AlCl3; Quercetin
hydrate (Acros, New Jersey, USA); Vitamin C; K2S2O8 (Merck, Darmstadt, Germany);
NaHCO3; FeCl3 x 6 H2O; FeSO4 x 7H2O (Lach-Ner, s.r.o., Czech Republic); Sodium acetate (Zorka Pharma a.d., Šabac, Serbia); Pyrogallol; TRIS (Carlo Erba Reagent, Italy); 4chloro-α-naphthol; Ammonium persulfate; PMSF (Sigma, Germany); Riboflavin; TEMED (Semikem, B&H); Glicine; NaOH (Lach-Ner, Czech Repulic); NaH2PO4 (Centrohem, Serbia); H2O2 (Sineks Laboratory, B&H); Acrylamide and bis-acrylamide (Carl
Roth GmbH, Germany). Antibiotics: Ampyciline; Erytromicine; Ciprofloxacine (HemofarminfectoLogica, Vršac, Serbia); Gentamicin (Alkaloid Skopje, FJR Macedonia);
culture media: Mueller Hinton broth (MHB) i Mueller Hinton agar plate (MHA) (Liofilchem, Italy). The bacterial cultures used in this study: Bacillus subtilis IP 5832 („Diastop“, Alkaloid, Skopje, FJR Macedonia); Lactobacillus plantarum 299v (Lp299v) („Flo258
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bion“, Abela Pharm in cooperation with Probi AB and Insitut Rosell, Italy); Pseudomonas aeruginosa and Escherichia coli isolated from food (Veterinary Institute “Vaso Butozan”, Banja Luka, Republic of Srpska, B&H) and Escherichia coli isolated from urine
samples (Institute for Health Protection of Republic Srpska, B&H).
Measurements were performed on the following instruments: 6305 UV-VIS spectrophotometer equipped with a thermal cell (Jenway, England), UV-VIS Spectrophotometer
(Shimadzu, Japan), Electrophoresis (BioRad, Germany), and pH meter (Hanna, USA).
Preparation of plant material
Top fresh nettle (Urtica diocia L.) leaves were collected for the purposes of this study from the Laktaši forest community in June of 2010. The plant material was authenticated by Dr Ljiljana Topalić-Trivunović of the Faculty of Technology, University of Banja Luka. The preparation of plant material was performed according to the following
analysis:
a) for the determination of protein solubility and peroxidase activity
Top nettle leaves (L1) and the first pair of leaves below the top (L2) were used in
the experiment (Figure 1). To obtain soluble proteins, the nettle leaves were airdried, chopped in small pieces and ground to powder with liquid nitrogen and
homogenized in 0.1 M sodium-phosphate buffer, pH 6.4 containing 1 mM phenylmethyl-sulfonyl fluoride (PMSF). The homogenate was centrifuged at 4000 x
g for 15 min at 4 ºC. After centrifugation, the supernatant was separated and marked as 'soluble protein fraction'.
b) for determination of chlorophyll and carotenoids
Chlorophyll a and b and carotenoids were extracted from the fresh nettle leaves in
acetone (0.5 g of the plant material in 5 mL acetone). After the centrifugation at
3000 rpm for 15 min, the supernatant was used and the absorbance of extract was
measured at 662 nm, 644 nm and 440 nm.
c) for determination of total phenols, flavonoids, and flavonols, non-enzymatic antioxidant and antimicrobial activity
The collected nettle leaves were dried at room temperature at a draft, protected
from direct light, for a few days. Then they were chopped up and stored until used
in a glass jar. The air-dried and ground material (20 g) was extracted (three times)
with 100 mL of 80% ethanol (v/v) at 25 oC, in ultrasonic bath (30 Hz) for 5
minutes. After that, the solution was continuously stirred for 30 minutes at the same temperature and then filtered. The combined extract was evaporated to dryness under reduced pressure at 40 oC. The obtained resin-like dry extract was dissolved in methanol and the contents of total phenols, flavonoids, and flavonols
were determined as well as antioxidant and antimicrobial activity.
Determination of soluble proteins, chlorophyll and carotenoids
Protein content was determined according to Lowry (12). The concentrations of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids were calculated according to
Holm (13) and Van Wattstein (14).
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Determination of total phenolic content
Total phenols were determined by a modified Folin-Ciocalteu method (15). The measurement was conducted by mixing 1.5 mL of working Folin-Ciocalteu solution (stock
Folin-Ciocalteu solution dissolved with water in 1:10 ratio), 1.5 mL of 7.5% NaHCO3
and 200 µL of the methanol extract solution (100 μg mL-1). The absorbance was measured after 30 minutes at 765 nm, along with the blank.
The standard gallic acid diagram was prepared by adding 200 µL of gallic acid of
different concentration (50-250 µg mL-1) instead of 200 µL of sample. The total phenolic
content was calculated as phenols equivalent to gallic acid (mg GAE/g dry extract) using the
following equation based on the calibration curve: y = 0.0016x + 0.0234; R² = 1, where y
is the absorbance and x the gallic acid concentration (µg mL-1).
Determination of total flavonoids
Total flavonols in the plant extracts were estimated by using the method of Kumaran
and Karunakaran (17). To 1 mL of sample solution (1 mg mL-1), 1 mL of 2% AlCl3 ethanol and 1.5 mL (50 g L-1) sodium acetate solutions were added. The absorption at 440 nm
was read after 2.5 h at 20°C. The standard quercetin diagram was prepared by adding 1
mL of quercetin of different concentration (10-80 µg mL-1) instead of 1 mL of sample.
The total flavonoid content was calculated as quercetin (mg QE/g dry extract) using the
following equation based on the calibration curve: y = 0.0214 x + 0.004; R² = 0.9993,
where y is the absorbance and x is the quercetin concentration (µg mL-1).
Determination of total flavonols
Total flavonols in the plant extracts were estimated by using the method of Kumaran
and Karunakaran (17). To 1 mL of sample solution (1 mg mL-1), 1 mL of 2% AlCl3 ethanol and 1.5 mL (50 g L-1) sodium acetate solutions were added. The absorption at 440 nm
was read after 2.5 h at 20°C. The standard quercetin diagram was prepared by adding 1
mL of quercetin of different concentration (10-80 µg mL-1) instead of 1 mL of sample.
The total flavonoid content was calculated as quercetin (mg QE/g dry extract) using the following equation based on the calibration curve: y = 0.0214 x + 0.004; R² = 0.9993, where
y-was the absorbance and x-was the quercetin concentration (µg mL-1).
Determination of antioxidant activity
a) enzymatic antioxidant activity
For the determination of peroxidase activity, pyrogallol (A430; ε = 12 mM-1 cm-1) was
used as hydrogen donor and the absorbance increase at 430 nm was measured. The reaction mixture consisted of 20 mM pyrogallol, 3.3 mM H2O2 in 100 mM sodium - phosphate
buffer (pH 6.4) and an aliquot of the extract.
Peroxidase (POD) isoforms were separated by native electrophoresis on a 10% polyacrylamide gel at 100V for 120 min. For the visualization of POD isoforms, the gel was
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incubated in a staining solution consisting of (5 mg 4-chloro-α-naphthol, 5 mL methanol
in 45 mL of 100 mM potassium-phosphate buffer (pH 6.5) and 0.03% H2O2.
b) non-enzymatic antioxidant activity
FRAP method (Ferric Reducing Antioxidant Power) is based on the reduction of Fe3+
ions to Fe2+ ions in the presence of an antioxidant (18). The obtained Fe2+ ions in the presence of TPTZ reagent [2,4,6 three(2-pyridyl)-S-triazine] make colored complex, with
the absorbance maximum at 593 nm. The reaction takes place in acid medium.
The reagents were: 10 mM TPTZ solution, 40 mM HCl, 20 mM FeCl3 x 6H2O
solution, 300 mM Na-acetate buffer, pH 3.6 and 20 mM FeSO4 x 7 H2O solution.
The working solution was prepared by mixing 25 mL of acetate buffer solution, 2.5
mL of TPTZ reagent and 2.5 mL of FeCl3 x 6H2O solution. The FRAP working reagent
must always be freshly prepared and kept until used in a water bath at 37 oC.
The measurement was performed by mixing 200 µL of Urtica dioica L. extract (500
µg mL-1) with 1.8 mL of FRAP working reagent, incubating for 10 minutes at 37 ºC and
finally measuring the absorbency at 593 nm, with a blank (1.8 mL FRAP working reagent + 200 μL distilled water).
The standard diagram for FeSO4 solution was prepared by adding 200 µL of FeSO4,
concentration 0.1-1.0 mM (the dependence is linear in the concentration range from 0.2
to 1.0 mM FeSO4) instead of 200 µL of extract.
The results were presented as mM Fe(II)/g of dried extract and compared with the
standard antioxidant compounds: BHA, BHT and vitamin C.
DPPH method. The DPPH method is based on the ability of stable free radical 2,2diphenyl-picrylhydrasyl (DPPH) to react with hydrogen donors, including phenol compounds. DPPH shows an intensive absorption in the visible part of the spectrum and is
easily determined spectrophotometrically (19).
The solution of 0.135 mM DPPH in methanol was used.
The measurement was done by mixing 2 mL of DPPH solution with 2 mL of extract
or standard compounds. The ranges of concentration of the compounds used were: gallic
acid 0.25-2.5 μg mL-1; vitamin C 2-10 μg mL-1; BHA 1.5-12 μg mL-1; nettle extract 20-80
μg mL-1. The reaction mixture was kept in the dark at room temperature and the absorbance was measured after 30 minutes at 517 nm, along with a blank.
The antiradical activity (AA%) was calculated from the following relation:
AA% =
Acontrol  Asample
Acontrol
 100
[1]
where:
Acontrol - DPPH working solution absorbency + methanol,
Asample - DPPH working solution absorbency + sample (or the standard solution).
Based on the diagram representing the antiradical activity vs. different sample concentrations or the reference compound, the value of IC50 was determined. This value represented the sample (or the reference compound) concentration needed for inhibiting
50% of DPPH radicals.
The results were also presented as antioxidant activity index (AAI) (20):
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AAI =
final
 gmL1 
cDPPH
IC50  gmL1 
[2]
AAI was compared with reference values, vitamin C, BHA and BHT.
ABTS+ radical scavenging assay. For the ABTS assay, the method of Re et al. (21)
was adopted. The stock solutions included 7 mM ABTS.+ solution and 2.4 mM potassium persulfate solution. The working solution was then prepared by mixing the two
stock solutions in equal volumes and allowed to react for 12 h at room temperature in the
dark. The solution was then diluted by mixing 1 mL ABTS.+ solution with 60 mL methanol to obtain an absorbance of 0.706 ± 0.001 units at 734 nm using the spectrophotometer. ABTS.+ solution was freshly prepared for each assay. Plant extracts (1 mL) were
allowed to react with 1 mL of the ABTS.+ solution and the absorbance was taken at 734
nm after 7 min, using the spectrophotometer. The radical scavenging capacity was
calculated by equation 3 as percentage of inhibition (I%) of ABTS.+ radicals..
I% 
( Acontrol  Asample )
Acontrol
 100
[3]
where Acontrol is the absorbance of ABTS.+ radical + methanol; Asample is the absorbance of
ABTS.+ radical + sample extract/standard.
The value of the IC50 was determined based on the diagram representing the percentage of inhibition vs. the concentration of the sample or the reference compound. This value represented the sample (or the reference compound) concentration needed for inhibiting 50% of ABTS.+ radicals.
The IC50 was compared with the reference values, BHT, BHA and vitamin C..
Determination of antimicrobial activity
The antimicrobial activity of ethanol nettle extract was screened by using macrodilution method with slight modifications. All analyzed cultures were incubated until
log-phase when the density of suspension was adjusted to 1.5 x 108 cfu mL-1 equal to that
of the 0.5 McFarland standard (22). Two rows of eight tubes were diluted with MHB
medium and then a two-fold serial dilution of the nettle extract concentration was made.
The last test tubes contained 1.13 mg mL-1 of the extract. One row of test tubes was filled
with the bacterial suspension. The density of each bacterial culture in the tubes was 5 x
105 cfu mL-1. The transport time was within 15 minutes of standardization, and the tubes
were incubated at 37 oC for 24 hours.
After the incubation this row of test tubes was compared with the row of test tubes
containing the same MHB and extract concentrations, but without the culture. The first
test tubes (the tubes were ordered from the lowest to the highest concentration) in which
the absence of visible bacterial growth occurred, represented the MIC. The MBC was
determined by subcultivation with a loop from all test tubes without visible growth of
microorganisms on the Petri dish with MHA. After the 24-hour incubation at 37 oC, the
lowest extract concentration contained in the test tube from which solid plates were
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cultured without bacterial colony growth, was recorded as MBC. In the same way, MIC
and MBC were determined for the appropriate antibiotics, with the antibiotics solutions
being prepared using an appropriate procedure (23). All experiments were carried out in
triplicates.
Among the leaves of different ages, there are differences in the chlorophyll content,
chlorophyll a/b ratio, carotenoids content, as well as the intensity of photosynthesis. It has
been shown that the level of chlorophyll increased in young expanding leaves and
decreased during senescence (24). Our result showed that the content of total chlorophyll
as well as the carotenoid content were higher in younger nettle leaves (L1) (Table 1).
Table 1. Content of chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll
Chl(a+b), carotenoids and soluble proteins in nettle leaves of different age.
Samples
Chla
(mg⁄gFW)
0.882±0.002*
0.698±0.053*
L1
L2
*
Chlb
(mg⁄gFW)
0.285±0.01*
0.320±0.03*
Chl(a+b)
(mg⁄gFW)
1.174±0.006*
1.02±0.026*
Carotenoids
(mg⁄gFW)
0.323±0.006*
0.216±0.05*
Protein content
(mg/gFW)
17.505±0.765*
14.365±0.1976*
mean values of three measurements ± SD
In the photosynthetic tissues, carotenoids are synthesized in the chloroplasts, where
they accumulate primarily in association with the light-harvesting complex and reaction
centres (25). Higher carotenoid content in L1 nettle leaves may be associated with their
photo-protective role. As the leaves of different ages represent different physiological
states, this may contribute to significant changes in the quantity and quality of the leaf
proteins. The obtained results showed higher protein content in younger nettle leaves
(Table 1). Yeoh and Paul (26) showed that young apical cassava leaves had a high protein
content.
Table 2. Content of total phenols, flavonoids and flavonols in extract of Urtica dioica L.
Sample
*
**
U. dioica
L
Total phenols
mg GAE/gDW**
Total flavonoids
mg QE/gDW
Total flavonols
mg QE/gDW
208.37 ± 4.39*
20.29± 0.48*
22.83 ± 0.30*
mean value of three measurements ± SD
DW- dry extract
Phenolic components are found in the natural world, especially in the plant kingdom,
and their diverse biological functions have been proven, including the antioxidant (27,
28) and antimicrobial activities (29). Many studies of phenolic components have reported
that the environmental, climatic, or geographic factors as well as extraction techniques
may significantly influence the quality and the quantity of phenolic components present
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in nettle (30,31,32). As shown in Table 2, the total phenolic content in ethanol extract of
nettle leavess is high (208.37 mg GAE/gDW), whereas the content of total flavonoids and
flavonols is relatively low (20.29 and 22.83 mg QE/gDW, respectively).
When the values of total phenolic components in ethanol extract of nettle leaves are
compared (Table 2), it is evident that they are considerably higher than the ones in methanol extract of nettle collected from the Nothern provinces of Iran (24.1mg GAE/gDW)
(31). On the other hand, the values of total phenolic components in ethanol nettle extracts
were lower than the values found by Soxhlet extraction with methanol from nettle leaves
collected from Turkey (332 mg GAE/gDW) (30). Accordingly, the content of total flavonoids in methanol nettle extract is significantly higher than that in ethanol extract (43.3
and 33.94 mg QE/gDW, respectively) (30,31) (Table 2)
Determination of antioxidant activity
Enzymatic antioxidant activity. Peroxidases (POD, EC 1.11.1.7.) are members of a
large group of heme-containing glycoproteins that catalyze oxidoreduction between H2O2
and various reductants. They are widely distributed in higher plant parts and are involved
in several physiological functions such as organogenesis (33), auxin catabolism (34),
lignification (35), suberization (36), cross-linking of cell wall structural proteins (37),
auxin catabolism (38), self-defense against pathogens (39), salt tolerance (40), and senescence (41).
The activity of soluble peroxidase with pyrogallol as electron donor was higher in
younger nettle leaves, sample L1 (Table 3). Using native electrophoresis, two peroxidase
isoforms, labeled as POD1 and POD2, were detected in both samples (Figure 1, b).
Figure 1. A) Nettle plant with marked leaves used for the experiment: L1- Top young
nettle leaves (L1) and the first pair of leaves below the top (L2) were used in the
experiment (Figure 1). B) Peroxidase pattern in L1 and L2 sample obtained on native
10% polyakryamide gel. Arrows indicate POD isomorfs
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Table 3. Peroxidase activity in nettle leaves of different age
Samples
L1
L2
*
Peroxidases activity
mol/mgprot /min)
1.174±0.145 *
0.675±0.171
mean value of three measurements ± SD
Peroxidases (POD, EC 1.11.1.7.) are members of a large group of heme-containing
glycoproteins that catalyze oxidoreduction between H2O2 and various reductants. They
are widely distributed in higher plant parts and are involved in several physiological
functions such as organogenesis (33), auxin catabolism (34), lignification (35), suberization (36), cross-linking of cell wall structural proteins (37), auxin catabolism (38), selfdefense against pathogens (39), salt tolerance (40), and senescence (41).
The activity of soluble peroxidase with pyrogallol as electron donor was higher in
younger nettle leaves, sample L1 (Table 3). Using native electrophoresis, two peroxidase
isoforms, labeled as POD1 and POD2, were detected in both samples (Figure 1, B).
Peroxidase isoforms differ in electrophoretic mobility (RfPOD1 = 0.82, RfPOD2 = 0.67).
Most higher plants possess several isozymes and their number and relative concentration
in different tissues vary according to the stage of plant development or to environmental
factors. Peroxidases are considered as ubiquitous enzymes in the vacuoles, which are also
the target compartment for the accumulation of secondary metabolites (42). It has been
shown that secondary metabolites, including phenols, may act as substrates for class III
peroxidases (POD) (43,44,45) and they consume excess of H2O2. Furthermore, it is
shown that phenols may form a co-operative regenerating cycle with POD and ascorbic
acid, enabling to scavenge high doses of H2O2 (42). Acting together, POD and phenols
are important part of the antioxidant metabolism in response to different types of biotic
and abiotic stresses.
Non-enzymatic antioxidant activity. To evaluate the antioxidant activity accurately,
only one method is not sufficient since many factors can affect the evaluation. It is
required to take more than one measurement and also to take into consideration different
mechanisms of antioxidant activity.
The total antioxidant capacity of ethanol extract of nettle was determined by FRAP
method and the results showed a weak antioxidant activity compared to the control
antioxidants such as vitamin C and BHA (20 times more powerful than the nettle extract),
whereas the BHT showed to have two times higher antioxidant activity than ethanol
extract of stinging nettle. (Table 4).
The stable free radical DPPH is often used to evaluate the antioxidant properties of
natural products. The stable free radical DPPH scavenging effect is otherwise expressed
as an IC50 value, i.e. the extract concentration required to inhibit 50% DPPH radicals. It
was shown, however, that the observed values of IC50 may vary depending on the initial
DPPH concentration, so that the AAI has been used instead and antioxidants are classified as weak, when AAI < 0.5, moderate, when AAI between 0.5-1.0, strong, when AAI
between 1.0-2.0, and very strong, when AAI >2.0 (20).
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Table 4. Antioxidant activity of U.dioica L. leaf determined by FRAP, DPPH
and ABTS methods
Samples
Vitamin C
BHA
BHT
Extract U. dioica L.
* DW- dry extract
** mean value of
FRAP
mM(Fe II)/g DW
143.09 ± 11.29**
147.28 ± 13.87
16.64 ± 0.30
7.50 ± 0.43
DPPH
AAI
IC50 (μg/g DW)
4.97 ± 0.01**
5.36 ± 0.01**
3.96 ± 0.17
6.58 ± 0.28
1.15 ± 0.04
23.16 ± 0.84
0.85 ± 0.003
31.38 ± 0.102
ABTS
IC50 (μg/gDW)
1.37 ± 0.01**
1.72 ± 0.08
6.27 ± 0.16
23.55 ± 0.64
three measurements ± SD
It can be seen from Table 4 that the AAI values for standard antioxidants, vitamin C
(4.97) and BHA (3.96), correspond to the antioxidants classified as showing "very strong
antioxidant activity“, whereas BHT (1.15) is classified as an antioxidant with "strong
antioxidant activity". The nettle extract has the AAI value of 0.82, and it is classified as
an antioxidant with "moderate antioxidant activity".
When the values of total antioxidant activity of nettle leaves were compared by using
stable free radical DPPH (values were expressed as IC50), it was shown that ethanol
extract of nettle leaves had significantly higher antioxidant activity (31.38 μg mL-1) than
some of the methanol extracts ranging from 1.45 mg mL-1 (32), and 105.16 μg mL-1 (46),
to 175 μg mL-1 (47).
ABTS method is also a common method for determination of antioxidant activity of
herbal extracts. The nettle leaves have the ability to inhibit ABTS radical and this method
is used to measure that. The results, (Table 4) show that ethanol extract of nettle leaves
had significantly lower antioxidant activity measured according to ABTS than the other
compared to the standard control antioxidants such as vitamin C, BHA and BHT.
The nettle extract IC50 value was 23.55 μg mL-1, vitamin C and BHA had 1.37 and
1.72 μg mL-1, respectively, and BHT 6.27 μg mL-1. These values show that nettle extract
has 17.2 times lower antioxidant activity than vitamin C, 13.7 times lower than BHA, and
3.8 times lower than BHT. Statistical discrepancy was observed between these results and
those obtained for methanol extracts of nettle leaves from Turkey (40.59 mMTE/g of dry
extract) (30) and Poland (17.3 μM TE/g of dry extract) (48) (TE-trolox equivalent),
which have been found to have significant antioxidant potential.
Antimicrobial activity
The ethanol extract of nettle leaves diluted with methanol showed a weak antibacterial activity. The nettle extract exibited best antibacterial activity against the cultures B.
subitilis IP 5832 and E. coli which were isolated from food, with the lowest MIC values
recorded (Table 5). All MIC values were above the highest tested concentration, except
for the culture P. aeruginosa (144.86 mg mL-1). P. aeruginosa is a naturally resistant
Gram-negative bacterium that causes various infections in humans, and is rather insusceptible to herbal extracts. Water extract of nettle (U. dioica L. WEN), at the doses of 250
μg per disk had no effect on the growth of P. aeruginosa ATCC 9027 (11), whereas P.
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aeruginosa (clinical isolate) was less susceptible to the combined fractions of hexane
extract of U. dioica in comparison to the other Gram-positive and Gram-negative bacteria
tested (49). The plant extract Ankaferd Blood Stopper®, which contains nettle extract,
also showed the lowest inhibitory activity against P. aeruginosa (50).
E. coli is a Gram-negative bacterium which is commonly found in the intestinal tract
of humans and animals as normal flora but which can cause urinary tract infections and
foodborne disease. The ethanol extract of nettle leaves diluted with methanol showed a
MIC value that was lower against E. coli isolated from food samples in comparison to the
bacterial strain isolated from urine samples. The values of MIC against both E. coli strains were higher than the highest tested concentrations. The ethanol extract of nettle leaves
did not inhibit the growth of E. coli ATCC 9837 (51), unlike the water extract, which
exhibited considerable antibacterial activity (11).
B. subtillis, an endospore-forming Gram-positive bacterium, showed a higer susceptibility to the ethanol extract of nettle leaves diluted with methanol in comparison to the
other tested bacteria, but no MIC values against this bacterium were determined. L.
plantarum 299v is a probiotic Gram-positive bacterium found in many fermented food
products and in the human intestinal tract. This bacterium has many beneficial effects on
human health (52), and the MIC value of the ethanol extract of nettle leaves diluted with
methanol showed a potent antibacterial activity against this bacterium, which is of importance since nettle leaves have been consumed as food for centuries.
Table 5. Antibacterial activity of ethanol extract of Urtica dioica L. leaves diluted with
methanol
a
Samples
Extract (mg/mL)
MIC
MBC
B. subtilis
b
E. coli
c
E. coli
P. aeruginosa
L. plantarum
36.21
72.43
36.21
72.43
72.43
>144.86
>144.86
>144.86
144.86
>149.93
MIC
Ea
MBC
AMPa
MIC
MBC
5
0.625
20
20
0.3125
20
1,25
40
40
1,25
0.078
8
64
0.078
0.156
16
256
1.25
CIPa
Ga
MIC
MBC
MIC
0.3125
<0,078
0.156
0.3125
2.5
1.25
0.078
0.625
0.625
-
0.325
1.25
1.25
1.25
0.156
MB
C
5
2.5
5
1.25
-
Antibiotics (μmL-1): Erythromycin (E); Ampicillin (AMP); Ciprofloxacin (CIP); Gentamicin (G)
Escherichia coli isolated from urine samples; cEscherichia coli isolated from food
b
CONCLUSION
The obtained results showed insignificantly higher content of chlorophyll, carotenoids, and proteins in young nettle leaves as well as an increase in the soluble POD activities. Native electrophoresis of the soluble fraction showed that the presence of two peroxidase isophorms were detected in the soluble protein fraction of nettle leaves. The total phenolic content in ethanol extract of nettle leaves is high (208.37 mg GAE/gDW),
whereas the content of total flavonoids and flavonols is relatively low (20.29 and 22.83
mg QE/gDW, respectively). Ethanol extract of U. dioica leaves has significant enzymatic
and moderate non-enzymatic antioxidative action compared to the control antioxidants
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(vitamin C, BHA and BHT). Acting together, POD and phenols are important part of the
antioxidant metabolism in response to different types of biotic and abiotic stresses.
Ethanol extract of nettle leaves diluted with methanol showed a weak antibacterial
activity against B. subtilis IP 5832 and E. coli isolated from food. The other tested bacteria strains, of E. coli isolated from urine, P. aeruginosa, and L. plantarum did not exhibit any antibacterial activity at test concentration of ethanol extract of U. dioica. A weak
antimicrobial activity of the tested extract could be of importance since nettle leaves have
a wide range of uses, for food, medicinal purposes, fibers, and may have other positive
effects on human health.
Acknowledgements
This work was supported by the Ministry of Science and Technology of Republic of
Srpska (Project contract no. 19/6-020/961-118/10).
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КАРАКТЕРИЗАЦИЈА АНТИОКСИДАТИВНЕ И АНТИМИКРОБНЕ
АКТИВНОСТИ ЛИСТА КОПРИВЕ (Urtica dioica L.)
Зоран З. Кукрића, Љиљана Н. Топалић-Тривуновића, Биљана М. Кукавицаб,
Сњежана Б. Матоша, Светлана С. Павичића, Мирела М. Боројаб
и Александар В. Савића,
а
Универзитет у Бањoj Луци, Технолошки факултет, Војводе Степе Степановића75, 78000 Бања Лука,
Република Српска, Босна и Херцеговина
б
Универзитет у Бањoj Луци, Природно-математички факултет, Младена Стојановића 2, 78000 Бања
Лука, Република Српска, Босна и Херцеговина
Коприва (Urtica dioica L.) за потребе овог рада прикупљена је у региону Бања
Луке. У свјежим листовима различите старости одређен је садржај хлорофила а,
хлорофила б, каротеноида и солубилних протеина као и активност пероксидаза
(POD, EC 1.11.1.7.). Суви листови коприве су коришћени за добијање етанолног
екстракта. Суви остатак етанолног екстракта је растворен у метанолу и у добијеном
раствору је одређен садржај укупних фенола, флавоноида, флавонола, неензимска
антиоксидативна и антимикробна активност. Неензимска антиоксидативна активност одређена је FRAP, DPPH и ABTS методом. Резултати су поређени са антиоксидативном активношћу стандардних једињења (витамин Ц, BHT и BHА). Антимикробна активност рађена је методом макроразређењa.
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Добијени резултати су показали незнатно повећање садржаја укупног хлорофила, каротеноида и протеина у млађим листовима. Такође је у млађим листовима
измерена већа активност солубилних пероксидаза. Нативном електрофорезом
детектоване су две пероксидазне изоформе у солубилној протеинској фракцији листа коприве. Садржај укупних фенола у коприви је износио 208,37 мг GЕА/г сувог
екстракта (dw), укупних флавоноида 20,29 мг QE/гdw,а укупних флавонола 22,83 мг
QE/гdw.
Антиоксидативна активност добијена FRAP методом износила је 7,50 mM
Fe(II)/gdw, док је за DPPH и ABTS методу, изражена као IC50 била 31,38 и 23,55 μg
mL-1, респективно. Резултати указују на слабу и умерену антиоксидативну активност коприве.
Екстракт коприве је тестиран на различите Грам-позитивне и Грам-негативне
бактерије (Bacillus subitlis IP 5832, Lactobacillus plantarum 299v (Lp299v), Pseudomonas aeruginosa и Escherichia coli која је изолована из узорака хране и Escherichia
coli која је изолована из узорака урина). Комерцијални антибиотици (ампицилин,
еритромицин, ципрофлоксацин и гентамицин) су коришћени као позитивна контрола. Резултати показују да је минимална инхибиторна концентрација (МIC) и минимална бактерицидна концентрација МBC екстракта листа коприве у распону од
9,05 до више од 149,93 mg mL-1.
Кључне речи: коприва, садржај хлорофила, каротеноида и солубилних протеина,
укупни феноли, флавоноиди и флавоноли, антиоксидативна и антимикробна активност.
272
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A CHEMOMETRIC APPROACH FOR PREDICTION OF ANTIFUNGAL
ACTIVITY OF SOME BENZOXAZOLE DERIVATIVES AGAINST
Candida albicans
Sanja O. Podunavac-Kuzmanovića*, Lidija J. Jevrića, Strahinja Z. Kovačevića
and Nataša D. Kalajdžijaa
a
Faculty of Technology, University of Novi Sad, 21000 Novi Sad, Bul. Cara Lazara 1, Serbia
The purpose of the article is to promote and facilitate prediction of antifungal activity
of the investigated series of benzoxazoles against Candida albicans. The clinical importance of this investigation is to simplify design of new antifungal agents against the fungi
which can cause serious illnesses in humans. Quantitative structure activity relationship
analysis was applied on nineteen benzoxazole derivatives. A multiple linear regression
(MLR) procedure was used to model the relationships between the molecular descriptors
and the antifungal activity of benzoxazole derivatives. Two mathematical models have
been developed as a calibration models for predicting the inhibitory activity of this class
of compounds against Candida albicans. The quality of the models was validated by the
leave-one- out technique, as well as by the calculation of statistical parameters for the
established model.
KEY WORDS: chemometricс, antifungals, benzoxazole derivatives, Candida albicans,
molecular descriptors
INTRODUCTION
Predictions of antimicrobial properties of molecules based on their structure are the
fundamental and most interesting objectives of chemistry. The conception that there
exists a close relationship between bulk properties of compounds and their molecular
structure is quite rooted in chemistry. This idea allows one to provide a clear connection
between the macroscopic and the microscopic properties of matter, and thus has been
firmly established as one of the central foundations of chemistry. Therefore, it is the basic
aim of chemistry to attempt to identify these assumed relationships between chemical
structure and physico-chemical properties and then to quantify them.
Benzoxazoles and their derivatives are well known to the chemists, mainly because of
the broad spectrum of the antimicrobial properties exhibited by this class of compounds
(1-12). Interest in the chemistry, synthesis and microbiology of this pharmacophore
* Correspodning author: Sanja Podunavac-Kuzmanović, University of Novi Sad, Faculty of Technology,
Bulevar cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]
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continues to be fuelled by their biological properties such as antifungal, antitubercular,
antioxidant, antiallergic, and antiparasitic. It is also well known that these molecules are
present in a variety of antitumoural, anthelmintic and herbicidal agents (1-12).
A large number of research studies are needed to analyze the pharmacophore present
in these compounds using the Three Dimensional QSAR (quantitative structure-activity
relationship) methods. The physicochemical properties predicted from structure are helpful in the search for new molecules of similar or increased biological activity. QSAR
studies enable the investigators to establish reliable quantitative relationships, to derive a
QSAR model, and predict the activity of novel molecules prior to their synthesis. These
studies reduce the trial-and-error element in the design of compounds by establishing mathematical relationships between physical, chemical, biological, or environmental activities of interest and measurable or computable physicochemical, electronic, topological, or
stereochemical parameters. The 3D-QSAR methodology has been successfully used to
generate models for various chemotherapeutic agents (13-20).
In view of above and in continuation of our studies on QSAR analyses (21-29), the
aim of this investigation was to study the quantitative effect of the structure on antifungal
activity of some benzoxazole derivatives against Candida albicans.
EXPERIMENTAL
The structures of the benzoxazoles investigated in this study are presented in Table 1.
Table 1. The structures of the compounds studied
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
274
R1
N(CH3)2
CH3
C2H5
OCH3
F
NHCOCH3
NHCH3
N(CH3)2
C2H5
NHCOCH3
NH CH3
Cl
NO2
H
C(CH3)3
NH2
NHCH3
C2H5
F
R2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
H
H
H
H
NH2
NH2
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The results of antifungal activity against Candida albicans (MTCC 183) for all the
benzoxazole derivatives were taken from the literature (30). Minimum inhibitory concentration (MIC) of tested benzoxazoles is defined as the lowest concentration of the compound
at which no growth of the strain. The negative logarithms of molar MICs (log1/cMIC) were
determined and used for further calculations.
Molecular Modeling
The molecular modeling study was performed using HyperChem 7.5 software (HyperCube Inc, Version 7.5) running on P-III processor (31). HyperChem includes a model
builder that turns a rough 2Dsketch of a molecule into 3D. The created 3-D models were
cleaned up and subjected to energy minimization using molecular mechanics (MM2). The
minimization is executed until the root mean square (RMS) gradient value reaches a
value smaller than 0.1kcal/molÅ. The Austin Model-1 (AM-1) method was used for reoptimization until the RMS gradient attains a value smaller than 0.0001kcal/molÅ using
MOPAC. The lowest energy structure was used for each molecule to calculate molecular
descriptors.
Generation of the Descriptors
The numerical descriptors for each compound in the data set were calculated using the
software HyperChem (31), Dragon (32) and CS Chem Office Software version 7.0 (33).
Since there was a 78 different descriptors for each compound (electronic, constitutional,
hydrophobic, and topological), Pearson's correlation matrix was used as a qualitative
model, in order to select the suitable descriptors for MLR analysis. One way to avoid data
redundancy is to exclude descriptors that are highly intercorrelated with each other before
performing statistical analysis.
Statistical Methods
The complete regression analysis was carried out by PASS 2005, GESS 2006, NCSS
Statistical Softwares (34).
The results of the antifungal studies of 19 benzoxazole derivatives against Candida albicans are summarized in Table 2. As is evident, all the compounds show noteworthy antifungal activities against the tested fungi. Consequently, the compounds with high
log1/cMIC (or low MIC) are the best antifungals.
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Table 2. Data of the experimental and predicted values of log1/cMIC
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
log1/cMIC exper.
4.005
3.950
3.977
3.980
3.958
4.027
3.979
4.004
4.013
4.059
4.015
4.024
4.040
3.892
4.001
3.924
3.952
3.979
3.960
Antifungal activity
log1/cMIC predict.
4.015
3.955
3.973
3.992
3.964
4.037
3.961
3.998
3.996
4.053
4.011
4.009
4.050
3.909
4.005
3.924
3.955
3.962
3.960
Residuals
0.010
-0.005
0.004
-0.012
-0.006
-0.010
0.018
0.006
0.017
-0.006
0.004
0.015
-0.010
-0.017
-0.004
0.000
-0.003
0.017
0.000
In order to identify the effect of the chemical structure on the inhibitory activity,
QSAR studies of title compounds were performed. A set of benzioxazoles consisting of
19 molecules was used for multilinear regression model generation. An attempt has been
made to find structural requirement for inhibition of Candida albicans using QSAR
Hansch approach on benzoxazole derivatives. Different physicochemical, steric, electronic, and structural molecular descriptors were used as independent variables and were
correlated with antifungal activity. From the QSAR study of the series of benzoxazoles,
two best biparametric models were derived. Both the models include lipophilicity descriptor (logP). The specifications for the best-selected MLR models are shown in Table
3.
Table 3. Best MLR models for the prediction of antifungal activity
Model
1
2
276
Coefficient
Intercept
0.7685
logP
0.9314
MR
0.0198
Intercept
0.0715
logP
0.8941
HE
0.0033
n
r
S
F
19
0.9758
0.0767
128.6125
19
0.9759
0.0774
127.3176
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But, only high correlation coefficient is not enough to select the equation as a model
and hence various statistical approaches were used to confirm the robustness and practical applicability of the equations. The statistical validity of the resulting models, as given in Table 3, is determined by r, s, and F. It is noteworthy that all these equations were
derived using the entire data set of compounds (n = 19) and no outliers were identified.
The F-value presented in Table 3 is found statistically significant at 99% level since all
the calculated F values are higher as compared to the tabulated values.
For the testing the quality of the predictive power of selected MLR models the LOO
procedure was used (Table 4). The PRESS value above can be used to compute an r2CV
statistic, called r2 cross-validated, which reflects the prediction ability of the model. This
is a good way to validate the prediction of a regression model without selecting another
sample or splitting the data. In this study, the r2adj and r2CV are taken as a proof of the high
predictive ability of the QSAR models. A high value of these statistical characteristic (>
0.5) is considered as a proof of the high predictive ability of the models. The adjustable
correlation coefficient (r2adj) tells us the statistical significance of incorporated physicochemical descriptor in MLR. It takes into account the adjustment of the conventional
correlation coefficient (r2). PRESS is an acronym for prediction of the sum of squares. It
is used to validate a regression model with regard to its predictability.
Table 4. Cross-validation parameters
Model
PRESS
SSY
PRESS/SSY
SPRESS
r2CV
r2adj
1
0.1201
1.6764
0.0716
0.0795
0.9284
0.9345
2
0.1449
1.6764
0.0864
0.0873
0.9127
0.9330
Thus, the high value of LOO r2CV is the necessary condition for a model to have a
high predictive power, but it is not a sufficient condition. The only way to estimate the
true predictive power of a model is to test its ability to predict accurately the inhibitory
activities of compounds.
In order to verify the predictive power of the developed model, the predicted
log1/cMIC values of benzoxazole investigated were calculated by using models 1 and 2
and compared with the experimental values (Table 2). The data presented in Table 2
show that the observed and the estimated activities are very close to each other. The residual activity (the difference between experimentally observed log (1/cMIC) and QSAR
calculated log (1/cMIC)) is less than or equal to 0.018. Further, Fig. 1 shows the plot of the
linear regression of the predicted versus experimental values of the antifungal activity of
the investigated benzoxazoles.
To investigate the existence of a systemic error in developing the QSAR models, the
residuals of predicted values of inhibitory activity were plotted against the experimental
values in Figure 2. The propagation of the residuals on both sides of zero indicates that
no systemic error exists in the development of the regression models, as suggested by
Jalali-Heravi and Kyani (35). It indicates that these models can be successfully applied to
predict the antifungal activity of this class of molecules.
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4.06
r=0,9621
4.04
log1/cMICteor.
4.02
4.00
3.98
3.96
3.94
3.92
3.90
3.88
3.90
3.92
3.94
3.96
3.98
4.00
4.02
4.04
4.06
log1/cMICexp.
Figure 1. Plot of the predicted vs. the experimentally observed antifungal activity against
Candida albicans
0.020
0.015
0.010
Residual
0.005
0.000
3.90
-0.005
3.92
3.94
3.96
3.98
4.00
4.02
4.04
log1/cMICexp.
-0.010
-0.015
Figure 2. Plot of the residual values against the experimentally observed log1/cMIC values
The positive contribution of logP in both the proposed equations thus suggests its
significant participation in the inhibitory activity. The results clearly indicate that the
compounds with higher lipophilicity values exhibited increased inhibitory action on the
growth of the tested fungi. The other descriptors, MR and HE, were effective if combined
with logP. Both the descriptors are the indicators of lipophilicity/hydrophobicity. They
may be related to the binding between drug and receptor because the polarity is an
essential factor to bind active site of the receptor molecule.
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The results indicate the possibility of applying the chemometric techniques for a successful prediction of antifungal activity of the investigated series of benzoxazoles against
Candida albicans. The results illustrate that the MLR technique is appropriate to create
fine QSAR models for predicting the inhibitory activity of different compounds, and that
is useful for drug design and medicinal chemistry.
CONCLUSIONS
From the results discussed above, it can be concluded that the different substituted
benzoxazole derivatives showed in vitro considerable inhibitory activity against Candida
albicans. Molecular modeling and QSAR analysis were performed to find the quantitative effects of the molecular structure of the compounds on their antifungal activity.
Various physicochemical parameters, especially partition coefficient, molar refractivity
and hydration energy can be used successfully for modeling antifungal activity of benzoxazoles. Two best QSAR mathematical models are used to predict inhibitory activity of
the investigated benzoxazoles, and close agreement between experimental and predicted
values was obtained. The low residual activity and high cross-validated r2 values (r2CV)
observed indicate the predictive ability of the developed QSAR models. This means that
these models can be successfully applied to predict the antifungal activity of this class of
molecules.
Acknowledgements
These results are part of the projects No. 114-451-2707/2012-01, financially supported by the Provincial Secretariat for Science and Technological Development of Vojvodina and No. 172012 and 172014 supported by the Ministry of Education, Science and
Technological Development of the Republic of Serbia.
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ХЕМОМЕТРИЈСКИ ПРИСТУП У ПРЕДВИЂАЊУ AНТИФУНГАЛНЕ
АКТИВНОСТИ НЕКИХ ДЕРИВАТА БЕНЗОКСАЗОЛА ПРЕМА
Candida albicans
а
Сања О. Подунавац-Кузмановића, Лидија Ј. Јеврића, Страхиња З. Ковачевића и
Наташа Д. Калајџијаа
Универзитет у Новом Саду, Teхнолошки факултет, Булевар цара Лазара 1, 21000 Нови Сад, Србија
Циљ овог рада је предвиђање антифунгалне активности испитиване серије бензоксазола према Candida albicans. Клинички значај ових испитивања је поједноставити дизајнирање нових антифунгалних агенаса који су узрочници многих озбиљних обољења код људи. QSAR (quantitative structure-activity relationship) aнализа изведена је на деветнаест деривата бензимидазола. Вишеструка линеарна регресија коришћена је за моделовање зависности између молекулских дескриптора и
антифунгалне активности деривата бензоксазола. Дефинисана су два математичка
модела за предвиђање инхибиторне активности ове групе једињења према Candida
albicans. Квалитет модела потврђен је LOO (leave one out) техником, као и израчунавањем статистичких параметара за постављене моделе.
Кључне речи: хемометрија, антифунгална активност, деривати бензоксазола,
Candida albicans, молекулски дескриптори.
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COMPARATIVE EVALUATION OF THE ESSENTIAL OIL TERPENOIDS IN
THE STEM AND LEAF OF Ziziphora Clinopodioides IN THE REGIONS OF
ALMEH AND SOJOUGH OF GOLESTAN PROVINCE, IRAN
Pouneh Ebrahimi1, Akram Mirarab-Razi2 and Abbas Biabani1
1
Department of Chemistry, Gonbad Kavous University, P. O. Box 163, Gonbad, Iran
2
Faculty of Basic Sciences, Tehran University of Payam-e-Noor, Tehran, Iran
Microwave-assisted hydrodistillation was used to isolate essential oil from the leaf
and stem of Ziziphora clinopodioides collected in the flowering stage on two locations,
Almeh and Sojogh, of the Golestan Province (Iran), in June of 2009. The total contents of
monoterpene and sesquiterpene fractions (52.45% and 1.08%, respectively) in the leaf oil
of Almeh plants were higher than those of Sojogh (46.64% and 0.12%, respectively). The
essential oil of the stem of the plants from Sojogh was characterized by the presence of
eight oxygenated monoterpenes (22.17%), while four oxygenated monoterpenes
(11.15%), one monterpene hydrocarbon (2.71%), and one oxygenated sesquiterpene
(0.21%) were found in the plants from the region of Almeh. The analysis of the essential
oil of dried aerial parts showed the presence of oxygenated monoterpenes pulegone and
menthol (the region of Sojogh) and pulegone, 1,8-cineol, D-neoisomenthol and chrysanthenone (the region of Almeh), as the main constituents. Also, chrysanthenone (9.75%),
found as the second major component of the leaf of Z. clinopodioides of Almeh, was not
identified as the oil component of the other region. The results obtained on the chemical
composition of Z. clinopodioides oil of two regions from the Golestan Province revealed
that in general, that there are some differences in the major components and their
relative concentrations. This may be probably due to the different environmental and
genetic factors, different chemotypes and the nutritional status of the plants, as well as
other factors that can influence the oil composition.
KEY WORDS: Ziziphora clinopodioides, Terpenoid compounds, Golestan Province,
Microwave-assisted hydrodistillation.
INTRODUCTION
The plant of Ziziphora clinopodioides (Lam.), which is a habitant of Iran, has been
used in Iranian traditional medicine for treatment of some infectious conditions. Aerial
parts of this plant are used as stomachic, anti-fever, anti-inflammatory, sedative and
* Corresponding author: Pouneh Ebrahimi, Gonbad-Kavous University, P.O. Box 163, Gonbad, Iran, e-mail:
[email protected]
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flavoring agent (1-2). Also, in Iran, it is added to a special cheese herby cheese, in
particular in the eastern part of Iran (3). The genus Ziziphora L. belongs to the family
Labiatae, which grows wild as an endemic subspecies in many parts of Iran (4). There
are four species of the plant (Z. clinopodioides Lam., Z. capitata L., Z. persica Bunge.
and Z. tenuior L.) that widespread all over Iran. The species of Z.capitata, Z. persica, Z.
tenuior are annual and Z. clinopodioides is perennial (4-5).
A number of studies (6-13) showed the oil of some Ziziphora species is rich in
pulegone. The main constituents found in the oil of Z. vychodceviana and Z. persica
collected from Kazakhstan were also pulegone (57.5-66%) and isomenthone (5.1-15.7%)
(8). The major constituent of Z. tenuior L. oil has been reported to be pulegone (87.1%)
(9). The essential oil of Turkish endemic Z. taurica subsp. clenioides contains pulegone
(81.9%), limonene (4.5%) and piperitenone (2.3%) (10). In the literature, there are some
reports on the chemical constituents of Z. clinopodioides growing in the former USSR
and west part of Turkey (12-13). The surveys showed that the major compounds of the
essential oil were pulegone (13.2%-31.86%), 1,8-cineole (2.3%-14.5%), limonene (1.8%10.48%), menthone (4.6%-6.73%) and isomenthone (2%-10.8%).
Microwave heating has an incontestable place in analytical and organic laboratory
practices as a very effective and non-polluting method. It is applicable in sample digestion, organic synthesis, analytical chemistry, phytochemistry and food industry (14-15).
Microwave energy, with a frequency of 2.45 GHz, is well known to have a significant
effect on the rates of a variety of processes. The main reason for the increased interest
lies in the much shorter operation times. Microwave-assisted extraction of natural compounds is also an alternative to conventional techniques. Essential oils are among the
products which have been extracted efficiently from a variety of matrices by this method
(16).
In the present work, microwave-assisted hydrodistillation was used for isolating the
stem and leaf essential oil of Z. clinopodioides obtained from two locations of the Golestan Province in Iran (Almeh and Sojogh), where people frequently use this plant in traditional medicine. The chemical composition of essential oils obtained from the plants in
the flowering stage was studied by gas liquid chromatography (GLC). To the best of our
knowledge, no information about the chemical composition of the essential oil of Ziziphora clinopodioides grown in Almeh (Golestan National Park) and Sojogh was published before. Therefore, these results can be considered as the first report about the subject
of this study.
MATERIAL AND METHODS
Plant material and isolation
The aerial parts of the plant were collected during the flowering stage in June of 2009,
in the Almeh Mountains of the Golestan National Park and Sojogh region (Golestan
Province, Iran). The air-dried material (about 50 g) was cut into small pieces and moistened prior extraction by soaking in water and then allowing the excess of water to drain.
This step is essential to give the material the initial moisture. The moistened plant mate284
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rial was hydrodistilled at the fixed power of 450 W for 30 min using a microwave distillation apparatus. The essential oils obtained were dried over anhydrous sodium sulfate
and stored in a sealed vial at 4◦C until used.
Microwave extraction apparatus and procedure
Microwave extraction was carried out on a Milestone DryDist microwave apparatus.
The maximum output power of the microwave apparatus was 1000 W with 2450 MHz of
microwave radiation frequency. The reactor was a 500 mL short-necked flask. During the
experiments, time, temperature and power were controlled using the „easy-WAVE“
software package. Temperature was monitored by a shielded thermocouple (ATC-300)
inserted directly into the sample container and by an external infrared (IR) sensor, and
controlled by a feedback to the microwave power regulator. The experimental variables
were optimized by the univariate method in order to maximize the yield of essential oil.
A cooling system outside the microwave cavity condensed the distillate continuously.
The condensed water was refluxed to the extraction vessel in order to provide uniform
conditions of temperature and humidity for extraction. The extraction was continued at
100oC and atmospheric pressure until no more essential oil was obtained.
Gas chromatography (GC)
The isolated oil was diluted with hexane. The GC analysis of the essential oil was
carried out on a Younglin-ACM600 gas chromatograph with FID detector and a HP-5MS
capillary column (30 m × 0.25 mm i.d., 0.25µm film thickness) (USA) with a 5% phenyl
methyl siloxane stationary phase. Helium was used as the carrier gas at the flow rate of
0.8 mL min-1; split ratio was 1:30, with the injection volume of 0.2 µl (10% hexane solution). The oven temperature was held at 50ºC for 5 min, then raised to 240ºC at a rate of
3oC min-1, 240-300oC at 15oC min-1and then held at 300oC for 3 min. The injector and
detector temperatures were set at 290oC and 220oC, respectively.
Gas chromatography/mass spectrometry (GC/MS)
For the identification of the components, GC/MS analysis was performed under the
same conditions using an Agilent 6890 gas chromatograph equipped with an Agilent
5973 mass selective detector with an ionization voltage of 70 eV on a capillary column
HP-5MS. The ionization source temperature was set at 220oC.
Qualitative and quantitative analysis
The constituents of the volatile oils were identified based on their Kovats Index,
calculated in relation to the retention time of a series of n-alkanes (C4-C28) as reference
products, in comparison with those of the chemical compounds gathered by Adams table
(17-18), and the similarity of their mass spectra with those gathered in the MS library
(Wiley 275), or reported in the literature (19-22).
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The hydrodistillation of the air-dried leaf and stem of Z. clinopodioides (Lamiaceae)
yielded about 0.90 and 0.44% (w/w) oil, respectively, for the samples from both locations. The oil of leaves and stems was clear yellowish. The terpenoide compositions of
essential oils were analyzed by GC and GC-MS. The results showed that different terpenoids were isolated in the stem and leaf of the plants from both regions. The essential
oils were characterized by a higher diversity of the terpenes in the plants collected on the
Almeh than on Sojogh region.
Table 1. Terpenoid constituents of the stem essential oil of Ziziphora clinopodioides in
the flowering stage from Almeh and Sojogh regions
No.
Component
tR
RIa
RAb (%) in
Sojogh
RA (%) in
Almeh
1
1,8–Cineole
15.46
1035
0.211
0.220
2
L-Menthone
22.12
1147
2.248
2.148
3
L-(-)-Menthol
23.47
1171
1.890
1.526
4
Pulegone
25.73
1220
16.038
7.258
5
Iso–pulegone
22.70
1175
0.357
-
6
(+)–Neoisomenthol
22.33
1163
0.981
-
7
(Z)-Dihydrocarvone
23.73
1186
0.319
-
8
Cis- dihydrocarveol
29.88
1194
0.124
-
9
Lyratol
34.69
1164
-
0.211
10
α–Fenchene
41.34
1049
-
11
12
13
14
Oxygenated monoterpenes
Monoterpene hydrocarbons
Oxygenated sesquiterpenes
Total terpenoids (%)
2.714
c
22.168 (8)
22.168
Type
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
Sesquiterpene
Monterpene
Hydrocarbon
11.152 (4)
2.714 (1)
0.211 (1)
14.077
a
RI: Retention index was determined by GC-FID on a HP-5MS column
RA: relative area (peak area relative to total peak area)
c
Number of compounds in the group
b
In the investigated stem samples from Sojogh and Almeh, oxygenated monoterpenes
were identified as the major class of compounds, containing 8 and 4 compounds representing 22.17% and 11.15% of total oil, respectively (Table 1). Among them, dominant
were: pulegone, L-menthone and L-(-)-menthol. No significant quantitative difference in
L-menthone and L-(-)-menthol between the examined samples from two regions was
found. However, oxygenated monoterpene pulegone was the dominant compound in this
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class, and its content in the Sojogh sample (16.04%) was higher than that obtained for the
Almeh sample (7.26%). Another compound which was present in an appreciable amount
in the stem essential oil obtained from the Almeh sample was monterpene hydrocarbon
α–fenchene (2.71%).
The results of the qualitative and quantitative analysis for the Z. clinopodioides leaf
essential oil showed that twenty two and nine terpenoid compounds were identified in the
Almeh and Sojogh samples, representing 53.53% and 46.76% of total oil, respectively
(Table 2). Oxygenated monoterpene pulegone was found in a considerable amount in the
essential oils, with the higher content of pulegone in the plant samples originated from
Sojogh (37.80%) than in those from Almeh (18.04%). The results of this study are in
accordance with the previous studies of Ziziphora species, showing also that this plant is
rich in pulegone (8-10, 23). Other representative components found in the leaf essential
oil of Sojogh sample were menthol (4.37%), (+)–neo-menthol (2.15%) and L-menthone
(1.13%).
The essential oil obtained from Z. clinopodioides of Almeh also contained chrysanthenone (9.75%), D-neoisomenthol (8.22%), 1,8-cineol (7.12%) and menthol (1.53%) as
the most important oxygenated monoterpenes. Monoterpenic hydrocarbons such as α-pinene (1.45%), sabinene (1.34%) and ß-pinene (1.81%) in this sample were also found in
appreciable amounts.
The results of the determination of chemical composition of essential oils in Z.
clinopodioides from two regions of the Golestan Province revealed that in general, there
are some differences in the major components, as well as in their relative contents. This
may be probably due to the different environmental and genetic factors, different chemotypes and the nutritional status of the plants, as well as to other factors that can influence
the oil composition. Also, chrysanthenone (9.75%) as the second major oil component of
Z. clinopodioides of Almeh, found in the flowering stage of leaf, has not been identified
as the oil component of the other regions or species.
Table 2. Terpenoid constituents of the leaf essential oil of Ziziphora clinopodioides in the
flowering stage from Almeh and Sojogh regions
No.
Component
tR
RIa
RAb (%) in
Sojogh
RA (%) in
Almeh
1
α-Thujone
10.36
1061
-
0.152
2
α-Pinene
10.71
947
-
1.450
3
Camphene
11.33
980
-
0.184
4
Sabinene
12.71
970
-
1.338
5
ß-pinene
12.83
982
-
1.813
6
ß-Myrcene
13.57
991
-
0.390
7
1,8-Cineol
15.74
1035
0.385
7.116
Type
Monterpene
Hydrocarbon
Monterpene
Hydrocarbon
Monterpene
Hydrocarbon
Monterpene
Hydrocarbon
Monterpene
Hydrocarbon
Monterpene
Hydrocarbon
Oxygenated
monoterpene
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Table 2. Continued
No.
Component
tR
RIa
RAb (%) in
Sojogh
RA (%) in
Almeh
8
γ-Terpinene
16.93
1062
-
0.070
9
Terpineol
17.37
1175
-
0.084
10
cis-iso Pulegone
23.17
1175
0.348
0.343
11
(+)–Neo-menthol
22.259
1159
2.149
-
12
Menthol
23.75
1171
4.369
1.527
13
Pulegone
25.97
1220
37.795
18.041
14
Germacrene D
36.52
1480
-
0.499
15
L-Menthone
21.68
1147
1.133
0.734
16
D-Neoisomenthol
24.41
1163
-
8.218
17
Endobornyl acetate
28.16
1276
-
0.235
18
1R-Menthyl acetate
28.58
1294
0.293
0.780
19
Thymol
29.36
1302
0.168
0.225
20
Chrysanthenone
31.02
1158
-
9.751
21
β-Bourbonene
32.50
1384
-
0.439
22
β-Cubebene
34.31
1395
-
0.073
23
Spathulenol
40.29
1589
24
25
26
27
28
Oxygenated monoterpenes
Monoterpene Hydrocarbons
Oxygenated sesquiterpenes
Sesquiterpene Hydrocarbons
Total terpenoids (%)
0.122
0.063
46.640 (8)c
0.122 (1)
46.762
47.054 (11)
5.397(7)
0.063 (1)
1.012 (3)
53.526
Type
Monterpene
Hydrocarbon
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Sesquiterpene
Hydrocarbon
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Oxygenated
monoterpene
Sesquiterpene
Hydrocarbon
Sesquiterpene
Hydrocarbon
Oxygenated
Sesquiterpene
a
RI: Retention index was determined by GC-FID on a HP-5MS column
RA: relative area (peak area relative to total peak area)
c
Number of compounds in the group
b
CONCLUSION
The present study has been concerned with the determination and comparison of the
chemical composition of stem and leaf essential oils of Z. clinopodioides, collected in the
flowering phase on two locations, Almeh (Golestan National Park) and Sojogh in the
Golestan Provine (Iran). The total amount of monoterpene and sesquiterpene fractions in
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the leaf oil of the Almeh sample (52.45% and 1.08%, respectively) was higher than those
of the Sojogh sample (46.64% and 0.12%, respectively). The essential oil of stem of
plants from Sojogh was characterized by the presence of eight oxygenated monoterpenes
(22.17%), four oxygenated monoterpenes (11.15%), one monterpene hydrocarbon
(2.71%) and one oxygenated sesquiterpene (0.21%) in the plant from the region of Almeh. Also, chrysanthenone (9.75%) as the second major oil component of Z. Clinopodioides of Almeh, found in the flowering stage of leaf, was not identified as the oil component of the other regions or species.
In general, the analysis of essential oils of dried aerial parts of Z. clinopodioides
collected in the flowering stage showed that their main oxygenated monoterpenes pulegone and menthol (the region of Sojogh) and pulegone, 1,8-cineol, D-neoisomenthol
and chrysanthenone (the region of Almeh).
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УПОРЕДНA КАРАКТЕРИЗАЦИЈА ТЕРПЕНОИДА ЕТЕРИЧНОГ УЉА
СТАБЛА И ЛИСТА БИЉКЕ Ziziphora Clinopodioides ИЗ РЕГИОНА АЛМЕХ И
СОЏОГ ПРОВИНЦИЈЕ ГОЛЕСТАН, ИРАН
Pouneh Ebrahimi1, Akram Mirarab-Razi2 и Abbas Biabani1
2
1
Департман за хемију, Универзитет Гонбад Кавоус, П.П. 163, Гонбад, Иран
Факултет основних наука, Техерански Универзитет Пајам-е-Ноор, Tехеран, Иран
Микроталсно-потпомогнута хидродестилација је примењена да би се изоловала
етерична уља из узорака листа и стабла биљке Ziziphora clinopodioides прикупљених у фази цветања на две локације - Алмех и Соџог у провинцији Голестан (Иран),
у јуну 2009. Укупни садржаји монотерпенских и сесквитерпенских фракција у уљу
листа биљака из Алмеха (52,45% и 1,08%, респективно) су виши него код биљака
из Соџога (46,64% и 0,12%, респективно). Етерично уље из стабла биљака из Со290
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џога садржи осам оксидованих монотерпена (22,17%), док су четири оксидована
монотерпена (11,15%), један монотерпенски угљоводоник (2,71%), и један оксидовани сесквитерпен (0,21%) нађени у биљкама из региона Алмех. Анализа етеричних уља осушених надземних делова биљака показала је присуство оксидованих
монотерпена, пулегона и ментола (Соџог регион) и пулегона, 1,8-цинеола, D-неоизоментола и хризантенона (Алмех регион), као главних састојака. Такође, хризантенон (9,75%), нађен као друга главна компонента уља из листа биљке Z. Clinopodioides из Алмеха, није био идентификован у другом региону или биљној врсти.
Резултати добијени одређивањем састава уља биљке Z. clinopodioides из два региона провинције Голестан показују генерално да постоје извесне разлике у главним
компонентама и њиховим релативним концентрацијама. Ове разлике вероватно потичу од различитих еколошких услова и генетских фактора, различитих хемотипова и нутриционог статуса биљака, као и других фактора који могу утицати на састав уља.
Кључне речи: Ziziphora clinopodioides, терпеноидна једињења, провинција Голестан, микроталасно-потпомогнута хидродестилација.
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ANTIOXIDANT AND SENSORIAL PROPERTIES OF ACACIA HONEY
SUPPLEMENTED WITH PRUNES
Vesna T. Tumbas*a, Jelena J. Vulića, Jasna M. Čanadanović-Bruneta, Sonja M. Djilasa,
Gordana S. Ćetković a, Slađana S. Stajčić a, Dubravka I. Štajner b and Boris M. Popovićb
a
b
University of Novi Sad, Faculty of Agriculture, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia
The changes in total phenol and flavonoid content, as well as antioxidant activity was
monitored in acacia honey supplemented with prunes in 20, 30 and 40% mass concentrations. The total phenolic content increased by 2.5 times (from 16.18 to 41.64 mg
GAE/100 g) with increasing concentration of prunes in honey, while the increase in flavonoid content was even higher, approximately 11.5-fold (from 2.65 to 30.86 mg RE/100
g). The addition of prunes also improved th eantioxidant activity of acacia honey. The
honey samples with highest content of prunes, 40%, exhibited the best antioxidant activity
measured by hydroxyl radical sacvenging assay (EC50•OH=4.56 mg/ml), 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging assay (EC50DPPH=16.48 mg/ml), and reducing power (EC50RP=81.17 mg/ml). Judging from the high correlation coefficients, ranging from 0.771 to 0.947 for total phenolics, and from 0.862 to 0.993 for total flavonoids,
it is obvious that these compounds were associated with the antioxidant mechanisms. On
the other hand, sensorial properties of supplemented honeys were lower than that of pure
acacia honey, where flavor of supplemented honey was the least affected. Our results indicate that the supplementation of honey with prunes improves antioxidant activity of honey by enriching the phenolic composition, with slight modifications in sensorial characteristics.
KEY WORDS: Acacia honey, prunes, phenolics and flavonoids, antioxidant activity,
sensorial properties
INTRODUCTION
Honey is a natural sweet substance that bees produce by transforming flower nectar or
other sweet secretions of plants (1). As an easily assimilable food, honey makes a
valuable nutritive product for children, athletes and convalescents (2).
It was reported that honey contains about 200 substances (3). It is essentially a concentrated aqueous solution of inverted sugar, but it also contains a very complex mixture
* Corresponding author: Vesna T. Tumbas, Faculty of Technology, University of Novi Sad, Bulevar cara
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of other saccharides, enzymes, amino and organic acids, polyphenols, carotenoid-like
substances, Maillard reaction products, vitamins and minerals (4).
Honey and other bee products have health-promoting properties which make them
usable in pharmacy and medicine, both as drug components, prophylactic agents and diet
supplements. Honey activity in gastrointestinal disorders has been reported, providing
gastric protection against acute and chronic lesions (5). Also, honey antimicrobial properties have been known for thousands of years and have been attributed to phenolic compounds derived directly from the honey (6).
It was reported that the composition and antioxidant activity of honey depend on the
floral source used to collect nectar by honeybee, seasonal and climatic factors. Also, processing may have an effect on honey composition and antioxidant activity (7, 8).
The components in honey responsible for its antioxidative effect are flavonoids,
phenolic acids, ascorbic acid, catalase, peroxidase, carotenoids, and products of the Maillard reaction. The content of these components varies widely according to the floral and
geographical origin of honey (8, 9).
Serbia has a very long tradition of beekeeping. Its favourable climate, good geographical conditions and a variety of botanical species provide a great potential for the development of apiculture (10). The most common unifloral honeys are the acacia (Robinia
pseudoacacia), sunflower (Helianthus annuus) and linden (Tilia cordata) honey.
In order to expand the range of bee products taking into account the interest and satisfaction of the consumers, new technological solutions are constantly being sought. One
method is to enrich the honey with some fruits which contain high-valuable organic compounds such as phenolics.
In view of this the purpose of the present study was to determine the total phenolic
and flavonoid content, as well as antioxidant activity of acacia honey supplemented with
prunes, by three different assays, hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
free radical scavenging assays and reducing power. Besides, a sensory analysis of the
supplemented honey was also performed.
EXPERIMENTAL
Chemicals and instruments
The chemicals used for these investigations were Folin-Ciocalteu reagent (Fluka Chemical Co., Buchs, Switzerland), trichloroacetic acid, 2,2-diphenyl-1-pichrylhydrazyl
(DPPH), 5,5-dimethyl-1-pyroline-N-oxide (DMPO), rutin and gallic acid (Sigma Chemical Co., St. Louis, Mo, USA). All other chemicals and reagents were of the highest analytical grade, obtained from J.T. Baker (Deventer, Holland).
The total phenolic, flavonoid, DPPH free radical scavenging assay and reducing power were determined using a UV-1800 spectrophotometer (Schimadzu, Kyoto, Japan),
while the antioxidant activity against reactive hydroxyl radicals was evaluated by electron spin resonance (ESR) spectroscopy (Bruker 300E ESR spectrometer, Rheinstetten,
Germany).
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Honey and prunes samples
The honey sample (AH), monofloral honey form of acacia (Rudnik region) (obtained
during 2009 from the honeybee farm, Simonović, Beograd) was supplemented with Stanley variety prunes (Blace region) (obtained from the producer Tehno-Božići, Šabac). Prunes were cut into four pieces and added to the acacia honey in mass concentrations of
20% (AH20), 30% (AH30) and 40 % (AH40).
Total phenolic content
The total phenolics were determined spectrophotometrically by the Folin-Ciocalteu
method (11). The content of total phenolics was expressed as mg of gallic acid equivalents per 100 g of honey sample (mg GAE/100 g).
Total flavonoid content
Total flavonoids were measured by the aluminium chloride spectrophotometric assay
(12). Total flavonoid content was expressed as mg of rutin equivalents per 100 g of honey
sample (mg RE/100 g).
Hydroxyl radical scavenging activity
Hydroxyl radicals (•OH) were generated in the Fenton reaction system obtained by
mixing 0.2 ml of 112 mM DMPO, 0.2 ml of H2O, 0.2 ml of 2 mM H2O2, and 0.2 ml of
0.3 mM Fe2+ (control) (13). The influence of honey samples, at the range of concentrations 5.0-25.0 mg/ml, on the formation and stabilization of hydroxyl radicals was investigated by ESR spin trapping method. The ESR spectra were recorded after 5 min, with the
following spectrometer settings: field modulation 100 kHz, modulation amplitude 0.226
G, receiver gain 5 x105, time constant 80.72 ms, conversion time 327.68 ms, center field
3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW and temperature 23°C. The SA•OH value of the honey samples was defined as:
SA•OH (%)= 100 × (h0 – hx) / h0
where h0 and hx are the heights of the second peak in the ESR spectrum of DMPO-OH
spin adduct of the control and the samples, respectively.
DPPH free radical scavenging assay
The scavenging activity of honey samples was determined spectrophotometrically
using the modified DPPH method (14). Briefly, honey samples were dissolved in methanol, and 1.5 ml of each sample or 1.5 ml of methanol (blank) was mixed with 3 ml of
DPPH in methanol (0.02 mg/ml). The range of investigated concentrations was 0.33166.67 mg/ml. The mixtures were left for 15 min at room temperature and then the absorbances was measured at 517 nm against reference mixtures that were prepared in the
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similar manner, by replacing the DPPH solution with methanol. The capability to scavenge the DPPH radicals, DPPH scavenging activity (SA), was calculated using the following equation:
SADPPH• (%) = (A0 – Ax)/A0 × 100
where A0 is the absorbance of the blank and Ax is the absorbance of the sample.
Reducing power
The reducing power of honey samples was determined by the method of Oyaizu (15).
For this puropse, the solution of honey samples (10–120 mg) in 1 ml of distilled water or
1 ml of distilled water (blank) was mixed with 1 ml of phosphate buffer (pH 6.6) and 1
ml of 1% potassium ferricyanide K3[Fe(CN)6]. The mixture was incubated at 50°C for 20
min and then rapidly cooled. Following this, 1 ml of trichloroacetic acid (10%) was added and the mixture was then centrifuged at 3000 rpm for 10 min. An aliquot (2 ml) of
the upper layer, mixed with 2 ml of distilled water and 0.4 ml of 0.1% FeCl3, was left to
stand for 10 min. The absorbance of the mixture was measured at 700 nm against the
blank.
Sensory analysis
The main sensory attributes evaluated in the analysis were: color, viscosity, aroma,
and flavor of honey samples, according to the Regulation on quality evaluation of bee
products in Novi Sad fair (16). The sensory analysis were carried out by means of analytical descriptive analysis method, based on points (grades) from 0 (unacceptable product)
to 3 (optimal quality level). The panel consisted of 5 expert descriptors.
Statistical analysis
All analyses were run in triplicate and the results were expressed as means ± standard
deviation (SD). Statistical analyses were done by using Origin 7.0 SRO software package
(OriginLab Corporation, Northampton, MA, USA, 1991–2002) and Microsoft Office Excel 2007 software. Significant differences were calculated by ANOVA test followed by
the least significant difference (LSD) test (p ≤ 0.05).
Supplemented acacia honey samples, AH20, AH30 and AH40, were subjected to
spectrophotometric analysis of total phenolic and flavonoid contents. The results were
compared to the contents in pure acacia honey, AH, obtained in our previous study (17)
and presented in Figure 1.
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Origin
ure 1. Total phenoolic and flavonoidd contents in pure (AH) and supplem
mented acacia
Figu
honeys (AH
H20, AH30 and AH
H40)
The concentrationss of both phenolics and flavonoidss in supplemented
d acacia honey
increased significantlyy (p ≤ 0.05) with rrising the share off prunes compared
d to pure acacia
honey
y. In comparison to raw honey, thee addition of 40% of prunes increased the contents
of tottal phenolic substtances by 2.5 timees (from 16.18 to 41.64 mg GAE/1
100 g), and the
flavo
onoid level was appproximately 11.55-fold higher (from 2.65 to 30.86 mg
m RE/100 g).
The flavonoids
f
contribbution to the total phenols increased with increase off prune content
(16.38%, 35.84%, 49.63% and 74.11%
% for AH, AH20, AH30 and AH40
0, respectively).
Our results
r
of total flavvonoids in acacia honey are in agreeement with the reeports of Amiot
et al. (18) (0.5-1 mg quercetin
q
equivaleent/100 g of Euroopean acacia honeey), but slightly
lowerr than the results of
o Meda et al. (199) (6.14 mg quercetin equivalent/10
00 g of Burkina
Faso acacia honey). However,
H
the ressults of total pheenolic contents arre significantly
higheer than that of Berretta et al. (20) (55.52 mg GAE/1000 g of commerciall acacia honey)
and Bertoncelj
B
et al. (221) (4.48 mg GAE
E/100 g of Sloveniian acacia honey)..
Ramanauskiene
R
et al. (22) have ideentified p-coumariic and ferulic aciids as the main
comp
ponents in acacia honey. Yao et al. (23) reported thhat myricetin, triccetin, quercetin
and luteolin, are dominnant flavonoid coompounds in Austtralian honeys. An
nother study on
Europ
pean honeys show
wed that in lime trree (Tilia europaeea) honey, the pro
opolis flavonoids, pinobanksin
p
and pinocembrin
p
are thhe most abundant flavonoids and th
he highest level
of pin
nobanksin was detected in the Euroopean acacia (Robinia pseudoacacia
a) honey sample (2..31 mg QE/100 g)) (24). The main ccompounds in fressh plum (Prunus domestica)
d
and
dried
d plum (prune) arre caffeoylquinic acids isomers (3--caffeoylquinic accid, 4-caffeoylquiniic acid, 5-caffeoyllquinic acid) (25). Plums also contain anthocyanins (cyanidin 3-Orutino
oside, cyanidin 3--O-glucoside, and peonidin 3-O-ruttinoside are predo
ominant), flavonols (quercetin 3-O-ruutinoside is predoominant) and proaanthocyanidins, which
w
represent
70% of total polyphennols (25). Fang ett al. (26) identifieed 42 compounds in commercial
dried
d plums, among which
w
the hydroxxycinnamic acids are essential. Th
he polyphenols
conteents are halved in commercial prunne compared to freesh plum due to the
t degradation
durin
ng drying (25). According to our rresults, flavonoidss have been transsfered to honey
moree rapidly than otheer polyphenols.
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In the recent years, there has been an increasing interest in the determination of the
antioxidant activity of honey. Beretta et al. (20) have reported that it is necessary to use a
combination of antioxidant tests, comparative analyses and statistical evaluation to determine the antioxidant behavior of honey. Therefore, we used three different antioxidant
tests to assess the activity of acacia honey samples. In our experiments, the decrease of
•
OH radical concentration, generated via Fenton reaction, was monitored using ESR spectroscopy, and the decrease in the DPPH radical concentration was monitored spectrophotometrically. Reducing power, another spectrophotometrical test, was used as a significant indicator of honey potential antioxidant activity. Antioxidant activities were expressed as EC50•OH and EC50DPPH• value (the amount of antioxidant necessary to decrease the
initial concentration of hydroxyl or DPPH radicals by 50%) and EC50RP value (the effective concentration assigned at 0.5 value of absorption) and presented in Table 1.
Table 1. EC50 values of pure (AH) and supplemented acacia honeys (AH20, AH30 and
AH40) measured by three antioxidant assays
Honey sample
AH
AH20
AH30
AH40
EC50•OH (mg/ml)
19.14  0.76
16.89  0.54
15.89  0.63
4.56  0.18
EC50DPPH• (mg/ml)
164.09  7.38*
24.48  1.05
18.65  0.84
16.48  0.69
EC50RP (mg/ml)
100.80  4.54*
96.75  3.82
92.13  4.25
81.17  3.95
* Taken form Savatović et al. (17)
The results of all three antioxidant tests presented the same trend. The supplemented
honeys, AH20, AH30 and AH40, exhibited higher antioxidant activity than pure honey,
AH. The antioxidant activity increased with increasing the concentration of prunes in the
honey. The greatest increase of the antioxidant activity was noted for the DPPH free
radicals, where EC50DPPH• value decreased 10 times compared to pure honey. The result of
the AH antioxidant acitivity was in agreement with Meda et al. (19). It has been shown
that dried plums have one of the highest ORAC values (5770) out of a group of 22 fruits
and vegetables studied, and that phenolic compounds appear to be the main contributors
to their antioxidant capacity (27).
Many studies have shown that the antioxidant activity is strongly correlated with the
content of total phenolics (7, 20, 8, 19). Gheldof et al. (4) stated that phenolic compounds
significantly contribute to the antioxidant activity of honey. Also, the authors suggested
that the antioxidant activity appeared to be a result of the combined activity of the honey
phenolics, peptides, organic acids, enzymes and Maillard reaction products.
For the correlation analysis, the EC50 values were transformed into their reciprocal
values (1/EC50). Table 2 shows Pearson’s correlation coefficients between the analyzed
antioxidant actvities of acacia honeys and composition variables.
Our results confirm that phenolic compounds in general, and especially flavonoids,
seem to be dominant compounds in honey-participating antioxidant reactions. The highest correlation coefficient of 0.993 reveals a very good correlation between the flavonoid
contents and reducing power of analyzed honey samples.
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Table 2. Correlation matrix (Pearson’s correlation coefficients) between the composition
variables and the antioxidant activities of pure and supplemented acacia honeys
Variable
1/EC50•OH
1/EC50DPPH
1/EC50RP
Total phenolics
Total flavonoids
Total
flavonoids
0.895
0.862
0.993
0.974
-
Total
phenolics
0.771
0.921
0.947
-
1/EC50RP
0.928
0.851
-
1/EC50DPPH•
1/EC50•OH
0.605
-
-
The relation between the three methods for the determination of antioxidant activity,
and between the results of total phenolics and total flavonoids, was also good (r was from
0.605 to 0.974). Cimpoiu et al. (28) found a significant correlation (r = 0.9928) between
the antioxidant activities determined by the DPPH and ABTS assays, suggesting that
these two assays are almost comparable and interchangeable in the case of honey, when
an evaluation of antioxidant activity is required. In our study, the antioxidant assays correlated very well except for the correlation between the DPPH and hydroxyl radical
assays, which was lower, but still good enough (Table 2). It is assumed that honeys
contain compounds that can quench hydroxyl radicals also by additional reactions. Gheldof and Engeseth (8) found that the antioxidant capacities of honeys, evaluated by the
oxygen radical absorbance capacity (ORAC) assay, showed a linear dependence on the
total phenolics content. Zalibera et al. (29) investigated radical-scavenging capacities of
15 Slovak honeys using ESR and cation radical of ABTS (2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt), DPPH and hydroxyl radicals generated by
the photochemical decomposition of hydrogen peroxide. They found good ABTS•+ and
DPPH free radical scavenging capacity of tested honeys, and that this capacity correlated
well with the phenolics content. However, no correlation was found between the monitored ability to scavenge hydroxyl radicals and either phenolics content. Beretta et al.
(20) found that the close interdependence between the phenolics content, FRAP, DPPH,
and ORAC indicates that the antioxidant capacity of the honeys is due to their phenolic
constituents, which are able to interact with Mo(VI) and Fe(III) with a H/e− transferring
mechanism, and less to other chemical entities.
Honey samples with and without prunes were evaluated by a 5-member trained expert
descriptive attribute sensory panel. The samples were evaluated for color, viscosity, aroma and flavor. Samples were scored using the 0 to 3 intensity scale and the results are
presented in Figure 2.
The consumer evaluations of acacia honey samples indicated that pure honey, AH,
exhibited best sensorial properties, while the addition of prunes affected viscosity at the
highest level, and only slightly affected the flavor (3.0, 2.6 and 2.5 for AH20, AH30 and
AH40, respectively) and aroma (2.0, 2.6 and 2.0 for AH20, AH30 and AH40, respectively) of the honey. However, the color of the supplemented honeys was rated lower, related
to the changes in pale yellow color of pure acacia honey due to the dark brown pigmentation of the dried plum (2.0, 2.6 and 2.0 for AH20, AH30 and AH40, respectively).
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Origin
h
(AH20,
Figure 2. Sensorial chharacteristics of puure (AH) and suppplemented acacia honeys
AH330 and AH40)
The color of honeyy is related to the content of phenollics, besides mineerals and pollen
(30, 31).
3 In this regardd, this result is in aagreement with a higher content off phenolic compounds in the supplem
mented honeys. Nuuñez de Gonzalez et al. (27) investig
gated the effect
of pru
unes inclusion in pork sausages on their sensorial chharacteristics. Thee levels of dried
plum
m puree treatments had no effect on flavor intensity, texture,
t
or level of
o juiciness, but
did in
nfluence perceptioons for overall likee/dislike and overrall flavor of the prroduct.
The correlation anaalysis was also em
mployed to analyzze the relationship
ps between the
senso
orial properties annd composition vaariables evaluatedd, i.e. the total ph
henolic and flavonoid contents (Tablee 3).
Ta
able 3. Correlationn matrix (Pearson’’s correlation coeffficients) between composition
variables and sennsorial characteristtics of pure and suupplemented acacia honeys
Variable
Color
Viscosity
Aroma
Flavor
Totaal flavonoids
-0.871
-0.582
-0.582
-0.934
Total phenolics
-0.738
-0.535
-0.535
-0.968
Close
C
relationshipss between the totall phenolics and flaavonoids of the ho
oneys and their
colorr and flavor were confirmed
c
by highh correlation coeffficients between these
t
variables.
The negative
n
values off the correlation ccoefficients mean that the rate of sen
nsory characteristic decreased with inncreasing total phhenolic/flavonoid value. The relatio
onship between
colorr and phenolic conntents in honeys w
was also confirmedd by some other authors
a
(21, 29,
32).
CO
ONCLUSION
This study shows thhat acacia honey ssupplemented witth prunes can add many healthgiving antioxidants to thhe diet. The suppplemented honeyss contain substanttial amounts of
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bioactive compounds, originating from honey itself and also from the added prunes. Experimental results and statistical analysis show that these phytochemicals increase the
honey’s antioxidant activity but also slightly affect sensorial properties. Considering these findings, the content of prunes could be optimized to obtain the best combination of
the antioxidant and sensorial properties of the product. Also, the future studies can be focused on the identification and quantification of phenolics and other compounds present
in the supplemented honeys, and revealing other biological activities of these products in
respect of health benefits that make these honeys a consumer-valuable product. This product can be recommended to complete other polyphenol sources such as vegetables and
fruits.
Acknowledgement
This research is part of the Project No. 114-451-2093/2011-03, which is financially
supported by the Provincial Secretariat for Science and Technological Development of
the Autonomous Province of Vojvodina, Republic of Serbia.
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32. Frankel, S., Robinson, G. E., Berenbaum, M. R.: Antioxidant capacity and correlated
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АНТИОКСИДАТИВНЕ И СЕНЗОРНЕ КАРАКТЕРИСТИКЕ БАГРЕМОВОГ
МЕДА СА ДОДАТКОМ СУВИХ ШЉИВА
Весна Т. Тумбаса*, Јелена Ј. Вулића, Јасна М. Чанадановић-Брунета, Соња М.
Ђиласа, Гордана С. Ћетковића, Слађана С. Стајчића, Дубравка И. Штајнерб
и Борис М. Поповићб
а
Универзитет у Новом Саду, Технолошки факултет Нови Сад, Булевар цара Лазара 1, 21000 Нови Сад,
Србија
б
Универзитет у Новом Саду, Пољопривредни факултет, Трг Доситеја Обрадовића 8, 21000 Нови Сад,
Србија
У багремов мед су додате суве шљиве у масеним концентрацијама 20, 30 и 40%.
Утицај сувих шљива на особине меда испитан је мерењем садржаја укупних фенолних једињења и флавоноида, као и антиоксидативне активности. Са повећањем
концентрације сувих шљива у меду укупан садржај фенолних једињења повећао се
2,5 пута (од 16,18 до 41,64 mgGAE/100g), док је повећање садржаја флавоноида још
веће, око 11,5 пута (од 2,65 до 30,86 mgRE/100g). Додатак сувих шљива утицао је и
на повећање антиоксидативне активности меда. Узорци меда са 40% сувих шљива
показали су највећу антиоксидативну активност, која је одређена на слободне хидроксил радикале (EC50•OH=4,56 mg/ml) и 2,2-дифенил-1-пикрилхидразил (DPPH) радикале (EC50DPPH=16,48 mg/ml), као и тестом редукционе способности (EC50RP=81,17
mg/ml). Судећи по утврђеним високим коефицијентима корелације, у опсегу од
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0,771 до 0,947 за фенолна једињења, односно у опсегу од 0,862 до 0,993 за флавоноиде, може се закључити да ова једињења учествују у механизмима антиоксидативног деловања узорака меда. Са друге стране, сензорне карактеристике меда са
додатком сувих шљива оцењене су нижом оценом од чистог багремовог меда, при
чему је додатак шљива имао најмањи утицај на укус. Резултати испитивања су
показали да се додатком сувих шљива у мед побољшава антиоксидативна активност меда повећањем садржаја полифенолних једињења у њему, уз мале модификације сензорних карактеристика.
Кључне речи: багремов мед, суве шљиве, фенолна једињења и флавоноиди, антиоксидативна активност, сензорне карактеристике
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SCREENING OF ANTIBACTERIAL ACTIVITY OF RASPBERRY
(Rubus idaeus L.) FRUIT AND POMACE EXTRACTS
Aleksandra S. Velićanski*, Dragoljub D. Cvetković and Siniša L. Markov
Antibacterial activity of fruit and pomace extracts (concentration 50 mg/ml) of two
raspberry (Rubus idaeus L.) cultivars (Meeker and Willamette) was tested against selected Gram-positive and Gram-negative bacteria (reference and wild strains). Disc diffusion method with 15 µl of extracts and agar-well diffusion method with 50 and 100 µl
were used. Antibiotic (cefotaxime/clavulanic acid) was used as a control. Both raspberry
fruit extracts showed the strongest antibacterial activity against Pseudomonas aeruginosa (wild strain) and Bacillus cereus, where the largest clear zones (without growth) appeared. Escherichia coli was the most resistant strain, with only zone of reduced growth.
The highest antibacterial activity of pomace extracts was against Staphylococcus aureus
and Staphylococcus saprophyticus. There were no differences in the antibacterial activity
between cultivars for both fruit and pomace extracts.
KEY WORDS: Rubus idaeus L., raspberry fruit and pomace extracts, antibacterial
activity, agar diffusion method
INTRODUCTION
Rubus fruit have long been collected and consumed worldwide, regardless of whether
they were recognised for their possible health benefits from their natural phytochemicals
or simply because they tasted good. Today, rubus fruit are considered a healthy and
nutritious food, containing phenolics, vitamin C, dietary fibre, α-tocopherol, tocotrienol,
calcium, potassium, magnesium, carotenoids, linoleic acid (1). Serbia is one of the largest
producers and exporters of raspberries (Rubus idaeus L.) in the world. Between 90 and
95% of cultivated raspberries in Serbia are North American Willamette cultivar, which is
characterized by the excellent taste and a dark red colour. Besides the Willamette, the
Meeker cultivar is also popular (2,3).
Berries contain a variety of phenolic compounds (phenolic acids, flavonoids, lignans
and polymeric tannins) located in plant tissues, often in the surface layer of the plant or
berry, which is in connection to their main natural function, to protect the plant against
environmental stress and pathogens. The main phenolic compounds in raspberries are fla* Corresponding author: Aleksandra S. Velićanski, University of Novi Sad, Faculty of Technology, 21000 Novi
Sad, Bulevar cara Lazara 1, Serbia, e-mail: [email protected]
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vonoid anthocyanins (coloured substances), ellagic acid and ellagitannins, complex water-soluble phenolic polymers. From the phenolic acids, hydroxycinnamic or hydroxybenzoic acid derivatives are the most common in berries (1, 4).
Various phytochemical constituents of berry fruits exhibit a wide range of biological
effects, including antioxidant, anticarcinogenic, antiinflammatory, antineurodegenerative,
antiviral, and antibacterial activities which are attributed to the phenolic compounds, such
as flavonoids, phenolic acids and tannins. Due to a high content and wide diversity of
phenolic compounds and their health-promoting properties, berries are often regarded as
natural functional products properties (4-6). Not only the fruit, but also the raspberry
leaves and roots have long tradition as medicinal agents. Infusion from the leaves are traditionally used for easing childbirth-related muscle spasms, morning sickness, for colds,
sour throats, diarrhoea, threat wounds, colic pain, uterin relaxant, etc. The roots of the
plant are traditionally used for wound cleaning and relief from sore throats (7, 8).
Berries have been used as natural antimicrobial pharmaceuticals. For example, cranberry has been reported to control the growth of Listeria monocytogenes and to possess
compounds suppressing adhesion and growth of Helicobacter pylori and bacteria causing
urinaty tract infections (4). Phenolic berry extracts inhibit the growth of selected Gramnegative intestinal bacteria, while they are not active against Gram-positive benefitial
probiotic lactic acid bacteria. Cloudberry and raspberry phenolic extracts were shown to
be strong inhibitors of a nonvirulent Salmonella strains (9).
By-products of plant food processing represent a major disposal problem for the industry concerned, but they are also promising sources of compounds which may be used
because of their favourable technological or nutritional properties (10). These phytochemicals from waste materials deriving from agro-industrial production may be used as
functional food ingredients and as natural antioxidants to replace their synthetic equivalents. So, pomace (peels, seeds, and flesh remaining after juice pressing) extracts of 20
cultivated and wild fruits and berries showed antioxidant and antimicrobial activity (11).
Taking into account biological activities of raspberry as well as possible potential of
plant by-products, in this study, fruit and pomace extracts from two raspberry (Rubus
idaeus L.) cultivars (Meeker and Willamette) were used to screen antibacterial activity to
eight reference cultures and wild strains. Two methods were used: disc diffusion method
with the limited capacity of discs and agar-well diffusion method with much higher volume of holes.
EXPERIMENTAL
Plant material
Two raspberry (Rubus idaeus L.) cultivars (Meeker and Willamette) were obtained
from “Alfa RS”, Lipolist, Serbia. Samples of the raspberry were stored at -20oC until analysis. Raspberry pomaces from both cultivars was obtained after juice separation. The
yields of the Meeker and Willamette pomaces were 126.89 g and 101.19 g, respectively.
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Preparation of raspberry fruit and pomace extracts
Samples of the raspberry cultivars (70 g) and their pomaces (70 g) were extracted two
times: 60 minutes (560 ml) and 30 minutes (280 ml) at room temperature using a homogenizer, Ultraturax, DIAX 900 (Heidolph Instruments GmbH, Kelheim, Germany). The
extraction was performed with an 80% methanol aqueous solution containing 0.05% acetic acid. The obtained extracts were combined and evaporated to dryness under reduced
pressure and lyophilisated (Alpha 2-4 LSC Martin Christ, Osterode, Germany).
The yields of the lyophilisated fruit extracts from Meeker and Willamette cultivars
were: 8.39 g and 7.74 g, and of the lyophilisated pomace extracts were 6.99 g and 6.6 g,
respectively.
Samples for the determination of antibacterial activity
Samples for the determination of antibacterial activity were two raspberry (Rubus
idaeus L.) fruit and pomace extracts, from Meeker and Willamette cultivars, which were
dissolved in sterile distilled water to a concentration of 50 mg/ml.
Bacterial strains
Bacterial strains for the determination of antibacterial activity were reference and wild
strains from foodstuffs and drinking water. Gram-negative bacteria were: Salmonella
typhymurium (ATCC 14028), Escherichia coli (ATCC 10536), Pseudomonas aeruginosa
(ATCC 27853) and Pseudomonas aeruginosa (isolated from drinking water). Grampositive bacteria were: Staphylococcus aureus (ATCC 11632), Bacillus cereus (ATCC
10876), Staphylococcus saprophyticus (isolated from the confectionery product) and Listeria monocytogenes (isolated from minced meat). Wild strains were identified using Vitek®2 Compact System (bioMérieux, France).
Antimicrobial assay
Antibacterial activity was determined by disc diffusion and agar-well diffusion method (12). Bacterial strains were grown on Müeller–Hinton slant (Himedia, Mumbai, India)
for 24 h at 37°C, except Bacillus cereus, which was grown at 30°C. Cells were then suspended in a sterile 0.9% NaCl solution. The suspension for inoculation was adjusted to a
concentration of 1×106 cfu/ml, which was estimated by Densichek (Biomerieux, France).
A volume of 2 ml of the suspensions for inoculation was homogenised with 18 ml of melted (45°C) Müeller–Hinton agar and poured into Petri dishes.
For disc diffusion method, sterile 6 mm discs (Himedia, Mumbai, India) were placed
on the inoculated agar plates and impregnated with 15 µl of extracts solution. The antibiotic (30 µg cefotaxime /10 µg clavulanic acid per disc, Bioanalyse®, Ankara, Turkey)
was used as control. For the agar-well diffusion method, wells of 9 mm diameter were
made with a sterile metal tube by means of a vacuum pump. The extracts solution (50 and
100 µl) was then transferred into the wells of inoculated agar plates. For both methods,
the test plates were refrigerated at 8°C for 1 hour to allow the extracts to diffuse into the
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medium, and then were incubated for 24 hours at 37°C or 30°C (Bacillus cereus). After
the incubation, the diameters of the inhibition zones were measured and recorded in millimeters (mm). The evaluation of antibacterial activity was carried out in triplicate.
RESULTS AND DISSCUSION
The results of antibacterial activity of two raspberry fruit and pomace extracts are
presented in Tables 1 and 2.
Table 1. Antibacterial activity of raspberry fruit extracts (mean of diameter of the inhibition zone (mm) including disc (6 mm) or well (9 mm) with standard deviation in
parentheses)
Method
Agar-well
Control(cefotaxime/
Group
Tested strains
Cultivar
diffusion
clavulanic acid)
50 µl
100 µl
G (–)
15.5**
34.87*
12.58**
M
nd
bacteria
(0.0)
(0.58)
(0.35)
Escherichia coli
ATCC 10536
16.5**
14.0**
W
nd
(0.82)
(0.58)
16.5*
16.5*
M
nd
Salmonella
(1.73)
(0.58)
35.6*
typhymurium
(0.85)*
15.0*
13.5**
ATCC 14028
W
nd
(1.73)
(0.0)
11.75**
14.25*
7.75**
M
Pseudomonas
(0.5)
(0.5)
(0.5)
20.17*
aeruginosa
**
*
(0.76)
15.25
12.25
ATCC 27853
W
nd
(0.5)
(0.5)
*
*
*
11.75
16.0
7.0
M
(0.0)
(0.5)
(2.31)
Pseudomonas
15.33*
aeruginosa***
(0.58)
13.0*
18.0*
7.0*
W
(0.0)
(0.0)
(0.0)
G (+)
14.5*
16.0*
37.3*
7.5**
M
Staphylococcus
bacteria
(0.55)
(0.58)
(0.0)
(0.75)
aureus
14.5*
16.0*
8.5**
ATCC 11632
W
(0.55)
(0.5)
(0.0)
13.5*
M
nd
nd
(1.0)
Staphylococcus
27.0*
saprophyticus***
(0.5)
13.5*
W
nd
nd
(0.58)
13.5*
16.25*
7.5*
M
Bacillus Cereus
(0.58)
(0.58)
(0.5)
35.5*
(1.5)
13.5*
17.0*
9.0*
ATCC 10876
W
(0.5)
(0.58)
(1.0)
12.5*
9.5*
M
nd
(0.58)
(0.58)
Listeria
12.25*
monocytogenes***
(0.55)
12.5*
10.0*
W
nd
(0.0)
(0.58)
M – Meeker cultivar; W – Willamette cultivar; nd - not detected inhibition zone; * - clear zone
around the disc/well; ** - zone of reduced growth; *** - wild strain.
Disc
diffusion
15µl
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Table 2. Antibacterial activity of raspberry pomace extracts (mean of diameter of the
clear inhibition zone (mm) including disc (6 mm) or well (9 mm) with standard deviation
in parentheses)
Method
Group
Tested strains
Cultivar
Disc diffusion
15µl
G (–)
bacteria
21.75
(0.5)
20.0
W
(0.85)
22.0
M
Salmonella
(0.82)
typhymurium
23.0
ATCC 14028
W
(1.73)
18.25
M
Pseudomonas
(0.5)
aeruginosa
16.75
ATCC 27853
W
(0.5)
15.75
M
(1.26)
Pseudomonas
*
aeruginosa
15.5
W
(1.0)
G (+)
34.75
M
bacteria
(0.96)
Staphylococcus aureus
ATCC 11632
33.25
W
(0.96)
11.75
M
(0.96)
Staphylococcus
saprophyticus*
12.25
W
(0.96)
22.5
M
(0.58)
Bacillus cereus
ATCC 10876
24.25
W
(0.96)
10.0
M
(0.05)
Listeria
*
monocytogenes
10.75
W
(0.5)
M – Meeker cultivar; W – Willamette cultivar, * - wild strain
Escherichia coli
ATCC 10536
M
Agar-well diffusion
50 µl
100 µl
37.33
(0.58)
25.67
(0.58)
29.5
(1.0)
30.0
(1.0)
22.67
(0.58)
22.0
(1.0)
20.67
(0.58)
21.33
(1.15)
41.5
(4.43)
39.0
(1.15)
23.0
(1.0)
21.0
(0.5)
32.0
(0.58)
28.75
(2.5)
16.67
(0.58)
17.33
(0.58)
34.32
(1.54)
31.67
(0.58)
35.5
(1.0)
34.0
(1.63)
25.0
(1.0)
24.67
(0.58)
24.0
(1.0)
24.67
(0.58)
48.5
(1.0)
46.0
(1.63)
39.0
(1.0)
38.0
(1.0)
36.0
(1.0)
36.0
(1.63)
30.0
(1.0)
30.33
(0.58)
The smallest inhibition zones of raspberry fruit extracts for all tested bacterial strains
were observed by disc diffusion method (Table 1). Clear zones (not higher than 9 mm)
were found only for Pseudomonas aeruginosa (wild strain) and Bacillus cereus. Similar
clear zones of reduced growth were found for Pseudomonas aeruginosa and Staphylococcus aureus. All strains except for Staphylococcus saprophyticus were susceptible to 50 µl
of extracts, and higher zones appeared with using 100 µl of both Meeker and Willamette
fruit extracts. The most susceptible strains tested by agar-well diffusion method, were
Pseudomonas aeruginosa (wild strain) and Bacillus cereus. From all tested strains, Escherichia coli was the most resistant strain because only zone of reduced growth appea309
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red, even when 100 µl of extracts were used. This result indicate bacteriostatic activity of
tested extracts against Escherichia coli. The smallest clear zones of 12.5-13.5 mm are
appeared for Listeria monocytogenes and Staphylococcus saprophyticus by applying
agar-well diffusion method (Table 1).
As for raspberry pomace extracts (Table 2), for both methods and extracts volumes
(15 µl, 50 µl and 100 µl) only clear zones appeared, indicating bactericidal activity of the
pomace extracts. Both Meeker and Willamette pomace extracts showed the highest
antibacterial activity against Staphylococcus aureus and Staphylococcus saprophyticus,
while the smallest zones appeared for Pseudomonas strains (both reference and wild
strains).
The inhibition zones of tested control (cefotaxime/clavulanic acid disc) were significant, and for all bacteria clear zones around the discs appeared. The antibiotic showed the
most pronounced activity against Escherichia coli, Salmonella typhymurium, Staphylococcus aureus and Bacillus cereus (more than 30 mm). The least clear zone (less than 15
mm) appeared for Listeria monocytogenes, the strain with the smallest clear zone in the
case of action raspberry fruit extracts. Raspberry pomace extract showed similar or
higher activity against tested strains compared with control.
Rauha et al. (13) tested the antimicrobial activity of Rubus idaeus L. extract (extracted
with 70% aqueous acetone) by cylinder diffusion method (500 µl of extract, concentration of 1 mg/ml). Only slight antibacterial activity was obtained against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Escherichia coli, and
moderate activity for Bacillus subtilis. Antibacterial activity of raspberry extract in concentration of 1 mg/ml (extracted with acetone:water 70:30 v/v), on the growth of microbial strains in liquid culture was tested by Nohynek et al. (4). Results showed the strong
activity of raspberry extract, as it caused death of the Helicobacter pylori, very strong inhibition of Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis, and
strong inhibition of Campylobacter jejuni and Clostridium perfringens. Strong antibacterial activity was attributed to phenolic compounds, especially ellagitannin fraction (4).
Study of Ryan et al. (7) showed absence of antimicrobial activity of water and ethanol
raspberry leaf extracts. On the other hand, raspberry juice and cordial displayed growth
inhibition (at 10% and 20% concentration) of Staphylococcus aureus, Escherichia coli,
Mycobacterium phlei, Clostridium perfringens, Alcaligenes faecalis, Enterococcus faecalis, Shigella soneri and three Salmonella serovars (7). Similarly, using 100 µl of raspberry leaf methanolic extract (concentration of 20 mg/ml), Bonjar (14) obtained inhibition zone of 10-14 mm only for Bordetella bronchiseptica, which was generally (taking
into account all 48 plant species tested) the most sensitive strain among eleven strains
tested.
Ördögh et al. (11) tested antibacterial activity of the raspberry juice, as well as water
and methanol extracts of the pomace against acne-inducing bacteria by broth microdilution assay. Raspberry juice showed antibacterial activity only against Staphylococcus
epidermidis (minimal inhibitory concentration at the pH 7 was 12.35 mg/ml, and 9.18
mg/ml at the pH 5.5). Juice did not affect the growth of S. aureus, Streptococcus pyogenes and Propionibacterium acnes, while pomace extracts did not show any antibacterial activity. The difference in the antibacterial activity of raspberry juices and pomace
extracts tested in this study (from 20 cultivated and wild fruits) may be due to their
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different components; soluble in aqueous and alcoholic media. Water extract contains the
majority of anthocyanins, tannins, starches, saponins, polypeptides and lectins, while methanol extracts also contain polyphenols, lactones, flavones, and phenons (11).
Pupponen-Pimiä et al. (9) tested antimicrobial activity of Nordic berries including
raspberry (2 and 10 mg of lyophilized berry powders/ml) and phenolic berry extracts
(concentration of 1 and 5 mg/ml) against eight human pathogens in liquid culture by plate
count method. Salmonella enterica sv. Typhimurium and Staphylococcus aureus were
strongly affected by lyophilized raspberry. However, no effects on the growth of Listeria
monocytogenes was obtained. Phenolic raspberry extracts showed strong inhibitory activity against Staphylococcus spp. Salmonella spp. were only partly inhibited by the raspberry phenolic extrscts (9).
Phenolic compounds affected the growth of different bacterial species by different
mechanisms, yet not well understood. It is assumed that not only phenolic compounds are
responsible for the antimicrobial activity. The fruits of the genus Rubus are rich in ellagitannins, which can permeate the outer cell membrane of Gram-negative bacteria (9, 11).
So, antimicrobial activity of berries is likely to be caused by multiple mechanisms and
synergies because they contain various compounds, for example, weak organic acids,
phenolic acids, and tannins and their mixtures of different chemical forms (4). Additionally, the effect of pH is very important in case of microbicidal acids of the fruits. These are
membrane-active substances which damage the inner cell membrane in their undissociated form. They alter the membrane permeability of the microbial cell and acidify the
cytoplasm (11). In the study of Ördögh et al. (11), the antibacterial effect of juices and
pomace extracts was more or less independent of the pH, suggesting that other, non-dissociable compounds were responsible for the growth inhibition.
It is obvious that there are differences between the results shown in Tables 1 and 2,
and other mentioned studies. The observed differences are probably caused by the different methods and concentrations of tested extract solutions, but also, results depend on
the susceptibility of tested wild strains, as well as the composition and amount of active
components extracted from tested materials originated from different geographic areas,
growth conditions of plant material as well as seasonal variations (7).
CONCLUSION
In this study, the antibacterial potential of a Rubus idaeus L. (raspberry) fruit and pomace extracts against selected Gram-positive and Gram-negative organisms has been demonstrated. These findings can form the basis for further studies to isolate active compounds, elucidate their structures, and also evaluate them against wider range of bacterial
strains with the goal to find new therapeutic principles.
Аcknowledgement
This research is a part of the Project of the Ministry of Education and Science of the
Republic of Serbia (Project TR 31044).
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cordial in vitro. Res. Vet. Sci. 71 (2001) 155-159.
8. Venskutonis, P.R., Dvaranauskaite, A. and Labokas, J.: Radical scavenging activity
and composition of raspberry (Rubus idaeus) leaves from different locations in Lithuania. Fitoterapia 78 (2007) 162-165.
9. Puupponen-Pimiä, R., Nohynek, L., Hartmann-Schmidlin, S., Kähkönen, M., Heinonen, M., Määttä-Riihinen, K. and Oksman-Caldentey, K.-M.: Berry phenolics selectively inhibit the growth of intestinal pathogens. J. Appl. Microbiol. 98 (2005) 9911000.
10. Schieber, A., Stintzing, F.C. and Carle, R.: By-products of plant food processing as a
source of functional compounds – recent developments. Trends Food Sci. Tech. 12
(2001) 401-413.
11. Ördögh, L., Galgóczy, L., Krisch, J., Papp, T. and Vágvölgyi, C.: Antioxidant and antimicrobial activities of fruit juices and pomace extracts against acne-inducing bacteria. Acta Biologica Szegediensis 54, 1 (2010) 45-49.
12. Mayo, W.J.: Chemical methods of control: Antimicrobial drugs, in Laboratory experiments in microbiology. Eds. Johnson, T. R. and Case, C. L., The Benjamin/Cummings Publishing Company, San Francisco (1998) pp. 179-181.
13. Rauha, J-P., Remes, S., Heinonen, M., Hopia, A., Kähkönen, M., Kujala, T., Pihlaja,
K., Vuorela, H. and Vuorela, P.: Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. Int. J. Food Microbiol. 56 (2000) 312.
14. Bonjar, S.: Evaluation of antibacterial properties of some medicinal plants used in
Iran. J. Ethnopharmacol. 94 (2004) 301-305.
312
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СКРИНИНГ АНТИБАКТЕРИЈСКЕ АКТИВНОСТИ ЕКСТРАКAТА ВОЋА
И ТРОПА МАЛИНЕ (Rubus idaeus L.)
Александра С. Велићански, Драгољуб Д. Цветковић и Синиша Л. Марков
Антибактеријска активност екстраката воћа и тропа две сорте (Meeker и Willamette) малине (Rubus idaeus L.), концентрације 50 mg/ml, испитана је на одабране
Грам–позитивне и Грам–негативне бактерије (референтни и дивљи сојеви). За испитивање су коришћене диск-дифузиона метода (са 15 µl екстраката) и метода бунарчића у подлози (са 50 и 100 µl). Екстракти воћа од обе сорте показали су највећу
активност према сојевима Pseudomonas aeruginosa и Bacillus cereus, док је Escherichia coli показала најмању осетљивост. Екстракти тропа обе сорте малине дали су
зоне без раста (показатељ бактерицидног деловања) за све тест микроорганизне,
што указује на значајан антибактеријски потенцијал тропа малине. Највећу антибактеријску активност екстракти тропа су показали према Staphylococcus aureus и
Staphylococcus saprophyticus. Сорта малине није утицала на антибактеријску активност испитаних екстраката.
Кључне речи: Rubus idaeus L., екстракти воћа и тропа малине, антибактеријска
активност, агар дифузиона метода
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OPTIMIZATION OF THE Ocimum basilicum L. EXTRACTION PROCESS
REGARDING THE ANTIOXIDANT ACTIVITY
Senka S. Vidovića*, Zoran P. Zekovića, Žika D. Lepojevića, Marija M. Radojkovića, Stela
D. Jokićb and Goran T. Anačkovc
a
University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology, Franje Kuhača 20, 31000 Osijek,
Croatia
c
University of Novi Sad, Faculty of Natural Sciences, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia
b
The levels of input variables (temperature and extraction solvent) that optimize a particular response (total phenols content, total flavonoids content and antioxidant activity)
of the Ocimum basilicum L. extraction process were determined by the response surface
methodology (RSM). The influence of theextraction temperature on extraction process
was investigated in the range from 33.8ºC to 76.2ºC, as well as of extraction solvent
ethanol, in the range of concentrations from 21.7% to 78.3%. For the preparation of
basil dry extract, characterized with minimal IC50 value, the calculated optimal values of
temperature and ethanol concentration were: 75.33ºC and 73.66% (w/w).
KEY WORDS: basil, extraction, antioxidants, RSM.
INTRODUCTION
Sweet basil (Ocimum basilicum L.) is a widely used herb with many properties and
applications. This herb is used not only for the cooking, but also in commercial fragrances, flavourings and for increasing the shelf life of food products (1). It is distributed
in different folk medicines for a treatment of insomnia, kidneys inflammation, cough,
asthma, inflammation of urinary tract, etc. Basil extract has a sedative and anticonvulsant
properties (2), as well as important antimicrobial and antifungal activity (3). Herbs are
considered as sources of different antioxidant compounds. These compounds are of great
importance in terms of their use for preventing oxidative stress that may cause several
degenerative diseases. Many epidemiological studies, prove the existence of a link between diets rich in antioxidants and a reduced risk of diseases, particularly of cancer and
cardiovascular diseases. Beside their importance due to their medicinal properties antioxidants are important as preservatives in the food industry.
The high antioxidant activity of basil and its extracts, and the majority of its medicinal
properties, have been attributed primarily to rosmarinic acid (4), but some other like caf* Corresponding author: Senka S. Vidović, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara
1, 21000 Novi Sad, e-mail: [email protected]
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feic acid derivates, such as cichoric acid (5, 6), are also found in substantial concentrations. Rosmarinic acid belongs to phenolic compounds which have been marked as main
antioxidant agent.
In a past few years, much attention has been paid to the extraction of antioxidants and
the creation of antioxidant supplements from different natural sources (herbs, fruits and
vegetables). This attention increases since some of synthetic antioxidants used in the food
and other industries have been reported to be toxic. Natural antioxidants could replace
synthetic once, which is a tendency in the modern pharmaceutical and food industries.
Extracts from natural plants, as carriers of antioxidant compounds and antioxidant activity, could be obtained by different methods, by using different solvents and at different
process conditions (temperature, time, pressure, etc.). Extraction with organic solvents
offers good recovery of polyphenols, which is related to the preparation of extracts with
high antioxidant activity. As solvent extraction can be performed by different solvents
and at different process conditions, to obtain the extracts with appropriate properties, the
analysis and selection of optimal process parameters is necessary. To find the levels of
input variables that optimize a particular response (total phenols content, total flavonoids
content, and antioxidant activity) response surface methodology (RSM), as one of the
best optimization tools, can be applied. The objective of this study was to employ RSM
to assess the effect of different combinations of temperature, in the range from 33.8ºC to
76.2ºC, and aqueous solution of ethanol as extraction solvent, in a range from 21.7% to
78.3%, on the total phenols content (TP), total flavonoids content (TF), and antioxidant
activity of O. basilicum extracts.
EXPERIMENTAL
Plant material and sample preparation
Basil samples were collected in Bosnia and Herzegovina, Republika Srpska, near
Banja Luka, in July 2009. The collected plant material has been naturally dried under sun
and then stored in paper bags at room temperature. Voucher specimens (Ocimum basilicum L. 1753 No 2-1792, Bosnia and Herzegovina, Banja Luka, Česma, ruderal habitats,
08.2008. det.: Goran Anačkov) were confirmed and deposited at the Herbarium of the
Department of Biology and Ecology (BUNS Herbarium), Faculty of Natural Sciences,
University of Novi Sad, Serbia (7). The material was ground in a blender just before the
extraction. The particle size, 0.726±0.13 mm, was determined using sieve sets (Erweka,
Germany). The ground plant material (10 g) was extracted by aqueous solution of ethanol
(100 ml) of specific concentration at different extraction temperatures. The extraction
process was carried out for 90 minutes. After filtration, the solvent was evaporated and
obtained dry extract was analyzed for the total phenols, total flavonoids and antioxidant
activity.
Determination of total phenolics and total flavonoids
The content of total phenolics (TP) in the O. basilicum extract was determined by the
Folin-Ciocalteu procedure (8, 9) and expressed as mg of gallic acid equivalent (GAE) per
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g of dry O. basilicum extract. The total flavonoids content (TF) was determined by aluminium chloride colorimetric assay (10) and it was expressed as mg of catehin equivalents
(CE) per g of dry O. basilicum extract.
DPPH assay
The free radical scavenging activity of O. basilicum extracts was determined as described by Espin (11). Briefly, dry O. basilicum extract was mixed with methanol (96%)
and 90 M of 2,2-diphenyl-1-picryl-hydrazyl (DPPH) solution to give different final concentration (from 0.0025 mg/ml to 0.008 mg/ml) of extract. After 60 min at room temperature, the absorbance was measured at 517 nm and expressed as radical scavenging capacity. Radical scavenging capacity (%RSC) was calculated by the following equation:
% RSC  100 
Asample  100
[1]
Ablank
where: Asample is the absorbance of the sample solution and Ablank is the absorbance of the
blank control.
This activity was also expressed as the inhibition concentration at 50% (IC50), the
concentration of test solution required to obtain 50% of radical scavenging capacity.
Experimental design
Response surface methodology (RSM) was employed to analyze the effects of two
factors on three responses and to identify the combination that will optimize the extraction process. The five-level design (Table 1) was used for fitting a second-order response
surface, and it was rotatable design.
Table 1. Investigated levels and coded values for each of the independent variable
Independent
variable
Ethanol (X1, %)
Temperature (X2, ºC)
(-1.414)
21.7
33.8
(-1)
30
40
Coded levels
(0)
50
55
(1)
70
70
(+1.414)
78.3
76.2
The experiments were carried out to study the effect of the solvent concentration
(ethanol-water mixture) and extraction temperature on the extraction of antioxidant compounds and on the antioxidant activity of O. basilicum extracts. Three responses in the
form of different components and antioxidant activity of the extracts were evaluated: total
phenols content (TP), Y1, total flavonoids content (TF), Y2, and antioxidant activity (IC50),
Y3. Nineteen runs, with six replications at the central point, were performed to cover as
more as possible combinations of the factor levels. The coded and uncoded independent
variables used in the RSM design and obtained responses are shown in Table 2. Experimental data were analyzed by RSM to fit the second order polynomial model (Eq. [2]),
where b0, bi, bii and bij are the regression coefficients; Xi are the coded variables; X2 is the
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temperature and X1 is the concentration of the ethanol solution. Y is the analyzed response
(total phenols content, total flavonoids content and antioxidant activity).
Y  b0   bi X i   bii X ii2   bij X i X j
[2]
In this study, the RSM was used to examine the functional relationship between the
investigated variables, solvent concentration and extraction temperature, and the outputs,
or responses: content of antioxidant compounds, total phenols and flavonoides, and the
extract antioxidant activity. The range of tested extraction temperature was between
33.8ºC and 76.2ºC. The ethanol-water solution was used in the range of concentration
from 21.7% to 78.3% (Table 1). To find the interactions between these two parameters
(temperature and ethanol concentration) on the targeted compounds a statistical analysis
was applied. Experimental data were obtained according to the design of the response
surface methodology (RSM) presented in Table 2.
Table 2. Total phenols content (TP), total flavonoids content (TF) and antioxidant
activity (IC50) of extracts
Run
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
318
Temperature
(°C)
40
40
40
55
55
55
70
70
70
55
55
33.8
76.2
55
55
55
55
55
55
Ethanol
(%)
30
50
70
30
50
70
30
50
70
21,7
78.3
50
50
50
50
50
50
50
50
TP
(g GAE/100 g)
10.39
14.20
16.83
12.58
15.98
17.99
11.88
13.61
17.31
10.40
17.04
13.56
12.61
15.98
15.98
15.98
15.99
15.98
15.98
TF
(g CE/100 g)
4.48
4.98
4.28
4.79
5.48
5.58
4.62
5.53
5.10
4.09
5.58
4.51
5.38
5.48
5.47
5.47
5.48
5.47
5.48
IC50 · 10-3
(mg/ml)
5.05
4.70
4.44
4.43
4.27
3.97
4.63
4.10
3.87
5.24
4.15
4.98
4.01
4.28
4.28
4.25
4.27
4.28
4.28
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Statistical analysis was performed using STATISTICA 8.0, StatSoft (Europe) Gmbh,
Hamburg, Germany. The estimated coefficients (bi) of the second-order response model,
generated from the statistical analysis for all responses, are shown in Table 3. Measured
fit of the model data (R2) for all responses were high. The R2 values for TP, TF and IC50
was 0.976, 0.932, and 0.964, respectively.
Table 3. Estimated coefficient (bi) of second-order polynomial models for the
investigated responses
Coefficient
b0
Linear
b1
b2
Quadratic
b3
b4
Interaction
b5
R2
15.90145
p-value
for Y1
0.000000
5.50691
p-value
for Y2
0.000000
0.00219
2.66758
0.991513
0.000000
0.273790
0.31741
-1.36558
-0.83433
0.000097
0.003945
-0.24990
0.970
0.448965
TP (Y1)
0.004237
p-value
for Y3
0.000000
0.003905
0.001444
-0.000295
-0.000338
0.000014
0.000004
-0.33577
-0.39453
0.002441
0.000773
0.000125
0.000164
0.019102
0.004188
0.16925
0.932
0.182691
0.000037
0.964
0.570562
TF (Y2)
IC50 (Y3)
According to the data shown in the Table 3 linear term of ethanol concentration (b2)
show a positive effect on the investigated responses, e.g. TP (p<0.05). Both quadratic
terms, of temperature (b3) and ethanol concentration (b4), affect negatively on the TP
(p<0.05). The interaction between these two parameters has no significant influence on
the obtained response. The effects of temperature and ethanol concentration on TP of O.
basilicum can be described by equation (3), which can be used for calculation of optimal
extraction parameters.
Y1=15.09145+0.00219X1+2.66758X2-1.36558X12-0.83433X22-0.2499X1X2
[3]
The influence of the parameters is visualized by the chart presented in Figure 1. It can
be seen that the increase of the temperature to about 55ºC is accompanied by an increase
in TP. Further increase in temperature leads to a decrease of the TP yield. Further, the
increase of ethanol concentration leads to an increase in the TP. The total phenols yield
achieves its maximum at the ethanol concentration of around 80%. Using Eq. (3), the optimal values of temperature and ethanol concentration, for obtaining the maximum yield
of total phenols of O. basilicum extraction were calculated as follows: temperature of
52.79ºC, and ethanol concentration of 82.41%. Using these values of the investigated parameters (experimentale set at 52.8ºC and 82.4%) the total phenol content was calculated
as 18.07 g GAE/100 g of dry extract. This calculation was experimentally confirmed and
the content of total phenols in dry extract obtained under the determined optimal conditions was 18.08 g GAE/100 g.
319
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Origin
gure 1. Influence of
o temperature annd ethanol concenttration on total phenols content
Fig
Both
B
linear terms (b
( 1 and b2), of tem
mperature and ethaanol concentration
n, have statistically significant influeence (p<0.05) andd influencee posittively the investig
gated responsetotal flavonoids yield. Also, both quadratic terms (b3 andd b4), of temperatu
ure and ethanol
conceentration, influencced significantly (p<0.05), but neggatively the yield of total flavonoidss. The interaction between these tw
wo parameters, as in the case of totaal phenols, had
no in
nfluence on the obbtained response. The response surfface was generateed based on the
secon
nd -order equationn:
Y2=5.50691+0.227379X1+0.317411X2-0.33571X12-0..39453X22-0.16925X1X2
[4]
where Y is the flavonooids content, X1 is the ethanol conceentration and X2 is the extraction
temperature.
The influence of thhe parameters on T
TF is illustrated byy chart presented in
i Figure 2.
Figu
ure 2. Influence off temperature and ethanol concentraation on total flavo
onoids content
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Origin
As
A can be seen from
m Figure 2, the tootal flavonoids conntent increases wiith the increase
in thee temperature, buut after reaching a certain point, aroound 65ºC, it sho
ows a decrease.
The TF
T yield increasess with the increasee of ethanol conceentration, from ab
bout 20 to 60%.
Furth
her increase of ethhanol concentratioon leads to a deccrease of the flavo
onoids content.
The optimal
o
process values
v
of temperature and ethanol concentration neeeded for preparation
n of the O. basiliccum extract with a maximal content of total flavon
noids calculated
from Eq [4] were 63.077ºC and 60.35%.
Under
U
these condittions, the calculatted TF yield was 5.66 g CE/100 g of dry extract.
This calculation was exxperimetally conffirmed, i.e. under these
t
conditions (experimentally
(
set paarameters at 63.1ººC and 60.3%) thee obtained TF yieldd was 5.66 g CE/1
100 g.
Fu
urthermore, both linear terms (b1 aand b2), of temperrature and ethanoll concentration,
have a significant influuence (p<0.05) onn the antioxidant activity
a
of O. basillium extract. In
contrrast to the priviouss cases, linear term
ms had a negativee, while both quad
dratic terms (b3
and b4) exhibited statisstically significant positive effect on
o IC50 value. Agaain, the interaction between
b
these twoo parameters had nno influence on obbtained response (Eq.
(
[5]).
Y3=0.00424-0.000029X1-0.00034X
X2+0.00012X12+0..00016X22-0.00004
4X1X2
[5]
The
T influence of the
t process param
meters on the antioxidant activity of
o O. basilicum
extracts is presented inn Figure 3. The opptimal values for temperature
t
and ethanol
e
concentratio
on needed for the preparation
p
of an extract characteriized with the miniimal IC50 value
(max
ximal antioxidant activity), calculatted from Eq (5), is 75.33ºC and 73.66%. The calculateed value of IC50, using
u
obtained vallues for the investtigated parameterss, was 3.84·10-3
mg/m
ml. This calculatioon was experimenntally confirmed (at the conditions set at 75.3ºC
and 73.7%),
7
and the obtained
o
O. basillicum dry extract had the IC50 valu
ue of 3.83·10-3
mg/m
ml.
Figu
ure 3. Influence of temperature andd ethanol concentration on the antiox
xidant activity
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CONCLUSION
To find the levels of input variables, temperature and solvent concentration that optimize a particular response (content of total phenols and total flavonoids, as well as antioxidant activity of the extract), surface methodology has been used as an efficient tool. In
the investigated range of temperatures (from 33.8ºC to 76.2ºC) and ethanol concentration
(from 21.7% to 78.3%) for the preparation of an O. basilicum extract with highest content
of total phenols the determined optimum values were 52.79ºC and 82.41%. Under these
conditions the content of total phenols was calculated to be 18.07 g GAE/100 g of dry extract. On the other hand, the optimal values corresponding to the highest content of total
flavonoids are 63.07ºC and 60.35%. Under these conditions the content of total flavonoids was 5.66 g CE/100 g of extract. Finally, the optimal values of the process parameters
corresponding to the minimal IC50 value were 75.33ºC and 73.66%.
Acknowledgement
Financial support of this work by the Serbian Ministry of Education and Science,
Project No. TR 31013 is gratefully acknowledged. We are grateful to Department of Biology and Ecology, Faculty of Natural Sciences, University of Novi Sad, for support in
term of confirmed and deposited investigated herb at the BUNS Herbarium.
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ОПТИМИЗАЦИЈА ЕКСТРАКЦИЈЕ Ocimum basilicum L. У ОДНОСУ НА
АНТИОКСИДАТИВНУ АКТИВНОСТ
Сенка С. Видовића*,Зоран П. Зековићa,Жика Д. Лепојевићa,
Марија Радојковићa,Стела Јокићb и Горан Аначковc
a
Универзитет Јосипа Јурја Штросмајера у Осијеку, Прехрамбено-технолошки факултет, Фрање Кухача
20, 31000 Осијек, Хрватска
c
Универзитет у Новом Саду, Природно-математички факултет, Трг Доситеја Обрадовића 3, 21000 Нови
Сад, Србија
b
Метода одзивне површине примењена је у анализи утицаја температуре и примењеног екстрагенса на екстракцију Ocimum basilicum L. Анализирани су: садржај
укупних фенолних компонената у добијеним екстрактима, садржај укупних флавоноида, као и антиоксидативно деловање добијених екстраката. Утицај температуре
на процес екстракције босиљка испитан је у опсегу температура од 33,8ºC до
76,2ºC. Утицај примењеног екстрагенса на процес екстракције испитан је у опсегу
концентрација етанола од 21,7% до 78,3%. На основу експерименталних резултата
добијене су вредности улазних параметара потребне за припрему екстраката босиљака са минималном IC50 вредности: температура екстракције од 75,33ºC и 73,66%
етанол као екстрагенс.
Кључне речи: Ocimum basilicum, босиљак, екстракција, антиоксиданти, RSM
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ETHANOL FERMENTATION OF MOLASSES BY Saccharomyces cerevisiae
CELLS IMMOBILIZED ONTO SUGAR BEET PULP
Vesna M. Vučurović* and Radojka N. Razmovski
Natural adhesion of Saccharomyces cerevisiae onto sugar beet pulp (SBP) is a very
simple and cheap immobilization method for retaining high cells density in the ethanol
fermentation system. In the present study, yeast cells were immobilized by adhesion onto
SBP suspended in the synthetic culture media under different conditions such as: glucose
concentration (100, 120 and 150 g/l), inoculum concentration (5, 10 and 15 g/l dry mass)
and temperature (25, 30, 35 and 40 oC). In order to estimate the optimal immobilization
conditions the yeast cells retention (R), after each immobilization experiment was analyzed. The highest R value of 0.486 g dry mass yeast /g dry mass SBP was obtained at
30oC, glucose concentration of 150 g/l, and inoculum concentration of 15 g/l. The yeast
immobilized under these conditions was used for ethanol fermentation of sugar beet molasses containing 150.2 g/l of reducing sugar. Efficient ethanol fermentation (ethanol
concentration of 70.57 g/l, fermentation efficiency 93.98%) of sugar beet molasses was
achieved using S. cerevisiae immobilized by natural adhesion on SBP.
KEY WORDS: immobilization, bioethanol, sugar beet pulp, molasses, Saccharomyces
cerevisiae
INTRODUCTION
In the recent years, research on improving ethanol production has been accelerated
for both ecological and economic reasons, primarily for its use as an alternative to petroleum-based fuels (1). Currently, the global ethanol supply is originated mainly from sugar
and starch feedstocks (2). The development of a fermentation medium based on industrial
substrates is economically desirable (3). In the bioethanol production, the composition of
the medium affects the physiological state and, consequently, the fermentation performance of the microorganism employed (4). Molasses from the sugar beet processing due
to the high content of fermentable sugars, which can be directly used for fermentation
without any modification, is a very good raw material which is traditionally used for
ethanol production (5). Among many microorganisms that have been exploited for ethanol production, Saccharomyces cerevisiae still remains as the prime species (2). Recent* Corresponding author: Vučurović M. Vesna, University of Novi Sad, Faculty of Technology, Bulevar cara
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ly, yeast cell immobilization techniques have become increasingly important and are
being successfully applied in production of ethanol as a method for improving the process productivity (6). Among the different immobilization technologies, entrapment of
microbial cells within the polymeric matrices (calcium alginate, agar agar, gelatin, k-carrageenan, etc.) has been studied widely. However, technically it is less suitable for ethanol production because the growth of the yeast cells is restrained and also the slowly
growing yeast cells are difficult to be removed from the systems (2). However, the use of
yeast immobilized by natural adhesion onto low-cost plant materials such as wood chips
(7), apple peaces (8), orange peel (9), sugar cane bagasse (10), sugar cane pieces (11),
corn cobs and grape pomace (12) and maize stem ground tissue (13), can effectively
overcome these drawbacks. Yeast cells immobilization by adhesion onto the solid supports is attractive in ethanol fermentation, due to the operational easiness and high ethanol productivity, thanking to the effective retention of cells within the bioreactor. Besides, the possibility of recycling cells for inoculum permits the fermentation to be profitably carried out in continuous or repeated-batch mode (14). The sugar beet pulp (SBP)
was found to be efficient support for immobilization of S. cerevisiae in the ethanol
production because of its heterogeneous structure, high porosity, biocompatibility, high
water swelling capacity, good mechanical properties, and high cells retention capacity.
This immobilization method is cheap, simple and easy (15).
The novelity of this work lies in the investigation of the optimal conditions (inoculum
concentration, glucose concentration and temperature) for the S. cerevisiae immobilization onto SBP. Further, the efficiency of immobilized yeast for batch ethanol fermentation of sugar beet molasses was investigated with the aim to achieve efficient ethanol
production.
EXPERIMENTAL
Dried sugar beet pulp (SBP) kindly provide by a sugar factory near the city of Senta
(Vojvodina province, Serbia) was used as the support for yeast cells. The SBP hydration
was carried out by placing an amount of 15 g of dry SBP into 1 l Erlenmeyer flasks
containing 500 ml of synthetic culture medium containing different amounts of glucose
(100, 120 and 150 g/l) and the same amount of the following constituents: (NH4)2SO4 (1
g/l), KH2PO4 (1 g/l), MgSO4 (5 g/l) and yeast extract (4 g/l) at the pH 5.5, and was sterilized by autoclaving at 121oC for 30 min. After the sterilization, the flasks were kept at
room temperature for 24 h. The working microorganism was a commercial S. cerevisiae
strain (Alltech-Fermin, Senta, Serbia), commonly used in Serbian baking industry, in the
form of pressed blocks (70 % w/w moisture). To immobilize cells onto hydrated SBP, the
flasks were inoculated with 5 g/l, 10 g/l and 15 g/l of yeast on dry basis, and placed on a
rotary shaker (120 rpm) in a thermostat and kept at 30 oC for 24 h. After the immobilization of the yeast, the mass of cells immobilized onto the support was quantified gravimetrically according to Santos et al. (10). Cell retention onto the support (R, g/g) was calculated as the ratio of dry matter of cells immobilized in the support (g) to the support dry
mass (g). Carl Zeiss optical microscope connected to a Cannon S50 camera was used to
capture yeast cells immobilized onto SBP. After the immobilization of the cells, the me326
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dium was decanted using sterilized gauze. The support without extra medium was then
dried at 105oC to constant weight. The identical procedure was conducted by using
support particles recovered from the cell-free medium, as a control, in order to avoid any
interference in weighing measurements. The selected support containing immobilized
yeast, with highest cells retention (R), was used for the batch fermentation of 500 ml of
the sugar beet molasses in 1 l Erlenmeyer flasks. The reducing sugar content of molasses
was 150.2 g/l, the pH was adjusted to 5.5 by addition of 10% (v/v) H2SO4 and it was
sterilized by autoclaving at 121oC for 30 min. The fermentation kinetics was monitored
by measuring the weight loss due to CO2 release at various time intervals from the beginning of the fermentation batch until to its end (24 h). Samples of fermented liquids
were analyzed for ethanol and reducing sugar. The fermented liquid was centrifuged at
3000 rpm for 15 min. The sample of supernatant was hydrolyzed in 33% HCl at 100°C
for 10 min and neutralized with NaOH solution, and reducing sugar content was determined using the 3,5-dinitrosalicylic acid (DNS) method (16). The ethanol concentration in
the distillate was determined based on the density of the alcohol distillate at 20oC, by
pycnometer method (17). Reducing sugar conversion (Su, %) was calculated as the ratio of
utilized reducing sugar to the initial and multiplying by 100. The ethanol yield (Yp/s, g/g)
was calculated as grams of ethanol produced per gram of utilized reducing sugar. The fermentation efficiency was calculated as the percentage of the maximal theoretical ethanol
yield (Ep/s, %). The volumetric ethanol productivity (Qp, g/lh) was calculated as grams of
ethanol produced per liter per hour.
The cell immobilization and ethanol productivity of immobilized yeast cells depends
on the surface characteristics of the support, such as pore size, water content, hydrophilic
properties and magnetism (18). The S. cerevisiae was found to be immobilized by natural
adhesion onto SBP due to the electrostatic interactions between support and the yeast
cells surface and due to the capillary forces which hold cells inside the SBP cavities (15).
The process of cell adhesion to solid supports by biosorption is believed to occur due to
electrostatic or van der Waals interactions between the cell membrane and the support.
These adhesion forces are affected by variations in the medium composition and component concentrations, because they can strongly influence the surface energy of the immobilization support (14).
A series of optical microscopic images (Fig. 1) were taken to visually explore the
yeast immobilization onto SBP. As is shown in Fig. 1, in the immobilization process, the
yeast cells penetrate into the interior of bibulous sugar beet tissue, enabling adsorption
onto the surface of the carrier, and meanwhile the SBP cavities are also filled with yeast
cells.
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Origin
gure 1. Optical microphotograph
m
of a) SBP and b) Saaccharomyces cerrevisiae cells
Fig
(400×) imm
mobilized onto the SBP
Cells
C
retention cappacities obtained aafter 24 h of yeastt immobilization at
a temperatures
of 25
5, 30, 35, 40oC aree shown in Figuress 2-5.
0.407
0.33
33
0.5
0.362
0.304
0.4
0.270
0.230
0.239
0.219
0.204
s
0.3 Yeast cells
0.2
retention
R (g/g)
0.1
15
10
Yeast (g/l)
0.0
5
150
125
100
0
Glucose (g/l)
Figure
F
2. Retentioon of S. cerevisiaee cells onto SBP after
a
immobilizatio
on at 25oC
0.486
95
0.39
0.5
0.405
0.314
0.341
0.299
0.4
0.280
0.3
0.259
0.213
Yeast cellls
retention
n
R (g/g)
0.2
0.1
15
10
Yeas
st (g/l)
0.0
5
100
0
125
150
0
Gluco
ose (g/l)
Figure
F
a
immobilizatio
on at 30oC
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Origin
0.441
0.5
60
0.36
0.385
0.332
0.4
0.264
0.220
0.3
0.190
0.2
0.155
0.144
Yeast cells
s
retention
R (g/g)
0.1
15
10
Yeas
st (g/l)
0.0
5
150
125
100
0
Gluc
cose (g/l)
Figure
F
a
immobilizatio
on at 35oC
0.312
0.4
64
0.26
0.225
0.240
0.3
0.197
0.177
0.147
0.150
0.148
Yeast cells
s
retention
0.2
R (g/g)
0.1
15
10
Yeas
st (g/l)
0.0
5
100
0
125
150
Gluco
ose (g/l)
Figure
F
a
immobilizatio
on at 40oC
By
B comparing reteention capacities oobtained for the diifferent yeast conccentrations and
gluco
ose concentration obtained at eachh immobilization temperature (25, 30, 35, 40oC)
(Fig. 2-5) it can be conncluded that the cells retention incrreased along with the increase of
the in
nitial yeast concenntration from 5 g//l to 15 g/l, and with
w the increase of
o glucose concentration in the mediium from 100 g/l to 150 g/l. Maxiimum cells retentiion capacity of
0.407
7 g/g, 0.486 g/g, 0.441
0
g/g and 0.3112 g/g for the resppective immobilizzation temperatures of 25, 30, 35, 400oC, were obtainedd at the yeast conncentration of 15 g/l
g and glucose
conceentration of 150 g/l.
g On the basis off the yeast immobilization results it can be concluded that
t
for all inoculuums and glucose concentrations, maximal
m
values off cells retention
capaccity (R) were obtaained at the immobbilization temperaature of 30oC. Afteer 24 h of yeast
immo
obilization at 30 oC the cells retentiion capacity rangeed from 0.213 g/g
g for yeast concentration of 5 g/l andd glucose concenttration of 100 g/ll to 0.486 g/g forr yeast concentratio
on of 15 g/l and glucose
g
concentrattion of 150 g/l. These
T
results imply
y that the most
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appropriate temperature for yeast cells growth and, consequently, for immobilization is
30oC, while the temperatures of 25, 35 and 40oC were less effective. These results are in
accordance with the influence of temperature on yeast growth. Considering firstly temperature, this is one of the most important physical parameters which influence yeast
growth. In most laboratories and industries, yeast is generally grown in the range of 2030oC (19).
Due to the highest yeast retention capacity of 0.486 g/g, the yeast immobilized onto
the support by natural cell adhesion at 30 oC, glucose concentration of 150 g/l and inoculum concentration of 15 g/l was used for sugar beet molasses fermentation. Table 1 summarizes the fermentation parameters such as reducing sugar utilization, ethanol productivity, ethanol yield and fermentation efficiency obtained at the end of the fermentation
batch of sugar beet molasses by immobilized S. cerevisiae obtained at 30oC. Since,
146.93 g/l reducing sugar was utilized, the fermentation efficiency was 97.83 %. This indicates that the cells immobilized onto SBP utilized almost all available reducing sugar
from the molasses, suggesting efficient exploitation of this raw material for ethanol production. The final ethanol concentration of 70.57 g/l and ethanol productivity of 1.47 g/lh
were achieved in the batch fermentation of molasses. The ethanol yield per consumed reducing sugar of 0.480 g/g was achieved, equal to 93.98% of its theoretical value expressed as the fermentation efficiency, indicating that almost all utilized reducing sugar was
converted to ethanol. On the basis of these results it can be concluded that yeast cells
immobilized onto SBP showed high fermentative activity and may be recommended for
the further use in repeated batch or continuous process.
Table 1. Parameters of sugar beet molasses fermentation by S. cerevisiae immobilized on
SBP
Parameter
Initial reducing sugar, So (g/l)
Utilized reducing sugar, Su (g/l)
Reducing sugar utilization, Su (%)
Ethanol concentration, P (g/l)
Ethanol productivity, Qp (g/lh)
Ethanol yield, Yp/s (g/g)
Fermentation efficiency, Ep/s(%)
Value
150.20 ± 0.36
146.93 ± 0.65
97.83 ± 0.63
70.57 ± 0.83
1.47 ± 0.02
0.480 ± 0.004
93.98 ± 0.84
CONCLUSION
The work demonstrated the potential use of SBP as a support for S.cerevisiae immobilization under different immobilization conditions such as: glucose (100, 120 and 150
g/l), inoculum concentration (5, 10 and 15 g/l dry mass) and temperature (25, 30, 35 and
40oC). Efficient cells immobilization was confirmed by optical microscopy. The support
was effective for yeast immobilization for each examined combination of conditions. Pre330
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sented results show that the cells retention increases along with the increase of initial
yeast concentration from 5 g/l to 15 g/l, and with the increase of glucose concentration in
the medium from 100 g/l to 150 g/l. Also, it was found that the most appropriate temperature for yeast cells growth and consequently immobilization is 30oC. The highest yeast
cells retention (0.486 g dry mass yeast /g dry mass SBP) was obtained at 30oC, glucose
concentration of 150 g/l, and inoculum concentration of 15 g/l. Efficient bioethanol production from the molasses containing 150.2 g/l of reducing sugar, was achieved using
thus immobilized yeast (ethanol concentration of 70.57 g/l, fermentation efficiency
93.98%).
Aknowledgements
Financial support of the Project TR-31002 from the Ministry of Science and Technological Development of the Republic of Serbia is highly acknowledged.
REFERENCES
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R.: Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst
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10. Santos, D.T, Sarrouh, B.F., Rivaldi, J.D., Converti, A. and Silva, S.S.: Use of sugarcane bagasse as biomaterial for cell immobilization for xylitol production. J. Food
Eng. 86 (2008) 542-548.
11. Liang, L., Zhang, Y., Zhang, L., Zhu, M., Liang, S. and Huang, Y.: Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses.
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12. Genisheva, Z., Mussatto, S.I., Oliveira, J.M., Teixeria, J.A.: Evaluating the potential
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13. Razmovski, R. and Vučurović, V.: Bioethanol production from sugar beet molasses
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14. Yu, J., Zhang, X. and Tan, T.: A novel immobilization method of Saccharomyces
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15. Vučurović, V. and Razmovski, R.: Sugar beet pulp as support for Saccharomyces cerevisiae immobilization in bioethanol production. Ind. Crops Prod. 39 (2012) 128134.
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АЛКОХОЛНА ФЕРМЕНТАЦИЈА МЕЛАСЕ ПОМОЋУ ЋЕЛИЈА
Saccharomyces cerevisiae ИМОБИЛИСАНИХ НА РЕЗАНЦИМА ШЕЋЕРНЕ
РЕПЕ
Весна М. Вучуровић* и Радојка Н. Размовски
Универзитет у Новом Саду, Технолошки факултет, 21000 Нови Сад, Булевар Цара Лазара 1, Србија
Природна адхезија Saccharomyces cerevisiae на резанцима шећерне репе (SBP) је
једноставан и јефтин метод имобилизације којим се одржава висока густина квасца
у ферментационом систему. У овом раду су ћелије квасца имобилисане адхезијом у
синтетском медијуму под различитим условима имобилизације: концентрација глукозе (100, 120 и 150 g/l), концентрација инокулума (5, 10 и 15 g/l) и температура
(25, 30, 35 и 40оС). У циљу процене оптималних услова имобилизације након сваког поступка имобилизације анализиран је остварени степен имобилизације (R).
Највиша вредност степена имобилизације биокатализатора од 0,486 g суве масе
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квасца по g суве масе SBP је остварена при температури 30оС, концентрацији глукозе од 150 g/l и концентрацији инокулума 15 g/l. Овако имобилисани квасац је
примењен за ферментацију меласе шећерне репе почетне концентрације редукујућих шећера 150,2 g/l. У овом раду је приказана ефикасна алкохолна ферментација
меласе (концентрација етанола од 70.57 g/l, ефикасност ферментације од 93.98%)
применом ћелија S. cerevisiae имобилисаних природном адхезијом на SBP.
Кључне речи: имобилизација, биоетанол, резанци шећерне репе, меласа, Saccharomyces cerevisiae
Received:04 June 2012
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BIOSORPTION OF COPPER(II) AND CHROMIUM(VI) BY MODIFIED TEA
FUNGUS
Marina B. Šćiban*, Jelena M. Prodanović and Radojka N. Razmovski

The tea fungus was found to have good adsorption capacities for heavy metal ions. In
this work it was treated with HCl or NaOH at 20 oC or 100 oC, with the aim to improve
its adsorption ability. The sorption of Cu(II) and Cr(VI) ions from aqueous solutions by
raw and treated tea fungus was investigated in the batch mode. The largest quantity of
adsorbed Cu(II), of about 55 mg/g, was achieved by tea fungus modified with NaOH at
100°C. For Cr(VI), the largest quantity of adsorbed anions, of about 58 mg/g, was achieved by the adsorbent modified with NaOH at 20 oC. It was shown that acid modification
of tea fungus biomass was not effective.
KEY WORDS: biosorption, copper(II), chromium(VI), tea fungus, modification
INTRODUCTION
Heavy metals are very harmful as they can accumulate in living tissues. Hence, the
pollution caused by them is one of the most critical environmental issues (1). Effluents
from various industries may contain excessive concentrations of copper(II) and chromium(VI). It is known that high concentrations of copper are toxic to humans and other
organisms (2) and the effects of acute copper poisoning in humans are very serious, with
possible liver damage. Chromium(VI) from the electroplating, tanning, painting and
similar industries has been reported to be toxic to animals and humans. It is carcinogenic
and its bioaccumulation into flora and fauna creates serious ecological problems (3,4).
There are many different methods for the removal of heavy metals from water and
wastewater. The existing chemical methods require a large excess of chemicals, giving
voluminous toxic sludges. Also, they are generally expensive. In the recent decade, biosorption methods have received considerable attention of researchers all over the world as
an economic and eco-friendly option for removal of heavy metals from water and wastewater. The heavy metal adsorption technology by waste microbial biomass is effective,
and can sometimes provide better results than natural zeolites and activated carbon, being
comparable to synthetic ion-exchange resins (5). In addition, it uses inexpensive biosorbent materials. The maintaining of living microbial populations requires the stable and
controllable environment conditions, which is problematic under highly variable conditi* Corresponding author: Marina B. Šćiban, University of Novi Sad, Faculty of Technology, Bulevar cara
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ons of wastewaters. However, some types of microbial biomass in non-living form can be
good biosorbent materials.
The tea fungal biomass is a by-product or waste material from production of kombucha beverage. The kombucha culture is a symbiotic culture of bacteria (genera Acetobacter) and one or more yeasts (Saccharomycodes ludwigii, Saccharomyces cerevisiae, Saccharomyces bisporus, Torulopsis sp., Zygosaccharomyces sp.) in floating cellulose mat.
It has been investigated as biosorbent for the removal of different heavy metals from water (6-9). This paper is concerned with the effect of different treatments of tea fungus on
its adsorption efficiency for copper(II) and chromium(VI). Such examination was done
bearing in mind that pretreatment of some adsorbent could improve its adsorption
properties, e.g. adsorption capacity, rate of adsorption, prevention of leaching of some
undesired substances from adsorbent during adsorption, etc (9, 10).
EXPERIMENTAL
Tea fungus as biosorbent
The tea fungus used for biosorption experiments was obtained from the Faculty of
Technology in Novi Sad (Republic of Serbia). The fungal biomass was washed with an
adequate amount of distilled water in order to free it from the media components. The
fresh fungal biomass was cut by scissors in pieces of 1 x 2 mm and used in the biosorption experiments or for modification.
Modification of the tea fungus
Modification of the tea fungus was performed with an alkali and an acid. The pretreatments with alkali were done in the following way:
1. An amount of 40 g of washed and cut tea fungus was treated with 200 ml of 1
mol/l NaOH at 100°C for 5 minutes,
2. The weighed amount of 40 g of washed and cut tea fungus was treated with 200
ml of 1 mol/l NaOH at 20°C for 1 hour.
The pretreatments with acid were done in the following way:
ml of 1 mol/l HCl at 100°C for 5 minutes,
ml of 1 mol/l HCl at 20°C for 1 hour.
After the treatments with NaOH and HCl the tea fungus was washed with distilled
water in order to remove the excesive alkali and the acid and dried at 105oC during 20
hours.
Preparation of metal solutions
The stock solutions of metal salts (0.25 mol/l) were prepared by dissolving
CuSO4·5H2O and K2Cr2O7 in distilled water. The initial metal concentrations of about 0.4
mmol/l were obtained by dilution of stock solutions, and a real concentration of metal
ions was measured before each biosorption experiment. The pH of the adsorbate solu336
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tions was adjusted to desired values with 0.1 mol/l HCl or 0.1 mol/l NaOH. Fresh diluted
solutions were used for each experiment. All the chemicals used were of analytical grade.
Biosorption experiments
The biosorption experiments were performed by mixing 0.15; 0.25 or 0.5 g/l of tea
fungal biomass in 200 ml of the metal ion solution at the optimal pH values, which are
pH 4 for Cu(II) and pH 2 for Cr(VI) (6). Batch experiments were carried out at 25°C in
Erlenmeyer flasks on a rotary shaker at 200 rpm. Samples were taken after 1 hour and filtered by using filter paper Watman N°1 to remove the suspended biomass and analyze for
the residual Cu(II) and Cr(VI). The concentrations of heavy metal ions before and after
biosorption were determined using an atomic absorption spectrophotometer (Varian AA10).
The sorption capacity of the tea fungal biomass was calculated based on the mass balance:
q(mg/g)=(C0-C)/m
[1]
where q is the amount of metal uptake per unit mass of biosorbent (mg/g); C0 and C
(mg/l) are the initial and residual concentrations of metal ion, respectively, and m is the
dry mass of the biosorbent (g/l).
Each batch experiment was carried out in duplicate and avarage results are presented.
Removal of Cu(II) by tea fungus treated with NaOH and HCl
Adsorption of copper ions by unmodified and differently modified tea fungus was
investigated. Figure 1 shows the amounts of copper(II) uptake by unmodified and alkali
modified tea fungus, while Figure 2 presents copper(II) uptake by unmodified and acid
modified tea fungus.
A very good adsorption efficiency of copper ions was achieved by both unmodified
and modified tea fungus; it was better than Cu(II) adsorption by Neurospora crassa (10)
and many other microbial biomass materials (11), and similar with the adsorption by Ulva biomass (12), agricultural wastes (13), poplar sawdust (14), and so on. When tea fungus was applied in a large dose, then a larger surface area was available for adsorption of
copper ions. As a result, a smaller amount of copper ions was adsorbed, and smaller differences between unmodified and modified tea fungus were observed than in the case of
the application of a smaller adsorbent dose, i.e. a smaller available adsorbent surface
area. The results presented in Figure 1 show that the modification of tea fungus by alkali
in any case improved adsorption of copper(II). The tea fungus modified with NaOH at
100oC had significantly higher quantity of adsorbed copper(II) than the unmodified tea
fungus or modified with NaOH at 20°C. Obviously, the modification at higher temperature was better, but it is energy consuming, too.
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60
Unmodified
Modification 1
q (mg/g)
Modification 2
40
20
0
0.15
0.25
0.50
m (g/l)
Figure 1. Removal of Cu(II) by unmodified tea fungus and tea fungus treated with
1 mol/l NaOH at 20°C for 60 minutes (Modification 1) and with 1 mol/l NaOH at 100°C
for 5 minutes (Modification 2)
60
Unmodified
Modification 1
Modification 2
q (mg/g)
40
20
0
0.15
0.25
m (g/l)
0.50
Figure 2. Removal of Cu(II) by unmodified tea fungus and tea fungus treated with
1 mol/l HCl at 20°C for 60 minutes (Modification 1) and with 1 mol/l HCl at 100°C
The modification of tea fungus by acid gave worse results compared to the adsorption
by adsorbent modified with alkali. Similar results were obtained when different agricultural adsorbents were modified by acid and alkali solutions (13). Khosravi et al. (15) found
that NaOH increased, while HCl decreased the adsorption capacity of Azolla filiculoides
for Pb(II), Cd(II), Ni(II) and Zn(II). On the other hand, it was found that acid modification of poplar sawdust did not improve adsorption of copper(II), but enhanced zinc(II)
adsorption by about four times (16).
It is well known that the solution pH can significantly influence adsorption of heavy
metal ions. In contrast to the slight increase of the pH after the adsorption on unmodified
poplar sawdust (16), and on the alkali modified one (14), it was observed that the pH of
the solution after adsorption of copper ions by acid modified poplar sawdust was accompanied by a slight decrease. Probably, that was the reason of lower adsorption ability of
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acid modified tea fungus. However, acid modification at 100oC slightly improved Cu(II)
adsorption, but less than the modification with alkali. After all, it can be said that the high
temperature affects the adsorption characteristics of tea fungus by changing its structure
and/or surface conditions.
Removal of Cr(VI) by tea fungus treated with NaOH and HCl
The Cr(VI) ions (from K2Cr2O7) in the solution of the pH 2 are predominantly in the
form of Cr2O72- anions (17). The adsorption of these anions is completely different compared to the adsorption of Cu2+. The chromium(VI) adsorption by unmodified and alkali
modified tea fungus is presented in Figure 3, and its adsorption by unmodified and acid
modified tea fungus in Figure 4.
60
Unmodified
Modification 1
Modification 2
q (mg/g)
40
20
0
0.15
0.25
0.50
m (g/l)
Figure 3. Removal of Cr(VI) by unmodified tea fungus and tea fungus treated with
1mol/l NaOH at 20°C for 60 minutes (Modification 1) and with 1 mol/l NaOH at 100°C
60
Unmodified
Modification 1
Modification 2
q (mg/g)
40
20
0
0.15
0.25
0.50
m (g/l)
Figure 4. Removal of Cr(VI) by unmodified tea fungus and tea fungus treated with
1 mol/l HCl at 20°C for 60 minutes (Modification 1) and with 1 mol/l HCl at 100°C
The adsorption of dichromate anions by both unmodified and modified tea fungus
was better than their adsorption by many other microbial biomass materials (11). Alkali
modification at 20oC led to the significantly improved adsorption of dichromate anions,
but the modification at 100oC suppressed the adsorption. Alkali modification at high temperature led to unwanted changes in the adsorbent, which decreased the chromium(VI)
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adsorption. Mild modification had a beneficial effect that was manifested even when the
large surface of the adsorbent was available (when the large quantity of adsorbent was
used).
Although the adsorption of dichromate anions was better at lower pH values, acid
modification of tea fungus did not contribute to a better adsorption. Contrary, the amount
of adsorbed anions was lower than in the case when the unmodified tea fungus was used.
CONCLUSIONS
This paper deals with the adsorption of copper(II) and chromium(VI) ions by unmodified and modified tea fungus biomass – a waste product from kombucha production. The
modification was done by 1 mol/l NaOH or 1 mol/l HCl at 100°C for 5 minutes, or at
20oC for 1 hour. When the tea fungus was applied in a larger dose, then the larger surface
area was available for adsorption, and consequently, a smaller amount of ions was adsorbed per mass unit of the adsorbent. In that cases there was not strong competition for
active sites on the adsorbent surface and, because of that, the lower differences between
unmodified and modified adsorbents were observed. It was shown that both unmodified
and modified tea fungus had significant adsorption capacity, similar to some other biosorbents. Modification with NaOH gave better results. The largest quantity of adsorbed
copper(II), of about 55 mg/g, was achieved using tea fungus modified with NaOH at
100°C. For chromium(VI), the largest quantity of adsorbed anions, of about 58 mg/g, was
achieved by the adsorbent modified with NaOH at 20oC. It was shown that acid modification of tea fungus biomass could not be recommended.
Acknowledgement
This research was supported by the Ministry of Education and Science of the Republic of Serbia as a part of the Project Number III 43005 and Project Number TR 31002.
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by Scenedesmus abundans. Chemosphere 47 (2002) 249-255.
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Industrial Wastewater. Chem. Technol. Biotechnol. 69 (1997) 376-382.
4. Jianlong, W., Zeyu, M. and Xuan, Z.: Response of Saccharomyces cerevisiae t. Chromium Stress. Proc. Biochem. 39 (2004) 1231-1235.
5. Matheickal, J.T. and Yu, Q.: Biosorption of Lead(II) from Aqueous Solutions by
Phellinus badius. Miner. Eng. 10 (1997) 947-957.
6. Razmovski, R. and Šćiban, M.: Biosorption of Cr(VI) and Cu(II) by Waste Tea
Fungal Biomass. Ecol. Eng. 34, 2 (2008) 179-186.
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7. Razmovski, N.R. and Šćiban, B.M.: Effect of Different Conditions on Cu(II) and
Cr(VI) Biosorption by Dried Waste Tea Fungal Biomass. APTEFF 38 (2007) 149156.
8. Razmovski, R. and Šćiban, M.: Kinetics of Copper(II) and Chromium(VI)
Biosorption by Dried Waste Tea Fungal Biomass from Aqueous Solutions. Rom.
Biotech. Lett. 12, 3 (2007) 3241-3247.
9. Murugesan, G.S., Sathishkumar, M. and Swaminathan K.: Arsenic Removal from
Groundwater by Pretreated Waste Tea Fungal Biomass. Bioresource Technol. 97
(2006) 483-487.
10. Kiran, I., Akar, T. and Tunali, S.: Biosorption of Pb(II) and Cu(II) from Aqueous Solutions by Pretreated Biomass of Neurospora crassa. Process Biochem. 40 (2005)
3550-3558.
11. Volesky, B. and Holan, Z.R.: Biosorption of Heavy Metals. Biotechnol. Progr. 11
(1995) 235-250.
12. Suzuki, Y., Kametani, T. and Maruyama, T.: Removal of Heavy Metals from
Aqueous Solution by Nonliving Ulva Seaweed as Biosorbent. Water Res. 39 (2005)
1803-1808.
13. Šćiban, M., Klašnja, M. and Škrbić, B.: Adsorption of Copper Ions from Water by
Modified Agricultural By-products. Desalination 229 (2008) 170-180.
14. Šćiban, M., Klašnja, M. and Škrbić, B.: Modified Softwood Sawdust as Adsorbent of
Heavy Metal Ions from Water. J. Haz. Mat. 136 (2006) 266-271.
15. Khosravi, M., Rakhshaee, R. and Taghi Ganji, M.: Pre-treatment Processes of Azolla
filiculoides to Remove Pb(II), Cd(II), Ni(II) and Zn(II) from Aqueous Solution in the
Batch and Fixed-bed Reactors. J. Haz. Mat. 127 (2005) 228-237.
16. Prodanović, J.M., Šćiban, M.B. and Kukić, D.V.: Improvement of adsorption characteristics of wood sawdust by acid treatment, XV International Eco-Conference, Novi
Sad, 21.-24. September 2011., Proceedings: Environmental protection of urban and
suburban settlements I, pp. 297-305.
17.Seepe, A.H.: Determination of chromic acid and sodium dichromate in a concentrated
electrolytic solution with the aid of Artifical Neural Networks, M.Sc. Thesis, Faculty
of Natural and Agricultural Sciences, Department of chemistry, University of Pretoria, 2009. БИОСОРПЦИЈА Cr(VI) И Cu(II) МОДИФИКОВАНОМ ЧАЈНОМ ГЉИВОМ
Марина Б. Шћибан*, Јелена М. Продановић и Радојка Н. Размовски
Познато је да су тешки метали штетни по здравље људи и да се могу акумулирати у живим ткивима. Стога, загађење животног окружења изазвано тешким металима представља једно од најбитнијих питања заштите животне средине. Ефлуенти
који потичу из различитих индустрија могу садржавати повећане концентрације
јона бакра(II) и хрома(VI). Бакар у повећаним концентрацијама је токсичан за људе
и друге организме, а ефекти акутног тровања бакром код људи су веома озбиљни,
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са потенцијалним оштећењем јетре код продужене изложености. За хром(VI) је такође утврђено да је токсичан за животиње и људе, канцероген и да се акумулира у
флори и фауни доводећи до еколошких проблема.
У ранијим истраживањима, потврђено је да чајна гљива поседује добар адсорпциони капацитет за јоне тешких метала. У овом раду чајна гљива је третирана хлороводоничном киселином и натријум-хидроксидом на 20oC и 100oC, са циљем да се
побољша њена адсорпциона способност. Адсорпција Cu(II) и Cr(VI) јона из воденог раствора сировом и третираном чајном гљивом је испитивана у шаржним условима. Највећа количина бакар(II) јона, око 55 mg/g, је адсорбована чајном гљивом
модификованом натријум-хидроксидом на 100oC. У случају хрома(VI), највећа количина ових јона, око 58 mg/g, је адсорбована чајном гљивом модификованом натријум-хидроксидом на 20oC. Такође, утврђено је да кисела модификација негативно утиче на адсорпциону способност чајне гљиве за адсорпцију Cu(II) и Cr(VI) јона.
Кључне речи: биосорпција, хром(VI), бакар(II), чајна гљива, модификација
342
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Припрема рукописа:
Језик: Рукопис мора бити написан на еглеском језику.
Припрема: Рукопис мора бити припремљен на максимално 10 страница А4 формата (односи се само на научне радове), у MS Word-у, Times New Roman фонтом са
величином слова 10 pt, двоструким проредом и са свим маргинама од по 3 cm. Све
странице рукописа морају бити нумерисане. Табеле и слике морају бити постављене на месту појављивања у тексту.
Oпшти изглед. Рукопис треба да је изложен јасно и да садржи: Насловну страну,
Извод рада, Кључне речи, Увод, Експериментални део, Резултате и дискусију, Закључак, Захвалницу и Литературу (све на енглеском језику), као и Извод рада и
кључне речи на српском језику.
Насловна страна: На првој страни рукописа треба да стоји наслов рада (без симбола, формула или скраћеница) написан великим болдираним словима. Наслов рада
треба да је концизан и јасан и да се односи на садржај рукописа. Испод наслова
рада италик словима написати пуна имена свих аутора (име, средње слово и презиме), без научних и професионалних звања. Име аутора који је задужен за сву коресподенцију, током свих фаза рецензије и објављивања рада, јасно означити звездицом. Такође, неопходно је навести и његову е-mail адресу, као и пуну поштанску
адресу. Институцију(е) у којој су аутори запослени (или ангажовани) навести испод
имена свих аутора. Уколико аутори нису из исте институције иза имена сваког аутора означити словом у индексу припадност институцији и исто слово у индексу
написати испред назива одговарајуће институције.
Извод рада (100-250 речи, италик слова) написати испод наслова рада и имена аутора. Извод треба да садржи циљ истраживачког рада, методе, резултате и дискусију.
Кључне речи (нормална слова, максимално 5 кључних речи) навести испод Извода
рада.
Увод треба да садржи податке везане за претходни истраживачки рад са одговарајућим референцама, као и проблем и циљ истраживања описаних у раду.
Експериментални део. Материјал и методе, који су коришћени у раду, треба да
буду јасно и детаљно изложени како би остали научници могли да их понове. Детаљно описати само нове технике и методе, док је за већ познате методе довољно
навести одговарајуће референце.
Резултати и дискусија. Резултати морају бити приказани концизно и јасно, у табелама или илустрацијама. Значајност резултата истраживања приказати без понављања материјала изложеног у Уводу. Број и величину табела, илустрација, графика
и хемијских формула свести на неопходан минимум.
Закључак треба да покаже значајан допринос проблематике рукописа и могућност
њене даље примене.
Захвалница. Текст захвалнице треба да буде што краћи.
Референце у тексту означити по редоследу појављивања арапским бројевима у заградама ( ). Све публикације наведене у рукопису рада навести и у листи референци на посебној страници текста. Скраћени називи часописа треба да буду написани у складу са International Codex for Abbreviations of Journal Titles (Chemical
Abstracts). Списак референци треба написати по редоследу њиховог појављивања у
тексту. Навести имена свих аутора (не користити „... и сарадници“, са њиховим
иницијалима иза одговарајућег презимена. Навести пун наслов рада објављеног у
часопису. Називе часописа треба скратити у складу са Chemical Abstracts Service
Source Index, 2005 edition, and supplements. Иза скраћених назива часописа означити свеску (болд), број (ако постоји), годину (у загради) и први и последњи број
странице рада.
Примери:
Часописи: Pascual, E.C., Goodman, B.A. and Yeretzian, C.: Characterisation of Free
Radicals in Solubile Coffee by Electron Paramagnetic Resonance Spectroscopy. J. Agric.
Food Chem. 50, 21 (2002) 6114-6122.
Књиге: Banks, W. and Greenwood, C.T.: Starch and its Components, Edinburgh University Press, Edinburgh (1975) p.98.
Књиге са више поглавља: Mercier C.: Extrusion Cooking of Starch, in Polysaccharides in Food. Eds. Blanshard J.M.V. and Mitchell, J.R., Butterworth, London (1978) pp.
152-170.
Књиге извода радова: Noe, W., Howaldt, M., Ulber, R. and Scheper, T.: Immunobase elution assay for process control, 8th European Congress on Biotechnology, Budapest,
17-21 August 1997, Book of Abstracts WE 163, p. 246.
Тезе: Linstead, J.B.: Effects of adding natural antioxidants on colour stability of paprika, Ph.D. (or M.Sc.) Thesis, University of Glasgow, 2006.
Патенти: Miller, B.O.: U.S. Pat. 2542356 (1962), Dow Chemical Comp.: Abstr. 51
(1961) 2870.
Необјављени (непубликовани) подаци: Треба да буду цитирани уз коментар „у
штампи“, „необјављени резултати“ „личне белешке“.
Подаци преузети са интернета: Треба да садрже аутора, наслов, интернет адресу и датум приступа подацима (пример: Wright, N.A.: The Standing of UK Histopathology Research 1997-2002. http://pathsoc.org.uk (accessed 7 October 2004)).
Извод и кључне речи на срском језику треба написати ћириличним писмом на
крају рукописа рада (после списка литературних података) и у проширеном облику
(највише 1 страница). Наслов рада написати нормалним, великим словима, а испод
њега италик словима написати имена аутора (име, средње слово и презиме) као и
назив институције у којој раде.
Хемијска номенклатура и јединице. Аутори су обавезни да користе SI систем
јединица и хемијску номенклатуру која је у складу са правилима Chemical Abstracta где год је то могуће.
Табеле. Свака табела треба да је нумерисана арапским бројем иза којег следи назив
табеле (Table 1. Result...). Ширина табеле не сме бити већа од 12,5 цм.
Графици и слике. Сваки график или слику такође треба нумерисати арапским бројем иза којег следи назив (Figure 1. Chromatogram of...). Графици морају бити припремљени помоћу програма Microsoft Excel, Origin или Statistica. Шеме морају бити
припремљене помоћу програма Microsoft Visio или Corel Draw. Све графике и шеме
неопходно је доставити као посебне фајлове у оригиналној екстензији (нпр. xls,
xlsx, vdr, cdr). Скениране црно-беле шеме доставити као фајлове са tif, wmf ili bmp
екстензијом. Фотографије у црно-белој техници доставити као посебне фајлове са
jpg екстензијом.
Хемијске формуле и математичке једначине. Написати хемијске формуле и математичке једначине јасно и прецизно и тачно поставити индексе на своја места.
Једначине у тексту означити арапским бројевима у угластим заградама [ ]. Значења
коришћених скраћеница и симбола треба детаљно објаснити приликом њиховог
првог појављивања у тексту или дати посебан списак на посебној страници на крају
рукописа.
Додатне информације
Рукопис треба послати уреднику часописа у два одштампана примерка на следећу
адресу: Проф. др Соња Ђилас, Технолошки факултет Нови Сад, Булевар цара Лазара 1, 21000 Нови Сад, са назнаком „Рад за часопис Acta Periodica Technologica“.
Електронску верзију рукописа послати на следеће e-mail адресе: [email protected]
или [email protected]. Од аутора се очекује да предложе категорију рукописа рада
(прегледни рад или оригинални научни рад).
Рецензија. Сви радови достављени уредништву часописа биће послати на рецензију код најмање два независна рецензента који ће бити замољени да рецензију достављеног им рукописа изврше у року од 4-6 недеља. Коначну одлуку о публиковању рукописа доноси Уређивачки одбор часописа. Рукопис може бити враћен ауторима на исправку и допуну, уколико је то неопходно. Исправљен и допуњен рукопис треба вратити уредништву часописа што је пре могуће (најдаље за 2 недеље)
након достављања примедби и коментара рецензената ауторима.
Рад припремљен за штампу: У последњој фази припреме рукописа аутору задуженом за коресподенцију електронском поштом биће достављен рад припремљен
за штампу на корекцију искључиво техничке природе и сагласност за штампање.
Све корекције аутори достављају електронском поштом у року од 48 сати од пријема рада припремљеног за штампу.
Сепарати рада. Аутор задужен за коресподенцију добиће 10 бесплатних сепарата
објављеног рада. Сва питања везана за објављивање радова у часопису слати на еmail уредника часописа.
FORMER EDITORS-IN-CHIEF
Prof. Dr. Adalbert Šenborn (1967-1970)
Prof. Dr. Radivoj Žakula (1972-1975)
Prof. Dr. Miroslava Todorović (1976-1994)
Prof. Dr. Biljana Škrbić (1995-1998)
THIS ISSUE OF ACTA PERIODICA TECHNOLOGICA
IS FINANCIALLY SUPPORTED BY:
Ministry of Education, Science and Technological Development
of Republic of Serbia
Editorial:
University of Novi Sad, Faculty of Technology Novi Sad,
Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
Phone: +381 21 485 3652
Fax:+381 21 450 413
e-mail: [email protected]
Text-Proof-Reader: Prof. Dr. Luka Bjelica
Typsetting: Branislav Bastaja
Cover design: Živojin Katić
Printed by VERZAL, Novi Sad
Copies: 200

acta periodica technologica - Tehnoloski fakultet Novi Sad

Transcription

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