acta periodica technologica - Tehnoloski fakultet Novi Sad

Transcription

FACULTY OF TECHNOLOGY – NOVI SAD
ТЕХНОЛОШКИ ФАКУЛТЕТ – НОВИ САД
ACTA PERIODICA
TECHNOLOGICA
APTEFF, 41, 1-203 (2010)
ACTA PERIODICA TECHNOLOGICA - Novi Sad (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, Referativnii zhurnal – Khimija, VINITI, Moscow, listed
in Ulrich’s International Periodical Directory, and indexed in the Elsevier Bibliographic data bases – SCOPUS.
YU ISSN 1450 – 7188
UDC 54:66:664:615
CODEN: APTEFF
Publisher
University of Novi Sad, Faculty of Technology
21000 Novi Sad, Bulevar Cara Lazara 1, 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
From Serbia
Dr. Ratko Lazarević, Academician
Prof. Dr. Slobodan D. Petrović
Prof. Dr. Erne Kiš
Prof. Dr. Petar Dokić
Prof. Dr. Spasenija Milanović
Prof. Dr. Vladimir Srdić
ACTA PERIODICA TECHNOLOGICA
APTEFF, 41, 1-203 (2010)
CONTENT
FOOD TECHNOLOGY
Gordana R. Dimić, Sunčica D. Kocić-Tanackov, Olivera O. Jovanov,
Dragoljub D. Cvetković, Siniša L. Markov, Aleksandra S. Velićanski
PRESENCE OF LISTERIA SPECIES IN FRESH MEATS
FROM RETAIL MARKETS IN SERBIA
1
Olgica S. Grujić, Jelena D. Pejin, Srbislav S. Denčić
THE APPLICATION OF TRITICALE VARIETY ODYSSEY
AS THE SUBSTITUTE FOR MALT IN WORT PRODUCTION
7
Predrag M. Ikonić, Ljiljana S. Petrović, Tatjana A. Tasić,
Natalija R. Džinić, Marija R. Jokanović, Vladimir M. Tomović
PHYSICOCHEMICAL, BIOCHEMICAL AND SENSORY PROPERTIES
FOR THE CHARACTERISATION OF PETROVSKÀ KLOBASÀ
(TRADITIONAL FERMENTED SAUSAGE)
19
Sunčica D. Kocić-Tanackov, Gordana R. Dimić, Jelena T. Lević,
Dušanka J. Pejin, Jelena D. Pejin, Igor M. Jajić
OCCURRENCE OF POTENTIALLY TOXIGENIC MOULD
SPECIES IN FRESH SALADS OF DIFFERENT KINDS OF
READY-FOR-USE VEGETABLES
33
Gordana B. Koprivica, Nevena M. Mišljenović, Ljubinko B. Lević,
Lidija R. Jevrić, Bojana V. Filipčev
OSMOTIC DEHYDRATION OF CARROT IN SUGAR BEET
MOLASSES: MASS TRANSFER KINETICS
47
Nevena T. Nemet, Vladislava M. Šošo, Vera L. Lazić
EFFECT OF GLYCEROL CONTENT AND pH VALUE OF
FILM-FORMING SOLUTION ON THE FUNCTIONAL PROPERTIES
OF PROTEIN-BASED EDIBLE FILMS
57
Tamara Đ. Premović, Etelka B. Dimić, Aleksandar A. Takači,
Ranko S. Romanić
INFLUENCE OF IMPURITIES AND HULL CONTENT IN THE
MATERIAL FOR PRESSING ON SENSORY QUALITY OF
COLD-PRESSED SUNFLOWER OIL
69
Sladjana P. Stanojević, Miroljub B. Barać, Mirjana B. Pešić,
Mirjana M. Milovanović, Biljana V. Vucelić-Radović
PROTEIN COMPOSITION IN TOFU OF CORRECTED QUALITY
77
Aleksandra N. Tepić, Zdravko M. Šumić, Mirjana B. Vukan
INFLUENCE OF PARTICLE DIAMETER ON THE COLOUR
OF GROUND PEPPER (Capsicum annuum L.)
87
Vladimir M. Tomović, Ljiljana S. Petrović, Žarko S. Kevrešan,
Natalija R. Džinić, Marija R. Jokanović
DETERMINATION OF NICKEL CONTENT IN THE
SEMIMEBRANOSUS MUSCLE OF PIGS PRODUCED
IN VOJVODINA
95
CHEMICAL TECHNOLOGY AND PROCESS ENGINEERING
Milan N. Sovilj
CRITICAL REVIEW OF SUPERCRITICAL CARBON DIOXIDE
EXTRACTION OF THE SELECTED OIL SEEDS
105
Jelena Đ. Marković, Nataša Lj. Lukić, Dragica Z. Jovičević
APPLICATION OF LATTICE-BOLTZMANN METHOD
AND ANALYSIS ON FLUID FLOW BETWEEN TWO
SINUSOIDAL PLATES
121
Tatjana J. Vulić, Goran C. Bošković
Mg-Cu-Al LAYERED DOUBLE HYDROXIDES BASED
CATALYSTS FOR THE REDUCTION OF NITRATES
IN AQUEOUS SOLUTIONS
131
Marina B. Šćiban, Mirjana A. Vasić, Jelena M. Prodanović,
Mirjana G. Antov, Mile T. Klašnja
THE INVESTIGATION OF COAGULATION ACTIVITY
OF NATURAL COAGULANTS EXTRACTED FROM
DIFFERENT STRAINS OF COMMON BEAN
141
BIOCHEMICAL AND PHARMACEUTICAL ENGINEERING
Slavenko Grbović, Dejan Orčić, Maria Couladis, Emilija Jovin,
Dušan Bugarin, Kristina Balog, Neda Mimica-Dukić
VARIATION OF ESSENTIAL OIL COMPOSITION OF
EUCALYPTUS CAMALDULENSIS (MYRTACEAE)
FROM THE MONTENEGRO COASTLINE
151
Lidija R. Jevrić, Gordana B. Koprivica,
Nevena M. Mišljenović, Bratislav Ž. Jovanović
CHROMATOGRAPHIC BEHAVIOR AND LIPOPHILICITY OF
s-TRIAZINE DERIVATIVES ON SILICA GEL IMPREGNATED
WITH PARAFFIN OIL
159
Katarina M. Penov-Gaši, Andrea R. Gaković, Jovana J. Ajduković,
Maja Dj. Djurendić-Brenеsel, Evgenija A. Djurendić, Marina P. Savić,
Marija N. Sakač
REACTIVITY OF 17-HYDROXY-17-SUBSTITUTED
ANDROSTANE DERIVATIVES
169
Sanja O. Podunavac-Kuzmanović, Sonja D. Velimirović
CORRELATION BETWEEN THE LIPOPHILICITY AND
ANTIFUNGAL ACTIVITY OF SOME BENZOXAZOLE
DERIVATIVES
177
Slađana M. Savatović, Gordana S. Ćetković,
Jasna M. Čanadanović-Brunet, Sonja M. Đilas
UTILISATION OF TOMATO WASTE AS A SOURCE
OF POLYPHENOLIC ANTIOXIDANTS
187
Vesna T. Tumbas, Gordana S. Ćetković, Sonja M. Djilas, Jasna M.
Čanadanović-Brunet, Jelena J. Vulić, Željko Knez, Mojca Škerget
ANTIOXIDANT ACTIVITY OF MANDARIN (Citrus reticulata) PEEL
195
INSTRUCTION FOR MANUSCRIPT PREPARATION
ACTA PERIODICA TECHNOLOGICA
APTEFF, 41, 1-203 (2010)
САДРЖАЈ
ПРЕХРАМБЕНА ТЕХНОЛОГИЈА
Гордана Р. Димић, Сунчица Д. Коцић-Танацков, Оливера О. Јованов,
Драгољуб Д. Цветковић, Синиша Л. Марков, Александра С. Велићански
ПРИСУСТВО LISTERIA ВРСТА У СВЕЖЕМ МЕСУ
ИЗ МАЛОПРОДАЈНИХ ОБЈЕКАТА У СРБИЈИ
1
Олгица С. Грујић, Јелена Д. Пејин, Србислав С. Денчић
ПРИМЕНА ТРИТИКАЛЕА СОРТЕ ОДИСЕЈ КАО
ЗАМЕНЕ ЗА СЛАД У ПРОИЗВОДЊИ СЛАДОВИНЕ
7
Предраг М. Иконић, Љиљана С. Петровић, Татјана А. Тасић,
Наталија Р. Џинић, Марија Р. Јокановић, Владимир М. Томовић
ФИЗИЧКО-ХЕМИЈСКЕ, БИОХЕМИЈСКЕ И СЕНЗОРНЕ
КАРАКТЕРИСТИКЕ ТРАДИЦИОНАЛНЕ СУВЕ
ФЕРМЕНТИСАНЕ ПЕТРОВАЧКЕ КОБАСИЦЕ
19
Сунчица Д. Коцић-Танацков, Гордана Р. Димић, Јелена Т. Левић,
Душанка Ј. Пејин, Јелена Д. Пејин, Игор М. Јајић
ПОЈАВА ПОТЕНЦИЈАЛНО ТОКСИГЕНИХ ВРСТА
ПЛЕСНИ У САЛАТАМА ОД РАЗЛИЧИТИХ ВРСТА
ПОВРЋА СПРЕМНИХ ЗА КОНЗУМИРAЊЕ
33
Гордана Б. Копривица, Невена М. Мишљеновић, Љубинко Б. Левић,
Лидија Р. Јеврић, Бојана В. Филипчев
ОСМОТСКА ДЕХИДРАТАЦИЈА МРКВЕ У МЕЛАСИ
ШЕЋЕРНЕ РЕПЕ: КИНЕТИКА ПРЕНОСА МАСЕ
47
Невена Т. Немет, Владислава М. Шошо, Вера Л. Лазић
УТИЦАЈ САДРЖАЈА ГЛИЦЕРОЛА И pH ВРЕДНОСТИ
РАСТВОРА ЗА ПРИПРЕМАЊЕ ЈЕСТИВИХ ПРОТЕИНСКИХ
ФИЛМОВА НА ЊИХОВЕ ФУНКЦИОНАЛНЕ КАРАКТЕРИСТИКЕ
57
Тамара Ђ. Премовић, Етелка Б. Димић, Александар А. Такачи,
Ранко С. Романић
УТИЦАЈ САДРЖАЈА НЕЧИСТОЋА И ЉУСКЕ У МАТЕРИЈАЛУ
ЗА ПРЕСОВАЊЕ НА СЕНЗОРНА СВОЈСТВА ХЛАДНО
ПРЕСОВАНОГ УЉА СУНЦОКРЕТА
69
Слађана П. Станојевић, Мирољуб Б. Бараћ, Мирјана Б. Пешић,
Мирјана М. Миловановић, Биљана В. Вуцелић-Радовић
ПРОТЕИНСКИ САСТАВ ТОФУА КОРИГОВАНОГ КВАЛИТЕТА
77
Александра Н. Тепић, Здравко М. Шумић, Мирјана Б. Вукан
УТИЦАЈ СТЕПЕНА УСИТЊЕНОСТИ НА БОЈУ МЛЕВЕНЕ
ЗАЧИНСКЕ ПАПРИКЕ (Capsicum annuum L.)
87
Владимир М. Томовић, Љиљана С. Петровић, Жарко С. Кеврешан,
Наталија Р. Џинић, Марија Р. Јокановић
ОДРЕЂИВАЊЕ САДРЖАЈА НИКЛА У М. semimembranosus
СВИЊА ПРОИЗВЕДЕНИХ У ВОЈВОДИНИ
95
ХЕМИЈСКА ТЕХНОЛОГИЈА И ПРОЦЕСНО ИНЖЕЊЕРСТВО
Милан Н. Совиљ
КРИТИЧКИ ПРЕГЛЕД ЕКСТРАКЦИЈЕ ОДАБРАНИХ
УЉАРИЦА НАТКРИТИЧНИМ УГЉЕНДИОКСИДОМ
105
Јелена Ђ. Марковић, Наташа Љ. Лукић, Драгица З. Јовичевић
ПРИМЕНА ЛАТИС-БОЛЦМАН МЕТОДА (ЛБМ)
И АНАЛИЗА СТРУЈАЊА ФЛУИДА ИЗМЕЂУ ДВЕ
СИНУСОИДАЛНЕ ПЛОЧЕ
121
Татјана Ј. Вулић, Горан Ц. Бошковић
КАТАЛИЗАТОРИ НА БАЗИ Mg-Cu-Al
СЛОЈЕВИТИХ ХИДРОКСИДА ЗА РЕДУКЦИЈУ
НИТРАТА ИЗ ВОДЕНИХ РАСТВОРА
131
Марина Б. Шћибан, Мирјана А. Васић, Јелена М. Продановић,
Мирјана Г. Антов, Миле Т. Клашња
ИСПИТИВАЊЕ КОАГУЛАЦИОНЕ АКТИВНОСТИ
ПРИРОДНИХ КОАГУЛАНАТА ЕКСТРАХОВАНИХ
ИЗ РАЗЛИЧИТИХ СОРТИ ПАСУЉА
141
БИОХЕМИЈСКО И ФАРМАЦЕУТСКО ИНЖЕЊЕРСТВО
Славенко Грбовић, Дејан Орчић, Maria Couladis, Емилија Јовин,
Душан Бугарин, Кристина Балог, Неда Мимица-Дукић
ЕТАРСКО УЉЕ EUCALYPTUS CAMALDULENSIS (MYRTACEAE)
СА ЦРНОГОРСКОГ ПРИМОРЈА
151
Лидија Р. Јеврић, Гордана Б. Копривица,
Невена М. Мишљеновић, Братислав Ж. Јовановић
ХРОМАТОГРАФСКО ПОНАШАЊЕ И ЛИПОФИЛНОСТ
s-ТРИАЗИНСКИХ ДЕРИВАТА НА ТАНКОМ СЛОЈУ
СИЛИКА ГЕЛА ИМПРЕГНИРАНОГ ПАРАФИНСКИМ УЉЕМ
159
Катарина М. Пенов-Гаши, Андреа Р. Гаковић, Јована Ј. Ајдуковић,
Маја Ђ. Ђурендић-Бренeсел, Евгенија А. Ђурендић, Марина П. Савић,
Марија Н. Сакач
РЕАКТИВНОСТ 17β-ХИДРОКСИ-17α-СУПСТИТУИСАНИХ
АНДРОСТАНСКИХ ДЕРИВАТА
169
Сања О. Подунавац-Кузмановић, Соња Д. Велимировић
KОРЕЛАЦИЈA ИЗМЕЂУ ЛИПОФИЛНОСТИ И
АНТИФУНГАЛНЕ АКТИВНОСТИ НЕКИХ
ДЕРИВАТА БЕНЗОКСАЗОЛА
177
Слађана М. Саватовић, Гордана С. Ћетковић,
Јасна М. Чанадановић-Брунет, Соња М. Ђилас
ИСКОРИШЋЕЊЕ ОТПАТКА ПАРАДАЈЗА КАО
ИЗВОРА ПОЛИФЕНОЛНИХ АНТИОКСИДАНАТА
187
Весна Т. Тумбас, Гордана С. Ћетковић, Соња М. Ђилас, Јасна М.
Чанадановић-Брунет, Јелена Ј. Вулић, Жељко Кнез, Мојца Шкергет
АНТИОКСИДАТИВНА АКТИВНОСТ КОРЕ
МАНДАРИНЕ (Citrus reticulata)
195
УПУТСТВО ЗА ПИСАЊЕ РАДА
FOOD TECHNOLOGY
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041001D
UDC: 637.514:579.86:658.87(497.11)
BIBLID: 1450-7188 (2010) 41, 1-6
Original scientific paper
PRESENCE OF LISTERIA SPECIES IN FRESH MEATS FROM RETAIL
MARKETS IN SERBIA
Gordana R. Dimić, Sunčica D. Kocić-Tanackov, Olivera О. Jovanov, Dragoljub D.
Cvetković, Siniša L. Markov, Aleksandra S. Velićanski1
Listeria spp. are Gram positive, short, non-sporing rods, microaerophilic. Of the six
species currently recognized, Listeria monocytogenes is the most important as it causes a
range of infections in humans and animals. The organism can be found in a wide variety
of habitats including the soil, food processing environments and raw foods. The ability of
the organism to grow at refrigeration temperatures is of major importance in food production. This study examines the presence of Listeria species in fresh meat. 29 samples
(chicken, pork and beef) meat. This bacteria was found in 82.7% of analyzed samples; 7
L. innocua, 8 L. monocytogenes and 9 L. welshimeri (of all isolates). L. innocua prevailed in pork meat (40%), L. monocytogenes in chicken and pork meat (30%), and L. welshimeri in beef meat (44.4%).
KEYWORDS: fresh meat, Listeria spp., L. monocytogenes
INTRODUCTION
Food producers are bound to deliver healthy, wholesome and safe products to the
market. Meat and meat products are classified as epidemiologically hazardous food
which may be contaminated with bacteria from the Listeria genus. These microaerophilic
Gram-positive rod-shaped bacteria can contaminate fresh meat during processing from
many sources: air, contaminated water and/or during the distribution. Studies have confirmed that feces of healthy humans contain Listeria organisms (1, 2, 3). Lymph glands
have been also shown to be sources of contamination (4). Some of the species such as L.
monocytogenes and L. ivanovii have been addressed as causative agents of severe food
infections. Listeriosis has been listed as a rare disease in relation to the other food-borne
infections but with exceptionally high fatality rate (20-30%) (5). The manifestations of
listeriosis include septicemia, meningitis (or meningoencephalitis), encephalitis, and intrauterine or cervical infections in pregnant women, which may result in spontaneous
abortion or stillbirth. At present the infective dose of L. monocytogenes is unknown,
Dr Gordana R. Dimić, Assoc. Prof., [email protected], Sunčica D. Kocić-Tanackov, M.Sc., Assist., Faculty
of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Olivera O. Jovanov, B.Sc.,
Jugoinspekt, Dunavska 23, 21000 Novi Sad, Dr Dragoljub D. Cvetković, Assis. Prof., Dr Siniša L. Markov,
Assoc. Prof., Aleksandra S. Velićanski, M.Sc., Assist., Faculty of Technology, University of Novi Sad, Bulevar
cara Lazara 1, 21000 Novi Sad, Serbia
1
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041001D
UDC: 637.514:579.86:658.87(497.11)
BIBLID: 1450-7188 (2010) 41, 1-6
although it is believed to vary with the strain and susceptibility of the patient (6). Sometimes in susceptible persons, fewer than 103 cfu/g or ml may cause disease (7). Listeriosis
is a serious disease, primarily transmitted through various foods: milk, milk products,
meat and meat products, fish, eggs and egg products, fruits and vegetables. It is particularly difficult to control, since it is ubiquitous and widespread in the environment, and
since it possesses physiological characteristics that allow growth under conditions that
are usually adverse for most other pathogenic bacteria. The consumption of contaminated
sausages lead to an outbreak in the USA during 1998, involving 110 cases with 4 deaths
(8). In another case in France, between 1999 and 2000, as a result of consumption of
pork, 7 deaths had been seen out of 26 listeriosis cases (8). Especially hazardous are undercooked meat products. L. monocytogenes is resistant to high salt concentrations and
low temperatures, which enables its survival in sausages and dried meat products.
Many studies are focused on the detection of Listeria spp. in various food commodities in order to prevent epidemic of human listeriosis. Much attention has been paid on
the incidence of pathogenic Listeria spp. strains. Taking into consideration that the existing data on the incidence of Listeria food contamination in Serbia are rather scarce, the
aim of this study was to investigate the occurrence of Listeria spp. in various types of
fresh meat from local retail markets.
EXPERIMENTAL
Material
For the experiment, 29 samples of fresh chicken, pork and beef meat were purchased
from retail markets in Novi Sad (Serbia). Growth media used in the experimental work
were obtained from HiMedia (Mumbai, India).
Isolation of Listeria species
For the isolation of Listeria species, a two-stage enrichment procedure in Fraser broth
was used (9). After sterilization, selective supplements (ferric ammonium citrate, acriflavin hydrochlorid, and nalidixic acid) were added to the broth. Meat samples were incubated in Erlenmeyer flasks with Fraser broth at 30°C for 24 h. After that, 0.1 mL of broth
were transferred into 10 mL tubes of Fraser broth for secondary enrichment and incubated at 37°C for the next 48 h. The content was transferred with inoculation loop to PALCAM and Oxford agar plates. The plates were incubated at 37°C for 48 h. The suspect
Listeria colonies were further confirmated on the basis of Gram stain, catalase, oxidase,
and motility tests.
Identification of the isolated species
The VITEK® 2 system (bioMerieux, France) and VITEK® 2 Gram-Positive (GP)
identification card were used for identification of Listeria spp. The GP identification card
is based on established biochemical methods and newly developed substrates. There are
2
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041001D
UDC: 637.514:579.86:658.87(497.11)
BIBLID: 1450-7188 (2010) 41, 1-6
43 biochemical tests measuring carbon source utilization, enzymatic activities and resistance. Final identification results are available in approximately eight hours or less.
RESULTS AND DISCUSSION
Listeria spp. were identified in 82.7% of the investigated meat samples. Their presence was significant in all types of meat investigated. The obtained data showed that pork
meat was the most contaminated (90%) (Table 1).
Table 1. Prevalence of the Listeria spp. in different types of meats
Meat type
Chicken
Pork
Beef
Number of contaminated samples
8
9
7
Listeria spp.
Prevalence (%)
80
90
77.8
From the samples of chicken, pork and beef the following species were isolated: L.
innocua (7 isolates), L. monocytogenes (8 isolates) and L. welshimeri (9 isolates). L. innocua was the most frequent in pork meat (40%). L. monocytogenes was more prevalent in
chicken and pork (30%) than in beef meat (22.2%), whereas L. welshimeri dominated in
beef meat (44.4%) (Fig. 1).
Figure 1. Distribution of L. innocua, L. monocytogenes and L. welshimeri in the analyzed
meat types
In general, L. welshimeri was the species with the highest incidence (isolated in 31%
of samples) followed by L. monocytogenes and L. innocua with slightly lower but still
significant incidence (27.6% and 24.1%) (Fig. 2).
3
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041001D
UDC: 637.514:579.86:658.87(497.11)
BIBLID: 1450-7188 (2010) 41, 1-6
Figure 2. Isolated Listeria species in the fresh meat
It is evident from the present study that the prevalence of Listeria was significant in
the fresh meat samples. It should be noted that pathogenic L. monocytogenes was present
in all meat types. In contrast to other pathogenic microorganisms, this bacterium has ability to grow and reproduce in a range of temperature, from - 1.5 to 45°C (10), which is a
serious problem for food manufacturers. According to the general opinion, meat contamination with bacteria occurs during the skin removal (11).
Listeria species were detected in swabs taken from skins of beef ribs and rounds as
well as in those taken after the skin removal (2). Rayser et al. (12) reported Listeria contamination in 89% of minced beef meat, over 70% of minced poultry and turkey meat and
96% of pork sausages. Besides L. monocytogenes, L. innocua and L. welshimeri were also identified. Skovgaard et al. (13) showed that the occurence of L. innocua was more
frequent than L. monocytogenes in beef and poultry meat. Also, L. innocua was the dominating species in poultry meat and poultry products in the study reported by Kosek-Paszkowska et al. (14).
L. monocytogenes was isolated in 16% of fresh sausages, 15.8% mechanically cut
poultry meat, 10.7% skinless sausage, 6.8% minced meet, and 4.3% fermented durable
sausages (15). Over the period 2000-2003, under the scrutiny of the USDA and FDA, 713
samples of various food commodities were recalled from retail markets (16). The recalled
products included fresh pork, poultry and beef meat, dairy products, seafood, ice creams,
fresh, and processed fruits and vegetables. Meat accounted for about one half of the recalled products. Microbial contaminants were the reason for 43.9% recall events. L. monocytogenes, E. coli, and Salmonella enteritidis were the main pathogens in the suspected
foods. L. monocytogenes accounted for 186 product recalls which accounted for 26% of
the total recall events. L. monocytogenes was present in 19.3% of tested chicken meat and
7.1% of beef meat (17). Especially concerning fact is that it is capable of not only surviving in vacuum packed meat products (18) but of reproducing under these conditions
(19).
CONCLUSION
This study has confirmed a high prevalence of Listeria in meat, which implies that
chances of contracting listeriosis are greatly increased when consuming inadequately
4
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cooked meat and meat products. Control for the Listeria presence in every stage of food
processing and distribution, including retail market, raising the hygienic and sanitary
standards are key prerogatives in the prevention of listeriosis outbreaks.
REFERENCES
1. T. I. Braun, D. Travis, R. R. Dee and R. E. Nieman: Liver abscess due to Listeria
monocytogenes: case report and review. Clin. Infect., Dis. 17 (1993) 267-269.
2. S. Bonardi, F. Brindani and E. Maggi: Isolation of Listeria monocytogenes and Listeria spp. from pigs at slaughter in Italy. Ann. Fac. Medic. Vet. di Parma. 22 (2002)
205-210.
3. S. Bonardi, A. Bottarelli, S. Fusaro, S. Bentley, A. Gnappi and A. Morini: Epidemiological investigation on Listeria spp. in bovine slaughterhouse. Industrie Alimentari
36, 2 (1997) 139-140.
4. N. Skovgaard and B. Norrung: The incidence of Listeria spp. in faeces of Danish pigs
and in minced pork meat. Int. J. Food Microbiol. 8 (1989) 59-63.
A. Schuchat, B. Swaminathan and C. V. Broome: Epidemiology of human listeriosis.
Clin. Microbiol. Rev. 4 (1991) 169-183.
5. S. Uhitil, Jakšić, T. Petrak, H. Medić and L. Gumhalter-Karolyi: Prevalence of
Listeria monocytogenes and the other Listeria spp. in cakes in Croatia. Food Control
15 (2004) 213–216.
6. J.M. Jay, M.J. Loessner and D.A. Golden: Modern food microbiology, Springer
Science+Business Media, New York (2005) pp. 591-617.
7. H. Colak, H. Hampikyan, B. Ulusoy and E.B. Bingol: Presence of Listeria monocytogenes in Turkish style fermented sausage (sucuk). Food Control 18 (2007) 30-32.
8. ISO 1129-1: Microbiology of food and animal feeding stuffs - Horizontal method for
the detection and enumeration of Listeria monocytogenes - Part 1: Detection method,
Geneva, Switzerland (2004) p. 1.
9. J. A. Hudson, S. J. Mott and N. Pennez: Growth of Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica on vacum and saturated carbon dioxide
controlled atmosphere-packaged sliced roast beef. J. Food. Prot. 57 (1994) 204-208.
10. S. Duraković, F. Delaš, B. Stilinović and L. Duraković: Moderna mikrobiologija
namirnica. Kugler, Zagreb, Hrvatska (2002) p. 93.
11. E. T. Rayser, S. M. Arimi, M. M. C. Bunduki and C. W. Donnelly: Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry
products with two primary enrichment media. Appl. Environ. Microbiol. 62 (1996)
1781-1787.
12. N. Skovgaard and C. A. Morgen: Detection of Listeria spp. in faces from animals, in
feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6 (1988) 229-242.
13. K. Kosek-Pasykowska, J. Bania, J. Bystron, J. Molenda and M. Czerw: Occurence of
Listeria spp. in raw poultry meat and poultry meat products. Bull. Vet. Inst. Pulawy
49 (2005) 219-222.
14. J. Marinšek and S. Grebenc: Listeria monocytogenes in minced meat and thermally
untreated meat products in Slovenia. Slov. Vet. Research 39, 2 (2002) 131-136.
5
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15. V. Salin, S. Darmasena, A. Wong and P. Luo: Food-Product Recalls in the U.S.,
2000-2003. J. of Food Distribution Res. 37, 1 (2006) 149-153.
16. S. S. Green: Listeria monocytogenes in meat and poultry products. Interim Rept. To
Nat’l Adv. Comm. Microbiol. Spec. Foods. FSIS/USDA, Nov. 27 (1990)
17. S. Buncic: The incidence of Listeria monocytogenes in slaughtered animals, in meat,
and meat products in Yugoslavia. Int. J. Food Microbiol. 12, 2-3 (1991) 173-180.
18. S. Bunčić, L. Paunović and D. Radišić: The fate of Listeria monocytogenes in fermented sausages and in vacuum-packaged frankfurters. J. Food Prot. 54 (1990) 413417.
ПРИСУСТВО LISTERIA ВРСТА У СВЕЖЕМ МЕСУ ИЗ МАЛОПРОДАЈНИХ
ОБЈЕКАТА У СРБИЈИ
Гордана Р. Димић, Сунчица Д. Коцић-Танацков, Оливера О. Јованов, Драгољуб Д.
Цветковић, Синиша Л. Марков, Александра С. Велићански
Listeria врсте су Грам позитивне, аспорогене, микроаерофилне бактерије koje су
способне да рaсту у опсегу температура од 4-37oC. Налазе се у земљишту, сировој
храни и производном окружењу. Најзначајнија врста као узрочник инфекција људи
и животиња (листериоза) je Listeria monocytogenes. За безбедност намирнице је
важна чињеница да је ова бактерија способна да се размножава на температури хлађења. Овај рад показује присуство Listeria врста у свежем месу. Укупно је испитано
29 узорака пилећег, свињског и јунећег меса. Ове бактерије су установљене у 82,7%
узорака; 7 L. innocua, 8 L. monocytogenes и 9 L. welshimeri (од свих изолата). L.
innocua је била доминантна у свињском месу (40%), L. monocytogenes у пилећем и
свињском месу (30%) и L. welshimeri у јунећем месу (44,4%).
Received 8 October 2010
Accepted 3 November 2010
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THE APPLICATION OF TRITICALE VARIETY ODYSSEY AS THE
SUBSTITUTE FOR MALT IN WORT PRODUCTION
Olgica S. Grujić, Jelena D. Pejin and Srbislav S. Denčić
The objective of this work was to investigate the possibility of triticale application as
the partial substitute for malt in wort production. For wort production, two series of
experiments were performed in which triticale variety Odyssey, from experimental fields,
Rimski Šančevi location (Serbia), was used as the substitute for barley malt in grist with
and without the addition of commercial enzyme Ultraflo Max (Novozymes, Denmark).
Triticale was added in each of the carried series of experiments as the substitute for malt:
0, 10, 20, 30, 40, 50, 60, 70 and 80% in grist. Experiments were carried out on laboratory scale by the application of infusion procedure for wort production. Regarding extract
content, triticale variety Odyssey could be used as the substitute for malt up to 60% without the addition of commercial enzyme Ultraflo Max. With the addition of this enzyme
triticale variety Odyssey could be used as a substitute for malt up to 80%, regarding the
extract content. With the increase in the content of triticale in the grist, viscosity increased. The addition of commercial enzyme Ultraflo Max significantly reduced wort viscosity. The obtained results indicate that worts produced with the addition of triticale variety Odyssey to grist yielded good analytical quality parameters.
KEYWORDS: Triticale, malt, wort
INTRODUCTION
According to the definition given by the Bavarian law of the purity of beer production
(“Reinheitsgebot”) from 1516, beer is the product which must be produced only from
malted barley, hops and water, using the brewer’s yeast for fermentation (1). Brewing is a
multistage process involving biological conversion of raw materials to final product (2).
In modern brewing the use of adjuncts (unmalted cereals) is a well-established procedure
(3).
From the technological point of view, an adjunct is primarily employed in brewing to
provide carbohydrates that can ultimately be broken down into fermentable sugars, and
maximum starch content is clearly desired in the use of a brewing adjunct (4). Adjuncts
are also used in beer production to increase beer stability, and possibly to reduce the
production costs (5). Despite of the undisputed economic role of adjunct utilization, beer
Dr. Olgica S. Grujić, Prof., Dr. Jelena D. Pejin, Assist. Prof., University of Novi Sad, Faculty of Technology,
Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia, Dr. Srbislav S. Denčić, Scientific Advisor, Institute of field and
vegetable crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia
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quality is based on wort composition rather than on wort price. Thus, the brewer needs to
ensure that wort prepared from mixed grists of malt and adjuncts does not diminish the
traditionally high quality standards (6). Maize and rice are the most commonly used
sources of starch-rich brewing adjuncts. Nevertheless, a wide range of other cereals, such
as unmalted barley, wheat, or sorghum have also been employed (3).
Triticale (Triticosecale spp. Wittmack), the first man-made cereal, is the product of a
cross between wheat (Triticum spp.) and rye (Secale spp.) (6). In modern times, it has
been reported that triticale is cultivated in more than 30 countries worldwide (7) on
araund 3.7 milion ha in total, yielding more than 12 milion tonnes a year (8). Triticale has
agronomic advantages, it can be grown on more marginal land (arid, acidic, etc.) and requires less agricultural chemicals (fertilizer, agronomic chemicals, etc.) (9). Recent studies have shown that unmalted triticale may be suitable as a brewing adjunct. Most nonmalt adjuncts do not contribute to enzyme activity. However, triticale goes beyond this
specification, since some triticale varieties already contain high levels of amylolytic activity in their unmalted natural form, in conjunction with lower levels of proteolytic activity (10). Because of this and the low gelatinization range (59-65ºC), triticale is capable
of degrading its own starch content with the efficiencies roughly equal to those of barley
malt (3). Besides amylolysis and proteolysis, the degradation of non-starch polysaccharides during mashing is considered another essential parameter in brewing. Arabinoxylan
and mixed linkage β-glucan have been recognised as factors that contribute to wort viscosity, decrease wort and beer filtration rates and cause subsequent problems such as
haze formation or reduced extraction efficiency (4). When using triticale, mixed linkage
β-glucans are negligible as a result of their minor content in the grains (11). In contrast,
the solubilisation of triticale arabinoxylans slightly increases wort viscosity and may
affect beer filtration rates (3, 6). In addition to these quality criteria, Glatthar et al. (6)
showed that substantial savings (up to 25% in costs for raw materials) could be achieved
by using triticale instead of brewing adjuncts currently in use.
The objective of this paper was to investigate the possibility of triticale application as
the partial substitute for malt in wort production. For wort production, two series of experiments were performed in which triticale variety Odyssey, from experimental fields,
Rimski Šančevi location (Serbia), was used as the substitute for barley malt in grist with
and without the addition of commercial enzyme Ultraflo Max (Novozymes, Denmark).
EXPERIMENTAL
For wort production, two series of experiments were performed in which triticale
variety Odyssey, from experimental fields, Rimski Šančevi location (Serbia), was used as
the substitute for barley malt in grist with and without the addition of commercial enzyme
Ultraflo Max (xylanase and β-glucanase) as recommended by the manufacturer (Novozymes, Denmark). Ultraflo Max is a beer filtration enzyme solution that lowers wort viscosity and levels of arabino-xylans, as well as levels of β-glucans. Triticale was added in
each of the carried series of experiments as the substitute for malt: 0, 10, 20, 30, 40, 50,
60, 70 and 80% in grist based on extract content. Experiments were carried out on laboratory scale by the application of infusion procedure (30 minutes at 45C; temperature in8
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crease to 70C in 25 minutes; 60 minutes at 70C, cooling to 20C) for wort production.
At the shares >40% of triticale, thermal treatment (temperature increase to 90°C; cooling
to 70-75°C, addition of 1g of malt; temperature increase to the boiling and kept at this
temperature for 10 minutes; cooling to 45°C) was used before the infusion procedure (for
starch gelatinization). Triticale, malt and wort analyses were performed using the standard European Brewery Convention, Analytica-EBC (12) and/or MEBAK (13) methods.
RESULTS AND DISSCUSION
Results of malt analyses are given in Table 1. According to analytical parameters of
malt analyses presented in Table 1, malt used in these experiments was of good quality.
Table 1. Results of malt analyses
Parameter
 Moisture content of grain, %
 Proteins, % DM
 Extract content, fine grist, % DM
 Saccharification, min
 Wort clarity
 Filtration, min
 Wort colour, EBC units
 Wort pH value
 Wort soluble nitrogen, mg/100 mL
 Viscosity, mPas, 8.6%e
 Wort formol nitrogen content, mg/100mL
 Extract difference, % DM
 Kolbach indice, %
 Hartong VZ 45°C, %
 Real attenuation, %
 Apparent attenuation, %
Malt
5.45
10.23
80.64
<10
Slightly opal
11
3.0
5.67
88.90
1.648
28.13
1.21
49.24
42.20
76.75
94.62
DM – dry matter
The results of triticale variety Odyssey analyses as an adjunct are given in Table 2.
9
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Table 2. Results of triticale variety Odyssey analyses (as an adjunct)
Parameter
 Moisture content of grain, %
 Protein content, %DM
 Extract content, fine grist, % DM
 Saccharification, min
 Wort clarity
 Filtration, min
 Wort colour, EBC units
 Wort pH value
 Wort soluble nitrogen, mg/100 mL
 Viscosity, mPas, 8.6%e
 Wort formol nitrogen content, mg/100mL
 Real attenuation, %
 Apparent attenuation, %
Odyssey
10.06
12.65
78.23
15-20
clear
20
3.5
5.62
80.51
1.959
26.85
63.11
77.93
DM – dry matter
The results of the worts analyses produced with triticale variety Odyssey are given in
Table 3.
Table 3. Results of worts analyses produced with triticale variety Odyssey
Triticale content
(%)
Wort extract
content, %
Saccharification,
min
0%
10%
20%
30%
40%
50%
60%
70%
80%
8.604
8.597
8.507
8.326
8.295
8.276
8.216
7.891
7.724
<10
<10
<10
10-15
10-15
15-20
15-20
>60
>60
Wort clarity
slightly
opal
slightly
opal
slightly
opal
clear
clear
clear
clear
clear
clear
Wort colour,
EBC units
3.0
3.0
3.0
3.0
3.0
3.5
3.5
3.5
3.5
Filtration, min
11
11
12
13
15
19
50
>60
>60
Wort pH value
5.67
5.65
5.65
5.64
5.64
5.63
5.63
5.62
5.62
88.90
88.77
86.80
80.15
78.40
77.70
74.90
54.60
51.10
1.648
1.648
1.741
1.810
1.891
1.972
2.146
2.303
2.474
28.13
27.85
27.39
27.13
27.06
26.88
26.69
26.41
26.22
76.75
71.26
68.93
66.38
63.57
62.86
62.04
60.42
60.10
94.62
88.03
85.11
81.92
78.44
77.51
76.57
74.51
74.18
Soluble nitrogen
content,
mg/100mL
Viscosity, mPas,
8.6%e
Wort formol
nitrogen content,
mg/100mL
Real attenuation,
%
Apparent
attenuation, %
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Substituting a share of malt with triticale variety Odyssey, the characteristic quality
parameters of worts changed compared to the control wort produced of malt only as
follows (Table 3):
- control malt yielded the highest extract content. Substitution of malt with triticale from 10% up to 80% resulted in a uniform extract decrease with increase of
triticale share in grist (10.23% decrease at 80% of triticale share compared to the
control wort);
- saccharification time was too high when the share of triticale was higher than
50%, whereas in case of 70 and 80% of triticale in grist the saccharification was
nonsatisfactory and unacceptable;
- substitution of malt with triticale, 30-80%, had possitive influence on wort clarity;
- substitution of malt with triticale from 10% to 80% resulted in wort colour increase with the increase of triticale share in grist;
- filtration rate significantly increased when 60, 70 and 80% of malt was substituted with triticale;
- wort pH value slightly decreased with the increase of triticale share;
- wort soluble nitrogen content decreased with the increase of triticale content in
wort (42.61% decrease at 80% of triticale share compared to the control wort);
- wort viscosity increased uniformly with the increase of triticale share (50.12%
increase at 80% of triticale share compared to the control wort), reaching very
high levels at 50, 60, 70 and 80%;
- wort formol nitrogen content slightly decreased with the increase of triticale share in grist (6.79% decrease at 80% of triticale share compared to the control
wort), and
- real and apparent wort attenuation decreased with the increase in triticale content
in wort.
The results of the worts analyses produced with triticale variety Odyssey and with the
application of enzyme Ultraflo Max are given in Table 4.
Table 4. Results of worts analyses produced with triticale variety Odyssey and enzyme
Ultraflo Max
Triticale content
(%)
Wort extract
content, %
Saccharification,
min
0%
10%
20%
30%
40%
50%
60%
70%
80%
8.604
8.626
8.535
8.432
8.419
8.400
8.243
8.234
8.085
<10
<10
<10
10-15
10-15
10-15
10-15
>60
>60
Wort clarity
slightly
opal
slightly
opal
slightly
opal
clear
clear
clear
clear
clear
clear
Wort colour,
EBC units
3.0
3.0
3.0
3.0
3.0
3.5
3.5
3.5
3.5
Filtration, min
11
11
11
11
12
12
13
13
15
Wort pH value
5.67
5.65
5.65
5.64
5.64
5.63
5.63
5.62
5.62
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Table 4. Continuation
Triticale content
(%)
Soluble nitrogen
content,
mg/100mL
Viscosity, mPas,
8.6%e
Wort formol
nitrogen content,
mg/100mL
Real attenuation,
%
Apparent
attenuation, %
0%
10%
20%
30%
40%
50%
60%
70%
80%
88.90
88.86
86.75
80.14
78.38
77.69
74.92
54.57
51.05
1.648
1.447
1.533
1.574
1.606
1.623
1.655
1.671
1.717
28.13
27.81
27.37
27.09
26.97
26.86
26.60
26.39
26.21
76.75
76.64
67.98
67.26
65.82
63.83
62.97
61.41
60.78
94.62
94.50
83.79
83.01
81.19
78.72
77.69
75.80
75.07
The use of triticale variety Odyssey as the substitute of malt for wort production with
the application of enzyme Ultraflo Max caused the changes of characteristic wort quality
parameters compared to control wort produced of malt only as follows (Table 4):
- extract content in worts produced with triticale was lower than in control one, and
the increase in triticale share caused a uniform extract content decrease (6.03%
decrease at 80% of triticale share compared to the control wort);
- saccharification time was satisfactory for triticale shares up to 60%, whereas at 70
and 80% content it was unsatisfactory i.e. it was too long. Saccharification time
was lower, up to the 70% malt replacement, comared to the worts produced without the application of Ultrflo Max enzyme;
- partial substitution of malt with triticale had a possitive effect on wort clarity
change compared to the control one, but no difference was observed compared to
the worts produced without the enzyme application;
- slight increase in wort colour was determined as a result of partial malt substitution with triticale. The results for the wort colour were identical to those of
worts produced without the addition of the enzyme Ultraflo Max;
- filtration time increased slightly at triticale shares 70 and 80%. The filtration time
was shorter comared to the samples produced without the addition of the enzyme,
especially at higher trticale ratios (60-80%);
- increase of triticale share resulted in a slight decrease of the wort pH but the results were similar to those obtained without the enzyme addition;
- wort soluble nitrogen content decreased with the increase of triticale content in
wort (42.58% decrease at 80% of triticale share compared to the control wort);
- 10-50% of triticale in grists caused wort viscosity decrease compared to the control wort, while the viscosity remained at the level of control wort at 60% of triticale. Wort viscosity increased uniformly with the furhter increase of the triticale
share,
- wort formol nitrogen content decreased with increase of triticale share in grist
(6.82% decrease at 80% of triticale share compared to the control wort) and
- substitution of malt with triticale from 10% to 80% resulted in uniform real and
apparent attenuation decrease. Real and apparent attenuation values were slightly
higher compared to the samples produced without the addition of Ultraflo Max.
12
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Comparative survey of the results of wort analyses produced by partial substitution
of malt with different shares of triticale without and with the addition of enzyme
Ultraflo Max
The influence of different triticale shares and the application of enzyme Ultraflo Max
on the wort extract content is presented in Figure 1.
Figure 1. Extract content of the produced worts
The worts produced without the enzyme Ultraflo Max yielded lower extract content
compared to those produced with the application of enzyme Ultraflo Max at the same
shares and conditions of malt substitution. The increase of triticale share resulted in extract content decrease with and without enzyme addition. With the application of the enzyme Ultraflo Max, at 80% of triticale share, wort extract content was by 4.67% higher
compared to the wort extract content with the same triticale share but without the enzyme
addition. Regarding extract content triticale variety Odyssey could be used as the substitute for malt up to 60% without the addition of commercial enzyme Ultraflo Max. With
the addition of this enzyme triticale variety Odyssey could be used as a substitute for malt
up to 80%, regarding extract content.
on the wort viscosity is presented in Figure 2.
It is obvious that the increase of triticale share affects viscosity increase which is in
agreement with the results obtained by Glatthar et al. (3, 6). Higher viscosity values were
found in worts produced without the application of enzyme Ultraflo Max. With the application of the enzyme Ultraflo Max, at 80% of triticale share, wort viscosity was by
30.60% lower compared to the wort viscosity with the same triticale share but without the
enzyme addition. Regarding viscosity, triticale variety Odyssey could be used as the substitute for malt up to 50% without the addition of the enzyme Ultraflo Max while with the
13
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addition of this enzyme triticale variety Odyssey could be used as a substitute for malt up
to 80%.
Figure 2. Viscosity of the produced worts
on the wort soluble nitrogen content is presented in Figure 3.
Figure 3. Soluble nitrogen content of the produced worts
The increase of triticale share resulted in a decrease of the wort soluble nitrogen content with and without enzyme addition, especially when triticale share was above 60%. It
can be assumed that enzyme inactivation took place during adjunct boilng in the thermal
pretreatment. Glatthar et al. (3) obtained similar results when using adjunct boiling du14
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ring thermal pretreatment. The addition of the enzyme Ultraflo Max did not have any influence on wort soluble nitrogen content. Regarding soluble nitrogen content, triticale variety Odyssey could be used as the substitute for malt up to 30% with and without the
addition of the enzyme Ultraflo Max.
on the wort formol nitrogen content is presented in Figure 4.
Figure 4. Formol nitrogen content of the produced worts
The worts produced with the enzyme Ultraflo Max yielded slightly lower formol
nitrogen content compared to those produced without the application of enzyme at the
same shares and conditions of malt substitution. The increase of triticale share resulted in
a decrease of the wort formol titration nitrogen content with and without enzyme addition. Regarding formol nitrogen content, triticale variety Odyssey could be used as a substitute for malt up to 80% with and without the addition of the enzyme Ultraflo Max.
CONCLUSIONS
Regarding extract content, triticale variety Odyssey could be used as a substitute for
malt up to 60% without the addition on commercial enzyme Ultraflo Max. With the addition of this enzyme, triticale variety Odyssey could be used as a substitute for malt up to
80%, regarding extract content. With the increase in the content of triticale in the grist,
viscosity increased. The addition of commercial enzyme Ultraflo Max significantly reduced the wort viscosity. The obtained results indicate that worts produced with the addition
of triticale variety Odyssey to the grist yielded good analytical quality parameters. Based
on the obtained results it is suggested that triticale may serve as a convenient brewing
adjunct.
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Acknowledgement
This work is a part of the Project No. 20139, which is financially supported by the
Ministry of Science and Technological Development of the Republic of Serbia.
REFERENCES
1. W. Kunze: Technologie Brauer und Mälzer, VLB Berlin (1998) p 20.
2. G. Walker: Yeast Technology in Yeast Physiology and Biotechnology, John Wiley
and Sons, Chichester (1998) pp. 283-284.
3. J. Glatthar, J. Heinisch and T. Senn: A study on the suitability of unmalted triticale as
a brewing adjunct. J. Am. Soc. Brew. Chem. 60 4 (2002) 181-187.
4. J. Glatthar, J. Heinisch and T. Senn: Unmalted triticale cultivars as brewing adjuncts:
effects of enzyme activities and composition on beer wort quality. J. Sci. Food Agric.
85 4 (2005) 647-654.
5. R. Agu: A comparison of maize, sorghum and barley as brewing adjuncts, J. Inst.
Brew. 108 (2002) 19-22.
6. J. Glatthar, J. Heinisch and T. Senn: The use of unmalted triticale in brewing and its
effect on wort and beer quality. J. Am. Soc. Brew. Chem. 61 4 (2003) 182-190.
7. M. Mergoun, H. Pfeffer, J. Peña, K. Ammar, S. Rajaram: Triticale crop improvement:
the CIMMYT programme in: Triticale improvement and production (FAO plant production and protection. 2004; Paper 179 (Edited by Morgoum M and Gómez-Macpherson H.), 11-26 Rome: Food and Agriculture Organization of the United Nations.
8. FAO. FAOSTAT, FAO statistical databases - agriculture [WWW document] URL
http://faostat.fao.org/site/567/default.aspx # ancor. Viewed on November 30, 2008.
9. I. Ciftci, E. Yenice and H. Eleroglu: Use of triticale alone and in combination with
wheat or maize: effects of diet type and enzyme supplementation on hen performance, egg quality, organ weights, intestinal viscosity, and digestive system characteristics. Anim. Feed Sci. Technol. 105 1-4 (2003) 149-161.
10. D. Goode and E. Arendt: Development in the supply of adjunct materials for brewing,
in: Brewing – New technologies. Ed. C. W. Bamforth, Woodhead Publishing Limited
and CRC Press LLC, Cambridge (2006) p 43.
11. G. Annemüller, B. Mietla, G. Creydt, F. Rath, R. Schildbach and T. Tuszynski: Triticale and triticale malts, part III: First brewing trails with triticale malts, Monatsschrift
für Brauwissenschaft. 52 (1999) 131-135.
12. European Brewery Convention, Analytica – EBC, EBC Analysis Committee, Verlag
Hans Carl, Getärnke-Fachverlag, Nürnberg (1998) Sections 3 and 4.
13. MEBAK-Methodensammlung der Mitteleuropäischen Brautechnischen Analysenkommission Band. 1, 3. Auflage, Neubearbeitet und ergänzt, Selbstverlag der
MEBAK, Freising-Weihenstephan (1997) pp 133-289.
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ПРИМЕНА ТРИТИКАЛЕА СОРТЕ ОДИСЕЈ КАО ЗАМЕНЕ ЗА СЛАД У
ПРОИЗВОДЊИ СЛАДОВИНЕ
Олгица С. Грујић, Јелена Д. Пејин, Србислав С. Денчић
Циљ овог рада је био да се утврди могућност примене тритикалеа као делимичне замене за слад у произовдњи сладовине. За производњу сладовине изведене су
две серије експеримената у којима је сорта тритикалеа Одисеј, из селекционих огледа са Римских Шанчева (Србија), коришћена уместо јечменог слада у усипку са и
без додатка комерицијалног ензима Ultraflo Max (Novozymes, Данска). Тритикале је
додаван уместо слада у усипку у следећим односима: 0, 10, 20, 30, 40, 50, 60, 70 и
80%. Експерименти су изведени у лабораторијским условима применом инфузионог поступка за производњу сладовине. У погледу екстракта, сорта тритикалеа
Одисеј може заменити 60% јечменог слада без додатка комерцијалног ензима Ultraflo Max, док се са додатком ензима Ultraflo Max тритикале сорте Одисеј може користити уместо слада и до 80%. С порастом удела тритикалеа у усипку вискозност
сладовина се повећавала. Додатак ензима Ultraflo Max је значајно смањио вискозност сладовинa. Добијени резултати показују да су сладовине произведене уз примену тритикалеа сорте Одисеј уместо слада имале добре аналитичке показатеље.
Received 15 September 2010
Accepted 6 October 2010
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PHYSICOCHEMICAL, BIOCHEMICAL AND SENSORY PROPERTIES
FOR THE CHARACTERISATION OF PETROVSKÁ KLOBÁSA
(TRADITIONAL FERMENTED SAUSAGE)
Predrag M. Ikonić, Ljiljana S. Petrović, Tatjana A. Tasić, Natalija R. Džinić,
Marija R. Jokanović and Vladimir M. Tomović
A study was carried out on a typical homemade Petrovská klobasá in order to characterize this traditional dry-fermented sausage, to provide a basis for establishing the
quality standard and protecting designation of origin. This paper reviews the chemical
composition, some physicochemical, proteolytic and sensory parameters of Petrovská
klobasá made by five manufacturers chosen as representatives. Beside the differences
between sausages made by different manufacturers the main properties of this traditional
product were though recognized. Compared to other dry-fermented sausages Petrovská
klobasá is characterized by a high content of protein (23.36-30.45%) and low contents of
NaCl (2.99-3.28%). With some minor exceptions, the values of other chemical parameters are within the range of those observed for various dry-cured sausages. Weight loss
during the processing is high (up to 45.71%) and pH value (~ 5.4) corresponds to the values for this parameter in other European traditional fermented sausages. Contents of
different nitrogen fractions show that Petrovská klobasá undergoes significant proteolytic changes. At the end of ripening, Petrovská klobasá is characterized by aromatic and
spicy-hot flavor, dark-red color and hard consistency.
KEYWORDS: Petrovská klobasá, dry-fermented sausage, composition, proteolysis,
physicochemical properties
INTRODUCTION
Traditional food is an important element of the European cultural heritage. Production
and sale of traditional food products, different from conventional and mass products, provide a decisive economic input to many regions and can contribute to the diversification
and prevention from depopulation of rural areas (1, 2).
Foods that are typical of any region or area have their own peculiar characteristics
that arise from the use of local ingredients and production techniques, which are deeply
Predrag M. Ikonić, B.Sc. [email protected], Institute for Food Technology, Bulevar cara Lazara 1,
21000 Novi Sad, Serbia; Dr. Ljiljana S. Petrović, Prof., Faculty of Technology, Bulevar cara Lazara 1, 21000
Novi Sad, Tatjana A. Tasić, B.Sc., Institute for Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Dr.
Natalija R. Džinić, Assist. Prof., Marija R. Jokanović, M.Sc., Assist, Dr. Vladimir M. Tomović, Assist. Prof.,
Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
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rooted in tradition and linked to the territory. Thus, there is a wide variety of dry-fermented meat products on the European market as a consequence of variations in the raw
materials, formulations and manufacturing processes, which come from the habits and
customs of the different countries and regions (3, 4).
Petrovská klobasá, traditional dry-cured sausage, is made in an area nearby town of
Bački Petrovac in the Autonomous Province of Vojvodina (Northern Serbia). It is a part
of Slovaks’ heritage, who inhabited Vojvodina in the second half of 18th century. Nowadays, they are producing it in traditional way according to the original recipe of their ancestors, without the use of nitrate/nitrite, glucono delta-lactone (GDL) and microbial starters. In village households, this sausage is made in the end of November and during December. This is when the temperatures are around 0oC or lower. Petrovská klobása is prepared by mixing ground pork meat and lard with addition of red hot paprika powder, salt,
crushed garlic, caraway and shugar. Well mixed compound is stuffed into natural casing,
back part of pig large intestines (colon), in the units of 35-45cm length. After stuffing it
undergoes smoking process for about 10 days with pauses, using specific kinds of wood.
Afterwards it is left to dry and ripen for a period of up to 4 months, until it achieves optimum quality. At the end of ripening Petrovská klobasá is characterized by specific savoury taste, aromatic and spicy-hot flavor, dark red color and hard consistency.
Aiming to establish quality standard and protect designation of the origin, traditional
process of manufacturing and gross characteristics of Petrovská klobasá have been investigated in a number of rural households. Some biochemical, physicochemical and sensorial characteristics of sausages, produced by five representative manufacturers, are presented in this paper.
EXPERIMENTAL
Samples
Sausages were produced during December and the processes of drying and ripening
lasted 120 days, when the manufacturers, according to their experience, estimated that the
production process was finished. Samples (n=3) from each batch (A, B, C, D, E) were taken on the 0, 50, 80 and 120th day after preparation, transported to the laboratory under
refrigeration (4oC) and analyzed on the same day.
Аnalysis
Moisture, fat, protein (Kjeldahl N x 6.25), NaCl and ash contents were quantified
according to the ISO recommended standards (5-9). Non-protein nitrogen (NPN) content
was determined in filtrate using Kjeldahl method (7) after quantitative precipitation of
proteins with 10% TCA. α-aminoacidic nitrogen (NH2-N) was determined according to
Sörensen’s formol titration (1907) modified by Petrov (1965) (10). Analyses for all samples were carried out in duplicate.
To determine weight loss, five sausages from each batch were weighed just after stuffing. The same sausages were reweighed on the 5, 15, 30, 60, 90 and 120th day. The differences in weight are expressed as the percentage of the initial weight.
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The pH values of sausages were measured using the portable pH meter (Consort
C931, Turnhout, Belgium) equipped with an insertion glass combination electrode (Mettler Toledo Greifensee, Switzerland). Means of three measurements are presented.
Color measurements of Petrovská klobása were carried out using photo-colorimeter
MINOLTA CHROMA METER CR-400 (Minolta Co., Ltd., Osaka, Japan). Color characteristics are given in the CIE L*a*b* system. Two measurements were taken on two fresh
cut surfaces of sausages (n=3) from each batch. Data presented are means of 12 measurements.
Instrumental texture measurements were performed using a Universal Testing Machine (Instron International LTD, High Wycombe, UK), model 4301. Firmness was determined as force needed for penetration (Fp) through 1cm thick slice, and expressed as
Newton (N). Measurements were performed using extension for penetration (needle with
5 arms) at a crosshead speed of 100 mm/min (11). For each sausage, 12 determinations
were carried out.
Sensory evaluation of Petrovská klobása was performed by panel consisting of 5 trained differently aged members. Evaluations were carried out according point system of
analytical descriptive test (12) using a scale from 0 to 5; 0 – visible mechanical or microbiological contamination, untypical product and similar; 5 – extraordinary, typical, optimal quality level. Following attributes were evaluated: external appearance of sausage,
appearance and composition of cut surface, color and color maintenance on the cut surface, odor and taste, texture and juiciness. For each evaluated sausage, mean scores of five
tasters are presented.
Differences between the sausages produced by different manufacturers were analysed
through an analysis of variance ANOVA (Statistica 9.1 - StatSoft, Inc. 2010). The Duncan’s post hoc test was used for comparison of mean values. Differences were considered
significant at p < 0.05.
The mean values of the chemical components of sausages during processing are presented in Table 1. The protein contents found in most of Petrovská klobása samples after
120 days of drying and ripening (~30%) were similar to those observed for Italian traditional dry sausage (13) or even higher compared to the reported values for the majority
of different fermented sausages (1419).
The fat contents in final products varied from 34.09% (E) to 46.01% (C). Although
significant differences (p < 0.05) in fat content (up to 12%) were observed, the highest fat
content was a little bit lower or similar to the registered values for Sremska sausage or
some traditional Spanish sausages (15, 17, 19).
The average moisture values were slightly higher than those stated for Sremska sausage (15), similar to the values found in some Italian sausages (13, 20), but lower if compared with traditional Spanish sausages Androlla, Botillo and Chorizo de cebolla (17, 19)
and Milano Salami (14).
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Table 1. Changes in chemical composition during the ripening of Petrovská klobása)
Time
(day)
0
50
80
120
a-e
Chemical
component
(%)
Protein
Fat
Moisture
Ash
NaCl
Protein
Fat
Moisture
Ash
NaCl
Protein
Fat
Moisture
Ash
NaCl
Protein
Fat
Moisture
Ash
NaCl
Sample
A
B
c
18.98±0.19
19.38±0.20b
57.97±0.18b
3.06±0.12b
1.84±0.10a
24.42±0.40a
33.44±0.73a
34.75±0.87b
3.96±0.10d
2.54±0.21a
26.87±0.68a
39.53±0.89a
26.75±0.50b
4.32±0.14c
2.83±0.14b
29.79±0.30ab
41.39±1.19a
22.14±1.00a
4.87±0.13b
3.01±0.10a
C
a
17.00±0.22
20.23±0.55c
54.74±0.59d
2.64±0.17a
1.75±0.08a
24.73±0.31a
33.09±0.67a
36.61±0.44c
3.72±0.17c
2.50±0.22a
26.97±1.14a
40.33±0.64a
28.39±0.49a
3.84±0.18b
2.67±0.18ab
29.62±0.57a
41.48±0.79a
23.19±0.47b
3.87±0.21a
3.28±0.14a
D
b
14.00±0.13
28.93±0.68e
51.10±0.71c
2.42±0.23a
2.02±0.07b
19.49±0.43b
36.36±0.52d
37.62±0.52d
3.27±0.16a
2.37±0.18ac
20.03±0.38b
45.47±0.82d
29.17±0.63a
3.39±0.23a
2.57±0.27ab
23.36±0.42c
46.01±0.56d
25.05±0.72c
3.99±0.13a
3.01±0.29a
E
a
17.24±0.20
22.36±0.19d
56.84±0.59a
2.53±0.32a
1.84±0.08a
21.91±0.32c
29.55±0.29c
41.75±0.41a
3.06±0.06b
2.05±0.03b
24.46±0.91c
36.96±0.63c
32.43±0.59d
3.62±0.34ab
2.35±0.26a
27.52±0.58d
37.71±0.52c
27.53±0.49d
5.26±0.13c
3.15±0.14a
19.51±0.34d
18.73±0.27a
57.34±0.20ab
2.74±0.10a
1.87±0.05a
24.81±0.36a
26.18±0.40b
40.99±0.37a
3.31±0.13a
2.13±0.09bc
29.14±1.01d
31.42±0.98b
31.51±0.48c
4.61±0.07c
2.35±0.20a
30.43±0.62b
34.09±1.15b
29.11±0.47e
4.74±0.15b
2.99±0.20a
Means within the same row with different superscript letters are different (p < 0.05)
The values of ash content at the end of ripening of Petrovská klobása (3.87 - 5.26%)
were similar to or a little bit lower than in some examinated fermented sausages (1520).
Production of Petrovská klobása is characterized by low addition of NaCl. In the final
products the NaCl content varied from 3.01% in A and C sausages, up to 3.28% in B sausage (p > 0.05), being lower than in the majority of fermented sausages (1315, 21). Similar NaCl contents were found in Botillo and in Italian sausage from the region of Friuli
Venezia Giulia (17, 18), but lower in Androlla, Chorizo de cebolla (17, 19) and Croatian
fermented sausage (21).
Weight losses of Petrovská klobása produced by five different manufacturers, during
smoking, drying and ripening processes, are shown in Fig. 1. It is evident that the values
for weight loss of sausages from the all five production batches after 120 days of manufacturing were more than 40%. The values for weight losses during the production process were similar to those reported for traditional Greek sausage with the same level of
fat content in basic formulation (22), or higher than those which were found for Italian
sausage of Felino type (20).
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50
45
Weight loss (%)
40
35
30
25
20
15
10
5
0
0
20
A
40
60
B
Time (day)
C
80
100
D
120
140
E
Figure 1. Changes in weight loss during the ripening of Petrovská klobása)
The pH value changes of sausages during processing are shown in Fig. 2. The initial
pH values ranged from 5.26 (A) to 5.56 (E). As can be seen from the figure, during the
first 15 days, the pH values in sausages B and E decreased. After that period the pH in
those sausages started to increase gradually, reaching after 120 days the values 5.36 and
5.42, respectively. Such changes of pH are in line with the findings of other authors that
have investigated physicochemical properties of fermented dry sausages (14, 21, 23, 24).
In sausages A, C and D, uncharacteristic variations of pH values were registered during
drying and ripening period, with a noted increase in the beginning of the process. Having
in mind that deeper microbiological examinations were not the subject of this investigation, based on experiences and theoretical knowledge it is possible to conclude that this
phenomenon could be a result of lower microbiological quality of raw materials (meat,
paprika, natural casings) and/or poor hygienic conditions during manufacturing (ambient
conditions). Moreover, this phenomenon could be an indicator of unsatisfactorily performed stuffing procedure, which resulted in the incorporation of higher quantities of air
into sausages. This probably enabled the development of dominant microflora of meat,
i.e. aerobic, Gram-negative, psychotropic, proteolytic bacteria (25), during the very first
couple of days. Degradation of proteins generates amino acids which can be decarboxylated, deaminated, or even further metabolized. Therefore, the generated ammonia and
amines cause an increase in pH (26). Thus, pH increased in A, C and D sausages. After
that initial period, in which aerobic bacteria consumed oxygen, an increase of lactic acid
bacteria number had probably took place, with beginning of fermentation, and pH value
started to decrease, preventing thus further growth of undesirable microorganisms. The
investigations of hygienic integrity of sausages manufactured by all five producers after
50, 80 and 120 days, showed that they fulfilled the demands of Serbian Regulations (27).
The final pH value of about 5.4 corresponds to the high sensory quality of Petrovská klobása. Hence, this pH value could be applied as a threshold value for the estimation of the
completion of the ripening process.
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5.9
5.8
5.7
5.6
pH
5.5
5.4
5.3
5.2
5.1
5.0
4.9
0
20
40
60
80
100
120
140
Time (day)
A
B
C
D
E
Figure 2. Changes in pH during the ripening of Petrovská klobása
As the results in Table 2 show, the highest NPN content was found in the raw sausage
mixture D (0.28 g/100g) and the lowest in the raw sausage mixture C (0.23 g/100g). At
the same time, it should be noted (Fig. 3) that the share of NPN in total nitrogen (NPN %
TN) of raw sausage mixture C was the highest (10.27%). After 120 days of ripening the
highest NPN content was found for E sausages, amounting to 0.74 g/100g. The lowest (p
< 0.05) absolute NPN content and the highest NPN % TN (Fig. 3) again were found for C
sausages, being 0.62 g/100g and 16.59%, respectively. Somewhat lower values for NPN
% TN were found in D (16.35%) and E (15.20 %) sausages. This indicates that during the
production processes of C, D and E sausages intensive proteolytic processes occurred.
Table 2. Changes in nitrogen fractions content during the ripening of Petrovská klobása
Time
(day)
Nitrogen
fraction
(g/100g)
Sample
A
B
c
C
a
D
b
E
a
TN
3.04±0.03
2.72±0.03
2.24±0.02
2.76±0.03
0
NPN
0.25±0.01a
0.27±0.01c
0.23±0.01b
0.28±0.00d
NH2-N
0.075±0.005a
0.085±0.007b
0.079±0.003ab
0.080±0.006ab
TN
3.91±0.06a
3.96±0.05a
3.12±0.07b
3.50±0.05c
a
d
b
50
NPN
0.51±0.01
0.59±0.01
0.48±0.01
0.50±0.01a
b
c
a
NH2-N
0.141±0.011
0.276±0.014
0.209±0.015
0.179±0.023a
a
a
b
TN
4.30±0.11
4.32±0.18
3.20±0.06
3.91±0.14c
b
e
a
80
NPN
0.53±0.01
0.60±0.01
0.49±0.01
0.54±0.01c
a
b
a
NH2-N
0.215±0.020
0.286±0.016
0.224±0.027
0.252±0.025ab
TN
4.77±0.05ab
4.74±0.09a
3.74±0.07c
4.40±0.09d
b
c
a
120
NPN
0.64±0.01
0.67±0.01
0.62±0.01
0.72±0.01d
b
a
b
NH2-N
0.239±0.045
0.353±0.047
0.278±0.049
0.372±0.020a
a-e
24
3.12±0.05d
0.26±0.01a
0.082±0.007ab
3.97±0.06a
0.53±0.01c
0.198±0.032a
4.66±0.16d
0.58±0.01d
0.243±0.036a
4.87±0.10b
0.74±0.01e
0.362±0.008a
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Results shown in Table 2 and Fig. 3 agree with findings of various other authors, and
they indicate that proteolysis process during the fermentation and ripening effects with
the increasing of the non-protein nitrogen contents in sausages (17, 19, 2831). García de
Fernando and Fox (28) recorded the increase of NPN % TN from the initial value of 8 to
the final value of 15, after 41 day of pork sausage production. Similar increase of NPN
share in the total N was registered by Beriain et al. (29) in the Spanish traditional sausage
Salchichon, from initial 9.40 to the final 15.95% after 30 days of manufacturing. Average
NPN content in Spanish traditional sausage Androlla after 60 days of drying and ripening
was 0.57 g/100g (18), which corresponds to the results registered for Petrovská klobása
in similar period of manufacturing.
18
16
NPN (%) TN
14
12
10
8
6
4
2
0
0
A
50
B
Time (day)
C
80
120
D
E
Figure 3. Changes in NPN shares in total nitrogen content during the ripening of
Petrovská klobása
The highest mean of α-aminoacidic nitrogen content (NH2-N) was determined in the
raw sausage mixture B (0.085 g/100g) and the lowest one in the mixture A (0.075
g/100g). After 120 days of ripening, the highest NH2-N content was registered in the D
sausages (0.372 g/100g) and the lowest content of this nitrogen fraction was again found
in the A sausages (0.239 g/100g). Mean values of α-aminoacidic nitrogen contents during
the traditional manufacturing process of Petrovská klobása, shown in Table 2, are higher
than those published for Spanish traditional sausages Androlla (0.096 g/100g), Botillo
(0.095 g/100g) and Chorizo de cebolla (0.136 g/100g (17, 19). However it must be remembered that ripening of the Petrovská klobása lasted longer.
After the previously shown results of NPN content and NPN % TN were viewed together with the changes of pH (Fig. 2) in the course of the manufacturing period. It was
concluded that during the first 10 days more intensive proteolysis occurred in C and D
sausages, probably caused by undesirable microorganisms. The consequence was an increase of pH value and non-protein nitrogen content. At the end of the manufacturing
process, the highest content of non-protein nitrogen was found in E sausages, which
correlates with the highest pH value of this product. Namely, in that sausage the pro25
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teolysis took place somewhat later, during ripening, after the fermentation and under the
influence of technologically useful microflora, which caused positive effects on sensory
properties of that product (Fig. 4).
As can be seen, the mean scores of the observed sensorial parameters have been
higher in the course of time, reaching their maximum after 120 days of processing, when
it was estimated that the sausages reached a very high sensory quality (Fig. 4). After 50
days, mean scores of individual sensory properties were, mainly, between 3.0 and 3.5,
with a marked minimum for texture and juiciness (about 2.7), indicating that the sausages
were not homogenized, i.e. gelling of the dissolved proteins and fastening of the sausage
structure were not finished (32). This sensory evaluation of Petrovská klobása after 50
days of processing is fully understandable as it is a slow fermenting product characterized
with a low NaCl content and absence of additives or supplements that would enable
shorter periods of drying and ripening. At the 120th day of drying and ripening, it was
estimated, that the sausages reached high sensory quality, being costly between 4.0 and
5.0, indicating optimal, typical quality with, occasionally, smaller deviations and deficiencies.
Figure 4. Changes in sensory properties of Petrovská klobása during the ripening
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Color characteristics of Petrovská klobása samples, expressed in CIE L*a*b* system,
are shown in Table 3. After 120 days of drying and ripening, the highest L* value was
determined on the cut surface of C sausages (36.38) and the lowest on cut surface of A
sausages (31.78). Such significantly different (p < 0.05) L* values can be explained by
different (p < 0.05) fat contents in these sausages, which is in concordance with the
results reported for traditional Greek sausage, which indicate that products with higher fat
tissue contents tend to have higher L* values. On the other hand, with the increased
weight loss during drying and ripening, the lightness decreases and sausage is becoming
darker (22).
Table 3. Changes in color during the ripening of Petrovská klobása
Time (day)
50
80
120
a-c
Sample
Color
parameter
A
B
C
D
E
L*
41.27±5.09ab
37.95±0.78a
42.87±1.05b
40.67±4.09ab
39.92±1.90ab
a*
25.39±2.47a
32.17±3.05bc
34.91±1.42c
26.08±3.32a
30.24±1.76b
b*
29.53±3.97a
35.59±2.72b
38.14±2.69b
27.28±5.90a
29.88±3.69a
L*
31.56±2.06
b
a
a
a
38.41±1.89a
a*
21.13±2.01c
33.96±1.58b
32.09±1.65b
24.82±2.67a
27.66±2.27a
b*
19.87±2.79
a
b
b
a
21.14±2.90a
L*
31.78±3.51a
34.09±1.64ab
36.38±2.55b
35.18±2.03ab
34.37±1.76ab
a*
21.59±4.50
a
b
b
a
25.08±2.39a
b*
19.40±5.50a
19.47±2.30a
20.23±2.21a
37.72±1.86
32.11±1.99
32.04±1.39
38.62±2.36
29.80±3.56
29.71±1.98
27.91±2.17b
25.07±3.64b
40.99±3.05
21.54±4.35
24.46±1.38
During the whole processing period of Petrovská klobása, very high shares of red
(a*) and yellow (b*) color on cut surfaces were registered for products from all five manufacturers. Most probably, this was a consequence of the addition of a high amount of
quality red hot paprika powder (capsantine content was not below 3.5 g/kg of dry matter
or 120 ASTA) to the basic formulation of sausage mixture.
Table 4. Changes in firmness - penetration force (N) during the ripening of Petrovská
klobása
Time (day)
50
a-c
Sample
A
4.28±0.75
B
a
a
80
10.70±0.86
120
14.48±1.04a
C
3.46±0.75
a
7.92±0.76
c
10.38±2.01c
D
1.90±0.10
c
b
E
b
5.43±0.57b
a
10.15±1.05a
5.30±0.59
4.38±0.37
9.95±0.90
6.18±0.48b
13.04±1.16a
13.53±0.82a
27
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Results for instrumental measurements of texture are shown in Table 4. After 120
days of drying and ripening, the highest penetration forces were 14.48 N and 13.53 N for
A and E sausages, respectively. Significantly lower firmness (p < 0.05) was determined
for C sausages (6.18 N). Hence, taking into account previous results (moisture and fat
content, weight loss, pH) great differences for firmness between sausages are fully logical
and expected. In spite of a high weight loss, C sausages were characterized with a high
fat content, which resulted in soft consistency and low penetration force. Sausages A lost
the highest quantity of water during the processing and became too firm and dry, which
reflected as high penetration force and the lowest score for this sensory property (Fig. 4).
Consistency of E sausages was sensory evaluated with very high scores, which can be
explained by its optimal chemical composition and weight loss, i.e. moisture content in
the final product (29.11%). In addition, the final pH, NPN and NH2-N contents in these
sausages indicate the correct fermentation and ripening process, intensive proteolysis and
homogenization of the mixture.
CONCLUSION
Production of Petrovská klobása in traditional conditions leads to a significant heterogeneity of final products. The quality standard for this type of sausage does not exist, and
beside the differences between sausages the main findings of this study will be a good
basis for its establishing. Compared to other dry-fermented sausages, Petrovská klobása
is characterized by high protein content, usual moisture, fat and mineral substances contents and lower NaCl content. It is also characterized by a high weight loss, and pH which
corresponds to the published values for this parameter for other traditional fermented
sausages. During ripening, Petrovská klobása undergoes significant proteolytic changes
that have positive effects on sensory properties, primarily due to the formation of characteristic taste and odor. At the end of ripening Petrovská klobasá is characterized by
aromatic and spicy-hot flavor, dark-red color and hard consistency.
Acknowledgement
The authors wish to express their sincere gratitude to the Ministry of Science and
Technological Development of the Republic of Serbia for its financial support (Project
Number: 20037).
REFERENCES
1. E. Dimara, A. Petrou and D. Skuras: Agricultural policy for quality and producers'
evaluations of quality marketing indicators: a Greek case study. Food Policy. 29
(2004) 485–506.
2. L. Guerrero, A. Claret, W. Verbeke, G. Enderli, S. Zakowska-Biemans, F. Vanhonacker, S. Issanchou, M. Sajdakowska, B. Signe Granli, L. Scalvedi, M. Contel and
M. Hersleth: Perception of traditional food products in six European regions using
free word association. Food Qual. Pref. 21 (2010) 225–233.
28
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041019I
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BIBLID: 1450-7188 (2010) 41, 19-31
3. L. Aquilanti, S. Santarelli, G. Silvestri, A. Osimani, A. Petruzzelli and F. Clementi:
The microbial ecology of a typical Italian salami during its natural fermentation. Int.
J. Food Microbiol. 120 (2007) 136–145.
4. R. Talon, S. Leroy and I. Lebert: Microbial ecosystems of traditional fermented meat
products: The importance of indigenous starters. Meat Sci. 77 (2007) 55–62.
5. ISO 1442:1997. International standards meat and meat products  Determination of
moisture content. Geneva: International Organization for Standardization, 1997.
total fat content. Geneva: International Organization for Standardization, 1973.
nitrogen content. Geneva: International Organization for Standardization, 1978.
8. ISO 1841-1:1996. International standards meat and meat products  Determination
of chloride content-Part 1: Volhard metod. Geneva: International Organization for
Standardization, 1996.
ash content. Geneva: International Organization for Standardization, 1998.
10. M. Džamić: Praktikum iz biohemije, Naučna knjiga, Beograd (1989) pp. 194491
11. J. Popov-Raljić: Tehnologija i kvalitet gotove hrane, Tehnološki fakultet, Novi Sad
(1999) p. 372.
12. R. Radovanović and J. Popov-Raljic: Senzorna analiza prehrambenih proizvoda,
Poljoprivredni fakultet, Beograd, Tehnološki fakultet, Novi Sad (2001) p. 331.
13. V. M. Moretti, G. Madonia, C. Diaferia, T. Mentasti, M. A. Paleari, S. Panseri, G.
Pirone and G. Gandini: Chemical and microbiological parameters and sensory attributes of a typical Sicilian salami ripened in different conditions. Meat Sci. 66, 4 (2004)
845–854.
14. E. Casiraghi, C. Pompei, S. Dellaglio, G. Parolari and R. Virgili: Quality attributes of
Milano salami, an Italian dry-cured sausage. J. Agric. Food Chem. 44 (1996) 1248–
1252.
15. S.Tojagić: Potreba standardizacije sremske kobasice kao nacionalnog proizvoda.
Tehnologija Mesa. 6 (1997) 265-267.
16. T. Stamenković, B. Dević, A. Đurković, A. Hromiš, D. Janković and Z. Orlić:
Sirovodimljene i sirovosušene kobasice (senzorna svojstva, hemijski pokazatelji i
bakteriološka ispravnost). Tehnologija Mesa. 2 (1998) 70-75.
17. J. M. Lorenzo, M. Michinel, M. López and J. Carballo: Biochemical characteristics of
two Spanish traditional dry-cured sausage varieties: Androlla and Botillo. J. Food
Compos. Anal. 13 (2000) 809-817.
18. G. Comi, R. Urso, L. Iacumin, K. Rantsiou, P. Cattaneo, C. Cantoni and L. Cocolin:
Characterisation of naturally fermented sausages produced in the North East of Italy.
Meat Sci. 69 (2005) 381–392.
19. A. Salgado, M.C. García Fontán, I. Franco, M. López and J. Carballo: Effect of the
type of manufacture (homemade or industrial) on the biochemical characteristics of
Chorizo de cebolla (a Spanish traditional sausage). Food Control. 17 (2006) 213-221.
20. E. Bianchi, S. Bergoni and A. Cantoni: Ripening of "Felino" type Italian salami in
small and big industries in Emillia. Meat Industry Review. 1 (1974) 7-12.
29
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041019I
UDC: 637.524(497.11):637.04/.05
BIBLID: 1450-7188 (2010) 41, 19-31
21. J. Gasparik-Reichardt, Sz. Tóth, L. Cocolin, G. Comi, E. Drosinos, Z. Cvrtila, L.
Kozačinski, A. Smajlović, S. Saičić and B. Borović: Technological, physicochemical
and microbiological characteristics of traditionally fermented sausages in
Mediterranean and central European countries. Meat Technology. 46 (2005) 143-153.
22. S. N. Papadima and J. G. Bloukas: Effect of fat level and storage conditions on
quality characteristics of traditional Greek sausages. Meat Sci. 51 (1999) 103-113.
23. S. Savić, O. Bunčić, B. Uzelac and J. Tripković: Mikroflora "Sremske salame"
proizvedene sa i bez starter-kulture. Tehnologija Mesa. 42 (2001) 71-74.
24. M. Spaziani, M. Del Torre and M. L.Stecchini: Changes of physicochemical,
microbiological, and textural properties during ripening of Italian low-acid sausages.
Proteolysis, sensory and volatile profiles. Meat Sci. 81 (2009) 77-85.
25. Z. Bem and J. Adamič: Mikrobiologija mesa i proizvoda od mesa. 1st ed. Tehnološki
fakultet, Novi Sad (1991) p. 152.
26. G. Kaban and M. Kaya: Effects of Lactobacillus plantarum and Staphylococcus
xylosus on the quality characteristics of dry fermented sausage “Sucuk”. J. Food Sci.
74 (2009) 58–63.
27. Serbian Regulations 26/93, 53/95, 46/02. Microbiological quality of food products on
market. Beograd: Službeni list SRJ (2002) 565-570.
28. D. G. García de Fernando and P. F. Fox: Study of proteolysis during the processing of
a dry fermented pork sausage. Meat Sci. 30 (1991) 367-383.
29. M. J. Beriain, G. Lizaso and J. Chasco: Free amino acids and proteolysis involved in
’salchichon’ processing. Food Control. 11 (2000) 41-47.
30. M. C. Hughes, J. P. Kerry, E. K. Arendt, P. M. Kenneally, P. L. H. McSweeney and
E. E. O'Neill: Characterization of proteolysis during the ripening of semi-dry
fermented sausages. Meat Sci. 62 (2002) 205-216.
31. Ü. Dalmış and A. Soyer: Effect of processing methods and starter culture
(Staphylococcus xylosus and Pediococcus pentosaceus) on proteolytic changes in
Turkish sausages (sucuk) during ripening and storage. Meat Sci. 80 (2008) 345-354.
32. I. Vuković: Osnove tehnologije mesa. 1st ed.: Infotek, Beograd (1992) p. 256.
ФИЗИЧКО-ХЕМИЈСКЕ, БИОХЕМИЈСКЕ И СЕНЗОРНЕ
КАРАКТЕРИСТИКЕ ТРАДИЦИОНАЛНЕ СУВЕ ФЕРМЕНТИСАНЕ
ПЕТРОВАЧКЕ КОБАСИЦЕ
Предраг М. Иконић, Љиљана С. Петровић, Tatјана A. Taсић, Наталија Р. Џинић,
Марија Р. Јокановић, Владимир М. Томовић
У овом раду су испитана својства традиционалне суве ферментисане Петровачке кобасице како би се дефинисали њени параметри квалитета и заштитила ознака географског порекла. Истраживања спроведена на узорцима кобасица произведених на традиционалан начин у пет репрезентативних домаћинстава, обухватала су испитивање хемијског састава, физичко-хемијских, биохемијских и сензорних параметара квалитета. Иако су уочене одређене разлике у квалитету испитаних
кобасица основне карактеристике овог традиционалног производа су ипак препоз30
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нате. У поређењу са другим сувим ферментисаним кобасицама Петровачка кобасица се одликује високим садржајем протеина (23,36–30,45%) и ниским садржајем
NaCl (2,99-3,28%). Уз мања одступања вредности осталих хемијских показатеља су
у складу са вредностима утврђеним за друге кобасице истог типа. Током производње уочен је велики губитак масе, до 45,71%, а вредност pH (~5,4) одговара вредностима овог параметра утврђенiм у другим традиционалним ферментисаним кобасицама. Повећање садржаја различитих фракција азота указујe на значајну протеолитичку активност током зрења Петровачке кобасице. Финални производ поседује препознатљиву арому, пикантан укус, тамноцрвену боју и чврсту конзистенцију.
Received 13 Octobar 2010
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OCCURRENCE OF POTENTIALLY TOXIGENIC MOULD SPECIES IN FRESH
SALADS OF DIFFERENT KINDS OF READY-FOR-USE VEGETABLES
Sunčica D. Kocić-Tanackov, Gordana R. Dimić, Jelena T. Lević, Dušanka J. Pejin,
Jelena D. Pejin and Igor M. Jajić
In the mycological survey of fresh salads of different vegetables, the collected samples were tested for total counts of moulds with special attention paid to the presence of
potentially toxigenic species. The survey also included the isolation and the identification
of species, as well as the evaluation of mycotoxin biosynthesis ability of potential producers of ochratoxin A (OA) and sterigmatocystin (STC). Mould counts ranged from 10.0 to
4.7102 cfu g-1. The most common moulds found in fresh salads were Cladosporium
(42.89%), Penicillium (25.78%), Aspergillus (14.67%) and Alternaria (6.89%). C. cladosporioides (40.44%), followed by A. niger (10.22%), P. aurantiogriseum (7.55%), A. alternata (6.89%) and Fusarium spp. (3.11%) were the most dominating species. Other
species were represented with 2.22% (Eurotium spp.), 1.56% (Botrytis spp.), 0.67%
(Phoma spp.), 0.44% (Geotrichum spp., Mucor spp., Phialophora spp.) and 0.22% (Emericella spp., Paecilomyces spp., Trichoderma spp., Xeromyces spp.). Twenty-two of 41
identified mould species were potentially toxigenic, which accounted for 46.18% of the
total isolated population. The most frequent were the potential producers of ochratoxin A
(17.77%). Potential producers of moniliformin were isolated in 3.11% of samples, while
producers of fumonisin and STC were found in 2.67% and 2.44% of samples, respectively. The tested isolates of OA producers did not demonstrate the ability to biosynthetise
this mycotoxins, but two out of five isolates of A. versicolor were found to biosynthesise
STC in doses of 109.2 ng mL-1 and 56.3 ng mL-1. The obtained results indicate that such
products may threaten human health, considering that isolated species were potentially
toxigenic, while isolates of A. versicolor also biosynthesised STS.
KEYWORDS: Toxigenic moulds, mycotoxins, fresh salads, vegetables.
INTRODUCTION
Moulds comprise a large group of microorganisms, which are frequent contaminants,
and causes of spoilage in many food commodities. They are not only responsible for the
Sunčica D. Kocić-Tanackov, MSc., Assist., [email protected], Dr Gordana R. Dimić, Assoc. Prof., Faculty of
Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Dr Jelena Lević, Sci. Advisor,
Maize Research Institute, Zemun Polje, Slobodana Bajića 1, 11185 Beograd, Dr Dušanka J. Pejin, Prof., Dr
Jelena D. Pejin, Assis. Prof., Faculty of Technology, University of Novi Sad, Dr Igor Jajić, Assis. Prof., Faculty
of Agriculture, University of Novi Sad, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia
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formation of offensive odours and off-flavours in food, but for the production of very
toxic secondary metabolites - mycotoxins and allergenic compounds. Moulds can excrete
mycotoxins into the substrate and then disappear due to unfavourable growth conditions,
but the excreted toxins remain in the substrate. By consuming such food, toxic metabolites are deposited in human and animal organisms where they can cause diseases called mycotoxicoses. Mycotoxicoses are manifested with carcinogenic, hepatotoxic, mutagenic, teratogenic, cytotoxic, immunosuppressive, estrogenic and anabolic effects (1, 2).
Large body of data reported worldwide and here confirms the occurrence of mycotoxins
in almost all types of food commodities of either animal or plant origin, as well as in
animal feed (2 -14).
By broadening our knowledge about mould species profiles, their properties, stimulatory or inhibitory factors that affect their growth, more effective methods for the control
and elimination of negative effects could be established. Therefore, the aim of this study
was to determine the profile of fungal population in different types of fresh salads.
EXPERIMENTAL
Materials
A mycological survey included samples of ready-to-use (RTU) salads made of differrent types of vegetables, which were diced and packed. Seventeen randomly chosen
samples were obtained from supermarkets in Novi Sad (Serbia).
Isolation and determination of total mould count
The isolation and mould counting was performed on Dichloran Rose Bengal Chloramphenicol agar (DRBC, Himedia, India). The presence of dichloran in the medium limited the growth of fast-growing moulds such as Mucor and Rhizopus, whereas chloramphenicol inhibited the bacterial growth. The dilution method by Koch was used to determine total mould counts. Serial dilutions were performed with 0.1% peptone. The inoculated medium was stored at 25C for 5-7 days. The total mould count represented a
mean of triplicate measurements.
Mould identification
In order to obtain pure cultures and perform the identification, after the determination
of total mould counts, colonies that were suspected to belong to genera Penicillium, Aspergillus, Eurotium and Emericella were re-inoculated on Chapek agar, whereas the
others were inoculated on Malt Extract Agar (MAE). The inoculated media were incubated at 25C for 7 days. Isolates belonging to the genus Fusarium were cultivated on Potato Dextrose Agar (PDA, Himedia, India) and Carnation Leaf Agar (CLA) to obtain monospore cultures (13-16). These cultures were incubated at 25C under an alternating 12h
UV light/12 h dark regime for 10-14 days, to stimulate the formation of conidiogenic
structures.
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The identification of Fusarium species was performed according to the methods and
keys described by Nelson et al. (15), Leslie and Summerell (16) and Lević (13). Criteria
proposed by Pitt and Hocking (17), Samson et al. (1), Samson and Frisvad (18) were used
to identify Penicillium species. Aspergillus, Eurotium and Emericella species were identified by the methods of Pitt and Hocking (17) and Klich (19).
Determination of OA and STC biosynthesis
Pure cultures of potentially toxigenic Penicillium, Aspergillus, Eurotium and Emericella species, isolated from the samples, were cultivated stationary in a liquid medium
with the yeast extract and saccharose at 25C for 21 days. Tests were performed in 300
mL Erlenmeyer flasks and the medium volume was 100 mL. The inocula were prepared
in the following manner: conidia were skimmed from the surface of cultures grown on
PDA, using approximately 5 mL of Yeast extract saccharose (YES) broth and the suspension was transferred to the Erlenmeyer flasks with YES broth. After 21-day inoculation,
cultures were filtered through Whatman filter No. 1. Then, the pH (potentiometric, pH
meter MA 5730 Iskra) of each filtrate was measured, as well as the mycelia weight (using
data on the content of dry solids). Total content of dry solids was calculated as the
difference in the mycelia weight before and after drying. Drying was conducted at 60C
6h and at 40C, overnight (20). The content of mycotoxin was determined in the obtained
filtrate.
From the potentially ochratoxigenic species, eight isolates of A. niger, 10 of P. aurantiogriseum, and three of P. chrysogenum were chosen to determine ochratoxin A. The
ability to synthetise STC was evaluated in two isolates of A. versicolor, two of E. herbariorum and one of E. nidulans.
Qualitative OA and STC determination
The qualitative determination of ochratoxin A (OA) was conducted according to the
multimycotoxin method described by Balzner et al. (21) and the STC determination was
performed by thin-layer chromatography (TLC) according to van Egmond (22). The
samples which were found positive to the presence of mycotoxins by the TLC method
were further analysed by HPLC, to quantitatively determine the content of toxins.
Quantitative STC determination
The STC content was determined by liquid chromatography coupled with tandem
mass spectrometry, using Agilent Technologies 1200 Series Rapid Resolution liquid
chromatograph with G6410A QqQ MS-MS detector with electrospray ionisation (ESI).
One µL of filtered samples was injected into the system, and components were separated
on Rapid Resolution HT Zorbax Eclipse XDB-C18 50 mm × 4.6 mm × 1.8 µm column
(Agilent Technologies) held at 30°C. The mobile phase A consisted of 0.04 % V/V aqueous formic acid with 2 mmol L-1 CH3COONH4, while phase B was acetonitrile. The mobile phase was delivered in the gradient mode: 0 min 75% B, 5 min 100% B, 7 min 100%
B, post time 3 min (run time 10 min) with a constant flow of 0.5 mL min-1. During the
35
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first 3 min, the eluate was forwarded to MS without any flow splitting, and afterwards it
was diverted to waste. The ESI parameters were as follows: drying gas flow (N2)
9 L min-1 , temperature 350°C, nebuliser gas pressure 40 psi, capillary voltage 4 kV (optimised). The signal was acquired in the MRM (multiple reactions monitoring) mode,
using a positive polarity (resulted in fewer unwanted adducts formation, as well as, better
sensitivity). The protonated ion m/z = 325 was used as a precursor, with products
[M+H-CO2]+ m/z = 281 and [M+H-CH3•]+ m/z = 310 chosen as the target and qualifier
ions, respectively. For both transitions, optimised conditions were: fragmentor voltage
120 V, collision cell voltage 35 V. Using reference standards, the calibration curve in 7.81000 ng mL-1 range was prepared and the STC concentration was determined by the external standard method. The peak identity was confirmed by using a qualifier-to-target
peak area ratio, which amounted to 0.52 ± 0.01 for standards and to 0.52 ± 0.03 for
samples.
Mycopopulation in tested samples
The presence of moulds was detected in all tested salad samples. Total mould counts
ranged from 10.0 to 4.7102 cfu g-1. Forty one species were detected and they belonged to
the genera of Alternaria, Aspergillus, Botrytis, Cladosporium, Geotrichum, Emericella,
Eurotium, Fusarium, Mucor, Paecilomyces, Phialophora, Phoma, Penicillium, Trichoderma and Xeromyces. The most frequently isolated mycopopulations were Cladosporium, Penicillium, Aspergillus and Alternaria (Table 1).
Table 1. Frequency of mould species in fresh salads of different vegetables
Genus
Alternaria
36
Frequency of
genus (%)
6.89
Aspergillus
14.67
Botrytis
1.56
Cladosporium
42.89
Geotrichum
Emericella
0.44
0.22
Eurotium
2.22
Species
A. alternata (Fr.) Keissler
A. glaucus Link
A. niger van Tieghem
A. restrictus G. Smith
A. versicolor (Vuill.) Tiraboschi
A. wentii Wehmer
B. cinerea Pers.
C. cladosporioides (Fres.) de Vries
C. macrocarpum Preuss
C. sphaerospermum Penzig
G. candidum Link
E. nidulans (Edam) Vuill.
E. amstelodami L. Mangin
E. chevalieri L. Mangin
E. herbariorum Link
E. rubrum Jos. König et. al.
Frequency of
species (%)
6.89
0.67
10.22
1.11
1.78
0.89
1.56
40.44
1.56
0.89
0.44
0.22
1.33
0.22
0.44
0.22
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Genus
Fusarium
Frequency
of genus
(%)
3.11
Mucor
0.44
Paecilomyces
Phialophora
0.22
0.44
Phoma
0.67
Penicillium
25.78
Trichoderma
Xeromyces
0.22
0.22
Species
F. oxysporum Schlecht.:Fr.
F. proliferatum (Matsushima) Nirenberg
F. subglutinans (Wollenw. & Reinking)
Nelson, Toussoun & Marasas
M. circinelloides v. Tieghem
M. hiemalis Wehmer
P. variotii Bain
P. fastigiata (Legerb. & Melin) Conant
Ph. glomerata (Corda) Wollenweber &
Hochapfel
P. aurantiogriseum Dierckx
P. bilaii Kucey
P. brevicompactum Dierckx
P. corylophilum Dierckx
P. chrysogenum Thom
P. citrinum Thom
P. commune Thom
P. expansum Link
P. funiculosum Thom
P. glabrum (Wehmer) Westling
P. implicatum Biourge
P. janthinellum Biourge
P. rugulosum Thom
P. solitumWestling
T. harzianum Rifai
X. bisporus L.R. Fraser
Frequency of
species (%)
0.44
1.78
0.89
0.22
0.22
0.22
0.44
0.67
7.55
0.67
3.55
0.22
0.44
0.89
0.44
3.55
1.33
2.67
2.67
0.44
0.22
1.11
0.22
0.22
The genus Cladosporium dominated with three isolated species or 42.89% of total
samples (Table 1). The most frequent was C. cladosporioides (40.44%). This species has
been frequently isolated from different foods including fresh salads, wheat, flour, barley,
rice, dry fish, etc. (17). Due to the psychrophilic nature of this species (able to grow at
minus 5ºC) it can spoil refrigerated food such as cheese and meat (23, 24, 25). It decomposes cellulose, pectin and lipids. It is capable to grow at water activity (aw) values below
0.86 at 25ºC (26), and shows resistance against microwave heating (27). Cladosporium
has not been known to produce mycotoxins (17). Besides this species, which dominated
in the tested samples, C. macrocarpum and C. sphaerospermum were also found, but
with much lower frequency (from 1.56% to 0.89%). They have not been recognised as
toxin producers.
The Penicillium population was present with the highest number of isolated species
(14) (Table 1). The most frequent were P. aurantiogriseum (7.55%), P. brevicompactum
(3.55%) and P. expansum (3.55%). Penicillium species widely occur in all food com37
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modities and environments. Some of them are markedly xerophilic organisms (P. brevicompactum, P. implicatum, P. chrysogenum). Some species (P. digitatum, P. expansum,
P. italicum) have been implicated in patogenesis of fruit diseases. Psychrotrophic species
can develop at refrigeration temperatures. Some of the species isolated from the samples
of fresh salads (P. aurantiogriseum, P. chrysogenum) have been recognised as potential
producers of mycotoxins (1, 17, 18). P. aurantiogriseum is one of the species with the
highest incidence in food and feed originating from our environment (28). It grows in a
wide temperature range, from –2 to 30ºC, with the optimal temperature at 23ºC and
minimal aw of 0.81 (17).
Species of the genus Aspergillus accounted for 14.67% of the total isolated mycopopulation (Table 1). The most frequently was A. niger. It occurs mainly as a storage
mould, however, it can be found in fields, too. It is considered more likely in warmer
climates as its optimum growth conditions range from 35 to 37ºC. This species is frequently isolated from dried commodities as it can grow at aw of 0.77. Its presence has
been well documented in numerous foodstuffs: fresh fruits, vegetables, spices, nuts, cereals, meat products, dried fish, cheese (1, 17, 19). Conidia of A. niger are resistant to
microwave and solar heating and UV irradiation. Some isolates demonstrated ability to
produce OA (19). Besides A. niger, other Aspergillus species contaminated the tested
samples: A. glaucus (sexual form E. herbariorum), A. restrictus, A. versicolor and A.
wentii. A. versicolor is a potential producer of STC. This species has been reported in
baby foods, cereals, nuts, foodstuffs obtained in health food stores, frozen and fermented
meat products, dried sardine (1, 17).
The genus Alternaria is field moulds that frequently contaminate cereals, fruits and
vegetables. One of the isolated species A. alternata grows over a wide temperature range
from -5 ºC to +36ºC, the optimum temperature being 25ºC and minimum aw of 0.88 (17).
It is a producer of many toxic metabolites.
Species of the genus Fusarium are field moulds and mostly contaminate cereals and
vegetables. F. proliferatum was the most frequently isolated species (1.78%) from the
Fusarium population (3.11%) (Table 1). It has been recognised as a pathogen in many
plants (13). It grows at aw and temperatures ranging from 0.97 to 0.92 and from 20 to
30ºC, respectively (17). It is known as a fumonisin producer (13, 16, 17). Other potential
mycotoxin producers of the genus Fusarium (F. oxysporum and F. subglutinans) were
also isolated from the samples.
Eurotium species were less prevalent in the mycopopulation of fresh salads (2.22%
frequency) (Table 1). E. amstelodami (1.33% frequency) was predominant Eurotium species. Similarly to P. aurantiogriseum, it is capable of decomposing cellulose and lignin,
therefore it is important in the biodegradation of plant materials and deterioration of plant
food (29). In the Eurotium population of fresh salads, three more species were isolated:
E. chevalieri, E. herbariorum and E. rubrum. They all are xerophilic (minimal aw of
0.70). They have been isolated from cereals, nuts, dried fruits and vegetables, cheese,
dried meat and fish (1, 17). E. herbariorum is a potential producer of STC.
Genera Botrytis, Geotrichum, Emericella, Mucor, Paecilomyces, Phialophora, Phoma, Trichoderma and Xeromyces were found in the lowest percentage of the tested samples (frequency 3.99%), represented with one species of each (Table 1).
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Presence of potentially toxigenic species in samples of fresh salads
According to literature data, 22 out of 41 identified species are potentially toxigenic,
(Table 2), which accounts for 46.18% of the total mycopopulation of fresh salads. Penicillium species showed the highest frequency (19.52%) among tested samples. Secondary
metabolites of Penicillium spp. differ in nature and many of them have been listed as mycotoxins. The most important toxins are ochratoxin A – OA (carcinogenic and nephrotoxic), citrinin (nephrotoxic), xantomegnin, viomellein and vioxantin (nephro- and heaptotoxic), nephrotoxic glycopeptides, verrucosidin (neurotoxin), patulin and penicillic acid
(general mycotoxins) and penitrem A (neurotoxin) (1). Some secondary metabolites with
unknown toxicity towards vertebrates can be used as indicators of toxigenic species. For
example, anicin and verucin A have been produced by only four Penicillium species,
which are known to produce nephrotoxins (OA, nephrotoxic glycopeptides): P. nordicum, P. verrucosum, P. polonicum and P. aurantiogriseum (30). Anicin and verucin A
(31, 32) are easily identified by HPLC as opposed to nephrotoxic toxins whose structure
remains unknown.
Potentially toxigenic Aspergillus species accounted for 12.0% of the total isolated
mycopopulation. Within this genus, producers of OA (A. niger) and STC (A. versicolor)
prevailed (Table 2).
A. alternata, the only isolated Alternaria species with a relatively high frequency
(6.89%) in tested samples (Table 2), is known to produce several mycotoxins, of which
the most important is tenuazoic acid. Toxins of A. alternate (AAT) are highly toxic metabolites with a structure similar to fumonisins. The production of one or more of the toxins
was reported in tomato, wheat, barley, maize, Chinese sugar cane, rape seed, olives and
spices (17). A maximal production of alternariol, its monomethylether and altenuene, was
established at 25ºC and 0.98 aw (33), and tenuazonic acid at 0.90 aw and 25ºC (34).
Potentially toxigenic Fusarium species were much less present (3.11%). They are potential producers of moniliformin and fumonisins (Table 2).
Out of four Eurotium species, three were potential producers of toxic metabolites
which constituted 2.09% of the mycopopulation of salads. In this genus, the potential
producers of echunilin and STC were dominant (Table 2). Another species, producers of
STC, were the species of the genera Emericella and E. nidulans with 0.22% frequency.
Among toxigenic species, Paecilomyces variotii (0.22%), a producer of patulin and viriditoxin, was also isolated (Table 2).
Generally, potential producers of OA were present in the highest percentage
(17.77%). Producers of moniliformin, fumonisins and STC consisted 3.11%, 2.67% and
2.44% of the mycopopulation, respectively. In tested samples, aflatoxigenic species were
not detected.
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Table 2. Frequency of potentially toxigenic species isolated from samples of fresh salads
and the list of their toxins (1, 13, 16, 17, 18, 19)
Species
Alternaria alternata
Aspergillus niger
A. versicolor
A. wentii
Eurotium amstelodami
E. chevalieri
E. herbariorum
Emericella nidulans
Fusarium oxysporum
F. proliferatum
F. subglutinans
Paecilomyces variotii
Penicillium
aurantiogriseum
P. brevicompactum
P. chrysogenum
P. citrinum
P. commune
P. expansum
P. glabrum
P. janthinellum
P. rugulosum
P. solitum
Toxin
alternariol, alternariol monomethyilether, alterotoxin I and II, altenuene, tenuazonic acid,
naphtho- 4-pyrones, malphormins, ochratoxin A
(few isolates)
sterigmatocystin, nidulotoxin
emodin, ventilacton
echinulin, physcion
echinulin, neoechinulin, physcion (according to
some authors)
echinulin, physcion, sterigmatocystin
sterigmatocystin, emestrin
moniliformin, zearalenone, beauovericins, enijatine, fusarin C, wortmannin, nivalenol, fusarenone X, sambutoxin, fusaric acid, naphthoquinone pigments, nectriafurone, gibepyrones
fumonisins B1, B2, B3, beauovericins, fusaroproliferin, fusaric acid, fusarin C, moniliforme,
naphthoquinone pigments, fusapyrone
moniliforme, fumonisin B1, fusaric acid,
fusaproliferin, hlamidosporol, beauovericins,
naphthoquinone pigments
patulin, viriditoxin
penicillic acid, verrucosidin, nephrotoxic glycopeptides, anacine, auranthine, aurantiomine,
ochratoxin A
botryodiploidin, mycophenolic acid, Raistrick
phenols, brevianamide A
roquefortine C, meleagrin, chrysogine,
penicilline, ochratoxin A
citrinin, tanzawaic acid A
cyclopiazonic acid, rugulovasine A i B,
cyclopaldic acid
roquefortine C, patulin, citrinin, communesins,
chaetoglobosin C
citromycetin
janthitrem
rugolosin
cyclopenin, cyclopenp. compactins
Mould
frequency (%)
6.89
10.22
1.78
0.89
1.33
0.22
0.44
0.22
0.44
1.78
0.89
0.22
7.55
3.55
0.44
0.89
0.44
3.55
2.67
0.44
0.22
1.11
Biosynthesis of OA and STC by mould originating from fresh salads
Although ochratoxigenic species were present in the highest percentage, some of the
examined isolates did not show OA biosynthesis capabilities (Table 3). Abarca et al. (35)
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mentioned impaired ochratoxin production by A. niger (two out of 19 isolates). P. aurantiogriseum and P. chrysogenum have been frequently reported as producers of penicillic
acid and roquefortin C (1, 17). These findings are in conformity with the results obtained
in the study of Dimić (1999). Tested isolates of P. aurantiogriseum (5) and P. chrysogenum (6) did not show capability to produce OA. However, ochratoxin producing activity
of P. aurantiogriseum was reported by Škrinjar et al. (36). Thirty eight percent of isolates
were found to be able to synthesise this toxin on sterilised wheat kernels in concentrations ranging form 40.0 to 65.0 g kg-1.
A small increase in the pH (6.6) in comparison to the initial value was observed in
two isolates of A. niger, whereas it slightly dropped in the isolates of Penicillium spp.
(Table 3). The differences in mycelia weights were negligible.
Table 3. Biosynthesis of ochratoxin A by Aspergillus niger and Penicillium spp.
originating from fresh salads of various vegetables
Species (isolates)
pH
A. niger (S1)
A. niger (S2)
A. niger (S3)
A. niger (S4)
A. niger (S5)
A. niger (S6)
A. niger (S7)
A. niger (S8)
P. aurantiogriseum (S1)
P. chrysogenum (S1)
P. chrysogenum (S2)
P. chrysogenum (S3)
4.80
6.62
7.25
2.61
6.60
7.13
4.30
3.80
3.25
3.73
4.07
3.45
4.00
4.15
4.20
3.75
4.34
4.78
5.10
4.78
5.60
Dry matter
(g 100 mL-1)
4.69
4.64
4.13
4.94
4.32
4.29
4.42
4.51
2.87
3.58
3.90
3.42
2.56
3.87
4.03
3.45
4.18
3.70
3.35
3.15
3.47
Ochratoxin A
(ng mL-1)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd – not detected
STC was synthesised by isolates of A. vesicolor, whereas other potential producers
did not exhibit this ability (Table 4). Current studies implied to frequent occurrence of
STC positive strains of A. versicolor. Eighteen out of total 58 tested isolates, showed the
ability to synthesise the toxin (37), whereas Mills and Abramson (38) found that 30 iso41
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lates of total 32 were toxigenic. A high toxigenic potential of this species was also
confirmed by Halls and Ayres (39). Škrinjar and Ač (36) found STC in seven isolates out
of total nine. Dimić (4) reported that 90% of ten A. versicolor strains produced STC. In
relation to the initial pH (6.6), a small drop of its value was observed in all strains.
Similarly to the OA synthesis, the differences between the mycelial weights were minor.
Table 4. Biosynthesis of sterigmatocystin by Aspergillus spp. and Eurotium spp.
originating from fresh salads of different vegetables
Mould isolates
A. versicolor S1
A. versicolor S2
E. nidulans S1
E. herbariorum S1
E. herbariorum S2
pH
5.43
5.11
5.7
5.25
4.93
Dry matter
(g 100 mL-1)
3.52
3.05
2.85
3.48
4.07
Sterigmatocystin
(ng mL-1)
109.2
56.3
nd
nd
nd
CONCLUSION
Moulds were detected in all salad samples. Considering that isolated species were potentially toxigenic, while isolates of A. versicolor also biosynthesised STS, consummation of such salads could endanger public health. A successful programme aimed at subduing their growth should include actions that prevent spore germination and their spread
in the environment. The formation of mycotoxins is in relation with the mycelial growth.
Upgrading the hygienic conditions during processing, lowering the humidity, pH, temperature, addition of natural and synthetic preservatives, are all means by which the mould
growth and consequently, the mycotoxin production, in raw materials and food commodities could be decreased.
REFERENCES
1. A.R. Samson, S.E. Hoekstra and C.J. Frisvad: Introduction to Food- and Airborne
Fungi. Centraalbureau vor Schimmelcultures, Ultech, The Netherlands (2004).
2. F. Galvano, A. Ritieni, G. Piva and A. Pietri: Mycotoxins in the human food chain, in
The Mycotoxin Blue Book. Ed. D. Diaz, Nottingham University Press, England
(2005) pp. 187-225.
3. A. Dalcero, C. Magnoli, S. Chiacchiera, G. Palacios and M. Reynoso: Mycoflora and
incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina. Mycopathologia 137 (1997) 179-184.
4. G. Dimić: Mikološki i mikotoksikološki aspekti pojave plesni u začinima, Ph.D.
Thesis, University of Novi Sad (1999).
5. S. Kocić-Tanackov: Growth of toxigenic Fusarium species and zearalenone synthesis
in barley for malt production. M.Sc. Thesis, Faculty of Technology, University of
Novi Sad (2004).
42
APTEFF,41, 1-203 (2010)
DOI: 10.2298/APT1041033K
UDC: 635.5:582.28
BIBLID: 1450-7188 (2010) 41, 33-45
6. L. Legzduna and H. Buerstmayr: Comparision of infection with Fusarium head blight
and accumulation of mycotoxins in grain of hulless and covered barley. J. Cereal Sci.
40 (2004) 61-67.
7. G. Dimić, Ž. Maletić and S. Kocić-Tanackov: Xerotolerant mycopopulations and mycotoxins in muesli components. Proc. Nat. Sci. Matica Srpska 109 (2005) 81-87.
8. S. Kocić-Tanackov, M. Škrinjar, O. Grujić, J. Lević and J. Pejin: Capacity of Fusarium species isolated from brewer’s barley to synthesise zearalenone. Proc. Nat. Sci.
Matica Srpska 108 (2005) 157-165.
9. A. Zinedine, C. Brera, S. Elakhdari, C. Catano, F. Debegnach, S. Angelini, B. De
Santis, M. Faid, M. Benlemlih, V. Minardi and M. Miraglia: Natural occurrence of
mycotoxins in cereals and spices commercialized in Morocco. Food Contr. 17 (2006)
868-874.
10. S. Kocić-Tanackov, D. Dimić and D. Karalić: Contamination of spices with moulds
potential producers of sterigmatocystine. APTEFF 38 (2007) 29-35.
11. B. Romagnoli, V. N. MennaGruppioni and C. Bergamini: Aflatoxins in spices, aromatic herbs, herbs – teas and medicinal plants marketed in Italy. Food Contr. 18
(2007) 697-701.
12. M. Weidenbörner: Mycotoxins in Foodstuffs, Springer Science+Business Media,
LLC, New York, USA (2008).
13. J.T. Lević: Vrste roda Fusarium. Institut za kukuruz “Zemun Polje” i Društvo genetičara Sbije, Beograd (2008).
14. A.Veršilovskis and S. De Saeger: Sterigmatocystin: Occurrence in foodstuffs and
analytical methods – An overview. Mol. Nutr. Food Res. 54 (2010) 136–147.
15. E.P. Nelson, A. T. Tousson, and O.F.W. Marasas: Fusarium species. An Illustrated
Manual for Identification. The Pennsylvania State University Press. University Park
and London (1983).
16. F.J. Leslie and A.B. Summerell: The Fusarium Laboratory Manual. Blackwell Publishing, USA (2006).
17. J.I. Pitt and A.D. Hocking: Fingi and Food Spoilage. 2nd ed. Blackie Academic Academic  Profesional, London (1997).
18. A.R. Samson and C.J. Frisvad: Penicillium subgenus Penicillium: New Taxonomic
Schemes, Mycotoxins and Other Extrolites. Centraalbureau vor Schimmelcultures,
Ultech, The Netherlands (2004).
19. A.M. Klich: Identification of Common Aspergillus Species. Centraalbureau vor
Schimmelcultures, Ultech, The Netherlands (2002).
20. I. Rassoli and P. Owlia: Chemoprevention by thyme oils of Aspergillus parasiticus
growth and aflatoxin production. Phytochemistry 66 (2005) 2851-2856.
21. I. Balzer, C. Bogdanic and S. Pepejnjak: Rapid thin layer chromatographic method
for determining aflatoxin B1, ochratoxin A, and zearalenone in corn. Journal of
AOAC 61 (1978) 584-585.
22. H.P. van Egmond, W.E. Deyll and W.E. Paulsch: Analytical Method 1 - Thin-Layer
Chromatographic Determination of Sterigmatocystin in Grains, in Environmental
Carcinogens-Selected Method of Analysis. Eds. H. Egan, L. Stoloff, M. Castegnaro,
P.M. Scott, I.K. O Neill, H. Bartsch, Vol. V Mycotoxins IARC Scientific Publication
(1982).
43
APTEFF,41, 1-203 (2010)
DOI: 10.2298/APT1041033K
UDC: 635.5:582.28
BIBLID: 1450-7188 (2010) 41, 33-45
23. M.D. Northolt, H.P. van Egmond, P. Soentoro and E. Deijl: Fungal growth and presence of sterigmatocystin in hard cheese. J. Assoc. Off. Anal. Chem. 63 (1980) 115119.
24. R. Ozari and N.K.M. Mansour: Vorkommen von Scimmelpilzen, Insbesondere der
Gattung Cladosporium Linx ex Fries. Untersuchungen bei der Schlachtung von Schafen auf dem Schlachthof. Fleischwirtschaft 68 (1988) 495-499.
25. A.D. Hocking and M. Faedo: Fungi causing thread mould spoilage of vacuum packaged Cheddar cheese during maturation. Int. J. Food Microbiol. 16 (1992) 123-130.
26. A.D. Hocking, B. F. Miscamble and J. I. Pitt: Water relations of Alternaria alternata,
Cladosporium cladosporioides, Cladosporium spaerospermum, Curvularia lunata
and Curvularium pallescens. Mycol. Res. 98 (1994) 91-94.
27. I. Dragoni, C. Balzaretti, E. Puzzi and A. Papa: The fungicidal action of microwaves.
Tec. Molitoria 41 (1990) 1035-1041.
28. M. Škrinjar and D. Tešanović: Hrana u ugostiteljstvu i njeno čuvanje. Prirodno-matematički fakultet, Univerzitet u Novom Sadu, Novi Sad (2007).
29. M. Muñtanola –Cvetković: Opšta mikologija. Književne novine, Beograd (1990).
30. F. Lund and J.C. Frisvard: Chemotaxnonomy of Penicillium aurantiogriseum and
related species. Mycol. Res. 98 (1994) 481-492.
31. J.M. Boyes-Korkis, K. Gurney, J. Penn, P.G. Mantle, J.N. Bilton. and R.N. Sheppard:
Anacine, a new benzodiazepene metabolite of Penicillium aurantiogriseum produced
with other alkaloids in submerged fermentation. J. Nat. Prod. 56 (1993) 1707-1717.
32. T.O. Lanser, H. Franzyk and S.R. Jensen: UV-guided isolation of verrucines A and B,
novel quinazolones from Penicillum verrucosum structurally related to anacine from
Penicillium aurantiogriseum. J. Nat. Prod. 62 (1999) 1578-1580.
33. N. Mangan, G.R. Cayley and J. Lacey: Effect of water activity and temperature on
mycotoxin production by Alternaria alternata in culturevand wheat grain. Appl. Environ. Microbiol. 47 (1984) 1113-1117.
34. M. Etcheverry, S. Chulze, A. Dalcero, E. Varsavsky and C. Magnoli: Effect of water
activity and temperature on tenuazonic acid production by Alternaria alternata on
sunflower seeds. Mycopathologia 126, 3 (1994) 179-182.
35. M.L. Abarca, M.R. Bragulat, G. Castella and F.J. Cabanes: Ochratoxin production by
strains of Aspergillus niger var. niger. Appl. Environ. Microbiol. 60 (1994) 26502652.
36. M. Škrinjar and M. Ač: Stvaranje sterigmatocistina pomoću plesni Aspergillus versicolor izolovanih iz trajnih proizvoda i smeše začina. Tehnologija mesa 6 (1992) 311313.
37. K. Miyaki, M. Yamazaki, Y. Horie and S. Udagawa: On the toxigenic fungi growing
on stored rice. Shokuhin Eiseigaku Zasshi. J. Fd Hyg. Soc. Japan 11 (1970) 373-380.
38. J.T. Mills and D. Abramson: Production of sterigmatocystin by isolates Aspergillus
versicolor from western Canadian stored barley and apseed/canola. Can. J. Plant Sci.
Pathol. 8 (1986) 151-153.
39. N.A. Halls and J.C Ayres: Potential production of sterigmatocystin on country-cured
ham. Appl. Microbiol. 26, 4 (1973) 636-637.
44
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ПОЈАВА ПОТЕНЦИЈАЛНО ТОКСИГЕНИХ ВРСТА ПЛЕСНИ У
САЛАТАМА ОД РАЗЛИЧИТИХ ВРСТА ПОВРЋА СПРЕМНИХ ЗА
КОНЗУМИРЊЕ
Сунчица Д. Коцић-Танацков, Гордана Р. Димић, Јелена Т. Левић, Душанка Ј. Пејин,
Јелена Д. Пејин, Игор M. Јајић
У узорцима свежих салата од различитих врста поврћа спремних за конзумирање одређен је укупан број плесни, извршена њихова изолација и идентификација,
са посебним освртом на потенцијално токсигене врсте. Одабрани потенцијални
продуценти охратоксина А (ОА) и стеригматоцистина (STC) су испитани на биосинтезу ових микотоксина.
Укупан број плесни у испитиваним узорцима се кретао од 10,0 дo 4,7102 cfu g-1.
У укупној микопопулацији доминирали су родови Cladosporium (42,89%), Penicillium (25,78%), Aspergillus (14,67%) и Alternaria (6,89%). Доминантна врста је била C.
cladosporioides (40,44%), затим следе A. niger (10,22%), P. aurantiogriseum (7,55%),
A. alternata (6,89%) и Fusarium spp. (3,11%). Остале врсте су биле заступљене са
2,22% (Eurotium spp.), 1,56% (Botrytis spp.), 0,67% (Phoma spp.), 0,44% (Geotrichum
spp., Mucor spp., Phialophora spp.) и 0,22% (Emericella spp., Paecilomyces spp., Trichoderma spp., Xeromyces spp.)
Од 41 идентификованe врстe плесни 22 су према литературним подацима биле
потенцијално токсигенe, што је чинило 46,18% укупно изоловане микопопулације.
У највећем проценту су били заступљени потенцијални продуценати OА (17,77%).
Потенцијани продуценти монилиформина су били заступљени са 3,11%, фумонизина са 2,67% и STC са 2,44%.
Испитивани изолати потенцијалних продуцената OА нису показали способност
синтезе овог микотоксина, док су изолати A. versicolor биосинтетисали STC у концентрацијама од 56,3 и 109,2 ng ml-1.
Добијени резултати показују да овакви производи могу представљати опасност
по здравље људи, с обзиром да су изоловане потенцијално токсигене врсте, а изолати A. versicolor су и биосинтетисали STC.
45
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OSMOTIC DEHYDRATION OF CARROT IN SUGAR BEET MOLASSES:
MASS TRANSFER KINETICS
Gordana B. Koprivica, Nevena M. Mišljenović, Ljubinko B. Lević, Lidija R. Jevrić,
Bojana V. Filipčev
The osmotic dehydration process of carrot in sugar beet molasses solutions (40, 60
and 80%), at three temperatures (45, 55 and 65o C) and atmospheric pressure, was studied. The main aim was to investigate the effects of immersion time, working temperature
and molasses concentration on mass transfer kinetics during osmotic dehydration. The
most important kinetic parameters were determined after 20, 40, 60, 90, 120, 180, 240
and 300 min of dehydration. Diffusion of water and solute was the most intensive during
the first hour of the process and the maximal effect was observed during the first 3 hours
of immersion. During the next two hours of dehydration, the process stagnated, which
implied that the dehydration time can be limited to 3 hours.
KEYWORDS: osmotic dehydration, carrot, sugar beet molasses, mass transfer kinetic
INTRODUCTION
The various methods for extending shelf life of fruits and vegetables are fermenting,
pickling, canning or cold storage, freeze drying, etc. Convective hot-air drying is extensively employed as a preservation technique. However, using this method, food materials
are exposed to elevated drying temperatures, which leads to an increase in shrinkage and
toughness, reduction of both the bulk density and rehydration capacity of the dried product, and also causes serious damage to flavor, color and nutrient content (1). So, there is
a need for simple and inexpensive alternative processes that are not only energy intensive
and low capital investment but offer a way to make available these low cost, highly perishable and valuable crops available for the regions away from the production zones and
also during off - season (1).
Osmotic dehydration (OD) is one of these methods (2). The OD is a method for partial removal of water from the plant tissue by direct contact of product with a hypertonic
medium. The process is governed by the osmotic pressure difference between the food
material (hypotonic medium) and concentrated osmotic solution (hypertonic medium)
Dr Ljubinko B. Lević, Prof., [email protected], Gordana B. Koprivica, B.Sc., [email protected], Nevena M.
Mišljenović, B.Sc., [email protected], Dr Lidija R. Jevrić, assistant professor, [email protected], Faculty of
Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia, Dr Bojana V. Filipčev,
[email protected], Institute for Food Technology, University of Novi Sad, Bul. cara Lazara 1,
21000 Novi Sad, Serbia
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(3). The choice of optimal hypertonic aqueous solution appears to be the key problem in
osmotic dehydration. For fruits and vegetables dehydration, the most commonly used osmotic agents are sucrose and sodium chloride, as well as their combination. Glucose,
fructose, maltodextrin and sorbitol also can be used for OD (4).
Recent research has shown that use of sugar beet molasses as hypertonic solution improves the OD process (5). Sugar beet molasses is an excellent medium for this purpose,
primarily due to the high dry matter (80%) and specific nutrient content: 51% sucrose,
1% rafinose, 0.25% glucose and fructose, 5% proteins, 6% betaine, 1.5% nucleosides, purine and pyramidine bases, organic acids and bases, which subsequently results in a high
osmotic pressure of the solution (6, 7).
Apart from these ingredients, sugar beet molasses is a significant source of numerous
micronutrients (vitamins and minerals), especially of K, Ca, Na and Mg. Of special importance is the fact that all mineral components of molasses are in the dissolved state and
that the potassium is in much greater quantity than all other cations with a share of 75%.
In Serbia, sugar beet molasses has not yet been used as an ingredient in food industry.
Hence, extensive research has been conducted with the aim of introducing molasses as a
valuable ingredient in bakery, confectionery and the meat processing industry (8, 9). Sugar beet molasses as the by-product of sugar production is a cheap source of nutrients, i.e.
saccharose, it is available in large quantities, and for osmotic dehydration previous treatment is not needed. During the OD, the tendency is to increase the diffusion of water
from the sample into the surrounding solution and to decrease the penetration of solids
from the solution into the plant tissue, on the other hand (10).
The rate and dewatering degree from the material and changes in its chemical composition depend on the sort of the osmotic solution used, the kind and the size of raw material, as well as the ratio of material to osmotic solution, temperature, dehydration time,
and type of apparatus. The rate of OD is the highest at the beginning of the process. It results from the largest difference in the osmotic pressure between the osmotic solution and
the cell tissue of the material and small mass transfer resistance at this stage of the process (11). During the OD operation, two main mass fluxes at counter - current take place:
water loss, (WL) and solid gain, (SG). So, the determination of the osmotic treatment
effectiveness can be evaluated via the WL/SG ratio, taking into account that water removal must be greater than solute acquisition (12). The objective of this work was to study
the influence of main process variables such as temperature, concentration of sugar beet
molasses and operation time on the OD of carrot in sugar beet molasses. Also, the aim
was to investigate the mass transfer during the OD and find out an appropriate mathematical model which would describes the investigated process.
Mass transfer model
A large number of factors influence the OD process. However, earlier research has
shown that the temperature and concentration of the osmotic solution are two most influential factors on the mass transfer rate during the dehydration. In the OD process, three
main process variables are usually measured: moisture content, change in the weight and
change in the soluble solids. Of these, water loss (WL), weight reduction (WR), solid
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gain (SG), normalized moisture content (NMC) and normalized solid content (NSC) are
calculated as follows (13):
g
W 0 W
WR   
W 0
g
[1]
 g  u  uo
SG   
W0
g
[2]
g
WL   WR  SG
g
X
NMC 
X0
NSC 
u
u0
[3]
[4]
[5]
where: Wo - initial sample weight (g), W - sample weight after OD (g), uo - initial solid
content in the fresh sample (g), u - solid content in the sample after OD (g), Xo - initial
moisture content of the fresh sample before osmotic treatment (g), X - moisture content in
the sample after OD (g).
The existence of a simple mathematic model is very important from a practical point
of view, because in that case it is possible, for example, to predict the duration of the process for the desired moisture content and vice versa. Modeling of the calculated values of
the main kinetic parameters (WL, SG, NMC and NSC) was achieved by employing an
empirical model suggested by LabFit Curve Fitting Software and based on our experimental data:
P = a · tb · Cc
[6]
where t is the immersion time, C is the concentration of osmotic solution, P represents
WL, SG, NMC or NSC; a, b and c are parameters in Eq. [6].
The purpose of this analysis is to obtain a simple equation that takes into account the
both variables (time, concentration) simultaneously.
EXPERIMENTAL
Carrot samples were purchased in a local market in Novi Sad, Serbia, and stored at
4ºC until the use. Initial moisture content, Xo, was 88.45 ± 0.99%. Prior to the treatment,
the carrots were thoroughly washed and cut into cubes, dimension 1x1 cm. Pure sugar
beet molasses (around 80% solid content) and sugar beet molasses solutions (with 40%
and 60% solid content) were used as osmotic agents. Solutions were made by mixing pure molasses with distilled water. Sugar beet molasses was obtained from the sugar factory
Pećinci, Serbia. Initial dry matter content in sugar beet molasses was 83.68%. In all experiments, a weight ratio of solution to carrot sample of 4:1 was used; it can be considered
high enough to neglect the concentration changes during the process. The experiments
49
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were conducted under atmospheric pressure and static conditions and at temperatures
45ºC, 55ºC and 65ºC.
Mass transfer studies lasted 5 hours and the samples were taken out from the osmotic
solution at different times (20, 40, 60, 90, 120, 180, 240 and 300 min). After removal,
carrot samples were washed with water and gently blotted to remove excessive water.
The samples were kept in an oven (Instrumentaria Sutjeska, Serbia) at 105°C for 24 h
until a constant weight was attained. Dry matter content was calculated from the samples
weights before and after drying. The solid content of osmotic solutions was determined
refractometrically. All analytical measurements were carried out in accordance to AOAC
(14).
All results were treated using Statistica 9 and Origin 6.1 software.
Water loss
One of the most important parameters used to describe the process of osmotic dehydration is WL from sample.
Fig. 1 displays the WL as a function of the immersion time in molasses solutions (40,
60 and 80%) at studied temperature 65oC, because the best OD result was achieved at this
temperature.
Figure 1. Dependence of WL on dehydration time for the different concentrations of
sugar beet molasses solutions (40, 60 and 80% by weight) at 65 ºC
As can be seen from Fig. 1, the highest water loss from the samples occurred during
the first 20 min of dehydration regardless of the solution concentration or temperature. At
this interval, 52.06% (at 65oC and 80%) of the initial water content was removed from
the sample. During the next 100 min of the process, 42.9% of water was additionally removed from the sample (at 65oC and 80%). After the second hour, WL remained almost
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constant because of the drop in the osmotic driving force between the carrot tissue and
surrounding hypertonic solution. During the following 3 hours, only 5% of water was removed, which leads to a conclusion that the duration of osmotic dehydration can be limited to 3 hours.
By applying the empirical model (Eq. 6) and by statistical processing of experimental
data, the model parameters were calculated and given in Table 1. High values of the correlation coefficients indicated that the suggested model is appropriate and describes well
the influence of concentration on the efficiency of the OD process.
Table 1. Correlation data for WL during OD of carrot according to equation [6]
Correlation data
a
b
c
R
45 ºC
1.46·10-2±0.0023
0.285±0.013
0.546±0.035
0.9940
55 ºC
0.91·10-2±0.0022
0.269±0.018
0.671±0.052
0.9883
65 ºC
0.92·10-2±0.0026
0.277±0.022
0.686±0.063
0.9836
On the basis of data displayed in Table 1, the value of WL at any time or for any molasses concentration at constant temperature can be determined.
Solid gain
The SG describes the penetration of the solute from osmotic solution into the sample.
The aim of the OD is to remove water from the plant tissue and simultaneously minimize
the uptake of solute from osmotic medium into the tissue. Table 3 presents the effects of
osmotic medium concentrations (40, 60 and 80%), temperatures (45, 55 and 65oC) and
process duration on the SG of carrot.
Table 2. SG values for the OD of carrot using different concentrations of sugar beet
molasses solutions at different temperatures
The process duration
(min)
Temperature of
osmotic solution (oC)
Concentration of
osmotic solution (%)
0
20
40
60
90
120
180
240
300
45oC
55oC
65oC
40
60
80
40
60
80
40
60
80
0
0.024
0.029
0.032
0.029
0.044
0.051
0.052
0.052
0
0.032
0.048
0.058
0.064
0.069
0.072
0.076
0.077
0
0.042
0.059
0.072
0.078
0.084
0.097
0.105
0.108
0
0.013
0.022
0.029
0.042
0.050
0.052
0.058
0.061
0
0.026
0.040
0.055
0.064
0.066
0.072
0.079
0.082
0
0.029
0.047
0.059
0.066
0.068
0.080
0.092
0.101
0
0.012
0.025
0.042
0.052
0.055
0.061
0.067
0.071
0
0.021
0.029
0.038
0.063
0.072
0.078
0.082
0.082
0
0.026
0.056
0.084
0.099
0.105
0.142
0.136
0.120
The results showed that rapid increase in solids was registered in the first 60 min of
the process at 80% and 65°C. Within this period, 59.5% of solids from the osmotic so51
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lution diffused into the sample as compared to total amount of solids eliminated from the
sample during the process.
Since the process efficiency is characterized by a minimal SG in the treated sample,
the temperature of 65°C cannot be considered as optimal. At 45°C, the diffusion of osmotic active substances from the medium to the carrot was minimal (SG = 0.052 g/g initial
sample) after 5 h of immersion in 40% solutions. On the basis of these data it comes out
that the temperature of 45°C can be considered as an optimum for the OD of carrot in
molasses as an osmotic medium.
Table 3. Correlation data for SG during osmotic dehydration of carrot according to
equation [6]
Correlation data
a
b
c
R
45 ºC
2.3·10-4±0.0001
0.297±0.017
1.023±0.052
0.9918
55 ºC
6.07·10-4±0.0002
0.385±0.025
0.670±0.067
0.9842
65 ºC
1.28·10-4±0.0001
0.392±0.048
1.084±0.140
0.9537
High coefficient of correlation (Table 3) confirms that the proposed empirical model
equation [6] predicted well the solid gain in relation to the solution concentrations.
Water loss and solid gain ratio
In general, the increased concentration of osmotic medium favors the diffusion of solids into the sample, which leads to decline in the WL/SG ratio. This ratio is considered
to predict the best efficiency of the osmotic treatment. High WL/SG ratios point to intensive water removal from the samples accompanied with minimal solid gain.
Table 4. Effect of temperature, concentration and process duration on the change of the
WL/SG ratio at temperature of 65oC
T (o C)
65oC
52
Time (min)
0
20
40
60
90
120
180
240
300
80
0
16.169
9.562
7.396
7.075
7.520
5.863
6.156
6.872
C (%)
60
0
12.809
11.771
11.399
8.444
8.490
8.254
7.977
8.336
40
0
18.636
11.521
8.466
8.296
8.743
8.432
8.174
8.254
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The WL/SG ratio gradually decreased during the osmotic dehydration of carrot, after
the initial rise within the first 20 minutes of the process. The values ranged from minimal
5.863 (registered at 65°C, 80% solution, after 180 min) to a maximum of 18.636 (at
65°C, 40% solution, after 20 min). Both minimal and maximal ratios were achieved at
65°C, suggesting that the concentration of osmotic solution has a more pronounced effect
on this parameter, probably due to its greater effect on solid gain.
Normal moisture content and normal solid content
Figure 3 presents the effect of concentration and process duration for the molasses
solutions on the NMC and the NSC values of carrots during the OD at 65°C. Expectedly,
increased molasses concentration and temperature intensified the process and caused an
NMC drop and NSC elevation. At all tested temperatures and concentrations, the most
drastic NMC and NSC changes occurred within the first 60 min of the process. Minimal
NMC was achieved when 80% molasses at 65°C was used. Within the first 60 min, 65%
of the initial water content was reduced (from 1 to 0.346 g/g), whereas during the next 2
h it was reduced by additional 20%. During the last 120 min, the NMC decreased by the
negligible 3%. This supports our statement that the dehydration duration can be limited to
3 h.
Figure 2. Effect of the process parameters (concentration, temperature) on NMC and
NSC during OD of carrot in sugar beet molasses at 65 ºC
CONCLUSION
It was shown that the process of OD of carrots in sugar beet molasses is the most
intensive within the first hour. In the next two hours, the mass transfer weakened due to
decreased concentration gradient between the osmotic medium and plant tissue, and consequently lowers osmotic driving force. The water removal within the third and fifth hour
of the process duration is negligible therefore it was concluded that the process can be
limited to 3 hours.
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Maximal water loss was achieved at 65°C in 80% molasses (0.835 g/g of initial
sample) which is by 0.06 g/g or 0.03 g/g higher as compared to the maximal moisture
loss at 45°C or 55°C, respectively. This suggests that the process conduction at higher
temperatures is more favorable since the main goal is to maximize the water removal
from the sample. However, higher temperatures (65°C) also cause an increased uptake of
solids by carrot tissue (SG= 0.136 g/g initial sample).
Higher efficiency of osmotic dehydration is related to a high WL/SG ratio, which can
be obtained by the proper choice of process conditions (concentration, temperature). The
highest WL/SG ratio (18.636) was achieved at 65 °C in 40% molasses solution.
Acknowledgement
This research is part of the project supported by the Ministry of Science and Technological Development, Republic of Serbia, TR – 20112, 2008-2010.
REFERENCES
1. M. Maskan: Microwave/air and microwave finish drying of banana. J. Food Eng. 44
(2000) 71-78.
2. J. Shi & M.L. Maguer: Osmotic dehydration of foods: Mass transfer and modeling
aspects. Food Rev. Int. 18 (2002), 305–335.
3. N. K. Rastogi and K.S.M.S. Raghavarao: Mass transfer during osmotic dehydration:
Determination of moisture and solute diffusion coefficients from concentration profiles. Food Biprod. Process. 82 (2004) 44 – 48.
4. A. Ispir and I. Togrul: Osmotic dehydration of apricot: Kinetics and the effect of process parameters. Chem. Eng. Res. Des. 87 (2009) 166 - 180.
5. N. Mišljenović, G. Koprivica, Lj. Lević and T. Kuljanin: Influence of mono- and
double- edible coating on improving of osmotic dehydration of apple in saccharose
solution and sugar beet molasses. J. process. energy agric. 13 (2009) 184 - 187.
6. LJ. Lević, V. Filipović and T. Kuljanin: Osmotski tretman oblikovanog korena mrkve
u saharozi i melasi. J. process. energy agric. 11 (2007) 132 - 135
7. G. Koprivica, N. Mišljenović, Lj. Lević, V. Pribiš: Changes in nutritive quality of apple osmodehydrated in sugar beet molasses and saccharose solutions. Acta Periodica
Technologica 40 (2009) 35-46.
8. B. Filipčev, Lj. Lević, V. Pribiš and D. Kabić: Sugar beet molasses as a favorable hypertonic solution for osmotic pretreatment of apple, XIII Conference about Biotechnology. Proceedings, Čačak, Serbia, March 28-29 13 (2008) 323-329.
9. B. Filipčev, Lj Lević, M. Bodroža-Solarov, N. Mišljenović, G. Koprivica: Quality
Characteristics and Antioxidant Properties of Breads Supplemented with Sugar Beet
Molasses-Based Ingredients. Int. J. Food Prop. 13 (2010) 1035-1053.
10. M. Matuska, A. Lenart and H. N. Lazarides: On the use of edible coatings to monitor
osmotic dehydration kinetics for minimal solids uptake. J. Food Eng.72 (2006) 85-91.
11. R. Moreira and A. M. Sereno: Evaluation of mass transfer coefficients and volumetric
shrinkage during osmotic dehydration of apple using sucrose solutions in static and
non-static conditions. J. Food Eng. 57 (2003) 25 – 31.
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12. F. Chenlo, R. Moreira, C. Fernandez-Herrer and G. Vazquez: Osmotic dehydration of
chestnut with sucrose: Mass transfer processes and global kinetics modeling. J. Food
Eng. 78 (2007) 765–774.
13. M. Maguer: Osmotic dehydration: review and future directions, Proceedings of the
symposium in food preservation process, Brussels (1988) 283-309.
14. AOAC (2000). Official Methods of Analysis. Washington, USA.
ОСМОТСКА ДЕХИДРАТАЦИЈА МРКВЕ У МЕЛАСИ ШЕЋЕРНЕ РЕПЕ:
КИНЕТИКА ПРЕНОСА МАСЕ
Гордана Б. Копривица, Невена М. Мишљеновић, Љубинко Б. Левић, Лидија Р.
Јеврић, Бојана В. Филипчев
У раду је испитиван процес осмотске дехидратације мркве у растворима меласе
шећерне репе применом различитих концентрација раствора (40, 60 и 80%) и различитих температура (45, 55 и 65о C) а извођен је при атмосферском притиску. Главни
циљ експеримента је био да се испита утицај времена имерзије, радне температуре
и концентрације меласе на кинетику преноса масе током процеса осмотске дехидратације. Најважнији кинетички параметри процеса су одређивани након 20, 40,
60, 90, 120, 180 240 и 300 мин. Дифузија воде и растворка је била најинтензивнија
током првих сат времена имерзије док целокупни учинак достиже максимум у току
прва 3h трајања процеса. Током последња два сата дехидратације, процес издвајање
воде из третираних узорака је стагнирао, на основу чега се може закључити да целокупни поступак може бити скраћен на 3h.
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EFFECT OF GLYCEROL CONTENT AND pH VALUE OF FILM-FORMING
SOLUTION ON THE FUNCTIONAL PROPERTIES OF PROTEIN-BASED
EDIBLE FILMS
Nevena T. Nemet, Vladislava M. Šošo and Vera L. Lazić
The work is concerned with the effects of glycerol content and pH value of film forming solution on the functional properties of protein-based films. The films were produced of chicken breast proteins, dissolved under either acidic (pH 3) or alkaline (pH 11)
conditions, with different concentrations of glycerol (35%, 50% and 65% w/w of protein
content). Glycerol content affected significantly mechanical properties, water vapor permeability, color at pH 3 and film solubility (p<0.05). The pH value had significant influence on light transmission, color, transparency and film solubility (p<0.05). Considering
the results of mechanical properties and film solubility, the obtained films are in the
acceptable range for the use as a packaging material. It was estimated that water vapor
permeability, color, light transmission and transparency need to be improved for the
application.
KEY WORDS: Edible films, packaging material, glycerol, pH value.
INTRODUCTION
Plastic packaging has come into widespread use, thanks to its good mechanical properties and effectiveness as a barrier to oxygen and water. However, synthetic packaging
materials have led to the serious ecological problems due to their non-biodegradability.
With the emphasis on limited resources and the environment, in the recent years research
attention has turned to developing biodegradable and/or edible packaging (1).
An edible film has been defined as a thin, continuous layer of edible material, which
can prevent the food from interaction with its environment, gains or losses of moisture or
aroma, taking up oxygen or contamination with microorganisms (2,3). Furthermore,
edible and biodegradable films can be used to incorporate various food additives, such as
flavorings, antimicrobial and antioxidant agents into foods at specific locations.
The basic materials for the film preparation are biopolymers. The biopolymers to be
used as raw material for edible films, such as proteins, polysaccharides and lipids, should
be capable of forming continuous matrix and normally are from renewable and abundant
Nevena T. Nemet, B.Sc., [email protected], Vladislava M. Šošo, B.Sc., [email protected], Dr Vera
L. Lazić, Assoc. Prof., [email protected], Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad,
Serbia
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resources (4). Recently, increasing attention is being paid to the use of biopolymers from
agro-industrial residues or their composites as packaging materials, since it could contribute to the reduction of environmental pollution (5). Among these materials, proteins
have been extensively used for the development of edible films because of their relative
abundance, film-forming ability and nutritional qualities (6). Protein-based films have
impressive gas barrier and mechanical properties, compared with those from lipids and
polysaccharides, but show the poor water-vapor barrier properties (7, 8, 9).
The objective of this study was to determine the functional properties of chicken protein-based films as influenced by plasticizer content and pH value of film forming solution.
EXPERIMENTAL
Preparation of protein-based film
Films were prepared from a film-forming solution based on myofibrilar proteins isolated from chicken breast muscles in distilled water, plasticizer and either HCl or NaOH.
The film-forming solution was prepared as described by Shiku et al. (2) with a slight
modification.
Chicken breasts, bought in supermarket (Hat Yai, Thailand) were used as raw material. Frozen muscles were thawed using running water (26-27°C) until the core temperature reached 0°C. Protein content of chicken muscles was determined by Kjeldahl’s
method (10). Meat was chopped into small pieces and connective tissues were removed
as much as possible. The right amount of meat, needed to obtain the final protein concentration of 2% (w/v) in 100 ml of film-forming solution, was measured and added with
a small amount of distilled water (up to 30 ml). The mixture was homogenized at 13000
rpm for 1 minute, using a homogenizer (Polytron PT - MR 2100, Kinematica AG, Switzerland). The pH value of mixtures was then adjusted to 3 (group of samples A) and 11
(group of samples B), using 1M HCl and 1M NaOH, respectively. The mixture obtained
was filtrated through a layer of nylon sheet. Plasticizer (glycerol) was then added at 35%,
50% and 65% (w/w) of protein content (obtained samples - A35%, A50%, A65%, B35%,
B50% and B65%). The volume was then adjusted to 100 ml with distilled water, and the
solution obtained was used for film casting.
The prepared film-forming solution (4 g) was cast onto a rimmed silicone resin plate
(50 x 50 mm) and air blown for 12 h at room temperature, prior further drying at 25°C
and 50% relative humidity (RH) for 24 h in an environmental chamber (WTB Binder,
Tuttlingen, Germany). The resulting films were manually peeled off and used for the analyses.
Deteremination of film properties
Film thickness was measured using a micrometer (Gotech, GT-313-A, Gotech testing
machines Inc, Tawai) at five random positions of each film of ten specimens.
Mechanical properties. Prior to the testing of mechanical properties, the films were
conditioned for 48 h at 25±0.5 °C and 50±5 % RH environmental chamber. Tensile
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strength (TS) and elongation at break (EAB) were determined using Universal testing
machine LR 30 K LLOYD Instruments Ltd., Farenham. Ten specimens (2 x 5 cm) with
initial grip length of 3 cm were used for testing. Cross-head speed was 3 cm/min. TS was
calculated by dividing the maximum force at break by the initial cross-sectional area of
specimen (film thickness x 2 cm). EAB was calculated as follows:
d after
[1]
100
dbefore
where dbefore was 3 cm (initial grip length) and dafter was the difference between distance
of grips after the break of specimen and initial grip length.
Water vapor permeability (WVP) was determined using a modified ASTM method,
as reported by Shiku et al. (2). The film in three replicates was sealed on an aluminium
cup containing silica gel (0% RH) with silicone vacuum grease and a rubber band to hold
the film in place. The cups were weighed, and then placed at 30°C in a desiccator containing the distilled water. Distilled water was placed at the bottom of desiccator for providing RH of 100% at 30°C. The cups were weighed at 1 h intervals over a 8 h period.
The water vapor transferred through films was determined from the weight gain of the
cups. WVP (g/m·s·Pa) of the film was calculated according to (11):
EAB 
WVP 
1
wl

A  t P2  P1
[2]
where w is the weight gain of the cup (g), l is the film thickness (m), A is the area of the
exposed film (m2), t is the time of gain (s) and (P2-P1) is the vapor pressure difference
across the film (Pa).
Color of the films was determined as L (lightness, 0=black, 100=white), a (-a=greenness, +a=redness) and b (-b=blueness, +b=yellowness), using CIE colorimeter (Hunter
associates laboratory, Inc., VA, USA). The color of the films was also expressed as the
total difference in color, ΔE*:
2
2
E *  L*  a *  b*
*
*
2
[3]
*
where ΔL , Δa and Δb are the differentials between the color parameter of the samples
and the color parameter of the white standard (L0*=94.8, a0*=-0.78 and b0*=1.43).
Light transmission and film transparency. The ultraviolet (UV) and visible light barrier properties of the films were measured at selected wavelengths between 200 and 800
nm using the UV-16001 spectrophotometer (Shimadzu, Kyoto, Japan). The transparency
of the films was calculated according to (12):
T
 log T600
l
[4]
where T600 is the fractional transmittance at 600 nm and l is film thickness (mm).
Film solubility (FS) is a parameter of biodegradability of films and it was expressed
as the percentage of film dry matter solubilized in distilled water (13). Dry matter of the
film was determined according to AOAC 2000 method (14). The conditioned film sam59
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ples (2 cm x 4 cm) were weighed and placed in 50 ml centrifuge tubes containing 10 ml
of distilled water with 0.1 % (w/w) sodium azide and then stored at 30°C for 24 h with
continuous gentle stirring (250 rpm, Heidolth Incubator 10000, Schwabach, Germany).
Undissolved dry matter was determined by centrifugation at 3000 x g for 20 min (Avanti
J-E, Beckman Coulter) and drying them at 105 °C for 24 h. The weight of solubilized dry
matter was calculated by subtracting the weight of unsolubilized dry matter from the
initial weight of dry matter and expressed as the percentage of total weight.
Statistical analysis. Data were subjected to the analyses of variance (ANOVA) and
mean comparisons were carried out by Duncan’s multiple range test (15). Analysis were
performed using StatSoft package (STATISTICA 9.1 StatSoft Inc., Tulsa, OK, USA).
Film formation
The obtained films were strong and flexible enough to be peeled and handled. Films
produced of proteins solubilized under the acidic conditions were generally transparent
and smooth, while the films produced by proteins solubilized under the alkaline conditions were yellow, thick and sandy. Visually, the differences were noticeable between
the films containing 35% and 65% of glycerol, which were rigid and sticky, respectively.
Thickness
Results for film thickness are shown in Table 1.
Table 1. Film thickness for A and B samples, with different content of glycerol
Sample
Thickness
[mm]
A35%
0.0285
±0.004*
A50%
0.0299
±0.003
A65%
0.0316
±0.003
B35%
0.0297
±0.002
B50%
0.0309
±0.003
B65%
0.0319
±0.002
(*) – Mean ± SD from 10 measurements
Samples with higher concentration of glycerol had a higher content of dry mater in
film-forming solution, so it is expected for resulting film to be thicker. However, the
results showed that this difference was not statistically significant (p>0.05).
Mechanical properties
At the extreme acidic or alkaline pH values, strong electrostatic repulsion of ionized
groups occurs in the film-forming solution, which leads to the solubilization of proteins.
Solubilization processes are a prerequisite for film preparation and have the impact on the
mechanical properties of the resulting films (16). The unfolded proteins obtained using
either acidic or alkaline solubilizing process underwent the aggregation through hydrogen, ionic, hydrophobic and covalent bondings, particularly when the water was remo60
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ved. (17). The distribution and extents of intra- and inter-molecular interactions, which
give rise to three-dimensional network structures of the films, could affect their mechanical properties (6). Results given in Table 2 showed that when the same plasticizer
content was used, TS of films prepared at the pH 3 was significantly different (p<0.05)
from that prepared at pH 11. However, no significant effect of pH was observed on EAB
of protein-based films (p>0.05).
Plasticizers are the basic additives for the film-forming polymers. They reduce intermolecular forces, which increase the molecular spacing and mobility of biopolymer
chains. Addition of a plasticizing agent is necessary in order to overcome brittleness of
the film and to improve its flexibility (18). Brandenburg et al. (19) found that films made
without plasticizer are extremely brittle and shattered upon handling. Polar groups (-OH)
along plasticizer chains are believed to develop polymer-plastic hydrogen bonds that
replace the polymer-polymer interactions in the biopolymer films. Due to its small size
and high polarity, glycerol is most commonly used as plasticizing agent (20).
Results in Table 2 show that TS decreased and EAB increased significantly (p<0.05)
with increase of glycerol content. Thus, the addition of glycerol increased the extensibility of protein-based films, while reduced its mechanical strength.
Table 2. Tensile strength and elongation at break for A and B samples, with different
content of glycerol
Sample
TS [MPa]
EAB [%]
A35%
4.3137
±0.98*
79.7410
±6.91
A50%
2.4263
±0.49
99.0243
±5.26
A65%
1.5445
±0.31
117.8600
±4.31
B35%
4.1866
±0.57
81.6633
±15.02
B50%
3.3196
±0.46
96.2100
±13.68
B65%
2.5571±
0.53
101.2367
±9.78
TS of chicken protein-based films is in the same range as TS of conventional polyolefin films: 3-10 MPa (21). EAB values are higher than that of cellophane (by about 20%)
and considerably lower than those of the most commercial synthetic polymer films, like
LDPE (by about 500%) or HDPE (by about 300%) (22).
Water vapor permeability
Water vapor permeability is another important and widely studied property of edible
films. The barrier properties of the films are influenced by the hydrophobic/hydrophilic
nature of the polymer and by the type, level and compatibility of the incorporated plasticizer (23, 24). Results shown in Table 3 indicate that the WVP of edible films are much
higher than WVP of plastic films (LDPE 0.0055·10-10 g/m·s·Pa; PVC 0.0071·10-10
g/m·s·Pa; (25)). Increased transmission of water vapor through a protein-based film is
caused by a high content of polar amino acid residues in the structure of the film, as well
as to the presence of hydrophilic plasticizer - glycerol. (26).
The WVP values significantly increase (p<0.05) with increasing of the content of glycerol in the film-forming solution. A higher amount of glycerol gives a higher amount of
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polar groups in the film, which could absorb more water from the surrounding atmosphere.
At the same glycerol content used, no differences were found between films prepared
at the values pH 3 and pH 11 (p>0.05).
Table 3. Water vapor permeability for A and B samples, with different content of
glycerol
Sample
g
WVP x ( 106 
)
m  s  Pa
A35%
0.2147
±0.01*
A50%
0.2664
±0.01
A65%
0.2874
±0.01
B35%
0.2267
±0.01
B50%
0.2455
±0.01
B65%
0.2709
±0.01
Color
Color attributes are of prime importance because they directly influence consumers
acceptability. The L, a, b and ΔE* values of tested samples are shown in Table 4.
Higher a and b and slightly lower L values were observed for films prepared under
alkaline conditions, in comparison with those prepared under acidic conditions (p<0.05).
It was estimated that the films prepared at alkaline pH were more likely yellowish than
those prepared at acidic pH, as evidenced by greater b value. The result indicated that
alkaline conditions might induce the formation of yellowish pigment, especially via Maillard reaction. Alkaline conditions probably induced the hydrolysis of proteins and sugars,
leading to the availability of amino group from amino acids and carbonyl group from
reducing sugar. In the alkaline medium, amino groups are deprotonated and, hence, have
an increased nucleophilicity. As a consequence, the Maillard reaction was favored,
particularly during drying of the film (27).
Table 4. Color for A and B samples, with different content of glycerol
Sample
A35%
64.18
±0.08*
A50%
64.54
±0.28
A65%
65.43
±0.37
B35%
64.75
±0.26
B50%
65.21
±0.19
B65%
64.91
±0.17
a
-1.21
±0.07
-1.19
±0.06
-1.25
±0.20
-1.49
±0.09
-1.73
±0.05
-1.47
±0.08
b
1.96
±0.19
1.96
±0.16
2.11
±0.38
3.59
±0.32
5.04
±0.33
3.54
±0.21
ΔE*
2.18
±0.12
2.16
±0.11
2.30
±0.18
3.23
±0.23
4.23
±0.21
2.55
±0.09
L
At the pH 3, glycerol did not affect total difference in color (p>0.05), but it showed a
significant effect when the pH 11 was used for film preparation (p<0.05). Since glycerol
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is a colorless component, this effect of plasticizer was probably related to a dilution effect
due to its increasing concentration in the film-forming solution, without any probable
association with the plasticizing effect of glycerol (28).
Light transmittance and transparency
Light transmittance (%T) in the UV-vis range and transparency (T) values of tested
samples are presented in Table 5.
Table 5. Light transmittance and transparency for A and B samples, with different
content of glycerol
Wave length
[nm]
200
280
350
400
500
600
700
800
600
%T
A35%
0
0.4
72.8
76.6
80.9
82.6
83.6
84.4
A50%
0
0.5
74.2
77.9
82.1
84.0
85.3
86.2
A65%
0
0.6
73.5
77.3
80.9
82.3
83.4
84.2
2.98
±0.46
2. 55
±0.22
2.69
±0.25
B35%
0
0.3
51.6
57.7
63.6
68.8
67.4
68.5
B50%
0
0.2
46.9
53.1
59.6
61.9
63.6
65.2
B65%
0
0.2
57.8
63.8
69.5
71.4
72.9
74.3
6.15
±0.52
6.78
±0.53
4.62
±0.43
T
All tested samples showed the excellent barrier for light transmission in UV-range
(200 nm – 0%), probably owing to the high content of aromatic amino acids in proteinbased structure, capable to absorb UV-light (29). In the visible range (350-800 nm), the
light transmittance of films prepared at pH 3 ranged from 72.8 % to 86.2 %, but the much
lower values were found for the films prepared at pH 11 (46.9% - 74.3%). At the same
pH, %T of film slightly increased with increasing glycerol content.
The transparency values of films prepared at pH 3 were lower than that prepared at
pH 11, indicating that the former was more transparent than the latter (p<0.05). Glycerol
content did not affect the transparency of the films (p>0.05).
Film solubility
Solubility in water is an important property of edible films, since potential applications may require water insolubility to enhance product integrity and water resistance.
However, in some cases water solubility of the film before consumption of the product
might be beneficial (30). The FS of tested samples is presented in Table 6.
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Table 6 – Film solubility for A and B samples, with different content of glycerol
Sample
FS [%]
A35%
3.4133
±0.87*
A50%
5.5533
±0.71
A65%
9.9900
±0.77
B35%
5.7900
±0.61
B50%
9.3300
±1.11
B65%
12.8933
±0.29
The myofibrilar protein-based films exhibit very low FS, in comparison to the other
edible films (lentil, soy, whey and pea protein-based films have FS approximately 38, 35,
30 and 39%, respectively (24)). This result suggested that the myofibrilar protein polymer
network was highly stable and that only small molecules (small peptides, monomers and
non-protein materials) were soluble.
At the same glycerol content, all films prepared at pH 3 had a lower FS value than
those prepared at pH 11 (p<0.05). This result suggests that the films obtained with alkaline solubilizing process had a lower level of cross-linking with the weaker bonding, which
was possibly associated with the shorter chain length of protein molecules. This leads to a
lowered interaction between the molecules, which resulted in a higher solubility of the
resulting films.
At the same pH, all films with greater glycerol content exhibited a higher FS
(p<0.05). As reported by Cuq (31), in general, hydrophilic plasticizers, such as glycerol,
enhanced water solubility. It is probably because increasing the plasticizer content in the
film increased the water-soluble dry content. The relationship between water-soluble dry
mater and hydrophilic plasticizer content is linear (32).
CONCLUSIONS
Considering the results obtained for mechanical properties, it seems that chicken
breast protein-based films fall in the acceptable range of quality for use as a packaging
material, such as individual wrappers in a large box of carton. Water vapor permeability
of tested samples was much higher than those of typical polymeric packaging materials,
such as low-density and high-density polyethylene films, which considerably limits the
application of films for food packaging. Light transmittance results suggested that the
films could retard lipid oxidation by UV-light in a food system, but not by visible light.
Considering color and transparency results and significant effect of pHs of the film-forming solution on these parameters, it might be concluded that the films prepared at pH 3
are more transparent and clear enough for packaging food product, in comparison with
the films prepared at pH 11.
Almost all studied properties were affected by the glycerol concentration; nevertheless the effect of the pH was not evident in all properties. Protein is a good source for
edible film formation and therefore the application of the film could be suggested, but
there is still a number of limitations to be overcome. The shortcomings of the films could
be minimized by further works to find out the best protein/plasticizer ratio, type of plasticizer, pH of the film-forming solution, etc.
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Acknowledgements
The authors acknowledge the Faculty of Agro-industry, (University Prince of Songkla, Hat Yai, Thailand) for providing technical assistance and the facilities used in this
research. We also gratefully acknowledge the generous assistance and warm hospitality
we received from the members of Professor Soottawat Benjakul’s laboratory.
REFERENCES
1. Goméz-Estaca, J., L. Bravo, M.C. Goméz-Guillén, A. Alemán and P. Montero: Antioxidant properties of tuna-skin and bovine-hide gelatin films induced by the addition
of oregano and rosemary extracts. Food Chem. 112 (2009) 18-25.
2. Shiku, Y., P.Y. Hamaguchi and M. Tanaka: Effect of pH on the preparation of edible
films based on fish myofibrilar proteins. Fish Sci. 69 (2003) 1026-1032.
3. Kester, J.J. and O.R. Fennema: Edible film and coatings: A review. Food Technol.
40, 12 (1986) 47-59.
4. Gontard, N. and S. Guilbert: Bio-packaging: Technology and properties of edible
and/or biodegradable material of agricultural origin. Boletim da SBCTA. 30, 1 (1996)
3-15.
5. Popović, S., D. Peričin, Ž. Vaštag, Lj. Popović and V. Lazić: Evaluation of edible
film-forming ability of pumpkin oil cake; effect of pH and temperature. Food Hydrocolloid. In press.
6. Shiku, Y., P.Y. Hamaguchi, S. Benjakul, W. Visessanguan and M. Tanaka: Effect of
surimi quality on properties of edible films based on Alaska pollack. Food Chem. 86
(2004) 493-499.
7. Lazić, V. and D. Novaković: Ambalaža i životna sredina, Tehnološki fakultet, Novi
Sad (2010), pp. 108-119.
8. Stuchell, Y.M. and J.M. Krochta:Edible coatings on frozen king salmon effect of
whey protein isolation and acetylated monoglyceride on moisture loss and lipid oxidation. J. Food Sci. 60 (1995) 28-31.
9. Ou, S.Y., K.C. Kwok and Y.J. Kang: Changes in in vitro digestibility and available
lysine of soy protein isolate after formation of film. J. Food Eng. 64, 3 (2004) 301305.
10. AOAC 1999. Official Method of Analysis. 14th ed. Association of Official Analytical
Chemists, Washington, D.C., USA
11. McHugh, T.H., R. Avena-Bustillos and J.M. Krochta: Hydrophilic edible films:
modified procedure for water vapor permeability and explanation of tickness effects.
J. Food Sci. 58 (1993) 899-903.
12. Han, J.H. and J.D. Floors:Casting antimicrobial packaging films and measuring their
physical properties and antimicrobial activity. J. Plast. Film Sheet. 13 (1997) 287298.
13. Gennadios, A., A. Handa, G.W. Froning, C.L. Weller and M.A. Hanna: Physical properties of egg white-dialdehyde starch films. J. Agric. Food Chem. 46 (1998) 12971302.
65
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041057N
UDC: 621.798:547.426.1
BIBLID: 1450-7188 (2010) 41, 57-67
14. AOAC 2000. Official Method of Analysis. 17th ed. Association of Official Chemists,
Gaithersberg, Maryland, USA.
15. Steel, R.G.D. and J.H. Torrie: Principles and procedures of statistics: A biometrical
approach, Mc-Graw Hill, New York.
16. Hamaguchi, P.Y., W.W. Yin and M. Tanaka: Effect of pH on the formation of edible
films made from the muscle proteins of blue marlin (Makaira mazara). Food Chem.
100 (2007) 914-920.
17. Krochta, J.M.: Edible protein films and coatings, in Food Proteins and Their Applications. Eds. S. Damodaran and A. Paraf, Marcel Dekker, New York (1997) pp. 529549.
18. Barreto, P.L.M., A.T.N. Pires and V. Soldi: Thermal degradation of edible films based on milk proteins and gelatin in inert atmosphere. Polym. Degrad. Stabil. 79, 1
(2003) 147-152.
19. Brandenburg, A.H., C.L. Weller and R.F. Testin: Edible films and coatings from soy
protein. J. Food Sci. 58, 5 (1993) 1086-1089.
20. (M.Sc.) Chinabhark, K.: Effect of pH, Lipids and Protein Crosslinkers on the Properties of Surimi Film from Tropical Fish, Prince of Songkla University, 2005.
21. Briston, J.H.: Plastic films, (3rd ed) Wiley, New York (1988).
22. Pol, H., P. Dawson, J. Acton and A. Ogale: Soy protein isolate/corn-zein laminated
films: transport and mechanical properties. J. Food Sci. 67, 1 (2002) 212-217.
23. Bamdad, F., A.H. Goli and M. Kadivar: Preparation and characterization of proteinous film from lentil (Lens culinars); Edible film from lentil (Lens culinars). Food
Res. Int. 39 (2006) 106-111.
24. Ghanbanzadeh, B and A.R. Oromiehi: Biodegradable biocomposite films based on
whey protein and zein: Barrier, mechanical properties and AFM analysis. Int. J. Biol.
Macromol. 43 (2008) 209-215.
25. Kester, J.J. and O. Fennema: An edible film of lipids and cellulose ethers: barrier properties to moisture vapor transmission and structural evaluation. J. Food Sci. 54
(1989) 1383-1389.
26. Cuq, B., C. Amjad, J. Cuq and S. Quilbert: Edible packaging films based on fish
myofibrilar proteins: formulation and functional properties. J. Food Sci. 60 (1995)
1369-1373.
27. Martins, S.I.F.S. and B. Van: A kinetic model of the glycose/glycine Maillard reaction pathways. Food Chem. 90 (2005) 257-269.
28. Sobral, P.J.A., J.S. dos Santos and F.T. García: Effect of protein and plasticizer concentrations in film forming solutions on physical properties of edible films based on
muscle protein of Thai Tilapia. J. Food Eng. 70 (2005) 93-100.
29. Limpan, N., T. Prodpran, S. Benjakul and S. Prasarpran: Properties of biodegradable
blend films based on fish myofibrilar protein and polyvinyl alcohol as influenced by
blend composition and pH level. J. Food Eng. 100 (2010) 85-92.
30. Perez-Gago, M.B., P. Nadaud and J.M. Krochta: Water vapor permeability, solubility
and tensile properties of heat-denatured versus native whey protein films. J. Food Sci.
64, 6 (1999) 1034-1037.
66
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041057N
UDC: 621.798:547.426.1
BIBLID: 1450-7188 (2010) 41, 57-67
31. Cuq, B: Formation and Properties of Fish Myofibrilar Protein Films and Coatings, in
Protein-based Films and Coatings. Ed. Gennadios A., CRC Press, New York (2002)
p.224.
32. Hernandez-Muñoz, P., R. Villalobos and A. Chiralt: Effect of cross-linking using aldehydes on properties of glutenin-rich films. Food Hydrocolloid. 18 (2004) 403-411.
УТИЦАЈ САДРЖАЈА ГЛИЦЕРОЛА И pH ВРЕДНОСТИ РАСТВОРА ЗА
ПРИПРЕМАЊЕ ЈЕСТИВИХ ПРОТЕИНСКИХ ФИЛМОВА НА ЊИХОВЕ
ФУНКЦИОНАЛНЕ КАРАКТЕРИСТИКЕ
Невена Т. Немет, Владислава М. Шошo и Вера Л. Лазић
У раду је испитан утицај садржаја глицерола и pH вредности раствора за припремање протеинских филмова на њихове функционалне карактеристике. Филмови
су произведени од протеина пилећег белог меса, у киселој (pH 3), односно базној
(pH 11) средини, уз различит садржај глицерола (35%, 50% и 65% м/м на садржај
протеина). Садржај глицерола је значајно утицао на механичке особинe, пропустљивост водене паре, боју при pH 3 и растворљивост филмова (p<0.05). Ниво pH је
имао значајан утицај на пропустљивост светлости, боју, транспаренцију и растворљивост филмова (p<0.05). Узимајући у обзир резултате механичких особина и растворљивости филмова, закључује се да добијени филмови имају прихватљиве особине за примену у својству амбалажног материјала, али пропустљивост водене паре, боја, пропустљивост светлости и транспаренција морају бити побољшане да би
се могли успешно применити.
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INFLUENCE OF IMPURITIES AND HULL CONTENT IN MATERIAL FOR
PRESSING ON SENSORY QUALITY COLD-PRESSED SUNFLOWER OIL
Tamara Đ. Premović, Etelka B. Dimić, Aleksandar A. Takači and Ranko S. Romanić
This paper analyzes influence of different percentage of impurities and hull in the
material for pressing on sensory quality (appearance, smell, taste, aroma and colour) of
edible nonrefined sunflower oil. It has been concluded that simultaneous presence of impurities and hull in the starting material, especially presence of bigger quantities of impurities, has a rather negative effect on sensory quality of sunflower oil, made by cold
pressing on a screw press. Apart from sensory analysis, the effect of impurities and hull
on oil colour has also been identified by transparency determining, i.e. content of total
pigments – carotenoids and chlorophyll. It has been concluded that presence of bigger
quantities of impurities and hull cause the increase of content of both carotenoids and
chlorophyll, and at the same time, decrease of transparency value in cold-pressed
sunflower oil.
KEYWORDS: cold-pressed sunflower oil; impurity; hull; sensory qualities
INTRODUCTION
Edible plant oils today make up a rather big proportion of our diet, in which they
occupy a special place, due to its multifunctional importance: they are the main source of
energy, liposoluble vitamins, essential fatty acids and other minor ingredients. Oils belong to the category of essential and necessary foods which are consumed directly as they
are, or are applied during different preparations of food (1).
Process of oil production is permanently enhanced by developing people awareness of
importance of leading a healthy life, as well as the need to consume food with positive
effect on their health, i.e. food rich with protective components. Accordingly, a need for
production of cold-pressed oils arose, in order to preserve and protect their existing, natural and highly valuable components (2).
New tendencies include production of edible nonrefined oils of seeds of different
oleaginous plants, for example sunflower, pumpkin, soybean, rape, then flax, safflor, corn
germ, etc. However, on this planet, cold-pressed sunflower oil is the most frequent, which
keeps winning over the market, even in those countries in which the priority is given to
Tamara Đ. Premović, B.Sc., [email protected], Dr. Etelka B. Dimić, Prof., Dr. Aleksandar A.
Takači, Assoc. Prof., Ranko S. Romanić, B.Sc., University of Novi Sad, Faculty of Technology, Bulevar Cara
Lazara 1, 21000 Novi Sad, Serbia
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olive oil (3, 4). Our market also has a relatively increased demand and consumption of
cold-pressed oil, especially sunflower oil.
Along with the increased use of plant oils, knowledge about their quality is required
(1). Although there are a lot of indicators, which can be objectively measured and which
can express chemical, biological, nutritive and health quality, at quality evaluation of
edible nonrefined oils very important, even the first place is given to sensory qualities (3).
In comparison to refined oils, cold-pressed oils have more prominent sensory quality,
therefore these oils contribute to creating a specific aroma of food and provide special
gastronomic pleasure (5, 6).
It is well known that choice and quality of raw material are one of the most important
quality parameters of oil made by cold pressing, apart from importance of other parameters. Some of numerous quality factors of cold-pressed oils are: technology of processing
(7), storage conditions of seeds, that is oil, presence of impurities, premises, as well as
hull in the material for pressing, etc. (3).
Taking into consideration the fact that cold-pressed oil is widely used on the market,
and since sensory quality and content of pigments, which influence the colour, are extremely important, both from the aspect of an oil producer, as well as consumers, this
paper analyzes sensory qualities and colour of cold-pressed sunflower oil, depending on
the different presence of impurities and hull in the starting material for pressing.
EXPERIMENTAL
Materials
Within the scope of this paper, five samples of cold-pressed sunflower oil were prepared. Oils were made from six-month old seeds of domestic hybrid Cepko from regular
growing 2009. Cold-pressed oils were produced in the mini oil plant by pressing of
sunflower seeds with the given content of impurities and hull, with the use of a screw
press „Anton Fries“, Germany, with the capacity of 6,1-9,5 kgh-1 and 35-40 min-1 rotations of the screw. Oil temperature immediately after taken out of the press was 55-60ºC.
Pressed oils were kept 24 hours at the room temperature (20-25ºC) for sedimentation of
residue, after which the top layer of oil was decanted and filtrated through an ordinary laboratory filter paper. Numbers and identification of analyzed samples are given in Table
1.
Table 1. Identification of oil samples
Oil sample
1
2
3
4
5
70
Content of impurities (%)
0
0
5
10
10
Content of hull (%)
0
32
16
0
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Methods
Sensory evaluation of samples was done by a three-member expert committee, who
evaluated the following quality parameters: appearance, smell, taste, aroma and colour
(8). For evaluation of individual sensory qualities, the system of analytical-descriptive
tests was used with points ranging from 0 (unacceptable quality) to 5 (optimal quality) (9,
10). Apart from sensory analysis, the oil colour was determined also by transparence
measuring at 455 nm in relation to carbon-tetrachloride (8), as well as by determining of
total carotenoids (as β-caroten) and content of total chlorophyll (11).
Results of determining of transparence and content of total carotenoids and chlorophyll are expressed as a mean value of three replication and their standard deviations
(xsr±SD). Furthermore, statistically significant difference between samples, obtained by
application of the factorial analysis of variance, are also shown (12).
Results which were obtained by sensory evaluation of samples are shown in Figure 1.
The results show that Sample 2 may be characterized as “delicious-gourmet oil“, i.e. as
oil with extremely pleasant taste and smell. Aroma of this oil is characteristic, optimally
defined as the aroma of dried and healthy raw kernels of sunflower. Sample 1 has a bit
weaker aroma due to its aroma being less characteristic of the raw material, due to the
fact that this oil was made by pressing totally cleaned and shelled seeds, raw kernel.
Other samples have aroma far worse than the characteristic one. Sample 3 has a characteristic smell and taste of the raw material, but with weak strange smell and prominent
strange taste. Samples 4 and 5 have the worst aroma of all examined samples. These oils
have uncharacteristic taste with very prominent strange taste, and at the same time, uncharacteristic smell, with the presence of prominent (Sample 5), that is very prominent
(Sample 4) strange smell. With these sensory qualities, these oils could not be approved
for direct consumption, pursuant to valid regulations pertaining to the quality of edible
oils (13).
According to the evaluation results of aromas of Sample 2 and Sample 4, that is Sample 3 and 5, it can be concluded that presence of impurities in the pressing material has
the most unfavourable influence on the oil aroma. Comparing further results of aromas of
Sample 2, Sample 3, Sample 4 and Sample 5, it can be seen that simultaneous presence of
impurities and hull in the pressing material has unfavourable effect on oil aroma. On the
other hand, however, the presence of hull has favourable effect on oil aroma (Sample 1).
Sample 4 and Sample 5 have the weakest aroma, especially taste, which can be explained
by the fact that presence of a certain part of impurities (in this case 10%), that is impurities and hull (32%) in the pressing material significantly lead to occurrence of unpleasant
bitter taste of cold-pressed sunflower oil.
Results of this research about favourable effect of hull on aroma of cold-pressed sunflower oil, are not in accordance with available reference information. A group of authors
(14) in previous research with fresh seed obtained different results about effects of impurities and hull in the starting material for pressing on aroma of cold-pressed sunflower oil,
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which can be explained by a difference in age of the starting sunflower seed and difference in the amount of impurities and hull.
Figure 1 shows that two samples have maximal number of points when appearance, as
a parameter of sensory quality, is concerned, namely Sample 1 and Sample 2. Contrary to
these samples, which are “brilliantly“ clear and transparent, Sample 3, Sample 4 and
Sample 5 have noticeable mild turbidity without a visible residue, therefore receiving fewer points for appearance. By comparing appearances of Sample 1 and Sample 2, it can
be concluded that the presence of total hull (32% in Sample 2), does not have a negative
effect on the appearance. In Sample 3, Sample 4 and Sample 5 quantities of impurities
and hull present in oils have equal negative effects on the appearance, causing reduced
clarity, i.e. occurrence of mild turbidity.
Appearance
Appearance
5
5
4
4
3
2
Aroma
Colour
1
Sample 2
0
Taste
3
Sample 1
Aroma
Sample 3
Smell
2
Colour
1
Sample 5
0
Taste
Sample 4
Smell
Figure 1. Score of sensory quality of cold-pressed sunflower oil
Of all analyzed oil samples, two have maximal number of points when colour, as a
parameter of sensory quality is concerned, namely Sample 1 and Sample 2 (Figure 1).
These two oil samples have a characteristic, attractive and completely specific yellow colour, while Sample 3, Sample 4 and Sample 5 have a characteristic colour with certain
deviations in shade, therefore receiving fewer points.
This fact may also be explained by negative effects of impurities, that is by simultaneous presence of impurities and hull in the starting material for pressing on oil colour.
The results of this research show that impurities present in the starting material for pressing have negative effects on oil colour.
Differences in oil colour of samples, determined visually, are also confirmed both by
values of transparence, as well as content of total carotenoids and content of total chlorophyll, shown in Table 2.
Minimal value of transparence is obtained with Sample 5 (24.54%) and maximal with
Sample 1 (61.67%). By statistical processing of results of transparence values in the analyzed oils, the existence of statistically significant differences can be noticed (α= 0.05).
Therefore, it is possible to classify oil samples in three groups (a, b, c): Sample 1 and 2
(a), Sample 2, 3 and 4 (b), and Sample 3, 4, and 5 (c) (Table 2).
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Table 2. Transparence and content of pigments of cold-pressed sunflower oil
SAMPLE
1
Transparence
(% at 455 nm with CCl4)
INDICATOR
Content of total
carotenoids (mg/kg)
Content of total
chlorophyll (mg/kg)
61.67±0.0058
a
4.29±0.0098
b
0.00±0.000
b
2
43.56±0.0153
ab
6.82±0.0108
b
0.01±0.0001
3
36.46±0.0058
bc
7.82±0.0174
ab
0.32±0.0053
ab
4
38.84±0.0208
bc
7.60±0.0100
ab
0.17±0.0052
ab
5
24.54±0.0058
c
11.31±0.0220
a
0.99±0.0120
a
b
Different superscript letters a, b, c present significant differences (p0.05) among oil samples
Information available in cited works show that transparence of cold-pressed sunflower oil, made by different producers, widely ranges from 15 to 70%, most often from 5060%, which gives oil characteristic golden yellow colour (3). According to Dimić and
Romanić (15), transparence of cold-pressed sunflower oil of oleic type was 41.3%, while
transparence of cold-pressed olive oil was 25.2%. Virgin olive oil, by these authors, has
relatively lower values of transparence, 2.1-7.7%.
In the analyzed samples of cold-pressed sunflower oil, the content of total carotenoids, expressed as β-carotene, ranges from 4.29 (Sample 1) to 11.31 mg/kg (Sample 5),
Table 2, which is a bit lower content of total carotenoids if compared to values stated in
related works, ranging from 6.52-15.30 mg/kg (16). By statistical analysis of results of
content of total carotenoids in the analyzed oils, statistically significant differences have
been detected (α= 0.05), therefore it is possible to classify the oil samples in 2 groups (a,
b): Sample 3, 4 and 5 (a), and Sample 1, 2, 3 and 4 (b) (Table 2).
Different values of carotenoids in the analyzed samples of oils can be explained by an
assumption that results do not represent only values of content of total carotenoids in the
analyzed oils, but that impurities and hull present in material for pressing also contain a
certain amount of the same or similar pigments.
Given the research results (Table 2), it has been noticed that values between transparence and content of total carotenoids in the analyzed samples of oil show negative correlation with high level of correlation (R²>0.9), Figure 2, which is in accordance with the
cited works (15, 16).
Higher amounts of total carotenoids are present in palm oil, oil of pumpkin seed, corn
germs, sunflower, etc (3). According to the research results of Tuberoso et al. (2), the
content of β-carotene in sunflower oil is 0.1 mg/kg, while the content of total carotenoids
is 2-4 mg/kg. In soybean oil and rapeseed oil, the content of total carotenoids is 20-35
mg/kg and 25-100 mg/kg, respectively (8).
The content of chlorophyll in the analyzed samples ranges from traces to 0.99 mg/kg
(Table 2). Sample 1 contains total chlorophylls in traces, Sample 2 and 4 have a bit higher amount of total chlorophylls, respectively: 0.01± 0.0001 mg/kg and 0.17±0.005 mg/kg.
Their highest content is in Sample 3 (0.32±0.005mg/kg) and Sample 5 (0.99±0.012
mg/kg). Comparing the values of content of total chlorophylls in the analyzed samples, it
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Content of total carotenoids, mg/kg
has been concluded that the presence of impurities in the pressing material gives higher
content of total chlorophylls in oil than the presence of hull, and that simultaneous presences of impurities and hull in the pressing material cause the highest content of total
chlorophylls.
12
Transparence= -0,182*Carotenoids + 15,037
2
R = 0,9454
10
8
6
4
2
20
30
40
50
Transparence, %
60
70
Figure 2. Correlation of carotenoids content and transparence of cold-pressed sunflower
oil
By a statistical analysis of results, it has been established that there are statistically
significant differences in the content of total chlorophylls in the analyzed oils (α= 0.05),
which can be therefore classified in 2 groups (a, b): Sample 3, 4 and 5 (a), and Sample 1,
2, 3 and 4 (b) (Table 2).
By further comparison of these results with the colour results obtained by sensory
analysis, it has been established that oil samples with lower and minimal content of total
carotenoids and chlorophylls have the maximal point for colour as a parameter of sensory
quality.
Higher amounts of total chlorophylls are mainly present in nonrefined olive oil and
grape seed oil (3). According to information taken from the cited works (15), content of
total chlorophylls in virgin olive oil from the Mediterranean countries ranged from 7.7315.08 mg/kg.
Content of total chlorophylls in sunflower oil ranges from 0.5-0.8 mg/kg (8). Content
of total chlorophylls in cold-pressed sunflower oil ranges from traces to 1.208 mg/kg
(16), while content of total chlorophylls in cold-pressed sunflower oil of oleic type is 5.15
mg/kg (15).
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CONCLUSION
It has been concluded that both presence of impurities, as well as simultaneous presence of impurities and hull in the pressing material, have negative effects on sensory
qualities of cold-pressed sunflower oil; the presence of impurities causing worse sensory
quality of cold-pressed oils.
By comparing results of sensory analysis of oil, it can be established that aroma, i.e.
smell and taste of oil, more heavily depend on presence of impurities, and simultaneous
presence of impurities and hull than appearance and colour of oil. Apart from this, presences of impurities and hull in the starting material for pressing have almost identical
effects on appearance and colour of oil.
Summing up the results of determining of sensory quality, it can be concluded that by
pressing of seed with the presence of hull (at the amount of 32%), the highest-quality
cold-pressed oil is produced in relation to aroma, i.e. smell and taste.
Furthermore, presence of impurities and hull in the starting material for pressing in
higher amounts, causes higher content of total carotenoids and chlorophylls, as well as
lower value of transparence in cold-pressed sunflower oils.
REFERENCES
1.
A. Kamal-Eldin: Effect of fatty acids and tocopherols on the oxidative stability of
vegetable oils. Eur. J. Lipid Sci. Technol. 58 (2006) 1051–1061.
2. C.I.G. Tuberoso, A. Kowalczyk, E. Sarritzu and P. Cabras: Determination of antioxidant activity in commercial oilseeds for food use. Food Chemistry. 103 (2007)
1494-1501.
3. E. Dimić: Hladno ceđena ulja, Monografija, Univerzitet u Novom Sadu, Tehnološki
fakultet, Novi Sad (2005) pp. 30-32, 208-212.
4. B. Matthäus: Virgin oils – The return of a long known product. Eur. J. Lipid Sci.
Technol. 110 (2008) 595-596.
5. Е. Dimić: Kontrola kvaliteta hladno presovanih ulja. Acta Periodica Technologica.
31A (2000) 165-174.
6. K. Šmit, E. Dimić, R. Romanić, K. Bjelobaba-Bošnjak i B. Mojsin: Uticaj ljuske na
kvalitet hladno ceđenog ulja suncokreta, 46. savetovanje industrije ulja: Proizvodnja
i prerada uljarica, Zbornik radova, Petrovac na moru (2005) 89-93.
7. E. Dimić, J. Turkulov, Đ. Karlović i N. Keser: Kvalitet jestivih nerafinisanih ulja
suncokreta u zavisnosti od načina dobijanja, 38. savetovanje industrije ulja: Proizvodnja i prerada uljarica, Zbornik radova, Budva (1997) 23-30.
8. E. Dimić i J. Turkulov: Kontrola kvaliteta u tehnologiji jestivih ulja, Univerzitet u
Novom Sadu, Tehnološki fakultet, Novi Sad (2000) pp. 17-32.
9. М. Filajdić, М. Ritz, i М. Vojnović: Senzorska analiza lipidnih namirnica. Uljarstvo.
25, 2 (1988) 140-143.
10. R. Radovanović i J. Popov-Raljić: Senzorna analiza prehrambenih proizvoda, Univerzitet u Novom Sadu, Tehnološki fakultet, Novi Sad, Univerzitet u Beogradu, Poljoprivredni fakultet, Beograd-Zemun (2000-2001) pp. 149-155.
75
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041069P
UDC: 665.334.9:664.3.032:543.92
BIBLID: 1450-7188 (2010) 41, 69-76
11. J. P. Wolff: Manuel d̀analyse des corps gras, Azoulay, Editeur, Paris, (1968) pp.
186-188.
12. S. Hadživuković: Statistički metodi s primenom u poljoprivrednim i biološkim istraživanjima, Poljoprivredni fakultet - Institut za ekonomiku poljoprivrede i sociologiju
sela, Novi Sad (1991) pp. 154-160.
13. Pravilnik o kvalitetu i drugim zahtevima za jestiva biljna ulja i masti, margarin i druge masne namaze, majonez i srodne proizvode („Službeni list SCG“, broj 23/2006).
14. T. Premović, E. Dimić, R. Romanić i A. Takači: Uticaj nečistoća i ljuske na senzorska svojstva i oksidativnu stabilnost ulja, XIV Međunarodna Eko – konferencija,
Zbornik radova, Novi Sad, (2010) 317-325.
15. E. Dimić i R. Romanić: Analiza kvaliteta maslinovog ulja i hladno ceđenog suncokretovog ulja oleinskog tipa. Uljarstvo. 35, 3-4 (2004) 17-26.
16. T. Premović, E. Dimić, R. Romanić i A. Takači: Uticaj sadržaja nečistoća i ljuske na
senzorska svojstva hladno presovanog ulja suncokreta, 51. savetovanje industrije
ulja: Proizvodnja i prerada uljarica, Zbornik radova, Herceg Novi (2010) 119-125.
УТИЦАЈ САДРЖАЈА НЕЧИСТОЋА И ЉУСКЕ У МАТЕРИЈАЛУ ЗА
ПРЕСОВАЊЕ НА СЕНЗОРНА СВОЈСТВА ХЛАДНО ПРЕСОВАНОГ УЉА
СУНЦОКРЕТА
Тамара Ђ. Премовић, Етелка Б. Димић, Александар А. Такачи
и Ранко С. Романић
У овом раду је испитан утицај различитог удела нечистоћа и љуске у материјалу
за пресовање на сензорски квалитет (изглед, мирис, укус, арому и боју) јестивог нерафинисаног уља сунцокрета. Утврђено је да истовремено присуство нечистоћа и
љуске у полазном материјалу, а нарочито присуство већих количина нечистоћа,
веома неповољно утиче на сензорски квалитет сунцокретовог уља, добијеног поступком хладног пресовања на пужној преси. Утврђено је, такође, да нечистоћа и
љуска утичу на повећање садржаја како каротеноида тако и хлорофила, а самим
тим и на смањење вредности транспаренције уља.
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PROTEIN COMPOSITION IN TOFU OF CORRECTED QUALITY
Sladjana P. Stanojević, Miroljub B. Barać, Mirjana B. Pesić, Mirjana M. Milovanović
and Biljana V. Vucelić-Radović
Soybeans are an inexpensive, high-quality protein source. Soybeans have long been a
staple of the human diet in Asia, especially as tofu, which is prepared from soymilk. In
this study, tofu was made using a new production method which includes hydrothermal
cooking (HTC) and rennin-pepsin coagulant. The effects of the addition of gallic acid to
the slurry during tofu processing were studied. Tofu was made from two soybean genotypes: Lana and Balkan. The observed genotypes are characterized by relatively high
content of total proteins in flour, from 45.88% to 48.83%. The prepared tofu samples are
characterized by extremely high content of total proteins (52.17% - Lana tofu and
56.08% - Balkan tofu). The presence of gallic acid significantly affects the solubility of
tofu protein. The applied modifications of traditional procedure of tofu production significantly improved sensory properties of soybean protein products.
KEYWORDS: Tofu; hydrothermal cooking; proteinases; sensory properties; gallic acid
INTRODUCTION
Soybean is an annual herbaceous and leguminous plant. People of ancient Asia knew
for this plant and cultivated it 4 000 years ago. Soya was imported to the European continent during the 18th century, and in our region, arrived sometime in the early 20th century. Modern research, in the first place, emphasizes its beneficial effects on the organism.
Processing of soybeans yields several key products, of which, in our region of a relatively more common use are tofu and soymilk. Tofu is a jellied protein product, with homogeneous composition, cream-colored with mild flavor, which is produced by the coagulation of heated soymilk. Tofu prepared by coagulation of soymilk by CaSO4 or MgCl2
contains about 8% of total proteins, 4-5% lipids and about 2% of carbohydrates on fresh
weight basis. Tofu has a special nutritional value due to the presence of dietary fibers
(about 1%) and the absence of cholesterol, as well as a very low energetic value. The
high content of vitamins and minerals also contributes to the physiological value of the
tofu. Analyzing tofu, Wang and Murphy (1) found that it content of total isoflavones is
0.532 mg/g of tofu. However, in our region, a wider use of soy products in human
Dr Slađana S. Stanojević, Assist. Prof., [email protected], Dr. Miroljub B. Barać, Prof., Mirjana B. Pesić,
M.Sc. Assist., Dr. Mirjana M. Milovanović, Prof., Dr. Biljana V. Vucelić-Radović, Prof., University of Belgrade, Faculty of Agriculture, Institute of Food Technology and Biochemistry, 11080 Belgrade – Zemun, Nemanjina 6, Serbia
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nutrition has started only recently. Therefore, the main goal of this study was to make
soybean more acceptable to the domestic consumers in the form of products of corrected
quality in terms of sensory characteristics that would increase mass consumption. It was
noticed that the flavor of „green bean“, so-called „legume smell and taste“ of soybean
products prepared in the traditional way does not suit the consumers from our population.
These sensory properties, regarding its taste and smell, are a consequence of lipid oxidation - catalyzed by lipoxygenase, during steeping and crushing of the soybeans. Recently, a production process has been developed with the aim to reduce the duration of
crushing the soybeans, as well as boiling the crushed grain under pressure, at high temperatures in the shorter period, the so-called hydrothermal cooking (HTC) that decreases
the activity of lipoxygenase (2).
With the intention of improving the flavor of the produced cheese tofu was prepared
using HTC and enzyme rennet, with the addition of gallic acid. Gallic acid (3,4,5-hydroxyl benzoic acid) is considered to have a strong antiseptic and antioxidant effects due to
the presence of three free ortho-phenolic groups in its molecule. Both isolated and in the
form of food ingredients the antioxidants prevent oxidation of lipids. They are used in
food products in order to prevent perishing, rancidity or discoloration caused by oxidation.
The protein quality of soybean has a direct impact on the protein content of tofu. Dominant storage proteins of soybeans are globulins (3). Based on the coefficient of sedimentation during ultracentrifugation in standard phosphate buffer, they are divided into
four fractions: 2S, 7S, 11S, and 15S fraction. Over 70% of soluble proteins of mature
soybeans are the components of 7S and 11S fractions. The 7S fraction represents slightly
more than 1/3 of total soybean proteins. It consists mainly of β-conglycinin, γ-conglycinin, basic-7S-globulin, lectins, and small quantities of enzymes such as lipoxygenase
and β-amylase. β-Conglycinin (7S-globulin, vicilin) as the main protein of the 7S fraction
and it makes 90% of the entire fraction. In the soluble proteins it is present with 16.8 to
20.9% and in total proteins of soybean, with about 30%. Glycinin (11S-globulin, legumin) makes 32% of total soybean proteins, and is considered as the main protein of the
11S fraction. When analyzed by PAGE, glycinin is registered with two main zones, one
originating from the monomer and the other from the dimmer.
Due to the implementation of the manufacturing process that is significantly different
from the traditional, and the application of gallic acid, it is realistic to expect the changes
in protein composition of the obtained product, as well as the changes of sensory characteristics. The aim of this study was to examine the changes in the content and composition of the main protein fractions of the produced tofu after obtaining satisfactory sensory evaluation results.
EXPERIMENTAL
Soymilk processing. Two soybean genotypes grown in field conditions were evaluated. One genotype (Balkan) was selected by the Institute of Field and Vegetable Crops
(Novi Sad, Serbia) and the other (Lana) by the Maize Research Institute Zemun Polje
(Belgrade, Serbia). Soymilk and тofu were made using the new production method which
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includes HTC (2) with slight modifications and rennin-pepsin coagulant. Soybeans were
soaked in water (soybeans:water =1:5) at 5-7oC, for 14 h. Soaked beans were ground and
cooked in water (soybeans:water = 1:6) at 110oC, and 1.8 bar, for 8 min. (SoyaCow VS
30/40, model SM-30, Russia) (4). The slurry was filtered through a muslin cloth and
squeezed manually to obtain filtrate (soymilk).
Tofu processing and quality modifications by gallic acid. The prepared soymilks were separated in two equal volume parts. When the cooked milk was cooled, to one part
gallic acid (1ml gallic acid/l soymilk; 100ppm) was added, and afterwards commercial
rennin-pepsin rennet (rennet-″Idealka″, Rennet workshop-Novo Selo, Serbia) 10 ml/l of
cooked soymilk was added. To the second part, commercial rennin-pepsin rennet was
added only. The soy milk-coagulants was stirred manually and kept 20 min. Afterwards,
fast and brief manual stirring was applied and the content left for 15 min. The curds was
pressed with manual press (model SM-30, Russia) for 60 min. The weight of freshly formed tofu was recorded after pressing (5). Samples were stored at 4ºC before analysis.
Extractable soluble protein content. To determine soluble protein it was extracted for
1h at room temperature from defatted meal and tofu in a 1:20 ratio with 0.03 M Tris-HCl
buffer, pH 8, which contained 0.01 M β-mercaptoethanol. The mixture was centrifuged at
17000 x g for 15 min at room temperature. The protein content in the supernatant was
determined by the procedure of Bradford (6) at 595 nm.
Protein solubility in tofu was calculated from the amount of extractable soluble protein divided by the amount of total protein and multiplied by 100. Protein solubility was
calculated using the following formula (7):
Solubility (%) = soluble protein content/total protein content 100
1
Polyacrylamide gel electrophoresis (PAGE). PAGE was performed according to the
method of Davis (8). The separating gels were 7% (wt/vol), pH 8.9 and stacking gels
were 5% (wt/vol), pH 6.7. A 25 µl sample of the extract (2 mg protein/ml) diluted with
sample buffer, pH 8.0 [0.03M Tris-HCl buffer with 0.01M -mercaptoethanol, 10%
(vol/vol) glycerol, 0.0025% (wt/vol) bromophenol blue] was loaded per well. The gels
were run in a buffer solution, pH 8.3 [0.05M Tris (hydroxymethyl) aminomethane,
0.19M glycine] at 90 mA for 4 h to completion. Gels were fixed, stained with 0.1%
(wt/vol) Coomassie blue R-250 [dissolved in 12% (vol/vol) acetic acid, and 50%
(vol/vol) methanol] for 45 min and destained with 7% (vol/vol) acetic acid and 10%
(vol/vol) methanol for 48 h. PAGE was performed on the electrophoresis unit LKB-2001100 in connection with the power supply LKB-Macrodrive 5 and LKB-Multitemp as a
cooling unit (LKB, Sweden). Electrophoresis of tofu was performed in duplicate. Namely, two aliquots of the same sample were analyzed at the same time. Two gels were
run simultaneously in the same electrophoretic cell. The identification was done using 7S
and 11S protein fractions obtained according to the procedure of Than and Shibasaki (9).
Densitometric analysis. The destained gels were scanned and then analyzed by SigmaGel software version 1.1 (Jandel Scientific, San Rafael, CA). The quantitative estimation of each identified subunit was calculated as the percentage of the corresponding area
of the subunit with respect to the total area of the densitogram.
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Headspace gaschromatography (GC) analyses were performed on a Hewlett-Packard Model 5890 Series II GC, on an EC-5-capillary column (30m x 0,53mm) with film of
1.2 m. A volume of 25 ml of gaseous phase from 5% tofu dispersion, preheated to 45ºC
was analyzed. Helium flow was 3 ml/min. Electron-ionization detector, set on 35-350
m/z was used. Components identification was done by comparision whit appropriate
standards.
The total protein content in the samples was determined by the micro-Kjeldahl method (10). A nitrogen to protein conversion factor of 6.25 was used and calculated on the
dry matter basis. The total proteins content in defatted flours was 45.88% for Lana flour
and 48.83% for Balkan flour (11). Moisture content was determined by a standard AACC
procedure (12). Soybean seeds were dehulled and ground in a mill. The meal was then
defatted with n-hexane (meal:n-heksan=1:20 wt/V) for 2 hours, at room temperature and
air-dried. Tofu was defatted by the Folch method (13), by the extraction with methanol/chloroform mixture (methanol:chloroform = 1:20; tofu: methanol/chloroform mixture=1:10) for 2 hours, at room temperature and air-dried.
Sensory evaluation. The sensory quality of tofu was evaluated by point system (from
1 to 5). Estimate was done by a panel consisting of 5 members. They gave the average
marks of tofu quality and average corrected mark of sensory characteristics. The average
mark of tofu quality is the average value of 6 quality parameters sum (smell, taste, color,
cut, consistency and general appearance of tofu). Average mark corrected for quality
parameter significance was obtained by dividing the corrected mark with the number of
panel members. The corrected mark was obtained by multiplying marks for single tofu
quality parameter with the appropriate coefficient of importance (for the appearance of
tofu, consistency and cut - coefficient is 2; for color and smell - coefficient is 3; for taste coefficient is 8). Then that sum of the results was divided with 20 (sum of coefficients).
The mark for smell and taste is an average sum of marks for smell and mark for taste.
Samples for sensory evaluation were stored at 4ºC and warmed to room temperature before evaluation. Tofu was cut into cubic samples (~6 cm  6 cm  4.5 cm) and placed on
a plastic plate. Replicated samples were evaluated on different days.
Statistical analysis. Experiments were performed in triplicates, except for electrophoretic analysis, which was carried out in duplicates. The data were analyzed using Statistical software version 5.0 (StatSoft Co., Tulsa, OK).
The way of tofu preparation essentially determines the content and composition of
proteins in the final product, which has a direct impact on its application in nutrition.
Considering that in this study hydrothermal cooking process, which is significantly different from the traditional way of preparation of tofu, as well as coagulation of proteins by
enzyme coagulant (rennin and pepsin) was used, the changes in content and in composition of major proteins could be expected. It was expected that the applied method of tofu
modification (addition of gallic acid) would lead to the changes both in composition of
manufactured product and its sensory characteristics.
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The investigated genotypes are characterized by a relatively high content of total
proteins in defatted flour (Table 1) (11) which indicates that they are suitable for the use
in production of protein products. The high content of proteins in grain is very important,
given that, the quality and production of tofu directly depend on the characteristics of
soybean as a starting material and conditions of production (14). In addition to high content of total proteins in seeds, the investigated varieties are characterized by a favorable
content of soluble proteins, which results in very high solubility (Table 1). Due to the
favorable total protein content of soybeans, the prepared tofu is characterized by an extremely high content of total proteins (52.17% - Lana tofu and 56.08% - the Balkans tofu;
Table 2).
Table 1. Total nitrogen and protein content and soluble protein content of soybean
defatted flour (11)
Content (%)
total nitrogen
total protein
soluble protein
solubility
moisture
Lana
7.34±0.05
45.88±0.33
23.33±0.41
51.10±1.02
15.03
Balkan
7.81±0.03
48.83±0.19
27.90±0.02
57.14±0.18
11.09
Table 2. Total and soluble protein content of tofu
Content
(% d.b.)
total protein
soluble protein
solubility
Lana - tofu
without
with gallic
gallic acid
acid
52.17±0.3
52.18±0.10
14.20±0.06
27.48±0.30
27.22
51.52
Balkan -tofu
without
with gallic acid
gallic acid
56.08±0.67
65.07±0.19
28.88±0.08
41.55±0.32
52.66
63.85
The procedure of addition of gallic acid does not significantly change total protein
content but it affects the content of soluble proteins. Specifically, soluble protein content
in tofu prepared with the addition of gallic acid is significantly different from the samples
without it. This might be explained by acidic hydrolysis of tofu proteins by gallic acid,
during which smaller molecular weight protein molecules are formed, that increases the
content of soluble proteins.
The produced tofu shows significant cultivar differences in terms of the content of
soluble proteins, where much higher content of soluble proteins is registered in the tofu
prepared from Balkan variety. Proteins of the Balkan tofu are characterized by higher
protein solubility compared to the protein solubility of Lana tofu Table 2). These data
confirm that in addition to the technological process, the variety and quality of soybean
significantly affect the characteristics of manufactured products.
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By electrophoresis separation of soybean flour protein extracts the major protein
bands, with the following Rf-values: 0.01; 0.03; 0.08; 0.16 and 0.26 are registered. The
remaining part is made of minor components and proteins belonging to the protein fraction 2S (Figure 1).
Figure 1. (A) Densitometric analysis of PAG electrophoregrams of soluble soybean flour
and PAG electrophoregrams of soluble tofu proteins made without (B) and with (C) gallic
acid
Right at the entrance into the gel a protein band is registered. It corresponds to the polymorphous form of β-conglycinin (Bo) and high molecular weight polymers of the dominant protein fraction (Rf=0.01). Both varieties are characterized by a high content of
protein polymers (31.70% for Lana and 36.68% for Balkan). Such high percentage of this
protein fraction indicates a high degree of association of the dominant proteins, so the
part of the protein fraction which corresponds to -conglycinin simply „remained trapped“ at the entrance into the gel. Then follows the zone of β-conglycinin (Rf=0.03) and
glycinin zone, which is characterized by two protein bands. The band with smaller electrophoresis mobility corresponds to the glycinin dimer form, while the next band corresponds to the monomeric form of the molecule (Table 3). In the Ornstein-Davis discontinuous electrophoresis system, -conglycinin migrates more slowly than glycinin, while glycinin has a higher relative mobility in comparision to glycinin. Based on that fact, the
band with Rf-value of 0.03 was identified as β-conglycinin, while the band with Rf-value
0.26 corresponds to -glycinin.
The key components of the 7S fraction represent 10.11% to 13.00% of flour soluble
proteins; β-conglycinin is present with 3.68-4.29%, and  - conglycinin with 5.82-9.40%.
The values obtained for the participation of  - conglycinin in soluble proteins of flour
correspond to the obtained literature data while the literature data indicate a much larger
share of β-conglycinin in soluble proteins of flour (15). However, in this study, a large
quantity of polymorphic forms of β-conglycinin was registered.
Based on the presented data (Table 3) it is clear that there are differences between the
genotypes regarding participation of major proteins. The most notable difference is in the
participation of monomeric and dimeric forms of glycinin.
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Table 3. Soluble protein composition of soy flours (%)
Protein
Bo and large molecule polimers
β – conglycinin
glycinin dimeric
glycinin monomeric
Rf = 0.21
γ – conglycinin
Rf = 0.32 - 0.47
Rf
0.01
0.03
0.08
0.16
0.26
Lana
31.7±0.05
4.29±0.12
8.59±0.55
17.2±0.11
8.61±0.10
5.82±0.19
7.15±0.06
Balkan
36.68±0.48
3.60±0.01
3.24±0.03
18.76±0.55
5.86±0.05
9.40±0.22
4.86±0.05
The results obtained by densitometry of polyacrylamide gel electrophoresis of tofu, in
native conditions, indicate a very low solubility of tofu proteins (Figure 1). Namely, in
the analyzed cultivars, the presence of only one or two protein bands is identified (Table
4). Different solubility of tofu proteins under the conditions of PAGE may indicate the
different nature of the links (which are primarily characterized by different strength) in
the gel structure obtained from the tested varieties. These results showed that the solubility of the major tofu proteins is very low, indicating their very strong incorporation into
the gel matrix. This can lead to the conclusion that in the formation of the gel solid participate covalent bonds (such as disulfide bonds).
By PAGE of the extract of the tofu prepared with the addition of gallic acid a large
number of components is separated, which cannot be observed on the electrophoregrams
of unmodified tofu (Figure 1). It is obvious that the addition of galic acid led to both
hydrolysis and changes in surface properties of major proteins resulting in increase of
their solubility. Right at the entrance into the gel is recorded a much higher amount of
high molecular weight proteins in Balkan tofu (74.96%) then Lana tofu (9.64%). On the
other hand, Lana tofu has shown a significant content of glycinin dimeric form (57.43%),
while in the Balkan tofu this component was present in traces (0.69%, Table 4). Such
high content of Bo-isoform in β-conglicinin in Balkan tofu can be explained by a significant decrease in solubility of the other protein components, as evidenced by low values
of their contents, some of which being found in traces.
Table 4. Soluble protein composition of tofu (%)
Protein
Bo and largemolecule
polimers
β – conglycinin
glycinin dimeric
glycinin monomeric
γ – conglycinin
Rf = 0.28
Lana tofu
without
with gallic
gallic acid
acid
Balkan tofu
without
with gallic
gallic acid
acid
0.01
27.86±0.03
9.64 ±0.47
27.86±0.03
74.96 ±0.49
0.03
0.08
0.16
0.26




66.76±0.02
6.28 ±0.23
57.43 ±1. 89
7.97 ±0.1
4.07 ±0.14
2.10 ±0.03





5.82 ±0.03
0.69 ±0.01
0.17 ±0.01
4.64 ±0.07
0.17 ±0.01
Rf
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In Lana tofu, a significantly increased solubility of the 11S fraction is registered,
where the compact diametric form of glycinin is dominant. In terms of γ-conglycinin
content, there are no differences among the varieties. On the electrophoregrams of these
samples a diffuse zone is revealed with very close Rf values to that of γ-conglycinin
(Rf=0.28). It can be assumed that a protein fraction of very similar molecular weights γconglycinin was formed by acidic hydrolysis. This protein fraction, as well as other major
protein components, was not registered in the samples of tofu prepared without gallic
acid (Table 4). Therefore, it can be concluded that the presence of gallic acid significantly
affects the structure of tofu protein.
Figure 2. Gas-chromatograms of tofu made with gallic acid of Lana (A) and Balkan (B)
soy genotypes
Gallic acid is an excellent antioxidant, and it has been added in the preparation of tofu
primarily to reduce the unwanted odor of legumes (16). This objective was achieved,
taking into account that, GC showed only several components registering acetaldehyde as
major volatile (Fig. 2). The improvement of sensory characteristics is also confirmed by
favorable sensor evaluation grades given to the samples prepared with the addition of
gallic acid (Table 5).
Table 5. Results of tofu sensory evaluation (the mean marks of tofu quality)
Genotypes
the category of tofu quality
smell and taste
the average
quality
marks
corrected mark
Lana
Balkan
without gallic acid
3.25±0.07
2.66±0.13
3.43±0.19
3.38±0.62
3.15±0.40
3.26±0.65
Lana
Balkan
with gallic acid
3.30±0.11
3.64±0.13
3.65±0.31
3.87±0.17
3.55±0.55
3.86±0.26
CONCLUSION
The applied technological process of production has a significant impact on the protein content and composition of storage of soy proteins in tofu, as well as on its sensory
characteristics. The supplement of gallic acid in the production of tofu, in addition to
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improving the nutritional characteristics, reduces significantly the so-called leguminous
smell and taste of produced cheese, as confirmed by gas chromatographic analysis of tofu
volatiles, since mainly acetaldehyde was registered. In addition to improvement of the
nutritional and sensory properties, gallic acid significantly improves the solubility of the
main protein components of tofu. For this study the tofu was prepared from local varieties of soybean, which are not selected as so-called “tofu varieties”, but they are very
suitable for the tofu production since both of them gave a high-protein product. Because
of its exceptional sensory and nutritive values, the tofu might be accepted by larger number of consumers.
Acknowledgement
This investigation was supported by the Ministry of Science and Technological Development of the Republic of Serbia. The authors are indebted to the Institute of Field
and Vegetable Crops, Novi Sad, Serbia, and the Maize Research Institute, Zemun Polje,
Serbia, for providing soybean genotypes.
REFERENCES
1. H.J. Wang and P.A. Murphy: Isoflavone content in commercial soybean foods. J.
Agric. Food Chem. 42 (1994) 1666-1673.
2. C. Wang, A.L. Johnson and A.L. Wilson: Calcium coagulation properties of
hydrothermally processed soymilk. J. Am. Oil Chem. Soc. 80, 12 (2003) 1225-1229.
3. W.J. Wolf, E-R. Peterson and L.M. Schaer: Preparative reversed-phase high-performance liquid chromatography of soybean proteins and characterization of fractions
by gel. J. Agric. Food Chem. 40 (1992) 1809-1816.
4. S. Stanojevic, M. Barac, B. Vucelic-Radovic, M. Pesic, and S. Jovanovic: Yield and
quality of soybean milk and tofu as affected by production method. Food Industry, 17
(2006) 57-64.
5. S. Stanojevic, M. Barac, B. Vucelic-Radovic and M. Pesic: Sensory properties of tofu
as affected by production method. Hotellink. 12 (2008) 431-439.
6. M.M. Bradford: A rapid and sensitive method for the quantitation of microgram
quatities of protein utilizing the principle of protein- dye binding. Anal. Biochem. 72
(1976) 248-254.
7. A.K. Khatib, T.J. Herald, F.M. Armouni, F. MacRitchie and W.T. Schapaugh: Characterization and functional properties of soy -conglycinin and glycinin of selected
genotypes. J. of Food Science. 67, 8 (2002) 2923-2929.
8. J. Davis and L. Ornstein: Disc electrophoresis, background and theory. Ann. N.Y
Acid. Sci. 121 (1964) 321.
9. V.H. Than and K. Shibasaki: Major proteins of soybean seeds. A straightforward
fractionation and their fractionation. J. Agric. Food Chem. 24 (1976) 1117.
10. Crude Protein-micro Kjeldahl method. In Approved methods of the AACC (Vol-II,
10th ed.) (2000-a) AACC method 46-13.
85
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DOI: 10.2298/APT1041077S
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BIBLID: 1450-7188 (2010) 41, 77-86
11. M. Pesic, B.Vucelic-Radovic, M. Barac and S. Stanojević: The influence of genotypic
variation in protein composition on emulsifying properties of soy proteins. J. Am. Oil
Chem. Soc. 82, 9 (2005) 667-672.
12. Moisture and volatile matter in soy flours. In Approved methods of the AACC (VolII, 10th ed.) (2000-b) AACC method 44-31.
13. W.W. Christie: Extraction of lipids from samples. Lipid Technology. 5 (1993) 18-19.
14. M. Tezuka, H. Taira, Y. Igarashi, K. Yagasaki and T. Ono: Properties of tofu and soy
milks prepared from soybeans having different of glycinin. J.Agric. Food Chem. 48
(2000) 1111-1117.
15. M. Barać, S. Stanojević, S. Jovanović and M. Pešić: Soy protein modification – A
REVIEW. Acta Periodica Technologica (APTEFF) 35 (2004) 3-16.
16. W.L. Boatright: Effect of gallic acid on the aroma constituents of soymilk and soy
protein isolates, J. Am. Oil Chem. Soc. 79, 4 (2002) 317-323.
ПРОТЕИНСКИ САСТАВ ТОФУА КОРИГОВАНОГ КВАЛИТЕТА
Слађана П. Станојевић, Мирољуб Б. Бараћ, Мирјана Б. Пешић, Мирјана M.
Миловановић и Биљана В. Вуцелић-Радовић
Тофу је желирани протеински производ, који се добија коагулацијом загрејаног
сојиног млека. Потрошачима наше популације не одговара арома „зеленог зрна“,
такозвани „легуминозни мирис и укус“, који се осећа у производима соје припремљеним на традиционални начин. Са намером побољшања ароме добијених сирева,
припремљен је тофу применом хидротермичког третмана и ензима за коагулацију
млека (химозин-пепсин) уз додатак галне киселина, која је јак антисептик и антиоксидант.
Захваљујући високом садржају укупних протеина сојиног семена (45,88-48,83%
у обезмашћеном брашну) и припремљени тофу се карактеришe високим садржајем
укупних протеина (52,17% с.м.- Лана тофу и 65,08 % с.м. - Балкан тофу). Поступак
додатка галне киселине мења садржај растворљивих протеина (27,48% - Лана тофу
и 41,55% - Балкан тофу) који се значајно разликује у односу на узорке без ње
(14,20% - Лана тофу и 28,88% - Балкан тофу). Полиакриламидном-гел електрофорезом екстракта тофуа припремљеног уз додатак галне киселине раздвојен је већи
број компонената, које се не уочавају на електрофореграмима тофуа немодификованог квалитета.
Гаснохроматографском анализом испарљивих компонената тофуа регистрован
је углавном ацеталдехид, тако да је постигнут циљ ублажавања легулинозног мириса. Побољшање сензорних карактеристика потврђују и повољне сензорне оцене
које су добили узорци припремљени са галном киселином (3,65 – Лана тофу и 3,87
- Балкан тофу).
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INFLUENCE OF PARTICLE DIAMETER ON THE COLOUR OF GROUND
PEPPER (Capsicum annuum L.)
Aleksandra N. Tepić, Zdravko, M. Šumić, Mirjana B. Vukan
The influence of particle diameter of ground paprika on its colour was examined in
this study. Six samples of industrial ground paprika, from various phases of milling, and
different particle size were purchased from the factory. Extractable colour (ASTA) and
surface colour (L, a*, b*, C, λ), as the most important colour indicators, were measured.
Increasing the grinding level caused the increase of ASTA and red coordinate (a*) value,
and decrease of yellow coordinate (b*) value, as well as lightness of the samples, as a
consequence of the increase of the specific surface area of sample particles.
KEYWORDS: Ground paprika, ASTA, surface colour, particle size
INTRODUCTION
Paprika (Capsicum annuum L.) is one of the most important crops in the world and in
Serbia. Apart from its use as raw vegetable, there are numerous food, pharmaceutical and
cosmetic products containing paprika, or some of its constituents.
During industrial production of ground paprika, dried paprika is coarsely milled using
hammer mills, to particles up to 2-3 mm in diameter. In order to produce powder, these
particles are further milled using stone mills. At the end of the process, the finest powder is
obtained by sieving ground paprika through the sieve.
The influence of fruit maturity, pre-treatment applied, drying conditions, methods and
kinetics on the quality of ground pepper had been the subject of interest to many authors (16).
The main quality parameter of ground spice paprika is its colour. It can be defined from
three different aspects: extractable colour, surface colour, and carotenoid profile.
The most common and very simple method for the determination of paprika colour is
the spectrophotometrical measurement of its extractable colour. It can be measured according to several procedures, described by Association of Official Analytical Chemists (7, 8)
and the American Spice Trade Association (9).
The colour of the specified product depends much on the variety, growing conditions,
dehydration and storage conditions, and powder coarseness as well. However, the personal perception of colour varies in dependence of the sensitivity of the eye, the size of the
Dr Aleksandra Tepić, assistant professor, [email protected]; Zdravko Šumić, BSc; Mirjana Vukan, BSc, University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
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object being viewed, background colour, illumination, etc. (10). Since the verbal description of colour could be difficult and confusing, its numeric quantification is of great use.
Surface colour represents the human visual perception of the colour.
A system that is most commonly used for colour measurements is the CIELAB system, established by the International Commision on Illumination (11). Sinc the CIELab
defines colours more closely to the human colour perception, this system is often used in
the quality control of coloured products. Three colour coordinates for the determination
of colour are lightness (L*), red/green coordinate (a*), yellow/blue coordinate (b*). Hue
angle (h°) and chroma (C) can be calculated from a* and b*, and they are simpler to conceptualize (10). Hue, as the arctangent b*/a* defines the kind of colour. Samples with
h°=0 are purplish-red, 90° are yellow, 180° - bluish-green and 270° - blue. Paprika samples usually have hue angle values from 30 to 45° (red to orange range on the colour
wheel). Chroma is a measure of colour saturation. The higher the chroma, the more vivid
the sample (10).
Apart from variety, degree of maturity, drying and storage conditions, particle size
plays an important role in paprika colour perception.
The aim of this work was to investigate the effect of particle size on the extractable
and surface colour of paprika.
EXPERIMENTAL
Six samples of commercial ground paprika, harvested in 2009, were obtained from
Aleva a.d. company, Novi Kneževac, the most important producer of ground paprika in
Serbia. Sample No.1 was coarsely milled, samples 2-6 were taken from different stages
of milling, and sample 6 was the final product, the finest powder.
The mean diameter of the particles was measured by sieving the sample through
Ewreka sieves (Germany) (12). The paprika that remained on the sieve was measured,
and the rest was further sieved through the sieve of smaller porosity. The mean diameter
of particles was calculated as follows:
m 
100
   i 
d
 di 
where: mi - mass percent of fraction i; di - mean diameter of the i fraction
Extractable colour measurements. Extractable colour was measured according to
ASTA 20 method (9). An amount of 0.07 – 0.11 g of paprika powder was put into a
graduated 100 ml flask. Aceton was added to the mark, the mixture was shaken and kept
in the dark for 4h. An aliquot of the transparent decanted extract was taken. The absorbance of the solution was measured using JENWAY 6105 UV/VIS spectrophotometer,
set at 460 nm and calibrated with an acetone blanc. The ASTA 20 colour units were
calculated from:
ASTA 20 
88
Absorbance  16.4  If
weight of sample  g 
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where If is a correction factor for the apparatus, which is calculated from the absorbance
of a standard solution of potassium dichromate, ammonium sulphate and cobalt sulphate.
Apparent (surface) colour measurements. Surface colour was measured using Minolta
Chromameter CR 400. The values L, a*, b*, h°, c and λ were determined.
All determinations were done in three replicates, and the results are presented as
mean values. SD values were determined using Excel 2003.
The quality of ground paprika is mainly determined by its seasoning power – capsaicin content (13), its natural colour, its particle size and water content (14, 15). The particle sizes of examined samples of ground paprika are presented in Table 1.
Table 1. Particle size of examined ground paprika samples
Sample
1
2
3
4
5
6
Mean diameter, d (mm)
0.479
0.339
0.312
0.244
0.229
0.220
Oil seed gives ground paprika fine, shiny and intensive colour, and some stability due
to the presence of natural antioxidants as well (16).
One of the most important indicators of paprika quality is its ASTA value. The ASTA
value dependence on the particle diameter is given in Figure 1.
90
80
ASTA
70
60
50
40
30
0.45
0.40
0.35
0.30
0.25
0.20
d (mm)
Figure 1. Effect of particle size on the extractable colour of ground paprika
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As can be seen, decreesing the particle size from 0.479 to 0.220 mm (2.17 fold) caused an increase in the ASTA value from 31.10 to 86.95 units (2.8 fold). This trend is a direct consequence of particle size change, i.e. of the increase in the particle specific surface, which caused better and more intensive colour extraction. The greatest difference
was noticed between samples 1 and 2, probably due to the largest increase in specific surface. Moreover, it should also be emphasized that ASTA value of the finest powder,
86.95 ± 0.916, which is still acceptable, was considerably lower than the values reported
by other authors (17). The reason for a relatively low extractable colour lies in the fact
that raw material used for its production was stored for almost a year, and it is well
known that many factors affect colour loss in paprika during the storage, with carotenoid
degradation as the most important, caused by exposure to heat, light or oxygen.
Visual evaluation of the product, besides extractable colour, is of fundamental interest, since it affects consumer's acceptability of the product and determines the real commercial value of the product (18). Sample with highest particle diameter was the lightest
(46.99 ± 0.229). Other samples, having smaller particle sizes, were darker (42.66 ± 0.456
- 43.62 ± 0.286), indicating that the increase of specific surface caused samples darkening
to some degree. The tested samples were somewhat lighter than reported in other studies
(17, 19, 20). The red/green coordinate (a) values were in the range (23.82 ± 0.569)-(31.06
± 0.421), and slightly differed in samples 4-6. The yellow/blue coordinate values were
highest in sample 1, with the smallest particle size (35.25 ± 0.20), and slightly differed
between samples 2-6. Changes in L, a* and b* values indicate that the decrease of the
ground paprika particle size causes the darkening of the samples, decrease of yellow, and
increase of red component at the same time (Figure 2).
45
L, a*, b*
40
35
30
L
a*
b*
25
0.45
0.40
0.35
0.30
0.25
0.20
d (mm)
Figure 2. Influence of particle size on lightness, a* and b*
As reported Wall and Bosland (10), hue angles of ground paprika are in the range of
30-45°. According to their statement, it is obvious that h° of ground paprika samples examined in this research were closer to yellow in the colour wheel of the CIELab system.
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60
56
52
h (°)
48
44
40
36
32
0.45
0.40
0.35
0.30
0.25
0.20
d (mm)
Figure 3. Influence of particle size on hue angle of ground paprika
On the basis of trichromatic coefficients, a* and b* values, chroma (C) and dominant
wavelength were calculated (Table 2). According to Wall and Bosland (10), samples 4-6
were more vivid. All samples had dominant wavelength in the range of orange colour.
Table 2. Chroma and dominant wavelength in paprika samples with different
particle size
Sample
1
2
3
4
5
6
C
42.54 ± 0.311
42.17 ± 0.244
42.08 ± 0.749
43.13 ± 0.406
44.26 ± 0.337
43.26 ± 0.312
λ
591.3 ± 4.163
593.2 ± 0.289
593.9 ± 0.115
595.3 ± 0.289
595.1 ± 0.321
595.2 ± 0.529
As valid Serbian Regulations for ground paprika demand measuring the extractable
colour of samples as the most important quality defining parameter, and as Duncans's
multiple range test showed that there was a significant difference between ASTA for
samples 5 and 6, grinding of dried paprika to particle size of 0.222 is completely justified.
CONCLUSION
Based on the results of this research, it can be stated that the decrease of ground paprika particle size caused better and more intensive colour extraction, and as a conesquence, the increase of extractable colour (ASTA). The increase of grinding level, i.e. the
decrease of particle diameter, also caused a decrease of the yellow, and increase of red
coordinate, as well as the darkening of the sample, making samples with smaller particle
size more acceptable from a sensory point of view.
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Acknowledgement
This work is a part of the Project No 114-451-02503/2010, financially supported by
the Provincial Secretariat for Science and Technological Development of Vojvodina.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
92
İ. Doymaz and M. Pala: Hot-air drying characteristics of red pepper. J. Food Eng. 55
(2002) 331-335.
V. Németh Kiss, T. Cserháti, E. Forgács, J. Holló and T. P. Mota: Stability of pigments of paprika (Capsicum annuum) powders during various processing steps. Pol.
J. Food Nutr. Sci.. 5 / 46, 3 (1996) 81-87.
E. Kavak Akpinar, Y. Bicer and C. Yildiz: Thin layer drying of red pepper. J. Food
Eng. 59 (2003) 99-104.
M. I. Mínguez-Mosquera, M. Jarén-Galán and J. Garrido-Fernández: Influence of
the industrial drying processes of pepper fruits (Capsicum annuum Cv. Bola) for paprika
on the carotenoid content. J. Agric. Food Chem. 42, 5 (1994) 1190-1193.
M. I. Mínguez-Mosquera and D. Hornero-Méndez: Changes in provitamin A during
paprika processing. J. Food Prot. 60, 7 (1997) 853-857.
H. G. Daood, M. Vinkler, F. Márkus, E. A. Hebshi and P. A. Biach: Antioxidant vitamin
content of spice red pepper (paprika) as affected by technological and varietal factors.
Food Chem. 55, 4 (1996) 365-372.
Assoc. Off. Anal. Chem. (AOAC). Official Methods Analysis. Virginia: AOAC
(1984).
J. E. Woodburry. J. Assoc. Anal. Chem. 60 (1977) 1-4.
Amer Spice Trade Assoc (ASTA). Official Analytical Methods. New Jersey: ASTA
(1985)
M. Wall and P. W. Bosland: Analytical methods for color and pungency of chiles
(capsicums), in Instrumental Methods in Food and Beverage Analysis, Eds. Wetzel
D. and Charalambous G., Elsevier Science B. V., (1998) pp. 347-373.
Commission Internationale de l'Eclairage (CIE). Supplement 2, CIE Publ 15
(E/1.3.1) Paris: Bureau Centrale CIE, 1978.
Ž. Lepojević: Praktikum hemije i tehnologije farmaceutskih proizvoda, Tehnološki
fakultet Novi Sad (2001).
A. Aczél: Comparison between HPTLC and OPLC for the determination of capsaicin in red pepper. Fres. J. Anal. Chem. 343 (1992) 154-157.
D. Dimitrov, R. Tenov and I. Stanchev: Effect of artificial moistering and oil content
in colour stability of ground pepper. Nauchni Trudove Nauchnoizaledvatelski institut po konservna Prommishlenost Plovdiv. 6 (1969) 79-82.
J. Kanner, S. Harel, D. Palevitch and I. Ben-Gera: Colour retention in sweet red
paprika (Capsicum annuum L.) powder as affected by moisture contents and
ripening stage. J. Food Technol. 12 (1977) 59-62.
M. I. Mínguez-Mosquera, A. Pérez-Gálvez and J. Garrido-Fernández: Carotenoid
content of the varieties Jaranda and Jariza (Capsicum annuum L.) and response
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041087T
17.
18.
19.
20.
UDC: 664.521:621.927.082/.086
BIBLID: 1450-7188 (2010) 41, 87-93
during the industrial slow drying and grinding steps in paprika processing. J. Agric.
Food Chem. 48, 7 (2000) 2972-2976.
A. Tepić: Promena boje začinske mlevene paprike i zaštitino delovanje antioksidanasa. M. Sc. thesis. Tehnološki fakultet Novi Sad (2005).
J. M. Nieto-Sandoval, J. A. Fernández-López, L. Almela and J. A. Muñoz: Dependence between apparent color and extractable color in paprika. Color Res. Appl. 24,
2 (1999) 93-97.
N. A. Tepić and L. B. Vujičić: Colour change in pepper (Capsicum annuum L.) during storage. APTEFF 35 (2004) 59-64.
O. Lj. Vračar, N. A. Tepić, L. B. Vujičić and S. Šolaja: Influence of the heat treatment on the colour of ground pepper (Capsicum annuum). APTEFF 38 (2007) 5358.
УТИЦАЈ СТЕПЕНА УСИТЊЕНОСТИ НА БОЈУ МЛЕВЕНЕ ЗАЧИНСКЕ
ПАПРИКЕ (Capsicum annuum L.)
Александра Н. Тепић, Здравко М. Шумић, Мирјана Б. Вукан
У раду је испитан утицај пречника честица млевене зачинске паприке на боју.
Из фабрике је набављено шест узорака индустријске паприке из различитих фаза
прераде (млевења), различитих величина честица. Испитани су садржај екстраховане боје (ASTA) и површинска боја (L, a*, b*, c, λ), као најважнији параметри боје.
Повећање степена уситњености изазвало је пораст ASTA вредности и удела црвене
боје (а*) и истовремено смањење удела жуте боје (b*), као и светлоће узорака, као
последица повећања специфичне површине честица испитиваних узорака.
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DETERMINATION OF NICKEL CONTENT IN THE SEMIMEMBRANOSUS
MUSCLE OF PIGS PRODUCED IN VOJVODINA
Vladimir M. Tomović, Ljiljana S. Petrović, Žarko S. Kevrešan, Natalija R. Džinić
and Marija R. Jokanović
The content of nickel was investigated in the M. semimembranosus of sixty-nine pigs
from ten different genetic lines produced in Vojvodina. Nickel was determined by flame
atomic absorption spectrometry after mineralization by dry ashing. The difference in the
nickel content in the analyzed muscle tissues among different genetic lines of pigs was not
significant (P > 0.05). Nickel levels ranged from 12.93 to 80.18 μg/100 g, with a general
average of 32.41 μg/100 g. The average level of nickel was found to be higher than the
levels observed in pork in some developed countries.
KEYWORDS: Nickel, M. semimembranosus, Pigs
INTRODUCTION
Meat quality is the sum of all sensoric, nutritive, hygienic-toxicological and technological factors of meat. The nutritive factors of meat quality comprise proteins and their
composition, fats and their composition, vitamins, minerals, utilization, digestibility and
biological value. The hygienic-toxicological factors of meat quality comprise microorganisms (bacteria, spores, moulds), shelf life (pH value, redox potential, water activity - aw
value, temperature of storage), residues (antibiotics, hormones, other pharmaceuticals)
and contaminants (pesticides, mycotoxins, heavy metals, nuclides) (1-3).
Red meat (beef, veal, pork and lamb) contains high biological value protein and
important micronutrients including iron, zinc and vitamin B12, all of which are essential
for good health throughout life (4-7). Also, meat contains useful amounts of copper,
magnesium, cobalt, phosphorus, chromium and nickel (7).
Nickel is a silvery-white, hard metal. Although it forms compounds in several oxidation states, the divalent ion seems to be the most important for both organic and inorganic substances, but the trivalent form may be generated by redox reactions in the cell
(8). Nickel is widely distributed in nature, forming about 0.008% of the earth’s crust. The
core of the earth contains 8.5% nickel, deep-sea nodules 1.5%; meteorites have been
found to contain 5-50% nickel (9). Agricultural soils contain nickel at levels of 3-1000
Dr. Vladimir M. Tomović, Assist. Prof., [email protected], Dr. Ljiljana S. Petrović, Full Prof., Dr. Natalija R.
Džinić, Assist. Prof., Marija R. Jokanović, B.Sc., Assist., Faculty of Technology, University of Novi Sad,
Bulevar cara Lazara 1, 21000 Novi Sad, Serbia, Dr. Žarko S. Kevrešan, Research Associate, Institute for Food
Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
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mg/kg; in 78 forest floor samples from the northeastern United States of America,
concentrations of 8.5-15 mg/kg were reported (10). The nickel content is enriched in coal
and crude oil. Nickel in coals ranges up to 300 mg/kg; most samples contain less than
100 mg/kg, but there is a large variation by region (11). The nickel content of crude oils
is in the range <1–80 mg/kg (10, 12).
In most food products, the nickel content is less than 0.5 mg/kg fresh weight. Cacao
products and nuts may, however, contain as much as 10 and 3 mg/kg, respectively (9).
According to Solomons et al. (13), dry beans, cocoa products, baking soda, and some
nuts contain high levels of nickel (>2.0 μg/g); wheat and wheat products, shellfish, processed meats and many vegetables contain intermediate levels (0.2-2.0 μg/g); and whole
and dried milk, fresh fruits, meat and eggs contain low levels of nickel (<0.2 μg/g).
Nickel concentrations in drinking-water in European countries of 2-13 µg/liter have been
reported (14). Nickel contents of pork have been reported in several studies. Reported
data showed the nickel concentrations in pork were 5.5 μg/100g in Canada (15), 2.5
μg/100g in Denmark (16) and less than 1.6 μg/100g in Sweden (17). The highest reported
nickel concentration in pork was 35 μg/100g in Nigeria (18).
Nickel is present in a number of enzymes in plants and microorganisms. In humans,
nickel influences iron absorption and metabolism, and may be an essential component of
the haemopoietic process. COMA (Committee on Medical Aspects of Food and Nutrition
Policy) and FDA (US Food and Drag Administration) were unable to set recommended
amounts for nickel intake. Based on extrapolation from animal data, the hypothetical
human requirement for nickel would be 16 to 25 μg/1000 kcal or about 75 μg of elemental nickel per day (13). Nickel deficiency has not been observed in humans (19).
On the other hand, acute nickel exposure is associated with a variety of clinical
symptoms and signs which include gastrointestinal disturbances (nausea, vomiting, abdominal discomfort and diarrhoea), visual disturbance (temporary left homonymous hemianopia), headache, giddiness, wheezing and cough. Approximately 7-10% of the population (predominately women) are affected by nickel allergic dermatitis. There is evidence
suggesting that nickel ingestion may contribute to the exacerbation of eczema in sensitised individuals (19).
The lowest reported oral dose associated with acute effects in humans was 0.05
mg/kg bw (1.2 mg in a 60 kg adult) (19). Total diet studies indicate a total average oral
intake of 200-300 µg/day (10). Early estimates of daily nickel consumption in the USA
ranged from 300 to 600 µg (20). Recovery studies indicate an absorption rate of less than
15% from the gastrointestinal tract (21). Dietary intake of nickel in food is not expected
to result in harmful effects (19).
On the basis of the available data from the Statistical Office of the Republic of Serbia,
the total consumption of meat in Serbia is 43 kg/p/yr. Pig meat is the most widely
consumed meat in the EU, as well as in Serbia, and the consumption has been steadily
increasing (4). The Autonomous Province of Vojvodina (the northern part of the Republic of Serbia) is a region where the number of animals of the porcine species and the
production of pork meat are of high economic importance. Most studies have focused on
the proximate compositions, vitamins and other essential nutrients. In this sense, the aims
of this work were: (i) to determine the content of nickel in pork muscle tissues (M. semimembranosus); (ii) to investigate the potential difference in content of nickel between ten
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different genetic lines of pigs (two pure and eight crossbred pigs) used nowadays in
Vojvodina for commercial pork production; (iii) to compare the results of our study with
results found in other studies, i.e. other countries, for M. semimembranosus (ham).
EXPERIMENTAL
Animals, sampling and preparing
The pigs used in the present study were produced in a pig (cross)breeding programme
provided by nucleus and multiplication farms in Vojvodina (GGP-GP traditional pyramid
structure of genetic programme) (22). In this breeding programme, five pig purebreds
were used. The Large White (LW) and Landrace (L) were used as female lines and Duroc
(D), Hampshire (H) and Pietrain (P), were used as male lines. The investigation was performed on sixty-nine pigs (castrates males and females) from ten different genetic lines
(two purebred and eight crossbred pigs): [LW, n = 8; L, n = 7; LWxL, n = 7; LxLW, n =
6; Dx(LWxL), n = 7; Dx(LxLW), n = 6; (DxP)x(LWxL), n = 8; (DxP)x(LxLW), n = 7;
(HxP)x(LWxL), n = 6; (HxP)x(LxLW), n = 7].
The pigs were fattened at the ten biggest production farms in Vojvodina. The pig
fattening involved the following phases: starting period (from 15 to 25 kg), growing
period (from 25 to 60 kg) and finishing period (from 60 to 110 kg). The diets were based
on corn and soybean meals, and were formulated to meet the nutrient requirements (23)
for the different growth phases. The finishers were housed in pens with fully slatted floor
and 0.80 m2 space allocation per pig. Each pen contained 10 animals. The environmental
temperature in the building was 22°C. All pigs had ad libitum access to diet and water.
The pigs were randomly selected at an individual live weight between 95 and 110 kg
and about six months old. One pig from each genetic line was taken at every six mounts
from the same farm.
All the pigs were slaughtered in the two biggest Vojvodinian slaughterhouses according to routine procedure. Carcasses were conventionally chilled for 24 h in a chiller at 24°C. After chilling, M. semimembranosus (SM) was removed from the right hind leg of
each carcass. The meat samples were trimmed of visible adipose and connective tissue.
The samples for chemical analysis (approximately 250 g) taken after the homogenization
of the whole SM muscle, were vacuum packaged in polyethylene bags and stored at 40°C until analysis.
Analytical methods and quality control
The total ash content was determined according to ISO method (24).
The nickel content of the meat was determined after dry ashing mineralization
according to the following procedure (25): a twenty-gram sample was weighed into a
porcelain crucible and dried in a laboratory oven at 105°C for 3 h. After drying the sample was charred on a hot plate and then incinerated in a muffle furnace at 450°C overnight (16 h). If necessary, the ash was bleached with nitric acid/deionized water (1:2,
v/v), evaporated to dryness and heated in the muffle furnace for 1 h. When a suitable ash
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was obtained it was moistened with a small amount of water, treated with 10 ml of hydrochloric acid/deionized water (1:1, v/v) and evaporated to dryness. Finally, the ash was
redissolved in 10 ml of hydrochloric acid/deionized water (1:9, v/v), transferred into a 25
ml volumetric flask and diluted to volume with deionized water.
Nickel was measured in the ash solution by flame atomic absorption spectroscopy
according to the manufacturer's instructions (26). Measurement was made under optimized parameters given in Table 1.
Table 1. Parameters for the elemental measurement by AAS
Element
Ni
Wavelength (nm)
232.0 nm
Band width (nm)
0.2 nm
Flame
Air-acetylene
Sensitivity
0.1 μg/ml
A strict analytical quality control programme was employed during the study. The
quality control of the analytical measurements for ash was performed using the following
standard reference material (SRM): SMRD 2000 (Matrix meat reference material, National Food Administration, Uppsala, Sweden). For the determination of the Ni content
the SRM samples were spiked with three different concentrations of this element. The results of the analytical quality control programme are presented in Table 2. In every series
of samples, 2 blanks and 2 samples of standard reference material were included.
Table 2. Results of the analytical quality control programme (n = 8) used in the
determination of total ash and nickel in M. semimembranosus
Certified concentration (g/100g)
Recovery (%)
Limit of detection (μg/100g)
Total ash
2.65  0.10
100
Ni
103.2  5.21
12.5
All chemicals used in the sample treatments were of high purity grade (Suprapur,
Merck GmbH, Darmstadt, Germany) and deionized Milli-Q water was used throughout.
The porcelain crucible and glassware were cleaned prior to use by soaking overnight in
10% v/v HNO3 and rinsed with deionized Milli-Q water. Standard stock solution of
analyzed metal was prepared immediately before use by dilution (with deionized water)
of a 1000 mg/l standard solution.
All analyses were performed in duplicate.
Statistical analysis
All data are presented as mean, standard deviation (SD) and range (minimum and maximum concentrations). The analysis of variance (one-way ANOVA) was used to test the
hypothesis about differences between a number of mean values. The software package
STATISTICA 8.0 was used (27) for the analysis.
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The average content, standard deviation and range for the total ash and Ni in the
investigated samples of the muscle tissues (SM) of ten different genetic lines of pigs are
presented in Table 3.
Table 3. Total ash and nickel content in the M. semimembranosus from the pigs in
Vojvodina
Genetic line of pigs
LW (n = 8)
L (n = 7)
LWxL (n = 7)
LxLW (n = 6)
Dx(LWxL) (n = 7)
Dx(LxLW) (n = 6)
(DxP)x(LWxL) (n = 8)
(DxP)x(LxLW) (n = 7)
(HxP)x(LWxL) (n = 6)
(HxP)x(LxLW) (n = 7)
All animals (n = 69)
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Mean ± SD
Range
Total ash (g/100g)
1.06 ± 0.02
(1.01–1.07)
1.05 ± 0.02
(1.03–1.07)
1.03 ± 0.03
(1.00–1.07)
1.03 ± 0.03
(0.99–1.08)
1.06 ± 0.02
(1.03–1.08)
1.03 ± 0.02
(1.02–1.06)
1.03 ± 0.03
(0.99–1.06)
1.05 ± 0.02
(1.02–1.07)
1.04 ± 0.04
(0.98–1.08)
1.06 ± 0.02
(1.02–1.08)
1.04 ± 0.03
(0.98–1.08)
Ni (μg/100g)
30.76 ± 14.91
(16.05–59.03)
37.06 ± 23.00
(12.93–80.18)
23.26 ± 8.38
(15.09–39.33)
39.47 ± 8.35
(31.23–49.60)
36.71 ± 12.48
(23.94–59.33)
31.36 ± 9.35
(23.04–43.88)
26.92 ± 11.02
(14.03–41.84)
31.85 ± 18.56
(16.12–66.91)
29.05 ± 19.10
(13.12–58.41)
38.07 ± 28.29
(17.45–75.08)
32.41 ± 16.23
(12.93–80.18)
The average total ash content in the SM muscles (Table 3) was 1.04 g/100 g [ranging
from 0.98, genetic line of pigs: (HxP)x(LWxL), to 1.08 g/100 g, genetic line of pigs:
LxLW, Dx(LWxL), (HxP)x(LWxL) and (HxP)x(LxLW)]. The content of the total ash
found in the present study did not differ significantly (F = 1.433; P = 0.202) among the
SM muscle tissue belonging to different genetic lines of pigs. The average total ash
values for the SM muscle in the present study are in agreement with the reported values
in the literature (28, 29).
The content of nickel found in this study accounted for only 0.003% of the total ash
of the SM muscle (Table 3). The order of the genetic lines of pigs according to nickel
content in the SM muscle samples (Table 3) in μg/100 g was: LWxL < (DxP)x(LWxL) <
(HxP) x (LWxL) < LW < D x (LxLW) < (DxP) x (LxLW) < D x (LWxL) < L < (HxP) x
(LxLW) < LxLW. The content of nickel found in the present study did not differ significantly (F = 0.566; P = 0.816) among the SM muscle tissue belonging to the different
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genetic lines of pigs (Table 3). On the other hand, animals belonging to the same genetic
line, from the same farm, raised under the same conditions, given the same feed, and
slaughtered at the same age had Ni content in the SM muscle that could differ up to six
times (Table 3). The lowest, average and highest nickel content in the SM muscle was
12.93 (genetic line of pigs: L), 32.41 and 80.18 μg/100 g (genetic line of pigs: L),
respectively. According to Greenfield and Southgate (30), meat, as a biological material,
exhibits natural variations in the amounts of nutrients contained and the limits of natural
nutrient variation are not defined. The Serbian regulation (31), as well as EC regulation
(32), did not set maximum levels for Ni in any meat type.
The nickel levels obtained in pork in this study were higher than in pork (M. semimembranosus) of Canada: 5.5 μg/100g (15), Denmark: 2.5 μg/100g (16) and Sweden:
<1.6 μg/100g (17). Levels which are equal to those in Vojvodinian pork have been obtained in Nigerian pork (140 μg/100g dry weight, approximately 35 μg/100g wet weight)
(18). The relatively higher and non-uniform nickel levels in Vojvodinian pork indicate
nickel availability in local agricultural environment.
CONCLUSION
The results of the present investigation show that the content of nickel determined in
the M. semimembranosus of pigs was not influenced by the genetic lines. Compared with
developed countries, the nickel content in the SM muscle tissue of the pigs from Vojvodina is higher. In addition, the obtained nickel composition could be used to provide
regular nutrient compositional data of the pork meat in Serbia.
Acknowledgement
The research was financed by the Ministry of Science and Technological Development, the Republic of Serbia, project TR – 20037.
REFERENCES
1.
2.
3.
4.
5.
100
K. Hofmann: Definition and measurements of meat quality, 36th International Congress of Meat Science and Technology, Havana, Cuba, August 27 – September 1
1990, Proceedings, Vol. III, pp. 941-954.
K.O. Honikel: Biochemical and physico-chemical characteristics of meat quality.
Meat Technology, 40, 3-5 (1999) 105-123.
V. Olsson and J. Pickova: The influence of production systems on meat quality, with
emphasis on pork. Ambio, 34, 4-5 (2005) 338-343.
C.S. Williamson, R.K. Foster, S.A. Stanner and J.L. Buttriss: Red meat in the diet.
Nutrition Bulletin, 30, 4 (2005) 323-355.
G. Lombardi-Boccia, S. Lanzi and A. Aguzzi: Aspects of meat quality: trace elements and B vitamins in raw and cooked meats. J. Food. Comp. Anal. 18, 1 (2005)
39-46.
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041095T
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
UDC: 636.47(497.113):[546.74:543.48]
BIBLID: 1450-7188 (2010) 41, 95-102
A.J. McAfee, E.M. McSorley, G.J. Cuskelly, B.W. Moss, J. M.W. Wallace, M.P.
Bonham and A.M. Fearon: Red meat consumption: An overview of the risks and
benefits. Meat Sci., 84, 1 (2010) 1-13.
J.D. Higgs: The changing nature of red meat: 20 years of improving nutritional
quality. Trends Food Sci. Technol. 11, 3 (2000) 85-95.
X. Huang, K. Frenkel, C.B. Klein and M. Costa: Nickel induces increased oxidants
in intact cultured mammalian cells as detected by dichlorofluorescein fluorescence.
Toxicol. Appl. Pharmacol. 120, 1 (1993) 29-36.
Nickel and nickel compounds, in Chromium, nickel and welding. Lyon, International
Agency for Research on Cancer (IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans, Vol. 49) (1990), pp. 257-445.
Nickel. Geneva, World Health Organization (1991) (Environmental Health Criteria,
No.108).
D.J. Swaine: Nickel in coal and fly ash, in Nickel in the Environment. Eds. J.O.
Nriagu, Wiley, New York (1980), pp. 67-92.
Nickel. Washington, DC, National Academy of Sciences (1975).
N.W. Solomons, F. Viteri, T.R. Shuler and F.H. Nielsen: Bioavailability of nickel in
man: effects of foods and chemically-defined dietary constituents on the absorption
of inorganic nickel. J. Nutr. 112, 2 (1982) 39-50.
R. Scheller, B. Strahlmann and G. Schwedt: Chemical and technological aspects of
food for a diet poor in nickel for endogenous nickel contact eczema. Hautarzt, 39,
(1988) 491-497 (in German).
R.W. Dabeka and D.A. Mckenzie: Survey of Pb, Cd, Fe, Ni, Co in food composites
and estimation of dietary intakes of these elements. J. AOAC Int. 78, 4 (1995) 897909.
National Food institute Denmark, Technical University of Denmark (2009).
Available at: http://www.foodcomp.dk/v7/fcdb_grpsearchres.asp?MainGrp=07
B. Sundström, J. Engman, C. Åstrand-Yates and I. Olsson: Levels of certain trace
elements in beef and pork imported to Sweden. Food Addit. Contam. 13, 7 (1996)
737-745.
P.C. Onianwa, J.A. Lawal, A.A. Ogunkeye and B.M. Orejimi: Cadmium and Nickel
Composition of Nigerian Foods. J. Food Comp. Anal. 13, 6 (2000) 961-969.
Nickel, risk assessment. Expert Group on Vitamins and Minerals (EVM). Food
Standards Agency, London, United Kingdom, (2003).
H.A. Schroeder, J.J. Balassa and I.H. Tipton: Abnormal trace metals in man: nickel.
J. Chronic Dis. 15, 1 (1962) 51-65.
F.W. Jr Sunderman, S.M. Hopfer, K.R. Sweeney, A.H. Marcus, B.M. Most and J.
Creason: Nickel absorption and kinetics in human volunteers. Proc. Soc. Exp. Biol.
Med. 191, (1989) 5-11.
P.M. Visscher, R. Pong-Wong, C.T. Whittemore and C.S. Haley: Impact of
biotechnology on pig (cross)breeding programmes. Livest. Prod. Sci. 65, 14 (2000)
57-70.
National Research Council. Nutrient Requirements of Swine, tenth revised edition.
National Academy Press, Washington, DC (1998).
101
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DOI: 10.2298/APT1041095T
UDC: 636.47(497.113):[546.74:543.48]
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24. ISO 936:1998. Meat and meat products. Determination of total ash. Switzerland:
International Organisation for Standardisation.
25. T.T. Gorsuch: The destruction of organic matter (first edition). Pergamon Press Ltd,
Oxford - New York -Toronto - Sydney - Braunschweig (1970).
26. Varian Flame atomic absorption spectrometry, Analytical methods. Varian Techtron
Pty Limited, Mulgvare Victoria, Australia (1989).
27. StatSoft, Inc. STATISTICA (data analysis software system), version 8.0 (2008).
Available at: http://www.statsoft.com/
28. Y.H. Hui, W.K. Nip, R.W. Rogers and O.A. Young: Meat science and applications,
Marcel Dekker, Inc., New York (2001).
29. J.R. Romans, C.W., Carlson, W.J., Costello, M.L., Greaser and K.W. Jones: The
meat we eat, Interstate publishers, Inc., Danville, IL (1994).
30. H. Greenfield and D.A.T. Southgate: Food composition data: production, management and use (second edition), FAO, Rome (2003).
31. Serbian Regulations: Content of Pesticides, Metals, Metalloids, and Other Toxic
Substances in Foods, 5 (1992) 67-85.
32. COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006 setting
maximum levels for certain contaminants in foodstuffs. Official Journal of the
European Union, L364, 5-24.
ОДРЕЂИВАЊЕ САДРЖАЈА НИКЛА У М. semimembranosus СВИЊА
ПРОИЗВЕДЕНИХ У ВОЈВОДИНИ
Владимир М. Томовић, Љиљана С. Петровић, Жарко С. Кеврешан, Наталија Р.
Џинић, Марија Р. Јокановић
У овом раду одређен је садржај никла у М. semimembranosus (н = 69) десет различитих генотипова свиња одгајаних у Војводини. Садржај никла је одређен пламеном атомском апсорпционом спектрофотометријом након „сувог спаљивања“ узорака. Садржај никла у мишићном ткиву није се значајно разликовао (P > 0,05) између различитих генотипова свиња. Одређени садржај никла био је у границама од
12,93 до 80,18 µг/100 г, са просечним садржајем од 32,41 µг/100 г. Просечни садржај никла, одређен у овом испитивању, већи је од садржаја никла који је у свињском месу утврђен у неким развијеним земљама.
Received 24 July 2010
Accepted 14 September 2010
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Review
CRITICAL REVIEW OF SUPERCRITICAL CARBON DIOXIDE EXTRACTION
OF SELECTED OIL SEEDS
Milan N. Sovilj
Supercritical carbon dioxide extraction, as a relatively new separation technique, can
be used as a very efficient process in the production of essential oils and oleoresins from
many of plant materials. The extracts from these materials are a good basis for the new
pharmaceutical products and ingredients in the functional foods. This paper deals with
supercritical carbon dioxide extraction of selected oil seeds which are of little interest in
classical extraction in the food industry. In this article the process parameters in the supercritical carbon dioxide extraction, such as pressure, temperature, solvent flow rate,
diameter of gound materials, and moisture of oil seed were presented for the following
seeds: almond fruits, borage seed, corn germ, grape seed, evening primrose, hazelnut,
linseed, pumpkin seed, walnut, and wheat germ. The values of investigated parameters in
supercritical extraction were: pressure from 100 to 600 bar, temperature from 10 to
70oC, diameter of grinding material from 0.16 to 2.0 mm, solvent flow used from 0.06 to
30.0 kg/h, amount of oil in the feed from 10.0 to 74.0%, and moisture of oil seed from 1.1
to 7.5%. The yield and quality of the extracts of all the oil seeds as well as the possibility
of their application in the pharmaceutical and food, industries were analyzed.
KEYWORDS:
supercritical extraction, seed oils, process parameters, global extraction
yield.
INTRODUCTION
Supercritical fluid extraction using carbon dioxide (SC-CO2) is a particularly suitable
isolation method for isolation of the valuable components from plant materials. A natural
plant extract, free from chemical alterations brought about by heat and water, and without
solvent residues and other artifacts can be obtained by this method. Carbon dioxide is
non-toxic, non-explosive, readily available and easily removed from the extracted products. All products characterized by their internal content are claimed by the market’s
consumers to have a reproducible and stable quality. Technological solutions are sought
that ensure these properties of the products and satisfy the standards in a more reproducible way. For extraction of oil seeds a variety of solvents, including alcohols, acetone
and hexane can be used. However, these organic solvents leave adsorbed residues behind
Dr Milan N. Sovilj, Prof., [email protected], University of Novi Sad, Faculty of Technology, 21000 Novi Sad,
Bulevar cara Lazara 1, Serbia
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Review
and elevated temperatures at the desolventization process can cause chemical transformation of the oleoresins. The solvent residues must be reduced to very small concentrations, generally in the range of 25-30 ppm or less (1).
Supercritical fluids are gases with properties between that of a „normal“ gas and a
liquid. Variation of pressure changes properties of supercritical fluids continuously from
more gas-like behavior to more liquid-like behavior. This behavior may lead to new
technologies in processing natural materials (extraction and purification), in processes
related to the environment (destruction of waste with supercritical water), and in reaction
engineering (hydrogenation with mixtures of hydrogen and supercritical carbon dioxide).
Supercritical fluids produce practically solvent-free products and avoid deteriorating
reactions. Process temperatures are low to very moderate. Solvent handling is favorable
because it can be easily and totally removed, it is readily available and cheap, and it is
accepted and part of the environment (water, carbon dioxide). Even then, solvent losses
are minimal when compared to ordinary men´s activities for these substances, since the
solvent is recycled. Situations where supercritical fluids may be useful emerge from the
effects which supercritical fluids have on pure compounds and mixtures. Supercritical
fluids change compound properties, phase equilibria, chemical equilibria, and related rate
processes, as it has been discussed in many publications.
ANALYSIS OF EXPERIMENTAL DATA
Almond fruits (Pinus dulcis) are important products from trees found in European
countries and elsewere. They are valued for their reamakable nutrition properties leading
to high economical value. They have high oil content, which contribute to their use as a
source of energy in the Mediterranean diet and exibit high levels of mono- and polyunsaturated fatty acids. Almond fruits have about 20% albumens, 50% lipid components, 17%
glucose, and some of minerals, as well as potassium, phosphorus, calcium, iron, and vitamin B3. The amount of oil in the almond fruits is about 50%. The almond oil has oleic,
linoleic, and palmitic acids. Almond fruits are rich a source of flavonic antioxidants, vitamin E, Mg, P, plant fiber, and very quality albumens. The almond fruit oil is light yellow
color and gentle odor. It can be used as an oil for care and massage. The high production
and competition among productive zones have hardened the commercialization of this
product. On the other hand, almond oil is very appreciated for alternative medicine, cosmetics, etc. Thus, production of almond oil may be an option for expanding the almond
market. In this way, using clean process such as supercritical fluid extraction for almond
oil extraction may also lead to an almond revaloraziton as a „light“ product (partially
defatted almonds).
Femenia et al. (2) presented the results of extraction of oil from crushed almond fruits
by supercritical carbon dioxide (SC-CO2) at 330 bar and 50oC, Table 1. The oil percentages in the feed ranged between 15.5 and 64.3% of extracted lipids. Moisture, oil fraction, protein, dietary fiber, ashes, and soluble sugars were determined for almond preparations before and after oil extraction. Samples of nontreated fresh almond seeds, fresh
almond kernels, and toasted seeds were analyzed to allow comparison with further analyses performed on partially defatted samples. As expected, oil extraction by SC-CO2
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promoted an increase of the percentages of the remaining fractions. Thus, defatted almond samples contained large amounts of protein and dietary fiber. This latter fraction
was particularly important in the case of whole almond seeds. From a nutritional point of
view, the reduced fat content, together with the increased protein and dietary fiber fractions, might provide almond kernels as an interesting food source. In fact, dietary recommendations have called for a reduction of total calories received from fat with an equivalent increase in calories from complex carbohydrates (2). Marrone et al. (3) examined the
extraction of oil from almond fruits with supercritical CO2 at 350 bar and 40oC. Almond
particles of three different mean sizes were tested (0.30, 0.70, and 1.90 mm). Extraction
of the smaller particles was performed at two different solvent flow rates (0.72 and 1.43
kg/h), Table 1. The quantity of oil extracted flow divided by the weight of the initial
charge (exctraction yield), is usualy given as a function of the ratio between the mass of
CO2 used and the mass of seeds charged in the extractor (specific mass of solvent). From
the comparison between the experimental data in this system it is possible to observe that,
during the first part of the extraction process, the mass of oil extracted is independent of
both the particle size and the solvent flow rate. These considerations strongly suggest that
the thermodynamic equilibrium of the oil between the solid and the fuid phases applies to
this part of the extraction. For extraction yields larger than 10%, the extraction curves
show a considerable spreading suggesting the onset of a mass transfer resistance. An
increase of particle size increases the extraction time as it is expected from a corresponding decrease of the mass transfer rate. For very large extraction times, it can be identify
an asymptotic value of the yield of about 50% by weight. This value is in good agreement
with the oil content measured in a Soxhlet extraction with hexane, which gave about 54%
of weight fraction of oil and was assumed to be the maximum possible yield, Y. Leo et
al. (4) gave the results of the SC-CO2 extraction of almond fruits at pressure from 350 to
550 bar, temperature from 35 to 50oC, and solvent flow rates from 10 to 30 kg/h, Table 1.
The results were compared with those obtained when hexane/methanol using as solvent.
The almond oil obtained from the various fractions, extracted with either hexane/methanol or supercritical CO2, was intensely yellow in colour having a variable content of tocopherols. After oil extraction, the residual defatted almond was a white flour with variable
grain size, more or less fine, depending on the degree of grinding of the fresh matrix and
on the effect of the operating conditions (pressure, flow rate, temperature) used to remove
the oil. The almond seeds were homogenized and sieved to obtain four representative
sub-samples: whole, broken (4-8 mm), milled (0.5-3.0 mm) and powered samples. The
decreasing of the particle causes an icrease in the oil content in the extract for the conventional extraction process of almond seeds. It was evident that the yield of SC-CO2 extraction not only depended on the utilized pressure, temperature and flow rate of the solvent
through the extraction bed, but also on the chemical and physical characteristics of the
matrix such as oil composition, moisture content and particle size (4). The influence of
pressure on the CO2 supercritical oil extraction from almond seeds was determined at
pressures of 350, 450 and 550 bar, respectively, and at a constant temperature of 50oC
and a constant flow rate of 20 kg/h. The extraction curves obtained at 450 and 550 bar
were characterized by an initial period in which the oil yield rose more steeply as the
pressure was increased and a second period characterised by a higher value of the plateau
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as the pressure increased. The extraction curve showed a linear period without a flex
point at low pressure (350 bar).
Table 1. Amount of oil in the feed and process parameters in the Supercritical carbon
dioxide extraction of the selected oil seeds
Oil seed
Amount of oil
in the feed, %
15.5 - 64.3
Almond fruits
(Prinus dulcis)
54.0
45.0 - 60.0
31.0
Borago seed
(Borago officinalis L.)
31.0
30.0
Corn germ
Grape seed
(Vitis vinifera L.)
45.0 - 55.0
10.0
12.0
Evening primrose seed
(Oenothere biennis L.)
28.0
56.0-60.0
Hazelnut
(Corylus avellana L.)
60.0
Linseed
(Linus usitatissimum L.)
35.0-45.0
Pumpkin seed
(Cucurbita peppo)
42,0-54,0
65.0
Walnut
(Juglans regia L.)
Wheat germ
74.0
8.0-14.0
11.0
Process parameter
o
P = 330 bar; T = 50 C; v = 20-40
kg/h; w = 1.1-5.5%
P = 350 bar; T = 40oC;
v = 0.72 - 1.43 kg/h;
dav = 0.30 - 1.9 mm
P = 350 - 550 bar; T = 35 - 50oC;
v = 10 - 30 kg/h; dav = 0.5 - 8.0 mm
P = 200 - 300 bar; T = 10 - 55oC;
v = 7.5 - 12.0 kg/h; w = 6.6%
P = 200 - 300 bar; T = 40 - 60oC;
v = 0.20 kg/h; dav = 1.125 mm;
w = 7.99%
P = 100 - 350 bar; T = 40oC;
v = 30 L/h
P = 300 bar; T = 42oC
P = 280 - 550 bar; T = 40oC;
dav = 0.39 - 0.97 mm; v = 0.36 kg/h;
w = 3.9%
P = 100-500 bar; T = 40-60oC
P = 200 - 300 bar; T = 40 - 60oC;
v = 0.17 kg/h; dav = 0.63 mm
P = 150 - 600 bar ; T = 40 - 60oC;
v =0.12 l/h; dav = 1.0 - 2.0 mm
P = 300 - 450 bar; T = 40 - 60oC;
v = 0.06 - 0.30 kg/h;
dav= 0.85 - 1.00 mm
P = 300–500 bar ; T = 47-52oC;
v = 8.8 kg/(kg h) ;
dav = 0.16 - 2.00 mm
P = 150 - 300 bar ; T = 40-60oC ;
v = 0.20 kg/h ; dav = 0.50 - 0.63 mm
P = 180 - 234 bar; T = 35 - 48oC;
v = 4.0 L/h; dav = 0.01 - 0.50 mm
P = 200 - 400 bar ; T = 25 - 70oC;
v = 10.5 kg/h; dav = 1.2 - 2.4 mm;
w = 2.5 - 7.5%
P = 200 - 350 bar; T = 40 - 60oC;
v = 15-25 L/h
P = 200 - 300 bar; T = 40oC
Ref.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(6)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
P – pressure, T – temperature, v – flow rate, dav – average diameter of grinding material, w – moisture content
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In view of these results it can be stated that at the beginning of the extraction process,
when the physical phenomenon of quantitative extraction mass depends mainly on the
available amount of oil extracted into the CO2, an increment of the extraction pressure
determines the yield increment of oil. On the other hand, at a constant flow rate an increas in pressure resulted in an increasing of extraction yield. As expected, using the
same quantity of CO2 per kg of matrix, as pressure increased the solubility of the almond
oil in CO2 increased. The results of the pressure effect on the oil yield suggest that the
increased yield resulted from the increasing density of the solvent and consequently from
increased solubility of almond oil in the CO2. The results obtained clearly indicate that
the extraction yield was promoted by an increase in temperature. At fixed conditions of
pressure and flow rates, an increase in temperature produced an almost fourfold yield
increment. An explanation of this phenomenon is that the oil solubility in the CO2 is
enhanced. However, the temperature effect on CO2 extraction is more difficult to assess
than the other parameters, pressure and solvent flow, because it influences both the solvent diffusion in the matrix and the oil dissolution in the solvent (4). From the experimental results it is evident that oil yield, at the initial stage of extraction, increased with
increasing CO2 flow rate from 10 to 30 kg/h, with constant pressure of 420 bar and
constant temperature of 50oC. It appered that flow the rate of supercritical fluid affects
directly the extraction rate. In conclusion, these experiments demonstrated that when
extracting a biological matrix at the described conditions, the oil yield increased with
increasing pressure, temperature and flow rate (4).
Borago seed (Borago officinalis L) contains about 30% of oil, i.e. lipid components.
The oil from borage seeds consists mainly of triglycerides consisting of C16 - C20 fatty
acids. The fatty acid composition of the oil expressed in percentage by weight, is as
follows: palmitic acid (9 - 12%), stearic acid (3 - 5%), oleic acid (15 - 20%), linoleic acid
(30 - 40%) and -linolenic acid (18 - 25%). One of this acids, -linolenic acid (GLA), is
the first intermediate formed during the conversion of linoleic acid to prostaglandins by
means of the dehydrogenation of the C-6 position by the enzyme A-6 dehydrogenase.
However, factors like stress or diabetes, can reduce, or even inhibit, the formation of
GLA causing an alteration of the metabolism and series of dysfunctions (1). Consequently, the possibility to restore the levels of GLA appropriately through a supplementary diet
has motivated a growing interest in the search of vegetables oils rich in this essential fatty
acid, such as borago seed oil, evening primrose oil, etc.
The extraction of borage seed oil using compressed CO2 was studied on a pilot plant
apparatus with the aim to of optimize plant performance and collect data for scale-up
purposes, (5). Effects of extraction pressure (200 - 300 bar) and temperature (10 - 55oC),
solvent flow rate (7.5 - 12.0 kg/h) and bed length (0.25 - 0.50 m) were examined, Table
1. It can be seen from experimental results that, in the early stages of the extraction, the
curves present a steady state situation (near-constant extraction rate) and follow the
solubility lines. After the extraction of around 70% of the oil (approximately 0.20 kg of
oil/kg of seed) the extraction rate reduces progressively and the curve tends to the maximum oil yield as indicated by the horizontal line. This gradual decrease in the extraction
rate is due to the exhaustion of the freely available oil and the mass transfer resistances
turned to the intraparticle diffusion of the last fraction of oil. The form of the extraction
curves is the same for all conditions despite of the considerable differences in solubility
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arising from the different pressures and temperatures (5). The investigation of the effect
of solvent flow rate on the extraction yield showed that the lower flow rate test resulted in
a slightly higher oil load, but the higher flow rate leads to a shorter extraction time to
reach the same yield as the lower flow rate test. Solvent flow rate affects both the residence time and the film coefficient. Within experimental ranges, the extraction rates were
found to be mainly controlled by the solubility of the oil in compressed CO2. The effects
of flow rate on the extraction curves was smaller than those of pressure and temperature
(5). Kotnik et al. (6) also investigated the high-pressure extraction of borage seed oil,
containing the valuable GLA, Table 1. Extraction was performed with supercritical carbon dioxide on a semi-continuous flow apparatus at pressures of 200 and 300 bar, and at
temperatures of 40 and 60oC. A constant flow rate of carbon dioxide in the range of 0.20
kg/h was maintained during extraction. Empirical results showed that the total yield and
extraction rate increased with increasing pressure from 200 to 300 bar. At 200 bar, the
temperature had a positive effect on the extraction yield and, oppositely, a negative effect
on the extraction rate. At 40oC, a higher extraction rate was observed than at 60oC, which
is a consequence of the higher density of CO2 and the higher solvent power at lower
temperature. At 300 bar, the temperature had no influence on the total extraction yield
and extraction rate, and the highest yields were obtained. The extraction yields obtained
using dense CO2 were similar to those obtained by conventional extraction using hexane
as solvent. The composition of extracted crude oil was determined by GC analysis. The
best results were obtained at 300 bar and 40oC for borago seed oil, where the quality of
oil was highest with regard to GLA content (6). Daukšas et al. (7) studied the influence of
different pressures of CO2 and the addition of caprylic acid methyl ester as an entrainer
for the extraction process of borage seed, Table 1.The increase of CO2 pressure from 100
to 350 bar resulted in the increase in extract yield from 0.14 to 24.29% (w/w) while the
changes in the extract composition were not so considerable. The highest solubility of
pure caprylic acid methyl ester in dense CO2 was determined at 100 and 300 bar. The
addition of this entrainer increased the yield of pure extract up to 47.8 times at 100 bar,
2.4 times at 200 and 300 bar. Due to the high solubility of caprylic acid methyl ester at
the lower (100 bar) pressure it is easy to separate the entrainer, which constituted only
4.22% of the total borage seed extract. The highest extract yield and the fastest extraction
rate were achieved after increasing the pressure above 250 bar. The extraction rate increased slightly with an increase of pressure from 250 to 300 and 350 bar; however, the
final yields were 21.89, 21.59 and 24.29%, respectively, which were quite comparable for
all these pressures. The appearance of the extracts obtained at different pressures was
very similar and can be characterized as a yellow oil-like transparent liquid. However,
some changes in the intensity of the color (darker at higher pressures) were observed (7).
Corn germ is a very quality product which contains a significant amount of oil. The
light-yellow oil is rich in the tocoferols (vitamin E), amino acids, fatty acids, as well as in
linoleic, oleic, palmitic acid, and contains small quantites of minerals: phosphorous, iron,
magnesium, and sodium. Corn germ oil can used use in the production of margarine, and
in some of culinary prescriptions. It is light-colored and has significantly lower refining
loss and phosphorous content. The corn kernel contains only 5% of oil, so processing it
for oil is uneconomical. Both wet and dry millers separate the lipid containing germ and
recover the crude oils by expeller pressing and/or solvent extraction with hexane. The
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literature reports that the dry-milled corn germ protein meal obtained by SC-CO2 extraction showed better nutritive and physicochemical properties than when the protein defatted by hexane. The SC-CO2 extraction dissolves only slightly polar lipids (phospholipids and glycolipids), so they essentially remain in the extracts. Thus increasing polar
lipid content can influence the functional properties of germ proteins (8).
Ronay et al. (8) investigated the extraction rate of corn germ with carbon dioxide+ethyl alcohol mixtures. The effect of the amount of ethyl alcohol entrainer on the
composition of oils as well as on the nutritive value and functional properties of defatted
proteins was examined. The laboratory experiments were carried out at constant pressure
(300 bars) and temperature (42oC) with amount of ethyl alcohol ranging from 0% to 10%
by CO2 weight, Table 1. Because of higher solubility of the oil with increasing alcohol
concentrations the extraction time was decreased significantly. The global extraction
yields were plotted against the specific CO2 solvent mass passed through the extraction
vessel. The extraction curves are characterized by a linear increase at the beginning and a
region in which the curve approaches asymptotically the horizontal line determined by
ultimate yield, and is controlled by diffusion in the solid phase at the end of the extraction. The former part represents the quasi-equilibrium conditions under which the extraction is conducted. The solvent is saturated with oil in this period. The alcohol content in
CO2 has a strong influence on the rate of the extraction. Increasing the amount of alcohol
in the fluid decreases the extraction time and the amount of CO2 consumption (8).
The grape seed oil (Vitis vinifera L.) is a product which is ever more appealing for its
large availability, as a by-product of the wine-making industry, and for its properties
which make it interesting for the food, cosmetic, and pharmaceutical industries. The industrial interest for grape seeds is demonstrated by their increasing cost and by some new
technologies developed to preserve their properties while processed in the alcohol industry. Grape seeds are traditionally sold to the oil extraction industry and more recently
they are asked for by cosmetic and pharmaceutical sectors for their use as a source of
antioxidants. Grape seed is a well known oilseed crop containing typically 8 - 15% (w/w)
of oil with recognized quality due to its high level of unsaturated fatty acids, namely oleic
and linoleic and antioxidant-rich compounds. In terms of applications, it is becoming
increasingly popular for culinary, pharmaceutical, cosmetics, and medical purposes. Grape seed oil contains a large percentage of free fatty acids (and high level of unsaturated
fatty acids, such as linoleic and oleic acids), mono and diglycerides and large amounts of
tannins. It is indicated for human consumption and in particular for infants and elderly
people: its pharmaceutical activities concern the ability to contrast free-radicals, cardiovascular diseases, cholesterol. The same properties emphasezeid (accentuated in
strength) are today commercially advertised in „new“ and costly products such as grape
seed extract and grape extract (9).
Fiori (9) presented the results on supercritical CO2 extraction of grape seed oil which
has been analyzed both experimentally and theoretically. Extractions of crushed seeds at
pressures varying in the range 280 - 550 bar and at a fixed temperature of 40oC were performed, Table 1. In the first part of extraction for all the pressure investigated the extraction yield increase (the slope of the curve is constant), then it lowers until the maximum yield is approached. The latter is about 8% for all the tests, except for the lowest
pressure. Considering the various tests in their linear portion, increasing the pressure the
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slope of the curve also increases. The initial linear portion of the experimental curves
yielded oil solubility data in the supercritical solvent. It was found that the maximum oil
yield appears to depend on the particle dimensions, the smaller the particle the greater the
final yield. This results are in accordance with other papers dealing with the similar
substrates. It is worth mentioning that small change in particle size (diameters of 0.49
and 0.51 mm) results in significant change in the extraction curve. This could be due to
the physical nature of the substrate or to the intrinsic approximation of considering the
particle dimensions as represented by an average value – the Sauter mean diameter (9).
Murga et al. (10) have studied supercritical extraction with CO2 extraction of grape seed
at pressures from 100 to 500 bar, and temepratures from 40 to 60oC, Table 1. They determined the solubility of some natural, low-molecular weight phenolic compounds, 3,4-dihydroxy benzoic acid (protocatechuic acid), methyl 3,4,5-trihydroxybenzoate (gallic acid
methyl ester or methyl gallate), and 3,4-dihydroxy benzaldehyde (protocatechualdehyde),
in SC-CO2. These phenolic compounds are contained in grape seeds and other natural
substrates. The data presented in this work indicate the possibillity are valuable to know
the possibility of separation from their natural matrices by SC-CO2.
Evening primrose seed (Oenothera biennis L.) contains about 28% of oil. Oil from
evening primrose seeds is rich in GLA, cis-6,9,12-octadecatrienoic acid), n-6 essential
fatty acid (FA), and free fatty acids (FFA) found in seeds oils, and thus have become a
major commercial source of this essential polyunsaturated fatty acid (PUFA). This fatty
acid is prone to oxidation and thermal rearrangement; therefore, the conventional recovery of the oil via mechanical expression and hexane extraction must be carried out under
very mild and controlled conditions. Studies have shown that GLA can help people with
breast cancer, skin problems, cardiovascular diseases, high blood pressure, and is useful
in treating neurological problems related to diabetes.
Kotnik et al. (6) studied the supercritical carbon dioxide extraction of the evening
primrose, Table 1. Extraction experiments were performed on a semi-continuous flow
apparatus with CO2 at pressures of 200 and 300 bar, and at temperatures of 40oC and
60oC. The flow rate of CO2 was measured at atmospheric pressure and room temperature:
approximately 0.20 and 0.17 kg/h for evening primrose seed extraction. The aim of this
work was also to investigate the differences between the compositions of oils obtained by
conventional extraction and SC-CO2; therefore, extraction by hexane was performed
using a Soxhlet apparatus. The median particle sizes of the extracted seeds of evening
primrose was 0.63 mm, determined by sieve analysis. The composition of the oils extracted was determined by GC analysis and compared with those published in the literature. These authors (6) concluded that for evening primrose seeds, the total yield and
extraction rate increased with increasing pressure from 200 to 300 bar. At 200 bar, the
temperature had a positive effect on the extraction yield and, oppositely, a negative effect
on the extraction rate. At 40oC, a higher extraction rate was observed than at 60oC, which
is a consequence of the higher density of CO2 and the higher solvent power at lower
temperature. At 300 bar, the temperature investigated had no influence on the total extraction yield and extraction rate, and the highest yields were obtained. The highest contents of total FFA and GLA in evening primrose extract were obtained by CO2 extraction
at 200 bar and 60oC. The total yield obtained by conventional extraction of evening primrose seeds using the Soxhlet apparatus was similar to that obtained by CO2 extraction,
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with much lower contents of total FFA and GLA. Generally, it can be observed that the
main difference is in the composition of FFA in the oil. The content of total and unsaturated FFA obtained by SC-CO2 was higher compared to conventional extraction. Consequently, the content of saturated FFA in the oil obtained using Soxhlet extraction was
higher.
Hazelnut (Corylus avellana L.) is the most produced in Mediterraenean, especially in
Turkey. The solubility of hazelnut particles (1-2 mm) in SC-CO2 under each condition
was deternined from the of extraction curves, drown as a g oil extrcated vs. CO2 at begining of a each extraction. The solubily values flow rate slopes of linear part It has a
high nutritional value, containing, generally, 65% oil, 14% protein, and 16% carbohydrates. More than 90% of its oil consists of unsaturated fatty acids, especially oleic (76 80%) and linoleic (6 - 14%) acids. It has been reported that high levels of mono- and
polyunsaturated fatty acids and the sterol and tocopherol contents play a preventive role
in many diseases, especially cardiovascular ones, because they contribute to lower the
low density lipoprotein cholesterol (11).
Ozkál et al. (11) explored the SC-CO2 extraction of hazelnut particle (1-2 mm) at
150-600 bar, and 40-60oC, Table 1. The solubility of hazelnut oil in SC-CO2 under each
condition was determined from the slopes of the linear parts of extraction curves drown
as g oil extracted vs. g CO2 observed the beginning of each extraction. The solubility values calculated by using SC-CO2 flow rate of 0.12 L/h were 10% lower than the ones
calculated by using 4 times lower flow rate of 0.03 L/h. Therefore, the flow rate of 0.03
L/h was considered to be low enough to ensure saturation during the solubility measurements. The solubility values were constant for the entire extraction time. The solubility of
an oil in SC-CO2 changes during the extraction only if its composition changes upon
fractionation. It was observed (11) that the solubility increased with pressure and temperature above 300 bar. However, at 150 bar the solubility showed a slight decrease with
temperature. This is consistent with the crossover phenomena generally observed for
some of the oils (12). The solubilities of oils in SC-CO2 increase both with the density of
SC-CO2 and the volatility of fatty acids. The crossover phenomenon is due to the competing effects of reduction in density of SC-CO2 and increase in the fatty acids volatility,
which accompanis the temperature rise. Therefore, it could be concluded that the crossover pressure for hazelnut oil was in between 150 and 300 bar. The extraction occurred
in two periods. The released oil on the surface of particles was extracted in the fast extraction period, and 39% of the initial oil was recovered under each conditions. However,
the duration of the fast extraction period decreased with increased pressure and temperature. The unreleased oil in the intact cells was extracted in the slow extraction period.
The maximum recovery was 59% at 600 bar and 60oC, for 180 min of extraction. The
fluid phase and solid phase mass transfer coefficients increased with increased pressure
and temperature. Ozkál et al. (12) used the Response Surface Methodology to determine
the effects of solvent flow rate (0.06, 0.18 and 0.30 kg/h), pressure (300, 375 and 450
bar) and temperature (40, 50 and 60oC) on hazelnut oil yield in SC-CO2 supercritical
carbon dioxide extraction, Table 1. Oil yield was represented by a second-order response
surface equation (R2=0.997) using Box-Bhenken design of experiments. Oil yield increased with increasing SC-CO2 flow rate, pressure and temperature. The maximum oil yield
was predicted from the response surface equation as 0.19 g oil/g hazelnut (34% of initial
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oil) when 4 g hazelnut particles (particle diameter<0.85 mm) were extracted with 0.30
kg/h SC-CO2 flow rate at 450 bar, and 60oC for 10 min. Total extraction time under these
conditions was predicted to be 35 min (13).
Linseed (Linum usitatissimum L.) is very rich in oil which contains a high level of the
esential fatty acids, quality proteins, soluble and insoluble fibers, flavonoids and phenolic
acids. More than 70% of linseed oil consists of the polyunsaturated fatty acids. The most
of acids constitute the -linoleic acid (ALA), essential -3 fatty acid, linoleic acid, and
essential -6 fatty acid.
Nikolovski et al. (13) investigated the SC-CO2 extraction of linseed oil at the pressures of 300, 400 and 500 bar, temperatures of 47oC and 52oC, extraction times of 4 h, solvent flow rate 8.8 kg/(kg h), and fractions of grinding material of 0.16-0.315 mm, 0.3150.80 mm and 0.80-2.00 mm, Table 1. The extraction yield increased with increasing pressure from 300 to 500 bar, if the temperature still constant, Figure 1. The extraction yield
decreased with increasing temperature, but this effect was of smaller significance than the
affect of the other process parameters. The maximum value of extraction yield was obtained when the smallest particle size of ground materials were used. This was caused by an
increse the specific area for mass transfer and a decreasing of diffusion path in the solvent.
Pumpkin seed oil (Cucurbita peppo L.) can be usually obtained by cold pressing and
it has a very good organoleptic properties, odor, flavor, and the color from darc green to
brown red. The seed contains from 42 to 54% of oil. The oil has high nutritional value,
containing, generaly, 50% linoleic, 25% oleic, 10% palmitic, and 5% stearic acids. It is a
rich source of micronutritients, as well as vitamin E, phytosterols and lignane (14). The
higher dietary intake of phytosterols from the diet, the lower is cholesterol absorption and
the lower is the serum cholesterol level.
Sovilj and Barjaktarović (14) studied SC-CO2 extraction of pumpkin seed on a laboratory scale at pressures of 150, 225 and 300 bar, temperatures of 40 and 60oC, solvent
flow rate of 0.20 kg/h, and diameter of ground materials of 0.56 mm, Table 1. The extraction yield obtained at 150 bar was rather low (18.4%) for an extraction time of 14 h.
However, extractions at higher pressures yielded greater quantities of the oil; at 225 bar
for 9 h, 36.3% of the oil and at 300 bar for 6 h, 41.0% of the oil, Figure 1. For comparison, hexane extraction of the same seed material yielded less than 40% of the oil. Temperature did not influence the extraction yield. At a pressure of 300 bar, the color of the
fraction yielded during successive extraction time intervals varied greatly, from pail
yellow (the first 2 h), through orange-yellow (from 2-4 h) to red (after 4 h).
Walnut (Juglans regia L.) is a crop of a high economic interest for the food industry.
Walnut fruit contains high level of oil (52 - 70%) and it is consumed, fresh or roasted,
alone or in other edible products. Among the by-products of the walnut industry the oil
has not yet obtained popularity, although it has been demonstrated that its consumption
has a lot of nutritional benfits. The major constituents in the walnut oil are fatty acids,
such as linoleic acid (56.5%), followed by oleic acid (21.2%) and linolenic acid (13.2%).
The presence of the other bioactive minor components, such as sterols, tocopherols and
phytosterols, has been documented in the literature (15,16). The main component of
sterols is β-sitosterol (85.16%), followed by campesterol (5.06%).
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40,0
150 bar
225 bar
300 bar
Y, %
30,0
20,0
10,0
0,0
0
50
100
150
m(CO2)/mmat, g/g
200
Figure 1. Dependence of the global extraction yield (Y) on the mass relation of carbon
dioxide (mCD) and ground particle bed (mmat) (specific solvent flow) in the extraction of pumpkin seed oil (the mean particle size 0.56 mm, temperature
40oC, flow of CO2, 0.2 kg/h and pressures of 150, 225 and 300 bar); Y =
(mE/mf) 100, mE - extract yield (kg), mf – feed (kg).
Oliviera et al. (15) examined the extraction of oil from walnut fruits with compressed
carbon dioxide in the temperature range of 35 to 48oC and in the pressure range of 180 to
234 bar. The influence of particle size was also studied at a solvent flow rate of 4.0
L/h,Table 1. Fatty acids, sterol, and tocopherol compositions were not different from those of oil obtained with n-hexane. Extraction yield increased with the increase in pressure
and under decrease in temperature. The particle size was also a very important parameter.
At the conditions studied, the yields increased with the increase in the median particle
diameter to an optimum value. At 150 min of extraction time, an optimal value of the
yield occurred for the particle diameter range studied, recommending values of about
0.25 to 0.3 mm. At 150 min, the yield exhibited high dependence on the pressure and
temperature, with high values for the yield at this extraction time at higher values of pressure and lower values of temperature (15). Martínez et al. (16) evaluated the oil extraction process from walnut seeds by pressing followed by extraction with supercritical CO2.
The pre-pressed material resulting from the treatment at 7.5% moisture content and 50oC
pressing temperature, was ground to 1.2 – 2.4 mm and then extracted with CO2 in a high
pressure pilot plant with single stage separation and solvent recycle. Extraction experiments were done at two pressures (200 and 400 bar) and temperatures (50 and 70oC) with
a CO2 mass flow rate of 10.5 kg/h, in an up-flow arrangement, Table 1. Pre-treatment of
moisture conditioning did not affect the oil quality as measured by free fatty acid content.
Increasing seed moisture content from 2.5% to 7.5% increased the extraction yield. The
highest extraction yield (89.3%) was obtained at 7.5% moisture and 50oC. At this moisture content, an increase of temperature from 50 to 70oC produced a significant reduction
in the extraction yield, due to the frequent choking problems during press operation. The
drop in the extraction yield at 7.5% and 70oC was common at the end of the press
operation (16). The oil quality data from walnut seeds pressed at different moisture and
temperature conditions indicated significant variations for all parameters evaluated. The
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most outstanding feature was the increase of tocopherol content and acid values with
increasing pressing temperature. The CO2 pressure had a pronounced effect on the extraction yield, the maximum recover being obtained at 400 bar. At this pressure, an increase
of temperature had no significant effect on the extraction yield (16).
Wheat germ is a by-product of the wheat milling industry. Germ constitutes about 2 3% of the wheat grain and can be separated in a fairly pure form from the grain during
the milling process. Wheat germ contains about 11% oil. Wheat germ oil is used in products such as foods, biological insect control agents, pharmaceuticals and cosmetic formulations. Wheat germ processing presents challenges due to its high content of polyunsaturated fatty acids and bioactive compounds. These compounds are prone to oxidation
and degradation under the conditions used for conventional edible oil extraction and refining methods. Resent studies have demonstrated that wheat germ oil has several important physiological effects, which include the ability to lower plasma cholesterol, to
reduce cholesterol absorption and to inhibit platelet aggregation (17).
Shao et al. (17) investigated the supercritical extraction of wheat germ with carbon
dioxide as a solvent. Supercritical CO2 extraction was carried out using a pilot plant extraction system. Thermostatic baths were switched on to reach the operating temperature
required for extraction. Gaseous CO2 was introduced into a compressor. The extraction
vessel was 1000 ml volume capable of operating up to 500 bar and 75oC with the circulation of heated water. The independent variables were temperature (40, 50 and 60oC),
pressure (200, 275 and 350 bar) and flow rate (15, 20 and 25 L/h), Table 1. After 200 g
sample was placed in extraction vessel, the extraction temperature, pressure and flow rate
were controlled automatically and maintained for 60 min. When the desired pressure,
temperature and flow rate were reached, the extraction was started. The oil dissolved in
the supercritical CO2 was separated from the carbon dioxide and collected in the separator. Conventional extraction was carried out using hexane in a Soxhlet apparatus for 20
h (with a fraction wheat germ size of 0.75 mm and humidity less than 0.35%) to ensure
maximum extraction efficiency. These values are considered very important to establish
an indisputable basis for comparison to the high-pressure process. Many parameters can
influence the separation performance of wheat germ oil extraction. It was shown that
wheat germ oil yield has a complex relationship with independent variables that encompass both first and second-order polynomials and may have more than one maximum
point. The best way of expressing the effect of any parameter on the yield within the experimental space under investigated was to generate response surface plots of the equation. The three-dimensional response surfaces were plotted as a function of the interactions of any two of the variables by holding the other one at a middle value. Contour plot
and response surface curve showing predicted response surface of oil yield as a function
of pressure and flow rate. It showed that at temperature 50oC the oil yield of wheat germ
increased with increase in pressure. The oil yield increased from about 7.13% to 10.00%
as the pressure was increased from 200 to 350 bar. The optimum pressure for the maximum yield of oil was around 350 bar. At lower pressure, the solubility of oil affected by
vapor pressure of the oil, apparently at this stage CO2 relatively act as an ideal gas that
does not have any special characteristic of a solvent. However, at high pressures, the
solubility of the oil increased due to the increase in density of CO2. As the density increases, the distance between molecules decreases and the interaction between oil and
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CO2 increases, leading to greater oil solubility in CO2. Piras et al. (18) explored the SCCO2 of wheat germ oil. The effects of pressure (200-300 bar at 40oC) and extraction time
on the oil quality/quantity of feed were studied, Table 1. A comparison was also made
between the relative qualities of material obtained by SC-CO2 extraction and by organic
solvent extraction. The extracts were analyzed for -tocopherol and polyunsaturated fatty
acid content. The maximum wheat germ oil yield at about 9% was obtained with SC-CO2
carbon dioxide extraction at 300 bar, while fatty acid and -tocopherol composition of
the extracts was not remarkable affected by either pressure or the extraction method. The
SC-CO2 gives an CO2 extraction yield not significantly that of different from organic
solvent extraction. A comparison is made between the relative qualities of the oils produced by SC-CO2 and by organic solvent extraction (hexane, methanol, chloroform-methanol 2:1 mixure) in terms of a much lower selectivity. CO2 yielded oil contained as
undesirable compounds.The effect of the specific mass of solvent, q (kg CO2/kg feed) on
the global extraction yield Y (%) for all the seed oils investigated is presented in Figure
2.
Y / 100 (kg extract yield/kg feed), %
70
Corn germ
0
300 bar, 42 C
60
Almond fruits
0
330 bar, 50 C
50
Pumpkin seed
0
300 bar, 40 C
40
Linseed
0
500 bar, 52 C
30
Hazelnut
0
600 bar, 60 C
Walnut
0
400 bar, 50 C
20
Wheat germ
0
350 bar, 50 C
10
Borago seed
0
300 bar, 40 C
Primrose seed
0
300 bar, 40 C
Grape seed
0
450 bar, 40 C
0
0
20
40
60
80
100
120
140
160
q / (kg CO2/kg feed)
Figure 2. Effect of the specific solvent flow (q) on the global extraction yield (Y) in
supercritical extraction of the selected oil seeds; q = kg CO2/kg feed, Y =
(mE/mf) 100, mE - extract yield (kg), mf - feed (kg).
RESULT AND DISCUSSION
The survey all the above papers related to the effect of extraction pressure on the
global extract yiled the SC-CO2 extraction showed that the extract yield increases with
increasing pressure due to the increase of the solvents density, if the temperature, the
solvent flow rate, and the diameter of the ground material were constant.
It was observed that the solubility increased with pressure and temperature above 300
bar. However, at 150 bar, the solubility showed a slight decrease with temperature (11).
This is consistent with the crossover phenomena generally observed for some of the oils,
which results presented in the literature.
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In general, the increase of the temperature resulted in the decrease of the extraction
yield, due to the decrease of the solvents density, whose effect seems to have dominated
over the increase of the solute vapor pressure. In some of the cases the temperature had a
positive effect on the extraction yield and, oppositely, a negative effect on the extraction
rate (6). At higher pressure (300 bar) it was shown that the temperature had no influence
on the total extraction yield and extraction rate, and the highest yields were obtained (6).
In the most of systems investigated solvent flow rate did not influence the extraction
yield. From the experimental results for the system almond fruits oil - CO2 (4) it is evident that oil yield, at the initial stage of extraction, increased increasing CO2 flow rate,
with constant pressure and constant temperature. On the other hand, in the system borage
seed - CO2, the investigation of the effect of solvent flow rate on the extraction yield
showed that the lower flow rate test resulted in a slightly higher oil load, but the higher
flow rate lead to a shorter extraction time to reach the same yield as the lower flow rate
test (3).
The extraction yield increases by decreasing the particle size of the groumd materials.
It is due to the higher amount of oil released as the substrate cells are destroyed by
milling, and this because of the amount of oil is easily extracted for direct exposure to the
supercritical CO2. Moreover, shorter diffusion paths in the milled solid matrix result in a
smaller intraparticle resistance to diffusion.
The moisture containing pre-treatment conditionig did not affect the oil quality as
measured by free fatty acid content. Increasing seed moisture content from 2.5% to 7.5%
increased the extraction yield (16).
In two of the systems, the addition of entrainers was achieved in the aim of enchancement the extract, effiency were added with aim to achive enchacement SC-CO2 extraction process (7,8). Caprylic acid methyl ester as an entrainer in the extraction process of
borage seed was added (7). The highest solubility of pure caprylic acid methyl ester in
dense CO2 was determined at 100 and 300 bar. The addition of this entrainer increased
the yield of pure extract at the pressures investigated. Due to the high solubility of caprylic acid methyl ester at the lower pressure it was easy to separate entrainer, which constituted only 4.22% of the total borage seed extract (7). In the ectraction extraction of
corn germ laboratory experiments were carried out at constant pressure (300 bar) and
temperature (42oC) with amount of ethyl alcohol (entrainer) ranging from 0% to 10% by
weight in CO2 (8). The alcohol content in the solvent had a strong influence on the rate of
the extraction. Increasing the amount of alcohol in the fluid decrease the extraction time
and the amount of CO2 consumption (8).
CONCLUSIONS
In this study, supercritical carbon dioxide extraction of some selected oil seeds are reviewed. Тhe influence of process parameters, such as pressure, temperature, solvent flow
rate, diameter of ground materials, and moisture of oil seed on the SC-CO2 extraction for
the selected seed oils: almond fruits, borage seed, corn germ, grape seed, evening primrose, hazelnut, linseed, pumpkin seed, walnut, and wheat germ was presented. The yield
and quality of the extracts all of the oil seeds investigated as well as the possibility of
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their application in the pharmaceutical and food industries were analyzed. The global extraction yield varied between 7.5 (grape seed) and 65.0% (almond fruits).
Acknowledgements
This research was supported by the Ministry for Science and Technological Development of the Republic of Serbia (Project No. 20022).
REFERENCES
1. E. Reverchon, and I. De Marco: Supercritical Fluid Extraction and Fractionation of
Natural Matter. J. Supercrit. Fluids 38, (2006) 146–166.
2. A.Femenia, M. Garcia-Marin, S. Simal, C. Rossello and M. Blasco: Effects of
Supercritical Carbon Dioxide (SC-CO2) Oil Extraction on the Cell Wall Composition
of Almond Fruits. J. Agric. Food Chem. 49, 12 (2001) 5828-5834.
3. C. Marrone, M. Poletto, E. Reverchon and A. Stassi: Almond Oil Extraction by
Supercritical CO2: Experiments and Modelling. Chem. Eng. Sci. 53, 21 (1998) 3713718.
4. L. Leo, L. Rescio, L. Ciurlia and G. Zacheo: Supercritical Carbon Dioxide Extraction
of Oil and α-Tocopherol from Almond Seeds. J. Sci. Food Agric. 85, (2005) 2167–
2174.
5. T. Lu, T., F. Gaspar, R. Marriott, S. Mellor, C. Watkinson, C., B. Al-Duri, J. Seville
and R. Santos: Extraction of Borage Seed Oil by Compressed CO2: Effect of
Extraction Parameters and Modelling. J. Supercrit. Fluids. 41, 1 (2007) 68-73.
6. P. Kotnik, M. Škerget and Ž. Knez: Kinetics of Supercritical Carbon Dioxide
Extraction of Borage and Evening Primrose Seed Oil. European J. Lipid Sci. Technol.
108, 7 (2006) 569-576.
7. E. Daukšas, P. R. Venskutonis and B. Sivik. Supercritical Fluid Extraction of Borage
(Borago officinalis L.) Seeds with Pure CO2 and Its Mixture with Caprylic Acid
Methyl Ester. J. Supercrit. Fluids. 22, (2002) 211–219.
8. E. Ronyal, B. Simandi, S. Tomoskoyi, A. Deak, L. Vigh and Zs. Weinbrenner:
Supercritical Fluid Extraction of Corn Germ with Carbon Dioxide–Ethyl Alcohol
Mixture. J. Supercrit. Fluids, 14, (1998) 75-81.
9. Fiori, L.: Grape Seed Oil Supercritical Extraction Kinetic and Solubility Data:
Critical Approach and Modeling. J. Supercrit. Fluids. 43, (2007) 43-54.
10. R. Murga, Sanz, M. T., Sagrario Beltrán, S. and Cabezas, H. L.: Solubility of Some
Phenolic Compounds Contained in Grape Seeds, in Supercritical Carbon Dioxide. J.
Supercrit. Fluids. 23, (2002) 113–121.
11. G. Özkal, U. Salgin and M. E. Yener: Supercritical Carbon Dioxide Extraction of
Hazelnut Oil. J. Food Eng. 69, 2 (2005) 217-223.
12. S. G. Özkal, M. E. Yener, U. Salgın and Ü. Mehmetoġlu: Response Surfaces of
Hazelnut Oil Yield in Supercritical Carbon Dioxide. Europen Food Res. Technol.
220 (2005) 74-78.
119
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DOI: 10.2298/APT1041105S
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BIBLID: 1450-7188 (2010) 41, 105-120
Review
13. B. G Nikolovski, M. N. Sovilj, M. Z. Djokić and S. S. Vidović: Kinetics and
Modeling of the Extraction of Flax Seed Oil (Linum usitatissimum l.) by Supercritical
Carbon Dioxide. Chem. Ind. 62, 5 (2008) 283–292.
14. M. N. Sovilj, and Barjaktarović, B. G.:Kinetics of the Extraction of Pumpkin Oil
(Cucurbita peppo L.) by Supercritical CO2 Chem. Ind. 59, 9-10 (2005) 238-242.
15. R Oliveira, M. F. Rodrigues and M. G., Bernardo-Gil: Characterization and
Supercritical Carbon Dioxide Extraction of Walnut Oil. JAOCS, 79, 3 (2002) 225230.
16. M. L. Martínez, M. A. Mattea and D. M. Maestri: Pressing and Supercritical Carbon
Dioxide Extraction of Walnut Oil. J. Food Eng. 88, 3 (2008) 399-404.
17. P. Shao, P. Sun and Y. Ying: Response Surface Optimization of Wheat Germ Oil
Yield by Supercritical Carbon Dioxide Extraction. Food Bioprod. Process 86, 3
(2008) 227-231.
18. Piras, A., A. Rosa, D. Falconieri, S. Porcedda, M. A.Dessì and Marongiu: Extraction
of Oil from Wheat Germ by Supercritical CO2. Molecules. 14 (2009) 2573-2581.
КРИТИЧКИ ПРЕГЛЕД ЕКСТРАКЦИЈЕ ОДАБРАНИХ УЉАРИЦА
НАТКРИТИЧНИМ УГЉЕНДИОКСИДОМ
Милан Н. Совиљ
Наткритична екстракција угљендиоксидом, као новија сепарациона метода, користи се као ефикасан поступак за добијање етарских и масних уља из великог броја биљних и лековитих сировина, при чему се добијају екстракти који служе као основа за производњу низа лековитих препарата и додатака прехрамбеним производима. У овом раду дат је преглед добијања уља помоћу наткритичног угљендиоксида из мање познатих уљарица, чији се производи такође могу користити као лековити препарати или служе као додаци у функционалној храни. Приказани су процесни параметри коришћени у наткритичној екстракцији угљендиоксидом за следеће уљарице: бадем, коштице грожђа, кукурузна клица, лешник, орах, пшенична
клица, семе боражине, лана, ноћурка и уљане тикве. Опсег вредности појединих
процесних параметара је био: притисак (150 - 600 bar), температура (10 - 70oC),
средњи пречник чврстих честица уситњеног материјала (0,16 - 2,00 mm), проток коришћеног растварача (0,06 - 30,0 kg/h), садржај уља у полазном материјалу (10,0 74,0%) и садржај влаге у полазном полазном материјалу (1,1 - 7,5%). Анализирани
су принос и квалитет добијених екстраката и могућност примене у фармацеутској и
прехрамбеној индустрији за сваку од испитиваних уљарица.
Received 8 February 2010
Accepted 21 May 2010
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APPLICATION OF LATTICE-BOLTZMANN METHOD AND ANALYSIS OF
FLUID FLOW BETWEEN TWO SINUSOIDAL PLATES
Jelena Đ. Marković, Nataša Lj. Lukić and Dragica Z. Jovičević
This paper is concerned with numerical study of fluid flow through a pair of corrugated platest. The aim was to observe and understand the behavior of the flow and vortex
formation through channels where the fluid is subjected to a periodic increase and
decrease in cross-section area. The plates modeled for the study had sinusoidal profiles.
A pair of plates with 180o phase shift was simulated in two-dimensional spaces. The Reynolds number was a function of the average plate spacing (Havg) and the laminar flow
velocity and it was in the range between 200 and 1000.
KEYWORDS: Lattice-Boltzmann method; plate heat exchangers; fluid flow
INTRODUCTION
Obtaining a good fluid mixing, reduction of heat transfer resistance and minimum
pressure drop are very important factors in heat exchangers design. Good fluid mixing
can be obtained in heat exchangers operating in turbulent regime, which, on the other
hand, has significant pressure drop as a negative effect. Heat exchangers operating in turbulent regime increase pumping cost, also the size of the pump is often a limiting factor,
especially in the case of compact heat exchangers or in heat exchangers with very viscous
fluids.
In order to obtain better heat transfer in plate heat exchangers several techniques have
been used so far, and one the most often applied techniques was the use of wavy instead
of flat plates (1).
Many investigations showed that use of sinusoidal instead of flat plates enhances heat
transfer without significant pressure drop (2-7).
Establishing a chaotic fluid flow can enhance fluid mixing in laminar regime, which,
as a result will lead to a better heat transfer. Chaotic advection occurs when there are
pathlines present in fluid which do not conform with laminar regime, and can be generated in ducts with periodically perturbed geometry in the downstream direction.
The aim of this study was to apply the lattice-Boltzmann method (LBM) in order to
simulate and analyze the fluid flow between two sinusoidal plates. The LBM is a
Jelena Marković, BSc, Assistant, email: [email protected], Nataša Lukić, BSc, Researcher, email:
[email protected], Dragica Jovičević, BSc, Junior Researcher, email: [email protected], University of Novi Sad, Faculty of Technology, Bul. Cara Lazara 1, 21 000 Novi Sad, Serbia
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relatively new simulation technique for complex fluid systems and has attracted interest
of researchers in computational physics. Unlike the traditional CFD (Computational Fluid
Dynamic) methods, which solve the conservation equations of macroscopic properties
(i.e., mass, momentum, and energy) numerically, LBM models the fluid consisting of
fictive particles, and such particles perform consecutive propagation and collision processes over a discrete lattice mesh. Due to its particulate nature and local dynamics, LBM
has several advantages over other conventional CFD methods, especially in dealing with
complex boundaries, incorporating of microscopic interactions, and parallelization of the
algorithm. A different interpretation of the lattice-Boltzmann equation is that of a discrete-velocity Boltzmann equation. The numerical methods of solution of the system of
partial differential equations then gives rise to a discrete map, which can be interpreted as
the propagation and collision of fictitious particles.
Mathematical formulation
Navier Stokes Equation. In a continuum domain, fluid flow is governed by Navier
Stokes equations, NSE.
For an incompressible flow, two-dimensional, the conservative form of NS equation
can be written in the Cartesian coordinate system, without body force, as,
x – momentum
p   u    u 
u  vu   vu 
[1]


     

x x  x  y  y 
t
x
y
y – momentum
v  uv   vv 
p   v    v 



     
t
x
y
y x  x  y  y 
[2]
The left-hand side represents the inertia and advection term, or the total acceleration
of fluid parcels. The first term on the right-hand side is the pressure gradient term. The
last two terms on the right hand-side represent the shear force due to viscous effect.
These two equations have three unknowns, u, v and p. the continuity equations is
needed to close the solution:
 u   v 

0
x
y
[3]
The main problem in solving fluid flow, using NS, are two folds. First, the advection
term is nonlinear, which needs special treatment to avoid instability and false “numerically introduced” diffusion problems. To overcome this problem, second or higher order
up-wind schemes have been used to approximate advection terms. Higher order schemes
reduce the numerical diffusion problem, however, it decreases the stability limit; or adds
a dispersion effect. In solving these problems, deferred correction method has been used.
The main idea is to use first order up-wind scheme and add an extra term as a source.
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Second, for incompressible flow there is no explicit equation for pressure, hence at
each time step in the updating process, the continuity equation needs to be satisfied. This
step requires the solution of the Laplace equation (pressure and pressure correction equations). Solving the Laplace equation consumes significant computing time. In fact, majority of the time needed to solve NS numerically is spent in solving the Laplace equation.

v
 v  v  p   2 v
t
[4]
For two-dimensional problems, it is possible to introduce stream function, however,
treatment of the boundary conditions is not straight forward (8).
The Lattice-Boltzmann. Lattice-Boltzmann equation (LBE) is linear. The nonlinearity of advection term (which is implicitly imbedded in the left hand side of the LBE) in
macroscopic approach is replaced by linear streaming process in LBM, similar to characteristic methods for solving compressible flows. Unlike in macroscopic approach, there is no need to solve Poisson equation to satisfy continuity equation which drastically
reduces the computational time, so LBM can be considered as an explicit method, which
bypasses solving simultaneous equations in every time step.
A short description of the lattice-Boltzmann equation (LBE) is given. Detailed information can be found in literature (8-10).
A statistical description of a system can be explained by distribution function f(r, c, t),
where f(r, c, t)drdc is number of molecules at time t positioned between r and r+dr
which have velocities between c and c+dc. An external force, F, acts on a gas molecule
of unit mass will change the velocity of the molecule from c to c+Fdt and its position
from r to r+cdt.
The number of molecules, before applying the external force is equal to the number
of the molecules after disturbance, if no collisions take place between the molecules.
Hence,
f (r  cdt , c  Fdt , t  dt )drdc  f (r , c, t )drdc  0
[5]
However, if collisions take place between the molecules, there will be a net difference
between the numbers of molecules in the interval drdc. The rate of change between final
and initial status of the distribution functions called collision operator, Ω. The equation
for evolution of the number of the molecules can be written as,
f (r  cdt , c  Fdt , t  dt )drdc  f (r , c, t )drdc  ( f )drdcdt
[6]
Dividing the above equation by dtdrdc and as the limit, yields
df
 ( f )
dt
[7]
Since f is a function of r, c and t, then the total rate of change, f with t, can be
expanded and when divided by dt, yields
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df f dr f dc f



dt r dt c dt t
[8]
The vector r can be expressed in 3-D Cartesian coordinate system r  xi  yj  zk ,
where i, j and k are unit vectors along x,y, and z direction, respectively.
Equation [8] can be rewritten as,
df f
F f f
c


dt r
m c t
[9]
where dc/dt,is equal to the acceleration and can be related to force F by Newton’s second
law.
Then the Boltzmann transport equation can be written as,
df f
F f

c

dt r
m c
[10]
The collision operator, Ω, is function of f and has to be determined to solve the
Boltzmann equation.
For system without external force the Boltzmann equations can be written as,
where c, and f are vectors.
f
 c  f  
t
[11]
After introducing the Bhatnagar-Gross-Krook (BGK) approximation to collision
operator (Ω), and after the Boltzmann equation is discretized it becomes (9):
f i r  ci t , t  t   f i r , t   
1

 f r, t   f
i
eq
i
r , t 
[12]
where fi(x,t) and fi eq(x,t) distribution function and distribution function at equilibrium,
respectively, ci discrete velocity vector and τ relaxation time defined by fluid’s kinematic
viscosity:
6  1

[13]
2
Discrete square lattice model with 9 particle velocities (D2Q9) is very common, especially for solving two dimensional fluid flow problems. It has nine velocity vectors, the
central speed is zero. Equilibrium distribution function for isothermal incompressible
fluid flow D2Q9 is defined as:
3
9
3


eq
2
[14]
f i  wi 1  2 ei  u  4 e  u   2 u  u 
2c
2c
 c

where wi and u are weight parameters and fluid velocity, respectively.
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Discrete vectors have the following values:
e0  0,0
ei   c,0 , 0, c 
i  1,2,3,4
[15]
ei   c,c 
i  5,6,7,8
Also c=Δx/Δt, where Δx and Δt are space between nodes and time step, respectively.
Weighting factors for distribution function are w0=4/9, w1,3,5,7 =1/9, and w2,4,6,8 =1/36.
Macroscopic density and fluid velocity can be found from the distribution function using
following equations:
8
   fi
i 1
8
u   ei f i
[16]
i 1
Boundary conditions
One of the important and crucial issues in LB simulation of flow is accurate modeling
of the boundary conditions. Adapting boundary conditions for Navier Stokes equations is
somewhat straightforward, which is not the case with LBM, where the inward distribution functions to the integration domain are unknowns. Therefore, it is necessary to
determine appropriate relationships for calculating those distributions functions at the
boundaries for a given boundary condition.
Boundary conditions used in this simulation are:
Upper plate – the bounce back boundary condition
Lower plate – the bounce back boundary condition
Inlet and outlet – the open boundary condition
Detailed description of the above conditions and their implementation can be found in
the literature (8,9).
Geometry
Geometry of the plates used in the simulation is shown in Fig.1.
Figure 1. Geometry of sinusoidal plates
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Height of the plate at a given coordinate x, is defined by the sine function:
h  Ax sin(
2x
),
x
Ax is a wave amplitude in the x direction, and Λx is a wavelength in the x direction.
Dimensions used in the calculations are Ax=0.45 cm, Λx=8.334 cm, Havg = 4 cm. The
dimensionless geometric parameters that describe the corrugated plate model are as
following:

A
 x  x and  x  x ,
H avg
H avg
where Havg is the separation between the corrugated plates that form the channel, which
was fixed at Havg =3 cm throughout this investigation.
The Reynolds number was defined as:
Re 
V in H
avg

[16]
where Vin is the average velocity at the inlet of the corrugated plates and υ is kinematic
viscosity.
The criterion used to determine if one wave has macroscopic mixing is the presence
of crossing paths in the central flow, broken recirculation regions or too big vortices
compared to the wave amplitude. Low Reynolds number was set at 200, while the upper
limit was 1000, because calculation procedure with Re>1000 was numerically unstable.
The flow pattern is very much like the one reported in previous investigations (6, 10).
At small Reynolds numbers there are steady recirculation regions along the sinusoidal
channel. These recirculation characteristics vary as the Reynolds number increases. When
the Reynolds number increases, the separation point is closer to the inlet wave, and the
reattachment point is closer to the outlet wave, as can be seen in Fig.2. The steady recirculation regions at the top and bottom sinusoidal plates are symmetrical with respect to
the midplane of the channel.
For waves further from the inlet, flow instabilities appear in the channel. As the Reynolds number increases, instabilities appear in waves closer to the channel inlet. At higher Reynolds numbers (it is known that in this type of channel mild flow instabilities
appear at Re=600) some waves present random particle paths that promote macroscopic
mixing over all the separation between the plates. According to the observations, it was
determined that the position of the first wave manifesting such behavior (number of wave
counted from the channel inlet) decreases as Reynolds number increases.
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Figure 2. Pathlines Havg=3 cm, Re=600
However, macroscopic mixing was rarely observed in wave’s number 1 and 2, as
shown in Fig.3.
Having analyzed the flow pattern carefully, other types of instabilities were observed
at the high-end of Reynolds number studied. These instabilities are rolling vortices that
appear in the limits between the principal flow and the upper part of the recirculation.
The vortices appear in the wave inlet and move downstream to the wave outlet, where
they join the principal flow. As the flow moves through the channel, the vortices grow. If
rolling vortices appear in some wave of the channel, downstream from this wave, steady
recirculation regions will not be observed and macroscopic mixing might appear. At a given Reynolds number, waves with rolling vortices and without macroscopic mixing may
appear, however, a wave with macroscopic mixing never appears if there are not waves
with rolling vortices upstream. According to this, it is believed that the macroscopic
mixing is caused by instabilities in the central core flow that are created by rolling vortices upstream from the wave with macroscopic mixing. Finally, it is important to mention that rolling vortices and macroscopic mixing move closer to the channel inlet as the
Reynolds number increases.
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Figure 3. Comparison between the position of the first wave that presents rolling vortices
and the position of the first wave that presents macroscopic mixing
CONCLUSIONS
The visualized flow pattern for given geometry of the heat exchanger agrees well
with previous numerical and experimental investigations (1).
Macroscopic mixing is an effective way to enhance the heat transfer and mixing in sinusoidal plates because of its random particle trajectories. Although it is in some way an
interesting phenomena, capable of improving the heat transfer and the stirring in sinusoidal plates, it does not appear in waves very near the channel inlet at the small Reynolds
number range.
Some previous investigations (3, 6, 11) showed that the enhancement of fluid mixing
and formation of recirculation regions improves heat transfer, although additional investigation on the heat transfer should be performed in order to obtain more information on
the influence of recirculation regions, vortex formation and chaotic mixing to heat transfer enhancement.
Aknowledgement
This research was financially supported by the Ministry of Science and Technological
Development of the Republic of Serbia (Project No. 142045)
REFERENCES
1. B. Giron-Palomares, A. Hernandez-Guerrero, R. Romero-Mendez, F. Oviedo-Tolentino: An experimental analysis of the flow pattern in heat exchangers with an egg carton configuration (parallel, convergent and divergent cases), International Journal of
Heat and Fluid Flow 30 (2009) pp.158-171
2. P. Gschwind, A. Reggele, V. Kottke: Sinusoidal wavy channels with Taylor-Goertler
vortices, Experimental Thermal and Fluid Science 11 (1995) pp. 270-275
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3. Y. Islamoglu, C. Parmaksizoglu: Numerical investigation of convective heat transfer
and pressure drop in a corrugated heat exchanger channel, Applied Thermal Engineering 24 (2004) pp. 141-147
4. J.Y. Jang, L.K. Chen: Numerical analysis of heat transfer and fluid flow in a three-dimensional wavy-Fin and tube heat exchanger, International Journal of Heat and Mass
Transfer 40 (1997) pp. 3981-3990
5. S. Mahmood, A.K.M. Sadrul-Islam,M.A.H. Mamun: Separation characteristics of fluid flow inside two parallel plates with wavy surface, International Journal of Engineering Science 40 (2002) pp. 1495-1509
6. T.A. Rush,T.A. Newell, A.M. Jacobi: An experimental study of flow and heat transfer
in sinusoidal wavy passages, International Journal of Heat and Mass Transfer 41
(1999) pp. 1541-1553
7. R. Sawyer, M. Sen, C. Hsueh-Chia: An experimental study of flow and heat transfer
in sinusoidal wavy passages, International Journal of Heat and Mass Transfer 41
(1998) pp. 3559-3573
8. A.A. Mohamad : Applied Lattice Boltzmann Method for Transport Phenomena, Momentum, Heat and Mass Transfer, Sure Print, Dalbrent, Canada, (2007).
9. S. Succi: The Lattice Boltzman Equation for Fluid Dynamics and Beyond, Oxford
University Press, Oxford, 2001
10. M.C. Sukop, D. T. J. Thorne, Lattice Boltzmann Modeling: An Introduction for
Geoscientists and Engineers, Springer, 2007
11. J. Zhang, J. Kundu, R.M. Manglik: Effect of fin waviness and spacing on the lateral
vortex structure and laminar heat transfer in wavy-plate-fin cores, International
Journal of Heat and Mass Transfer 47 (2004) pp. 1719-1730
12. C.C. Wang, C.K. Chen, C.K: Forced convection in a wavy-walled channel, International Journal of Heat and Mass Transfer 45 (2002) pp. 2587-2595
ПРИМЕНА ЛАТИС-БОЛЦМАН МЕТОДА (ЛБМ) И АНАЛИЗА СТРУЈАЊА
ФЛУИДА ИЗМЕЂУ ДВЕ СИНУСОИДАЛНЕ ПЛОЧЕ
Јелена Ђ. Марковић, Наташа Љ. Лукић, Драгица З. Јовичевић
У овом раду симулирано је струјaње флуида између две синусоидалне плоче.
Циљ рада је био да се уочи и анализира понашање тока флуида и формирање вртлога у каналима где долази до периодичног смањења и повећања попречног пресека. Дводимензиона симулација је урађена за модел синусоидалних плоча са фазним померањем 180о. Рејнолдсов број, као функција средњег растојања између
плоча ((Havg), је вариран у ламинарном режиму, од 200 до 1000.
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Mg-Cu-Al LAYERED DOUBLE HYDROXIDES BASED CATALYSTS FOR THE
REDUCTION OF NITRATES IN AQUEOUS SOLUTIONS
Tatjana J. Vulić and Goran C. Bošković
The secondary waste and bacterial contamination in physico-chemical and biological
separation processes used today for nitrate removal from ground water make novel catalytic technologies that convert nitrates to unharmful gaseous nitrogen, very attractive
for scientific research. The Mg-Cu-Al layered double hydroxide (LDH) based catalysts
with different Mg/Al ratio were investigated in water denitrification reaction in the
presence of hydrogen and with solely copper as an active phase. Since LDHs have ion
exchange properties and their derived mixed oxides possess memory effect (restoration of
layered structure after thermal decomposition), their adsorption capacity for nitrates was
also measured in the same model system. All studied samples showed nitrate removal
from 23% to 62% following the decrease in Al content, as well as the substantial adsorption capacity ranging from 18% to 38%. These results underlie the necessity to take
into account the effects of the adsorption in all future investigations.
KEYWORDS:
Mg-Cu-Al anionic clays, nitrate removal, nitrate adsorption, water
denitrification
INTRODUCTION
Nitrate concentration in drinking water supplies in many world regions (EU, USA,
Canada, India, etc.) has increased in the last decade (1, 2). Due to its high toxicity, removal of nitrate has become an environmental issue (2, 3, 4), which has increased the drinking water quality standards (50 mg/L in the European Union and 25 mg/L in the USA)
(1, 4). Effective removal of nitrate ions from ground water is presently achieved by conventional physicochemical methods like ion exchange, reverse osmosis and electrodialysis. However, generation of undesirable nitrates reach secondary waste streams, as a
draw-back of these methods, raises a need for the development of new technologies (1,
3). Selective reduction of nitrates to nitrogen is the most environment friendly method for
nitrates removal and can be realized by biological digestion and catalytic reduction. The
most widely used denitrification process today is biological denitrification; however, possible bacterial contamination, the presence of residual organics and the possible increase
in chlorine demand in purified water limit the application of this process (3). Therefore
Dr. Tatjana Vulić, Assistant Professor, [email protected], Dr. Goran Bošković, Professor, University of Novi
Sad, Faculty of Technology, 21000 Novi Sad, Bulevar Cara Lazara 1, Serbia
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the catalytic reduction of nitrates appears as the best solution because of efficient chemical reaction, flexibility, simple configuration of the reactor, etc. In this catalytic process, nitrate ions are reduced using hydrogen and converted into nitrites as an intermediate, while nitrogen, hydroxyl ions and ammonia are the final products (3). The advantage
of layered double hydroxides (LDHs) based catalysts is their ability to concentrate first
the NO3 ions from water solution in the LDH interlayer, and after complete conversion
to OH and either N2 or NH4+, exchange OH ions left in the interlayer with new NO3
ions from the solution and start another catalytic cycle (1). Typically, metal-oxide supported bimetallic catalysts, combining a noble metal, usually Pd or Pt, and another metal,
such as Cu, Sn, or In, supported on alumina have been applied for this reaction (1, 3-6).
The LDHs, also called anionic clays or hydrotalcite like materials, have brucile-like
layers consisting of octahedrally coordinated M(II) ions surrounded with OH. When
some M(II) ions are isomorphically substituted with M(III) ions a positive layer charge is
induced and compensated by the anions in the interlayer. Since there is no limitation to
the nature of interlayer anions, these anions can be exchanged, resulting in a substantial
anion exchange capacity of the LDH. After thermal decomposition/calcination of LDHs
the layered structure collapses and mixed oxides are formed having an interesting property called “memory effect”, an ability to restore the original layered structure in contact
with water and different anions. This property is the basis for the high adsorption capacity of LDHs. Due to the possibility to tailor catalytic properties of LDHs by varying
their composition, synthesis method and parameters, LDHs are suitable catalyst supports
(7, 8). When loaded with Pd and Cu, these catalysts have shown high activity in the reaction of nitrate reduction, which makes them very attractive for further scientific studies
(1, 3, 5). The catalytic activity of copper in LDHs as sole active phase has not been investigated so far, which justifies the present study. In order to estimate the net effect of
the catalytic reduction, the adsorption capacity of LDH based catalysts was also measured in the same model system, without the presence of hydrogen.
EXPERIMENTAL
For the synthesis of LDHs of the general chemical formula
[M(II)1−xAlx(OH)2]x[(CO3)x/2]·yH2O (where M(II) are divalent ions (Mg, Cu); x is the ratio
between Al content and the total metal content), low supersaturation method has been
chosen, explained in detail elsewhere (9). The composition of metals was chosen inside
and outside the optimal range (0.25x0.33) for the single phase LDH synthesis, with the
intention to compare single phase and complex, multiphase, metastabile systems. The
loading of Cu as the active component, constant in all samples, was 10 mol%. The constant flow of 4 ml min-1 of Mg(NO3)2·6H2O, Cu(NO3)2·6H2O and Al (NO3)3·9H2O in aqueous solutions with the total molar metal amount nMg + nAl + nFe = 1 mol, was introduced
to the distilled water and vigorously stirred (300 rpm). The constant pH value (pH 9.710.2) was maintained by adding of Na2CO3 and NaOH solutions.
The chosen denotation of samples shows the starting amount of all constituting metals
in molar percent. For example, the sample with starting 60 mol% of Mg, 10 mol% of Cu
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and 30 mol% of Al is denoted as Mg 60-Cu 10-Al 30. The calcined samples have an additional letter C, for example C-Mg 75-Cu 10-Al 15.
X-ray diffraction analysis (XRD) was used to determine the phase composition and
crystal structure using Seifert MZ-IV diffractometer (anticathode CuKα wavelength:
λ=0.15406 nm, halt time 60 s, at 40 kV and 55 mA) in 2θ range from 5o to 70o.
The morphology of the samples was studied by scanning electron microscopy (SEM)
using JEOL, JSM-6460LV instrument, at the working voltage of 20 kV and working distance of 10 mm.
The same instrument was used for the qualitative and preliminary quantitative analysis of mixed oxides surface by energy dispersive spectroscopy (EDS).
Preceding the reaction catalyst precursors were calcined 5h at 500oC and then reduced/activated in a hydrogen flow (60 ml min-1) at 200oC during 2h. Finely powdered catalyst samples (0.2 g) were introduced into a pyrex batch reactor and mixed with 100 ml
of aqueous nitrate solution (KNO3) with NO3 concentration of 100 mg dm-3 (100 ppm).
The mixture was stirred with magnetic stirrer (350 rpm) at room temperature and saturated with hydrogen at a constant flow of 50 cm3 min-1 (Brooks Instruments Division,
Emerson Electric CO, USA flow controller). After 1 h of reaction time, the hydrogen
flow was stopped, the catalyst was separated using Hettich, Rotofix 32 centrifuge (2200
rpm, 5 min) and the remaining solution was used to determine the concentration of nitrate, nitrite and ammonium ions. The Jenway ionselective electrode was used to determine the nitrate ion concentration and Cole-Palmer ionselective electrode for ammonium
ion concentration. UV-VIS CECIL CE 2021 2000 Series spectrophotometer was used to
measure nitrite ion concentration at 538 nm, applying the ISO 2918:1975 referent method. Catalysts performance is presented as NO3 conversion and selectivity towards undesirable product NH3, calculated as the ratio of NH3 concentration and conversion.
The adsorption experiments were performed applying the same catalyst-adsorbens/solution ratio, following the same model solution concentration and temperature, as
well as identical conditions of adsorption and separation sequences as used in the catalytic tests excluding the presence of hydrogen.
The diffractograms (Fig. 1) show the presence of characteristic LDH patterns (7, 9) in
all as-synthesized samples, sharp, symmetric (003), (006), (110) and (113) reflections and
asymmetric (102), (105) and (108) peaks. In the sample Mg 60-Cu 10-Al 30, synthesized
within the optimal range for the single LDH synthesis (0,22 <x < 0,33), having the most
intensive XRD peaks, only the LDH phase was identified. The same was found in the case of the sample Mg 75-Cu 10-Al 15 having the Al content near to the optimal range. In
the samples Mg 40-Cu 10-Al 50 and Mg 20-Cu 10-Al 70, with the enhanceed Al amount,
besides the LDH phase, an additional aluminum phase, Bayerite - Al(OH)3, was observed. The reflections of the other single hydroxide, Mg(OH)2 and Cu(OH)2, were not detected in any sample regardless of metals loading. It was observed that with an increase in
the departure from the optimal metals range for single LDH phase synthesis the intensities of XRD patterns characteristic for LDH phase decreased.
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Figure 1. XRD patterns of as-synthesized samples
(+ - LDH phase, * - Bayerite (Al(OH)3) phase)
After the calcinations and in accordance with previous investigations (9, 10), the
layered structure collapses with the formation of mixed oxides solid solution (Fig. 2). In
all samples, only the characteristic patterns of mixed oxides, MgCu(Al)O, with regular,
dense-packed, cubic, NaCl-type oxygen lattice were observed, with decreasing intensities
following the increase in the aluminum content. The characteristic XRD patterns of additional aluminum or copper phases were not detected after calcinations/thermal decomposition of samples.
Figure 2. XRD patterns of calcined samples (o – mixed oxide Mg(Al)O phase)
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Morphology of of calcined samples was analyzed by SEM. The images given in Fig.
3 indicate the presence of agglomerates of plate-like particles connected at the edges at
various angles. The size of particles and agglomerates within the studied series varied
depending on sample composition. The dimensions of particles decreased with the increase in Al content, which is in accordance with the results of our previous investigation
(9, 10) showing that aluminum ions act as nucleation centers during the coprecipitation.
The EDS analysis detected the presence of Mg, Cu, Al and O in all samples. Considerable amounts of C, N and Cl were also present, implying the necessity for a prolonged
calcination and more excessive washing of the synthesis solution, to eliminate carbonates
and nitrates. The presence of Cl probably originates from the poorly distilled water used
for washing. In order to give a quantitative analysis of surface composition, multiple EDS
analysis per sample, having statistically valid analysis of several surface spots is necessary.
Figure 3. SEM micrographs of calcined Mg -Cu -Al samples, magnification 50 000 x
The results of catalytic and adsorption efficiency are presented in Fig. 4 show the values for both catalytic reaction possibly coupled with adsorption and sole adsorption. The
catalytic nitrate conversion, ranging from 23% to 62%, depends on the catalyst composition, decreasing with the increase in aluminum content. All samples also show a substantial NO3 adsorption capacity, ranging from 18% to 38%. The highest catalytic conversion and the lowest adsorption capacity was observed for the Mg 75-Cu 10-Al 15 catalyst
having the lowest amount of Al. In contrast, the Mg 20-Cu 10-Al 70 catalyst, with the
highest amount of Al, has the lowest catalytic conversion and the highest adsorption capacity. The result is a bit ambiguous, showing a negative effect of the presence of hydro135
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gen on the adsorption efficiency (NO3 depletion due to the catalysis coupled with
possible simultaneous adsorption is lower compared to the sole adsorption). This may indicate that some catalyst properties change in the hydrogen environment, which may not
be preferential for NO3 adsorption. However, the conclusion about the possible mechanism of adsorption hindering in the presence of hydrogen should be based on a more detailed investigation of the observed phenomena. The best catalytic performance in the
studied series of Mg-Cu-Al LDH-based catalysts were observed for the samples having
single LDH phase precursors (Mg 75-Cu 10-Al 15 and Mg 60-Cu 10-Al 30). It could be
concluded that the larger amount of aluminum and the presence of an additional Al phase
besides LDH phase in the catalyst precursors has a negative effect on the catalyst behavior. That is in accordance with the earlier explained mechanism of water denitrification
on LDHs-based catalysts, in which the LDH phase is the sole environment for the catalytic cycle.
For a detailed analysis of catalytic activity, a supplementary investigation of redox
and acid-base properties are required. Substantial adsorption capacity underlies the
necessity to take into account the effects of adsorption in all future investigations.
Figure 4. Nitrate removal due to catalytic reduction and adsorption
Besides nitrate ion reduction the concentrations of products other than nitrogen (nitrite and ammonium ions) were also measured, as well as the pH value at the end of the
reaction (Fig. 5). The presence of traces of nitrite ion in all samples regardless of their
composition (in the range from 0.685 to 1.025 ppm) shows that nitrate to nitrite reduction
as the first intermediate step of the reaction, is not a limiting step. This step may have
happened on the copper active site, with different activities for the constant Cu concentration explained by various active site environments (different Mg/Al ratio). The following
NO2 transformation, either to desirable nitrogen or to harmful NH3 may have happened
on other active sites belonging to Mg or Al phases as it seems that various Mg/Al ratio in
different samples may be responsible for different NH3 concentrations (Fig. 5). It has to
be mentioned, however, that such dramatic changes in the Mg/Al ratio (from 75/15 to
20/70) bring only small differences in the absolute amount of NH3 produced, as shown in
Fig. 5. Nevertheless, substantial selectivity differences, measured by factor of 4 and mo136
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re, occur due to the substantial changes in NO3 conversion among catalyst samples (Fig.
5). Thus, the least selective catalyst towards undesirable ammonia (please note this is the
best catalytic performance), was the sample with the lowest amount of Al, Mg 75-Cu 10Al 15, which also exhibited the highest NO3 conversion. In general, selectivity to ammonia increased with the increase in Al content.
Figure 5. Total amount of NH3 produced and selectivity towards NH3. (corresponding
pH values have been measured at the end of the reaction)
The achieved selectivity towards ammonia in relation to pH observed at the end of the
reaction (Fig. 5) should also be discussed. The problem of high pH conditions as favorable for the enhanced NH3 production has been discussed in the literature (11) and
correlated to the catalyst pore structure (12). It was noticed that diffusion restricted OH
ions, produced in the course of the reaction, may temporarily block active sites and
diminish the catalyst activity. Besides, they can impose a permanent pH-gradient inside
the small pores, making suitable conditions for the nitrite transformation to ammonia.
Also, a high specific surface area coupled with beneficial mesopore size has been shown
in our previous work as a prerequisite for both high activity and high selectivity (13, 14).
The performance of the most active and selective catalyst sample, Mg 75-Cu 10-Al 15,
corresponds to the mentioned studies, at least when the observed pH value is concerned.
This sample shows advantageous performances working at the highest pH in a series,
which could be a consequence of an efficient exchange of OH ions inside of LDH layers
by NO3. Thus the majority of produced OH ions is driven out of the LDH interlayer into
the water solution, leaving new NO3 reactant near active sites under the favorable pH
conditions for desirable NO2 transformation to N2. In contrast, in the case of samples
with additional Al phases, OH ions remain trapped inside the pores cavities, pushing the
reaction towards NH3 production. A detailed textural characterization is required in order
to prove the mechanism leading to the observed selectivity differences. Also, for a further
investigation it could be recommended to confirm the results using another analytical
method for the measurement of the nitrate and ammonium ions concentration because the
measurements with the ionselective electrodes are very sensitive and tentative.
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CONCLUSION
The synthesis of Mg-Cu-Al layered double hydroxides having 10% Cu and different
Mg/Al ratios was successful, as confirmed by XRD analysis. In the samples with a
composition having substantial deviation from optimal single LDH phase synthesis, an
additional aluminum phase, Bayerite-Al(OH)3 was identified. The morphology of the
samples depends on sample composition. All samples have agglomerates of plate-like
particles with dimensions decreasing with the increase in Al content. EDS analysis detected the presence of Mg, Cu, Al and O in all samples, as well as a considerable amount
of C, N and Cl, suggesting that prolonged calcination and more excessive washing of
synthesis solution are needed to eliminate carbonates and nitrates from the samples. All
studied samples were active in the catalytic nitrate reduction reaction with hydrogen,
showing NO3 reduction from 23% to 62%, following the decrease in Al content. The
highest conversion was measured for the single-phase samples with high amounts of
magnesium and potential base properties. All studied samples showed substantial adsorption capacity, ranging from 18% to 38%. For the future investigation, it would be necessary to take into account the effects of the adsorption. It should be noted that Mg-CuAl LDHs have been successfully used as a catalyst support with palladium as active
phase. The observed activity of Mg-Cu-Al LDHs based catalysts showed that the support
also has a catalytic function and that careful design of Mg-Cu-Al with Pd as active phase
may lead to very efficient catalysts for the studied reaction.
Acknowledgement
The authors gratefully acknowledge the financial support of Matica srpska.
REFERENCES
1. A.E. Palomares, J.G. Prato, F. Rey and A. Corma: Using the “memory effect” of hydrotalcites for improving the catalytic reduction of nitrates in water. J.Catal. 221
(2004) 62–66.
2. M. Islam and R. Patel: Nitrate sorption by thermally activated Mg/Al chloride hydrotalcite-like compound. J. Hazard. Mater. 169 (2009) 524–531.
3. Y. Wang, J. Qu and H. Liu: Effect of liquid property on adsorption and catalytic
reduction of nitrate over hydrotalcite-supported Pd-Cu catalyst. J. Molecular Catal. A:
Chemical 272 (2007) 31-37.
4. U. Prüsse and V. Klaus-Dieter: Supported bimetallic palladium catalysts for waterphase nitrate reduction. J. Molecular Catal. A: Chem. 173 (2001) 313–328.
5. A.E. Palomares, J.G. Prato, F. Márquez and A. Corma: Denitrification of natural
water on supported Pd/Cu catalysts. Appl. Catal. B: Environm. 41 (2003) 3-13.
6. A. Pintar, J. Batista and I. Muševic: Palladium-copper and palladium-tin catalysts in
the liquid phase nitrate hydrogenation in a batch-recycle reactor. Appl. Catal. B:
Environm. 52 (2004) 49–60.
7. F. Cavani, F. Trifiro and A. Vaccari: Hydrotalcite-Type Anionic Clays: Preparation,
Properties and Application. Catal. Today 11 (1991) 173-301.
138
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DOI: 10.2298/APT1041131V
UDC: 546.175:66.097
BIBLID: 1450-7188 (2010) 41, 131-139
8. A. Vaccari: Preparation and catalytic properties of cationic and anionic clays. Catal.
Today 41 (1998) 53-71.
9. T. Vulić, M. Hadnadjev and R. Marinković-Nedučin.: Structure and morphology of
Mg–Al–Fe-mixed oxides derived from layered double hydroxides. J. Microscopy
232, 3 (2008) 634–638.
10. T. Vulić: Gline kao katalizatori, Zadužbina Andrejević, Beograd (2008) p. 62-65.
11. R. Gavagnin, L. Biasetto, F. Pinna and G. Strukul: Nitrate removal in drinking
waters: the effect of tin oxides in the catalytic hydrogenation of nitrate by Pd/SnO2
catalysts. Appl. Catal. B: Environm. 38 (2002) 91-99.
12. M. D’Arino, F. Pinna and G. Strukul: Nitrate and nitrite hydrogenation with Pd and
Pt/SnO2 catalysts: the effect of the support porosity and the role of carbon dioxide in
control of selectivity, Appl. Catal. B: Environm. 53 (2004) 161-16.
13. M. Kovacevic, A. Zarubica and G.Boskovic: Specific surface area – key factor
determining the catalytic activity of Pd/SnO2 catalyst in nitrate hydrogenation.
J.Optoelec.Advanc.Materials 9, 11 (2007) 3614-3618.
14. M. Kovacevic, E. Kis, and G. Boskovic: Benefits of mesopores in nanocristalline
Pd/SnO2 catalysts for nitrate hydrogenation, Acta Periodica Technologica EFF, 38
(2007) 69-74.
КАТАЛИЗАТОРИ НА БАЗИ Mg-Cu-Al СЛОЈЕВИТИХ ХИДРОКСИДА ЗА
РЕДУКЦИЈУ НИТРАТА ИЗ ВОДЕНИХ РАСТВОРА
Татјана Вулић и Горан Бошковић
Секундарни отпад и контаминација бактеријама, присутни код тренутно заступљених физичко-хемијских и биолошких процеса сепарације нитрата из водених
раствора, чини нове каталитичке технологије које конвертују нитрате до нештетног
гасовитог азота интересантним за истраживање. Катализатори на бази Mg-Cu-Al
слојевитих хидроксида (LDH) са различитим Mg/Al уделима су испитивани у реакцији денитрификације у присуству водоника са само бакром као активном фазом.
Пошто LDH имају јоноизмењивачки капацитет, а њихови мешовити оксиди поседују ефекат памћења (врећање слојевите структуре након термичке разградње),
испитиван је и адсорпциони капацитет у односу на нитрате у истом модел систему.
Сви узорци су показали уклањање нитрата од 23% до 62% које прати опадање Al
садржаја, као и значајни адсорпциони капацитет у опсегу од 18% до 38%. Ови резултати истичу неопходност узимања у обзир ефеката адсорпције при свим будућим истраживањима.
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THE INVESTIGATION OF COAGULATION ACTIVITY OF NATURAL
COAGULANTS EXTRACTED FROM DIFFERENT STRAINS OF COMMON
BEAN
Marina B. Šćiban, Mirjana A. Vasić, Jelena M. Prodanović, Mirjana G. Antov
and Mile T. Klašnja
Coagulation and flocculation by adding chemicals are the methods that are usually
used for removal of water turbidity. This study is concerned with the coagulation activity
of extracts of various strains of bean. The aim was to ascertain if bean varieties influence
coagulation activity. Active components were extracted from 1 g of ground sample with
100 ml distilled water. Contents of dry matter and nitrogen were specified in the solid
samples, and the content of soluble nitrogen was determined in the extracts. These data
were used to calculate the efficiency of extraction of nitrogen- containing compounds.
The coagulation activity was assessed by jar test using synthetic turbid water, of the
initial pH 9 and turbidity 35 NTU.The jar test was carried out by adding different
amounts of extracts to model water, and stirring the content. After sedimentation for 1 h,
residual turbidity was determined by turbidimeter and coagulation activity was
calculated. The increment of organic matter concentration after the coagulation was also
determined. These experiments confirmed that extracts of all investigated strains of bean
could be used successfully as natural coagulants.
KEYWORDS: Water clarification, natural coagulants, various strains of bean
INTRODUCTION
Natural water is usually turbid to some extent. Coagulation and flocculation are
commonly used methods for water turbidity removal, and are usually conducted by adding chemicals such as salts of aluminium and iron and polyelectrolytes. The first investigations about harmful influence of these chemicals on human health were published in
the 60's of the 20th century. Those and later publications showed that the residues of
aluminium salts in the water can cause Alzheimer’s disease (1, 2, 3). Also, there are studies that indicate that some of synthetic organic polymers, such as acrylamide, have
strong neurotoxic and carcinogenic effects (4).
Dr. Мarina B. Šćiban, Assist. Prof., Jelena M. Prodanović, M.Sc., Res. Assist., Dr. Mirjana G. Antov, Assist.
Prof., Dr. Mile T. Klašnja, Prof., University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000
Novi Sad, Republic of Serbia, Dr. Mirjana A. Vasić, Sen. Res. Assoc., Inst. of Field and Vegetable Crops,
Maksima Gorkog 30, 21000 Novi Sad, Serbia
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In view of the above problems, intensive investigatioins of natural coagulants have
been conducted in the last years in order to replace chemical coagulants in water treatment. It is believed that natural coagulants, that can originate from plants, animals or
microorganisms, are not harmful, and besides, the resulting biodegradable sludge can be
disposed in the nature without any adverse influence.
The idea of water clarification by natural coagulants is many centuries old. There are
written documents from India in which the seed of Strychnos potatorum tree was mentioned as water clarifier (5). In the XVI and XVII century, militaries of Peru used roasted
and ground corn beans (Zea mays) for this purpose. Recently, the most investigated plant
is Moringa oleifera, whose ground seeds are used for water clarification by women in
rural areas of Sudan. Results of these investigations confirmed that Moringa oleifera seed
extract is very efficient for water clarification (6, 7).
Considering the fact that M. oleifera is a plant that originates from tropical areas, we
wanted to investigate the possibility of preparing natural coagulants from sources that are
cheap and easily available in this region. Our previous investigations confirmed the fact
that extracts of various strains of Leguminose could be used as natural coagulants (8).
The aim of this study was to obtain natural coagulants from different strains of bean, in
order to compare their coagulation activity.
EXPERIMENTAL
Model water. The coagulation activity was assessed by jar test using synthetic turbid
water. As first, kaolin was ground in a ceramic mortar and sieved through the sieve with
pore size of 0.4 mm. Smaller fraction was then taken to prepare a 10 g/l suspension in tap
water. The suspension was stirred for 60 minutes on a magnetic stirrer, and left for 24
hours in order to achieve complete hydration of kaolin. Model water was prepared just
before performing the coagulation test, by adding this 1% kaolin suspension to tap water
in an amount of 5 ml/l to obtain the water with initial turbidity of 35 NTU (nephelometric
turbidity units).
Coagulants. Natural coagulants were extracted from four strains of bean, cultivated
in the Institute of Field and Vegetable Crops, Novi Sad:
 Sample 1 – Levač bean
 Sample 2 – Sremac bean
 Sample 3 – Zlatko bean
 Sample 4 – Balkan bean.
Natural coagulants were obtained in the following way: seeds were ground and sieved
through the sieve with pore size of 0.4 mm. An amount of a 10 g/l of the smaller fraction
was suspended in destilled water. This suspension was stirred 10 minutes on a magnetic
stirrer in order to extract active coagulants. After that, the suspension was filtered through
filter paper Macherey-Nagel MN 651/120. Obtained filtrates, called crude extracts, were
stored in a frigerator at 5ºC.
Contents of dry matter and nitrogen were specified in the solid samples, and the content of soluble nitrogen was determined in the extracts. These data were used to calculate
the efficiency of extraction of a nitrogen-containing compounds.
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Analytical methods. Dry matter was determined by standard method at 105ºC (9).
Content of nitrogen in ground samples and crude extracts was determined by the Kjeldahl
method (9). The pH was measured using a pH meter. Water turbidity was measured by
nephelometric method on a turbidimeter (10) and expressed in NTU. Content of organic
matter before and after coagulation was determined as permanganate demand (10).
Coagulation test. The coagulation activity was assessed by jar test using synthetic
turbid water, with the kaolin concentration of 50 mg/l and turbidity 35 NTU. The pH of
the model water was adjusted to pH 9 by adding 1 mol/l NaOH just before performing
coagulation test. The jar test was carried out by adding different amounts of extracts to
300 ml of model water. Fast stirring at 200 rpm for 1 min was followed by slower stirring
at 80 rpm for 30 min, and after that the system was left to sediment for 1 h. The same
coagulation test was conducted with no coagulant (blank). After sedimentation for 1 h,
residual turbidity was determined in 50 ml of upper clarified liquid, using turbidimeter
and coagulation activity was calculated:
Coagulation activity (%) = (Mb – Ms) х 100 / Mb
[1]
were Mb and Ms are the turbidities of the blank and the sample, respectively.
Considering the fact that several different strains of bean were available as source for
natural coagulants, the first step was to analyse them. Results of analyses of solid samples
of beans are presented in Table 1.
As can be seen from these results, all of four bean strains have similar contents of dry
matter. A little bit higher content of dry matter was determined in sample 2, and a little
bit higher content of nitrogen in sample 4. Previous investigations showed that proteins
exhibited coagulation activity (11), and this was the reason why we analysed the content
of proteins. The protein content varied significantly between bean strains, and it could be
said that sample 4 had by about 25 % higher content of proteins than sample 3. The fact
that different strains of bean had different contents of proteins and other compounds was
confirmed in previous investigations (12).
Table 1. Results of the analysis of solid bean samples
Dry matter (%)
Content of nitrogen (% dm)
Content of proteins (% dm)
1
88.9
3.6
22.4
Bean sample
2
3
92.1
88.5
3.2
3.1
20.2
19.7
4
88.5
4.1
25.6
Table 2 gives the results of the analysis of extracts obtained from solid samples. The
highest content of soluble nitrogen was found in sample 1. Also, the best extraction effi143
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ciency (97.5 %) was obtained with sample 1. The amount of extracted proteins was not
proportional to the content of proteins in solid samples. The efficiency of extraction
determined for sample 4 was not so good, although this sample had the highest content of
proteins.
Table 2. Results of the analysis of bean extracts
1
Bean sample
2
3
4
Content of soluble nitrogen (mg/l)
350
268
239
315
Efficiency of extraction of nitrogen- containing
compounds (%)
97.5
82.8
75.9
77.0
Figure 1 shows the influence of dose of different bean extracts on the coagulation
activity. It can be seen that sample 2 and sample 4 showed very similar behavior, with
maximum of coagulation activity at applied doses of extracts of 3.35 mg/l and 3.94 mg/l
respectively. Sample 3 showed maximum of coagulation activity at a dose of 1.49 mg/l.
In comparision with other samples, sample 1 had different behavior. This extract was
efficient at lower, as well as at higher doses. It had maximum of coagulation activity at a
dose of 6.56 mg/l, and with six times lower dose (1.09 mg/l) the coagulation activity
decreased by 25 % in comparision with the maximum. Good coagulation activity was
achieved for all of the samples when an optimal dose of the coagulant was applied.
Similar results were also obtained with extracts of other Leguminose species (8), while
natural coagulants obtained from Moringa oleifera seeds showed a little bit higher
coagulation activity (6).
Figure 1. Effect of dose of different bean strains extracts on coagulation activity (CA)
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Organic matter in water has a bad influence on water quality. Its presense in water
can change color and clarity, and can lead to the appearance of specific smell and taste of
water. Since natural coagulants are of organic nature, in the next step we wanted to
investigate how they influence the content of organic matter after performing coagulation
tests.
Content of organic matter in the water after performing coagulation tests was assessed
by determining the permanganate demand. As first, jar tests with a dose of 3 ml/l of different coagulants were performed. After separating the upper clear parts, permanganate demand was determined in each of them. Simultaneously, permanganate demand was determined for the blank, and it was 10.6 mg KMnO4/l. The obtained results are shown in
Table 3. Content of organic matter increased about two times in comparision with the
blank. The reason for this were high doses of coagulants that were applied.
Table 3. Permanganate demand in water after performing coagulation tests
1
Permanganate demand (mg KMnO4/l)
22.3
Bean sample
2
3
22.1
22.6
4
22.6
CONCLUSION
Considering performed experiments and results obtained in the investigation of the
coagulation activity of the extracts of natural coagulants obtained from various strains of
bean, it is possible to derive the following conclusions:
 All investigated strains of bean showed potential to be used for preparing coagulants for water clarification.
 Turbidity of model water was decreased by 5 – 50 % by using natural coagulants
obtained from different strains of bean.
 Samples 1, 2 and 4 showed maximum of coagulation activity (about 45%) in the
range of applied doses of coagulants from 3.5 mg/l to 4.5 mg/l.
 Sample 3 showed a lower coagulation activity (maximum about 33%) in comparision with other samples, but at a significantly lower dose of coagulant – 1.5 mg/l.
 Content of organic matter in the water after coagulation tests performed with all
samples was high, twice higher than it was in the blank.
Acknowledgement
This research was supported by the grant number BTN 20009 from the Ministry of
Science and Technological Development of the Republic of Serbia.
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REFERENCES
1. D.R. Crapper, S.S. Krishnan and A.J. Dalton: Brain Aluminium Distribution Alzheimer’s Disease and Experimental Neurofibrillary Degeneration, Science, 180 (1973)
511-513.
2. C.N. Martyn, D.J.P. Barker, C. Osmond, E.C. Harris, J.A. Edwardson and R.F. Lacey:
Geographical Relation between Alzheimer’s Disease and Aluminium in Drinking
Water, The Lancet, 1 (1989) 59-62.
3. R.G. Miller, F.C. Kopfler, K.C. Kelty, J.A. Stober and N.S. Ulmer: The Occurence of
Aluminium in Drinking Water, J. AWWA, 76 (1984) 94-101.
4. D.D. Mccollister, E. Oyen and V.K. Rowe: Toxicology of Acrylamide, Toxicol. Appl.
Pharmacol., 6 (1964) 172-181.
5. Sutherland J.: M. oleifera in water treatment,
www.le.ac.uk/engineering/staff/Sutherland/moringa/moringa.htm
(accessed
14
September 2010).
6. S.A. Muyibi and L.M. Evison: Optimizing Physical Parameters Affecting Coagulation
of Turbid Water with Moringa oleifera Seeds, Water Res. 29, 12 (1995) 2689-2695.
7. T. Ocuda, A.U. Beas, W. Nishiima and M. Okada: Improvement of Extraction
Method of Coagulation Active Components with Moringa oleifera Seed, Water Res.,
33 (1999) 3373-3378.
8. M. Šćiban, M. Klašnja and J. Stojimirović: Investigation of Coagulation Activity of
Natural Coagulants from Seeds of Different Leguminose Species, Acta Periodica
Technologica, 36 (2005) 81-87.
9. MEBAK: Brautechnische Analysenmethoden, MEBAK, Freising-Weihenstephan
(1997), Bd. I, p. 171.
10. Drinking water (in Serbian), Savezni zavod za zdravstvenu zaštitu, NIP ”Privredni
pregled”, Beograd, 1990.
11. M. Antov, M. Šćiban and N. Petrović: Proteins from Common Bean (Phaseolus
vulgaris) Seed as a Natural Coagulant for Potential Application in Water Turbidity
Removal, Bioresource Technology 101, 7 (2010) 2167-2172.
12. A. Tepić, B. Vujičić, M. Vasić and A. Lučić: Amino Acids and Phytic Acid in some
Serbian Varieties of Dry Beans (Phaseolus vulgaris); 2nd International Congress on
Food and Nutrition, Istanbul, 24-26 October 2007, Abstrac Book, p. 180.
ИСПИТИВАЊЕ КОАГУЛАЦИОНЕ АКТИВНОСТИ ПРИРОДНИХ
КОАГУЛАНАТА ЕКСТРАХОВАНИХ ИЗ РАЗЛИЧИТИХ СОРТИ ПАСУЉА
Марина Б. Шћибан, Мирјана A. Васић, Јелена М. Продановић, Mирјана Г. Антов,
Миле Т. Клашња
Коагулација и флокулација представљају најчешће примењиване методе за бистрење воде, и углавном се изводе додатком хемијских коагуланата. У овом раду је
испитивана коагулациона активност екстраката добијених из семена различитих
сорти пасуља, a с циљем дa сe утврди могућност њиховe применe кao природних
146
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коагуланатa зa уклањањe мутноћe водe, и дa сe утврди утицаj сортe пасуљa нa коагулациону aктивност. Eкстракциja aктивних компоненти je изведенa сa 100 ml дестилованe водe из 1 g самлевеног узоркa. У чврстим узорцимa je oдређен садржaj
сувe материje и азотa, a у екстрактимa садржaj растворљивог aзотa, нa oснову чегa
je oдређенa eфикасност eкстракциje jeдињењa сa aзотом. Зa oдређивањe кoaгулационe aктивности eкстракатa изведен je џар-тест, додавањем различитих дозa eкстракатa модел води, чији je pH 9 и почетнa мутноћa 35 NTU. Након мешањa у
oдређеном временском интервалу, чашe су oстављенe дa сe њихов садржaj исталожи, a затим je у бистром делу одређиванa мутноћa, и прекo њe коагулационa aктивност. Taкођe je утврђен допринос сваког oд eкстракатa порасту садржaja oрганских материja у води након њеног бистрењa у oдносу нa слепу пробу, a прекo перманганатног брoja. Oвим eкспериментимa потврђенo je дa сe eкстракти свих испитиваних сорти пасуљa могу успешнo применити кao природни коагуланти.
147
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VARIATION OF ESSENTIAL OIL COMPOSITION OF EUCALYPTUS
CAMALDULENSIS (MYRTACEAE) FROM THE MONTENGERO COASTLINE
Slavenko Grbović, Dejan Orčić, Maria Couladis, Emilija Jovin, Dušan Bugarin,
Kristina Balog and Neda Mimica-Dukić
In the current study the essential oil obtained from the leaves of Eucalyptus camaldulensis plants collected from five localities of the Montenegro coastline was analyzed.
The oil yield varied from 0.63 % (Kotor) up to 1.59% (Tivat). The chemical composition
of the leaf essential oil was analyzed using GC-MS technique. Monoterpene hydrocarbons were a major class of compounds. Among them, dominant compounds were p-cymene (17.38-28.60%), -phellandrene (12.35-14.47%) and -pinene (0.94-11.48%). The second largest group was oxygenated monoterpenes with cryptone (4.97-7.25) and terpinene-4-ol (2.75-4.21%) as predominant. Besides high content of sesquiterpene alcohol spathulenol (7.83-14.15%) was found. According to the results obtained E. camaldulensis
from Montenegro can be classified in the chemotype with low 1,8-cineole and high p-cymene and cryptone ratio.
KEYWORDS: 1,8-cineole, cryptone, GC-MS, Eucalyptus camaldulensis, p-cymene
INTRODUCTION
In recent years, natural extracts have been in high demand by the food manufacturers,
cosmetics, and pharmaceuticals due to the growing interest of consumers in the ingredients from the natural sources. Spices and essential oils are also well known for their various beneficial effects on human health. The use of aromatic plants and spices in phytotherapy is mostly due to the essential oils and their various biological activities, such as
antimicrobial, spasmolytic, carminative, hepatoprotective, antiviral, and anticarcinogenic
(1,2). However, besides having a wide spectrum of well-known biological and pharmacological activities, essential oil composition is frequently used in distinguishing particular
chemoraces among the species and genera.
Eucalyptus (Myrtaceae) is one of the most important and most widely planted genera.
Although being Australia’s native, more than 700 species wildly grow in many parts of
the world. In fact, Eucalyptus species are one of the most-extensively planted pulpwood
Slavenko Grbović, PhD student, Emilija Jovin, PhD student , Dejan Orčić, PhD student, Kristina Balog, PhD
student, Dušan Bugarin PhD student and Dr Neda Mimica-Dukić, Prof., Department of Chemistry Faculty of
Scineces, University of Novi Sad, Trg Dositeja Obradovića 3, 21 000 Novi Sad, Serbia. Dr Maria Couladis,
Department of Pharmacognosy and Natural Product Chemistry, Faculty of Pharmacy, University of Athens,
Greece
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species. The trees are planted largely for their leaves that are rich in essential oils and are
exploited commercially for their use in food, flavors, pharmaceutical and perfumery
industries (3). Used for centuries as a traditional Aboriginal herbal remedy, eucalyptus
leaves and their essential oils have found various applications in everyday life due to their
antiseptic, anti-inflammatory and antipyretic properties (4, 5).
Around 15 eucalyptus species grow in the Mediterranean region, out of which less
than 10 species were introduced in the coastal area of Montenegro at the beginning of the
20th century. Among them, Eucalyptus camaldulensis Dehn. (syn. Eucalyptus rostrata
Schl.) is the most commonly found in Montenegro. This species is used in the indigenous
system of medicine to cure various human ailments such as diarrhea, chronic dysentery,
malaria, infection of upper respiratory tract, and certain skin diseases (6). Essential oils
obtained from the leaves are of particular commercial interest. However, no information
about the chemical composition of Eucalyptus camaldulensis grown in Montenegro had
been published before we initiated the corresponding studies. Here, for the first time, we
report the composition of essential oil from the leaves of Eucalyptus camaldulensis plants
collected from the different locations of the Montenegro coastline.
EXPERIMENTAL
Plant material and chemicals
Plant material: For essential oil analysis leaves were collected from the trees of
Eucalyptus camaldulensis Dehn. growing at five locations at Montenegro coastline: Bar
(No 2-1812), Sutomore (No 2-1813), Tivat (No2-1814), Kotor (No2-1815), and Herceg
Novi (No2-18-16), in August 2006. Voucher specimens were prepared and identified by
Goran Anačkov, PhD, and deposited at the Herbarium of the Department of Biology and
Ecology, Faculty of Sciences, University of Novi Sad.
Essential oil isolation and analysis
Essential oils isolation: Air-dried plant materials were submitted to hydrodistillation
according to Eur. Pharm. 4 (7), using n-hexane as a collecting solvent. The solvent was
removed under vacuum, and the quantities of the essential oils were determined gravimetrically.
GC-MS analysis of essential oil: Qualitative analysis of essential oils was performed
by gas chromatography-mass spectrometry (GC-MS). Agilent Technologies 6890N5975B system was used, with data acquisition parameters as follows: carrier gas - He,
flow rate 1.0 mL/min, constant flow mode; injection volume 0.2 µL (split 50:1), inlet
temperature 250°C; Agilent Technologies HP-5MS 30 m  0.25 mm  0.25 µm column,
temperature program: 50°C for 1 min, 5°C/min to 100°C, 9°C /min to 200°C, hold 7.89
min; transfer line temperature 280°C; electron ionization, electron energy 70 eV, scan
mode, mass range 35-400 Da, quadrupole temperature 150°C, source temperature 230°C.
Acquired data were analyzed by Agilent Technologies MSD ChemStation software in
conjunction with AMDIS (Automated Mass Spectral Deconvolution and Identification
152
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System) and NIST MS Search software. Two different mass spectra libraries were used
for mass spectra identification: Wiley Registry of Mass Spectral Data 7th Edition (338000
spectra, 289000 unique compounds) (8), NIST/EPA/NIH Mass Spectral Library 05 with
190825 spectra, 163198 unique compounds (9). Identity is confirmed by comparison of
Kovat`s retention indices.
The amount of essential oil obtained by hydrodistillation from the dried leaves is presented in Table 1. The oil quantity ranged from 0.63 % (Kotor) up to 1.59% (Tivat).
Comparison with available literature data (10, 11, 12, 13) shows that only plants collected
from the Tivat location, have a satisfactory oil yield, whereas the other plants are rather
poor in oil content.
Table 1. Essential oil content (%) in dried leaves of Eucalyptus camaldulensis Dehn.
collected from five locations from Montenegro coastline.
Sample
Oil Content %
Bar
0.67
Sutomore
0.70
Tivat
1.59
Kotor
0.63
Herceg Novi
0.68
In all the investigated samples monoterpene hydrocarbons were identified as the major class of compounds (Table 2). Among them dominant were: p-cymene (17.38-28.60%)
and -phellandrene (12.35-14.47%). The next largest group was the one of oxygenated
monoterpenes that ranged from 20.63% (Kotor) up to 25.62% (Tivat). Interestingly, the
major volatile compound in all oils was irregular monoterpene cryptone (Figure 1) whose
content ranged from 4.97% (Kotor) to 7.25% (Tivat).
O
H
l
o
4
e
n
e
n
i
p
r
e
t
e
n
e
r
d
n
a
l
e
h
p
e
n
e
m
y
c
p

H
H
H
O
H
O
e
n
o
t
p
y
r
c
l
o
n
e
l
u
h
t
a
p
s
Figure 1. Molecular structures of major compounds in Eucalyptus camaldulensis
essential oil
153
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Table 2. Percentage content of volatile compounds in essential oil of the leaves from
examined Eucalyptus camaldulensis Dehn. from Montenegro
Compound
α-Thujene
α-Pinene
Sabinene
β-Pinene
β-Myrcene
α-Phellandrene
α-Terpinene
p-Cymene
β-Phellandrene
Eucalyptol
γ-Terpinene
α-Terpinolene
Linalool L
cis-p-menth-2-en-1-ol
menthenol isomer +
trans-Pinocarveol
n.i.
Pinocarvone
Terpinene-4-ol
Cuminyl alcohol
Cryptone
α-Terpineol
Myrtenal
Cuminaldehyde
Phellandral
Cumyl alcohol
Thymol
α-Terpineyl acetate
Aromadendrene
Allo-aromadendrene
n.i.
Bicyclogermacrene
Spathulenol
sesquiterpene
Viridiflorol
Sesquiterpene
sesquiterpene
Lepidozenal
Total identified
Monoterpene hydrocarbons
Oxygenized monoterpenes
Sesquiterpene hydrocarbons
Oxygenized sesquiterpene
a
KIa
927
934
974
978
992
1006
1018
1026
1030
1033
1060
1090
1101
1126
Tivat
2.68
1.66
1.26
0.94
0.77
4.26
0.87
28.60
13.99
2.89
1.12
0.56
0.81
0.85
Sutomore
3.01
5.01
1.47
11.48
0.93
3.81
0.98
20.02
14.46
2.03
1.31
0.57
0.96
0.69
Kotor
3.04
3.88
1.05
6.34
0.84
2.36
0.73
24.63
12.35
1.70
0.95
0.42
0.85
0.83
Herceg Novi
2.28
3.75
1.28
9.94
0.77
3.43
0.68
17.38
13.79
1.65
1.02
0.44
0.76
0.70
Bar
2.19
3.59
1.02
9.54
0.87
4.00
1.04
18.15
14.47
1.78
1.36
0.65
0.99
0.68
1144
0.66
0.98
0.97
0.89
1.11
1155
1168
1182
1188
1190
1194
1199
1246
1281
1294
1302
1355
1451
1474
1501
1509
1593
1599
1606
1639
1642
1648
0.38
tr
4.21
0.47
7.25
0.58
0.25
2.57
1.62
0.41
0.33
tr
0.30
1.79
0.44
3.73
7.83
0.90
0.14
0.30
0.38
0.12
95.92
57.37
25.62
6.06
8.30
0.38
0.33
3.11
0.28
5.27
0.66
0.73
1.69
1.54
0.31
0.32
tr
tr
1.63
0.38
3.88
8.52
0.83
tr
0.38
0.51
0.28
98.73
61.95
21.04
5.58
8.62
0.48
0.24
3.99
0.29
4.97
0.48
0.61
1.81
1.59
0.28
0.34
tr
tr
2.18
0.57
3.77
11.63
1.12
0.14
0.51
0.52
0.42
96.89
56.09
20.63
6.04
11.95
0.25
0.30
2.75
0.31
6.12
0.62
0.71
1.59
1.67
0.42
0.53
tr
tr
1.47
0.38
3.64
14.15
1.34
0.18
0.93
0.77
0.49
97.38
54.73
21.07
5.19
14.56
0.51
0.41
3.50
0.32
5.78
0.71
0.86
1.87
1.88
0.37
0.43
tr
tr
1.99
0.44
3.60
9.77
0.94
0.16
0.58
0.69
0.42
96.65
56.77
23.48
5.78
10.37
Retention indices relative to C9-C24 n-alkanes on the HP 5MS column. GC, identification based on retention
times of authentic compounds on HP 5MS column; MS, tentatively identified on the basis of computer matching
of the mass spectra of peaks with the NIST/NBS and Wiley libraries; tr - ratio in essential oil below 0.1%; n.i.
non identified
154
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Oxygenated sesquiterpenes were also found in a considerable amount. The highest
ratio was found in the essential oil obtained from the plants collected from Herceg Novi
(14.15%) and the lowest from plants from the vicinity of Tivat (7.83%). Sesquiterpene
alcohol spathulenol was the dominant compound in this class. Sesquiterpene hydrocarbons did not significantly vary between the samples. Allo-aromadendrene was one of the
most abundant compounds. Evidently, no significant qualitative and quantitative difference in volatile composition between the samples examined was found. Only the plants
from the Tivat region can be distinguished as containing the highest portion of p-cymene
and the lowest amount of -pinene.
The composition of the essential oil from E. camaldulensis, especially from the leaves, has been widely studied. By surveying the data reported we found a great diversity
of oil composition, which was effected by many factors such as: geographical origin,
tissue explored, date of harvest, genetic factors etc. (14, 15). Two groups of E. camaldulensis essential oils can be distinguished: those that contain 1,8-cineole as the main
compound, which include E. camaldulensis from Mali, Mozambique, Nigeria, Egypt and
Iran (16-21) and those that contain spathulenol, p-cymene and cryptone as main compounds, and small quantities of 1,8-cineol, like the oil examined here, and which are similar to E. camaldulensis from the south of Florida, Jerusalem and Greece (22, 13, 14).
Our results certainly suggest that only one chemotype of E. camadulensis exists on
the Montenegro coastline. The main characteristic of this chemotype is a high amount of
aromatic monoterpene hydrocarbon p-cymene, followed by monoterpenes: -phellandrene, -pinene (except for the plants from Kotor), cryptone, -phellandrene, terpinene-4-ol
and sesquiterpene, spathulenol. According to the high p-cymene, cryptone and terpinene4-ol ratio, a great similarity was found with the plants cultivated in Spain-Valencia (23)
and the south of Florida (22).
As already mentioned, the essential oil of eucalyptus species is of great commercial
value. In the south-eastern countries such as Thailand, E. camaldulensis is mainly planted
for the use as a pulpwood. Evidently, during the process of papermaking, a large amount
of waste such as leaves is disposed. Therefore, the possibility of exploiting the leaves as a
source of oil production is being extensively investigated (24).
Some recent studies show that E. camaldulensis oil exhibits a great antimicrobial and
repellent activity (25, 26). Eucamalol and epi-eucamalol were identified as potent repellents. Interestingly enough, these compounds can be synthesized from irregular monoterpene cryptone (25). Cheng et al., 2009. (27) found that the volatile oil obtained from E.
camaldulensis had an excellent mosquito larvicidal activity. The oil composition was
similar to the one examined here, in respect to high p-cymene and phellandrene portion.
Having these facts in mind, one can conclude that leaves of E. camaldulensis from Montenegro should be exploited as a source of a valuable essential oil with considerable
commercial value.
CONCLUSION
The GC-MS analysis of essential oil yield and composition in Eucalyptus camaldulensis Dehn., samples collected from five different locations of the Montenegro coast155
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line shows that all plants belong to one chemotype which is characterized by the high
portion of p-cymene, -phellandrene, -pinene, cryptone, spathulenol and a low ratio of
1,8-cineol. This is the first report on chemical study of the eucalyptus species from Montenegro. Investigations of the biological activity of oil and extracts of Eucalyptus species
in Montenegro are in progress.
Acknowledgement
This study was financially supported by the grant No 142036 from the Ministry of
Science and Technological Development of the Republic of Serbia.
REFERENCES
1. G. Wang, G., W. Tang, and R.R. Bidigare: Terpenoids as therapeutic drugs and pharmaceutical agents, in Natural Products: Drug Discovery and Therapeutic Medicine.
Eds. L. Zhang, A.L. Demain, Humana Press: Totowa (2005) pp. 197–227.
2. F. Bakkali,, S. Averbeck, D. Averbeck, and M. Idaomar: Biological effects of essential oils – A review. Food Chem. Toxicol. 46 (2008) 446–475.
3. M.I.H Brooker, and D.A. Kleinin: A field Guide to Eucalyptus. Vol. 1. South-Eastern
Australia. Bloomings Melbourne, Australia, 3rd edition, 1992.
4. S. Jeane S., A. Worku, S.M. Sousa, V.G. Duarte, M.I.L. Machado and F.J.A. Matos:
Analgesic and anti-inflammatory effects of essential oils of Eucalyptus. J Ethnopharmacol 89 (2003) 277–283.
5. A. Kumar, V.D. Sarma, A.K. Sing and S. Kamla: Antimicrobial properties of different Eucalyptus oils. Fitoterapia 59 (1988) 141–144.
6. S. Begum and B.S. Siddiqui: Triterpenoids from the leaves of Eucalyptus camaldulensis var. obtuse. J. Nat. Prod. 60 (1997) 20-23.
7. European Pharmacopeia, 4th ed.; Council of Europe: Strasbourg Cedex, France
(2002) 2.8.12, pp 183-184.
8. Wiley Registry of Mass Spectral Data, 7th Edition (2005).
9. NIST/EPA/NIH Mass Spectral Library with Search Program: (Data Version: NIST
05, Software Version 2.0d), National Institute of Standards and Technology, 2005.
10. M. Dethier, A. Nduwimana, Y. Cordier, C. Menut and G. Lamaty: Aromatic plants of
tropical central Africa. XVI. Studies on essential oils of five Eucalyptus species
grown in Burundi. J. Essent. Oil. Res. 6 (1994) 469-473.
11. M. Dang, M. Takacsova, D.V. Nguyen and K. Kristianova: Antioxidant activity of
essential oils from various species. Food/Nahrung 45 (2001) 64.
12. British Pharmacopoeia. London: HMS Stationary Office, 2 (1980) A109.
13. J.P Chalchat, T. Kundakovic and M.S. Gorunovic: Essential oil from the leaves of
Eucalyptus camaldulensis Dehn. Myrtaceae, from Jerusalem. J. Essent. Oil Res. 13
(2001) 105-107.
14. D. Tsiri, D., O. Kretsi, I. B. Chinou, C.G. Spyropoulos: Composition of fruit volatiles
and annual changes in the volatiles of leaves of Eucalyptus camaldulensis Dehn.
growing in Greece. Flavour Fragr. J. 18 (2003) 244–247.
156
APTEFF, 41, 1-203 (2010)
DOI: 10.2298/APT1041151G
UDC: 582.883.4:665.526(497.16)
BIBLID: 1450-7188 (2010) 41, 151-158
15. A. Giamakis, O. Kretsi, I. Chinou, C.G. Spyropoulos: Eucalyptus camaldulensis: volatiles from immature flowers and high production of 1,8-cineole and -pinene by in
vitro cultures. Phytochemistry 58 (2001) 351–355.
16. J.C. Chalchat, R.P. Garry, L. Sidibe and M. Harama, M. Aromatic plants of Mali (V):
chemical composition of essential oils of four eucalyptus species implanted in Mali:
Eucalyptus camaldulensis, E. citriodora, E. torelliana and E. tereticornis. J. Essent.
Oil Res. 12 (2000) 695–701.
17. F.P Pagula, K.H.C. Baser and M. Kurkcuoglu: Essential oil composition of Eucalyptus camaldulensis Dehn. From Mozambique. J. Essent. Oil Res. 12 (2000), 333–335.
18. A.O Oyedeji, O. Ekundayo, O.N. Olawore and W.A. Koenig: Essential oil composition of two varieties of Eucalyptus camaldulensis Dehn. from Nigeria. J. Essent. Oil
Res. 12 (2000) 102–104.
19. S.L. Maximous: Effect of harvest date and steam distillation time on essential oils of
three Eucalyptus species growing in El-Kassasin region. Bull. Faculty of Agr., Cairo
Univ. 55 (2004) 71–84.
20. F. Sefidkon, M.H. Assareh, Z. Abravesh and M. Mirza: Chemical composition of the
essential oils of five cultivated Eucalyptus species in Iran: E. intertexta, E. platypus,
E. leucoxylon, E. sargentii and E. camaldulensis. J. Essent. Oil Bearing Plants 9
(2006) 245–250.
21. H. Fadel, F.Marx, A. El-Sawy and A. El-Ghorab: Effect of extraction on the chemical
composition and antioxidant activity of Eucalyptus camaldulensis var.brevirostris leaf
oils., Zeitschrift fur Lebensmittel-Untersuchung und -Forschung A. 208 (1999) 212216.
22. R.S. Pappas and S. Sheppard-Hanger: Essential oil of Eucalyptus camaldulensis
Dehn. From south Florida: a high cryptone/low cineole eucalyptus. J. Essent. Oil Res.
12 (2000) 83–384.
23. M. Verdeguer, A. Bla´zquez, and H. Boira: Chemical compositions and larvicidal activities of leaf essential oils from two eucalyptus species. Phytotoxic effects of Lantana camara, Eucalyptus camaldulensis and Eriocephalus africanus essential oils in
weeds of Mediterranean summer crops. Biochem. System. Ecol. 37 (2009) 362-369.
24. P. Siramon and Y. Ohtani: Antioxidative and antiradical activities of Eucalyptus
camaldulensis leaf oils from Thailand. J. Wood. Sci. 53 (2007) 498-504.
25. K. Watanabe, Sh. Yoshinori, A. Kakamizu, A. Okada, N. Matsuo, A. Satoh and H.
Nishimurag: New mosquito repellent from Eucalyptus camaldulensis. J. Agric. Food
Chem. 41 (1993) 2164-2166.
26. B.C. Akin- Osanaiye, A.S. Agbaji and M.A. Dakare: Antimicrobial activity of oils
and extracts of Cymbopogon citrates (Lemon grass), Eucalyptus citriodore and Eucalyptus camaldulensis. J. Med. Sci. 7 (2007) 694-697.
27. S.S. Cheng , Ch. G. Huang , Y. J. Chen, J.J. Yu, W.J. Chen and Sh.T. Chang: Chemical compositions and larvicidal activities of leaf essential oils from two Eucalyptus
species. Bioresource Technology 100 (2009) 452–456.
157
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ЕТАРСКО УЉЕ EUCALYPTUS CAMALDULENSIS (MYRTACEAE)
СА ЦРНОГОРСКОГ ПРИМОРЈА
Славенко Грбовић, Дејан Орчић, Maria Couladis, Емилија Јовин, Кристина Балог и
Неда Мимица-Дукић
Иако аутохтоне у Аустралије и Тасманији, врсте еукалиптуса гаје се широм
света као украсне биљке али и као значајна индустријска сировина. Етарско уље
врста еукалиптуса је од посебног комерцијалног значаја и широко се примењује у
козметичкој, фармацеутској и прехрамбеној индустрији. Хемијски састав и биолошка активност етарског уља условљени су бројним факторима од генетских,
климатских, времена брања, до старости биљака и др. Од преко 700 врста еукалиптуса на Медитерану има око 15, а у Црној Гори мање од 10 врста. У овом раду по
први пут су представљени резултати о хемијском саставу етарског уља еукалиптуса
са црногорског приморја, и то врсте Eucalyptus camaldulensis Dehn. која је најраспрострањенија у Црној Гори. Испитивани су количина и хемијски састав етарског
уља добијеног хидродестилацијом из осушених листова биљака сакупљених са пет
различитих локалитета: Котор, Тиват, Сутоморе, Херцег Нови и Бар. Садржај уља
кретао се од 0,63% (Котор) до 1,59% (Бар). У етарском уљу доминирала су једињења из класе монотерпенских угљоводоника од којих су доминантни били: p-цимен
(17,38 – 28,60%), -феландрен (12,35-14,47%), -пинен (0,94-11,48%). Од оксидованих монотерпена доминантни су били ирегуларни монотерпен криптон (4,977,25%) и терпинен-4-ол (2,75-4,21%), док је од сесквитерпена доминантна компонента био сесквитерпенски алкохол спатуленол (7,83-14,15%). Добијени резултати
показују да на црногорском приморју доминира специфичан хемотип врсте Eucalyptus camaldulensis Dehn, сиромашан у 1,8-цинеолу (најчешћа доминантна компонента еукалиптуса), а богат у p-цимену и криптону.
Received 14 January 2010
Accepted 23 February 2010
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CHROMATOGRAPHIC BEHAVIOR AND LIPOPHILICITY OF s-TRIAZINE
DERIVATIVES ON SILICA GEL IMPREGNATED WITH PARAFFIN OIL
Lidija R. Jevrić, Gordana B. Koprivica, Nevena M. Mišljenović, Bratislav Ž. Jovanović
The chromatographic behavior of four group of s-triazine derivatives (14 compounds)
has been studied by TLC on silica gel impregnated with paraffin oil. Retention mechanism has been determined using the following mobile phases: water-acetone, water-acetonitrile, water-dioxane, water-tetrahydrofuran, water-methanol and water-ethanol, by
changing the volume fraction of modifier in the mobile phase. On impregnated silica gel,
a reversed-phase chromatographic process occurs. Good correlation was obtained between the retention constants, RM0 (determined by linear extrapolation), and slope, S, of
chromatographic equations. There was also satisfactory correlation between these retention constants and logP values calculated using different theoretical methods. The study
showed that the retention constants can be used as a measure of lipophilicity of investigated compounds.
KEYWORDS:
s-Triazine derivatives, RP-TLC, impregnated silica gel, multiple linear
regression analysis, lipophilicity
INTRODUCTION
Considerable attention has been paid to the analysis of chemical in the s-triazine
group, due to their widespread use in agricultural chemistry and their subsequent degradation in biological syistems (1, 2). For initial chemical screening of the activity of newly
synthesized compounds it is recommended first to determine their lipopholicity, an
important physico-chemical property in relation to biological activity. Lipophilicity is
difficult to quantitate, but the most widely accepted measure of lipopholicity is the octanol-water partition coefficient, defined as the ratio of the concentrations of the solute in
the two phases of a saturated 1-octanol-water system. Measurement of the octanol-water
partition coefficient is achieved by an alternative method, reversed-phase liquid chromatography (3). Reversed-phase thin-layer chromatography (RP TLC) has been found to
offer a rapid method for the analysis of a large number of s-triazine type compounds.
Certain relationships between the structure of s-triazine compounds and their mobility on
silica gel impregnated with paraffin oil have recently been demonstrated (4). The retenDr Lidija R. Jevrić, assist. prof., [email protected], Gordana B. Koprivica, B.Sc., [email protected], Nevena M.
Mišljenović, B.Sc., [email protected], Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad,
Serbia, Bratislav Ž. Jovanović, prof., [email protected], Faculty of Technology and Metallurgy, University of
Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia
159
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tion behavior of compounds in various chromatographic systems is believed to be different by nature, i.e. the different physico-chemical properties of an analyte can influence
its retention. Most recently, much effort has been done with the major aim of finding a
mathematical model relating the retention of a given analyte to physico-chemical and
structural parameters (descriptors) of test molecules. These correlations are known as
quantitative structure-retention relationships (QSRR) (5-7). Besides practical application
in optimization strategies, QSRR studies can significantly contribute to getting some
insight into the molecular mechanism of separation. The QSRR equations describing RM0
determined for different mobile phase organic component in terms of logarithms of n-octanol-water partition coefficients were derived. The partition coefficients (AlogP, IAlogP,
ClogP, logPKowin, XlogP, ACDlogP) were calculated by using different software packages. The purpose of the work described in this paper was, therefore, to select the logP
data and TLC system that best characterize octanol/water partitioning, and thus the lipophilicity of the investigated molecules.
EXPERIMENTAL
Fourteen derivatives of s-triazine (Table 1) were investigated. The compounds were
synthesized in the laboratory of the Department of Organic Chemistry, Faculty of Technology and Metallurgy, University of Belgrade (8, 9). Standard solutions (1 mg cm-3) were prepared in methanol, acetone, or chloroform. Samples were spotted on the plates by
means of a micro-pipette.
TLC was performed on 20 × 20 cm glass plates precoated with impregnate silica gel.
The thin-layer of impregnate silica gel was prepared by suspending 25 g silica gel 60
GF254 (Merck) in 100 ml diethyl ether containing 2.5 % paraffin oil. To ease the visualization, fluorescent indicator F254 (Merck) was incorporated into the layers (10).
Impregnate silica gel layer was developed using the following mobile phases:
Aprotic solvents: Acetonitrile-water ( =0.2-0.6; v/v), Acetone-water ( =0.5-0.8; v/v),
Tetrahydrofuran-water ( =0.45-0.7; v/v), Dioxane-water ( =0.5-0.8; v/v).
Protic solvents: Methanol-water ( =0.5-0.8; v/v), Ethanol-water ( =0.5-0.8; v/v).
The plates were developed to a distance of 15 cm by the ascending tchnique at room
temperature without previous saturation of the chamber with mobile phase. Dark spots
were observed under UV light (=254 nm). RM values were calculated from RM = log
((1/Rf) - 1). All calculations were performed using the computer software Origin, Version
6.1. The partition coefficients AlogP, IAlogP, ClogP, logPKowin and XlogP, were calculated for the compounds by applying different theoretical procedures (11, 12). ACDlogP
was calculated using commercial software and the other partition coefficients were obtained from the internet (13).
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Table 1. Chemical structures of the s-triazines studied
Cl
N
N
R
R
N
H
N
N
H
Series I
Compound
I.1
I.2
I.3
I.4
R
-CH(CH3)-C6H5
-CH(CH3)-C6H4-4-CH3
-CH(CH3)-C6H4-4-Cl
-CH(CH3)-C6H4-4-Br
Series III
Compound
II.1
II.2
II.3
Series II
R
CH2
-C(CH3)-(CH2)n
Series IV
Cl
N
n
3
4
5
Cl
N
N
R
N
R
N
N
Cl
R1
R2
Compound
III.1
III.2
III.3
III.4
R
N
R
C6H11
C6H11
C6H11
C6H11
R
H
CH3
C6H5
C6H5
R
H
CH3
H
C6H5
Compound
IV.1
IV.2
IV.3
N
N
H
R
CH2
n
3
4
5
-C(CH3)-(CH2)n
Determination of Retention Constants, RM0, TLC Equations
When the RM values calculated from Rf values (retention factor defined as the distance
migrated by the sample from the origin compared with the distance migrated by the
solvent front from the origin) were plotted against mobile phase composition for each
compound there was a range in which a linear relationship was observed between the RM
values and organic modifier concentration in the mobile phase, which can be expressed
by the equation RM  R 0 0  S , indicative of the reversed-phase chromatography, were
M
 is the amount (%) of organic compounds in the mobile phase (14). The obtained slopes,
S, and intercept values, RM0, of TLC equation for each solute are presented in Table 2.
The correlation coefficients of the TLC equations were satisfactory.
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Table 2. Extrapolated RM0 values, slopes, S, and correlation coefficients, r, obtained on silica gel impregnated with
paraffin oil
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Correlation Between Retention Constants, RM0, and Slope, S
A linear relationship was observed between the intercept, RM0, and slope, S, for protic
and aprotic solvents, as shown by the equations given in Table 3. The best correlation
was obtained for aceton as mobile-phase modifier (r = 0.994). There is a good correlation
between RM0 and S, which might reflect the suitability of the systems examined for estimating the lipophilicity of the compounds. The RM0 values, which are chromatographic
data describing the partitioning between a non-polar stationary and a polar mobile phase,
may therefore be appropriate for the assessment of lipophlicity.
Table 3. Equations for the relationships between the retention constants, RM0,
and slope, S.
Mobile phase
Acetone-water
Acetonitrile-water
Dioxane-water
Tetrahydrofuran-water
Methanol-water
Ethanol-water
Equation
RM0 = -0.572 – 1.121S
RM0 = -1.717 – 1.356S
RM0 = -1.895 – 0.965S
RM0 = -0.231 – 1.231S
RM0 = -1.278 – 1.911S
RM0 = -0.786 – 1.152S
r
0.994
0.954
0.920
0.985
0.987
0.962
sd
0.101
0.590
0.392
0.140
0.110
0.283
n
14
14
14
14
14
14
Based on the results obtained on silica gel impregnated paraffin oil, RM0 is directly dependent on the nature of mobile phase modifiers. In other words, the selectivity of separation of the tested substances are the result of specific interactions with the mobile phase.
Figure 1. Relationship between the extrapolated retention constants, RM0, and the number
of phenyl groups compounds of series III derivatives s-triazine molecules
As can be seen from Figure 1, of structural parameters, the most pronounced effect
on RM0 values has phenyl group. Namely, a linear relationship was observed between the
RM0 values and the number of phenyl groups, n(Ph), in the substituents at position 4 and 6
in the compounds of series III derivatives of s-triazine. A decrease in the polarity of the
molecule (increase in the number of phenyl groups) resulted in the increased retention
(Figure 1).
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Correlation of Retention Constants, RM0 and logP
Lipophilic character often seems to be the most important physico-chemical parameter in determining the biological activity of chemical agents. Lipophilicity can be expressed in terms of many different descriptors (logP, logkw, RM, RM0), obtained experimentally
or calculated. The experimental parameters most frequently used are the retention constants RM0 (RPTLC) and logkw (RPHPLC), whereas the calculated quantity is logP. The
partiton coefficient, logP, of a given compound between a non-aqueous and an aqueous
phase can be used as an expression of its lipophilic character (15).
Because the retention of a compound in reversed-phase chromatography is governed
by hydrophobic interactions, linear relationships between the retention constant, RM0, and
logP could be expected (16).
The partition coefficients (AlogP, IAlogP, ClogP, logPKowin, XlogP, ACDlogP) of striazine derivatives are listed in Table 4.
Table 4. Partition coefficients calculated by different theoretical methods
Comp.
I.1
I.2
I.3
I.4
II.1
II.2
II.3
III.1
III.2
III.3
III.4
IV.1
IV.2
IV.3
AlogP
5.25
5.65
5.94
6.18
4.96
5.78
6.55
4.91
5.55
6.07
6.98
3.83
4.21
4.64
IAlogP
5.06
5.13
7.08
5.89
5.15
5.96
6.65
4.76
4.81
5.80
7.98
3.63
4.08
4.48
ClogP
4.85
5.85
6.28
6.58
5.32
6.44
7.55
5.40
5.48
7.21
9.02
3.12
3.67
4.23
logPKowin
5.07
6.16
6.36
6.85
5.88
6.86
7.85
5.96
6.86
8.17
10.39
3.81
4.30
4.79
XlogP
4.83
5.70
6.07
6.43
3.71
4.85
5.99
4.01
5.14
6.14
8.27
2.37
2.94
3.51
ACDlogP
3.87
4.79
5.06
5.42
3.70
4.82
5.95
3.74
4.82
5.36
7.07
2.85
3.41
3.98
Table 5. Correlation coefficients (r) for the correlation between RM0 and different logP
values
RM0
Acetonitrile
Acetone
Dioxane
Tetrahydrofuran
Methanol
Ethanol
164
AlogP
0.835
0.763
0.747
0.570
0.702
0.855
IAlogP
0.705
0.708
0.624
0.533
0.604
0.795
ClogP
0.858
0.828
0.815
0.648
0.794
0.822
logPKowin
0.877
0.875
0.900
0.756
0.895
0.838
XlogP
0.795
0.671
0.681
0.532
0.665
0.900
ACDlogP
0.804
0.757
0.770
0.707
0.750
0.825
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By comparing the calculated values to define the lipophilicity of the investigated molecules, it is evident that ethanol as a modifier gives the highest correlation (calculated
average correlation coefficient is 0.839).
Retention Constants, RM0 for QSRR
The QSRRs are statistical models which quantify the relationship between the structure of a molecule and its chromatographic retention parameters in different kinds of
chromatography. The application of QSRR allows the prediction of the retention of a new
solute, identification of the most informative structural descriptors, elucidation of the molecular mechanisms of separation in a given chromatographic system, evaluation of complex physico-chemical properties of solutes and estimation of biological activities. The
relationship between the retention and the structural characteristics of a molecule explains the effect of chemical structure on the retention behavior in a more accurate way (17).
The use of multiple linear regression (MLR) analysis for fourteen s-triazine derivatives led to statistically significant equations relating lipophilicity (estimated by RM0 values (dependent variable) to different theoretically calculated six types of log P namely
AlogP, ClogP, ACDlogP, logPKowin, X logP and IAlogP values for each compound (independent variable). The specifications for the best-selected MLR models are shown in
Table 6 and Table 7.
These relationships were analyzed and the best model was selected on the basis of various statistical parameters like correlation coefficient (r), and standard deviation (SD).
In the first phase of work, the multilinear relationships between the retention constant
and two variable lipophilicity descriptors was examined.
Table 6. Statistical parameters for multilinear dependence between RM0 and two variables
descriptors lipophilicity
Modifier
RM0
Acetone
Dioxane
Methanol
Descriptors
logP1
logP2
logPKow XlogP
AogP
logPKow
logPKow XlogP
RM0 = a logP1 + blogP2 + c
a
b
c
r
SD
0.0993 0.648 -0.313 0.916 0.329
0.312 0.813 -0.410 0.949 0.306
0.571 0.672 -0.357 0.953 0.234
From the data in Table 6, it is evident that the retention constant correlates best with
XlogP combined with logPkowin (r = 0.953) - modifier methanol and logPkowin with AlogP
(r = 0.949) - modifier dioxane.
The multilinear dependence of the retention constant, RM0, and the three lipophilicity
descriptors is shown in Table 7. The best correlation was observed for the combined
logPKow, XlogP and IAlogP (r = 0.964)
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Table 7. Statistical parameters for multilinear dependence between RM0 and three
variables descriptors lipophilicity
Modifier
RM0
Acetone
Dioxane
Acetonitrile
Tetrahydrofuran
Methanol
Ethanol
logP1
AogP
logPKow
AogP
logPKow
logPKow
logPKow
Descriptors
logP2
logP3
logPKow
XlogP
XlogP
IAlogP
logPKow
ACDlogP
XlogP
ACDlogP
XlogP
IAlogP
XlogP
ClogP
RM0 = a logP1 + blogP2 + clogP3 +
a
b
c
d
r
-1.969 0.811 0.524 -0.632 0.948
0.731 0.853 -0.292 -0.231 0.957
-2.713 1.401 0.756 -1.203 0.901
0.664 0.271 -0.609 0.791 0.904
0.948 0.708 -0.251 -0.208 0.964
0.625 0.408 0.537 -0.503 0.907
d
SD
0.274
0.294
0.659
0.280
0.216
0.398
The analysis of these results indicates that the proposed models can correctly represent the relationship between the retention parameters of the investigated compounds on
silica gel and different log P values calculated for various compound solely from the molecular structure. These models are suitable for prediction of the retention of structurally
similar compounds under the same chromatographic conditions.
CONCLUSION
Experimentally obtained RP RM0 values depend on the nature of organic modifier in
the mobile phase. A linear relationship between RM0 and slope, S, values was found for all
mobile phases. Satisfactory linear correlation was obtained between the retention constants and AlogP, ClogP, ACDlogP, logPKowin, X logP and IAlogP. According to the correlation coefficients, RM0 is a useful property for evaluation of the relative lipophilicity of
the examined compounds.
The correlations between the retention constants, RM0, and selected lipofilicity parameter (different logP values) of the solutes were expressed by multiparametric equations
of high statistical significance, indicate that these models can be used to predict the retention constants of these molecules.
REFERENCES
1.
2.
3.
4.
5.
166
T. Kodoma, L. Ding, M. Yoshida and M. Yajima: Biodegradation of an s-triazine
herbicide, simazine. J. Mol. Catal., B Enzym. 11 (2001) 1073-1078.
S. Navarro, N. Vela, M.J. Giménez and G. Navarro: Persistence of four s-triazine
herbicides in river, sea and groundwater samples exposed to sunlight and darkness
under laboratory conditions. Sci. Total Environ. 329 (2004) 87-97.
J. Takel and J. Kovačičova: Chromatographic methods in the determination of
herbicide residues in crops, food and environmental samples. J. Chromatogr. A. 643
(1993) 291-303.
N. Perišić-Janjić and B. Jovanović: Reversed-Phase Thin-Layer Chromatographic
behaviour of Some s-triazine derivatives. J. Planar Chromatogr. 16 (2003) 71-75.
N. Perišić-Janjić, T. Đaković-Sekulić, L. Jevrić and B. Jovanović: Study of Quantitative Structure-retention Relationships for s-triazine derivatives in different RP
HPLC Systems. J. Planar Chromatogr. 18 (2005) 212-216.
APTEFF,41, 1-203 (2010)
DOI: 10.2298/APT1041159J
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
UDC: 632.954:543.544
BIBLID: 1450-7188 (2010) 41, 159-168
W.J. Lyman, W.F. Reehl and D.H. Rosenblatt: Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York (1982).
A. Chiriac, D. Ciubotariu and Z. Simon: QSAR – Quantitative Relationships, Mirton
Publishing House, Timişoara (1996).
G. Bončić-Caričić, Ž. Tadić and D. Jeremić: Electron impact mass spectrometry of
some 2,4,6-substituted s-triazines. Effect of the ring size. Int. J. Mass. Spectrom.
Ion. Phys. 47 (1983) 451-454.
G. Bončić-Caričić, B. Jovanović and D. Jeremić: Electron impact mass spectrometry
of some 2,4,6-substituted s-triazines. Effect of α-and β-substitution. Int. J. Mass.
Spectrom. Ion. Phys. 47 (1983) 455-458.
N. Perišić-Janjić, K. Popov-Pergal and B. Gordić: Normal- and reversed-phase thinlayer chromatography of some 3-(2 '-furoyl)-5-X-2,4-dioxotetrahydro-1,3-thiazole
derivatives. J. Planar Chromatogr. 12 (1999) 435-439.
C. Hansch and A.J. Leo: Substituent Constants for Correlation Analysis in Chemistry
and Biology, John Wiley, New York 1979.
J. Chou and P.C. Jurs: Computer-Assisted Computation of Partition Coefficients
from Molecular Structures Using Fragment Constants. J. Chem. Inf. Comput. Sci. 19
(1979) 172-178.
M. Kraszni, I. Bányai and B. Noszál: Determination of Conformer-Specific Partition
Coefficients in Octanol/Water Systems. J. Med. Chem. 46 (2003) 2241-2245.
K. Valko, R.S. Lloyd and L.G. Joseph: Retention in reversed-phase liquid chromatography as a function of mobile-phase comosition. J. Chromatogr. A. 656 (1993)
501-520.
R. Kaliszan, P.R. Brown and E. Grushka: (Eds) Advances in Chromatography, Marcel Decker, New York 1993.
R. Mannhold, G.I. Poda, C. Ostermann and I.V. Tetko: Calculation of molecular lipophilicity: state of the art and comparison of logP methods on more than 96.000
compounds. J. Pharm. Sci. 98 (2009) 861-893.
G. Romanelli, J. Autino and E. Castro: Application of Quantitative Structure-Retention Relationships (QSRR) to a Set of Organic Bromo and Nitrile Derivatives.
Turk J. Chem. 26 (2002) 335-343.
ХРОМАТОГРАФСКО ПОНАШАЊЕ И ЛИПОФИЛНОСТ с-ТРИАЗИНСКИХ
ДЕРИВАТА НА ТАНКОМ СЛОЈУ СИЛИКА ГЕЛА ИМПРЕГНИРАНОГ
ПАРАФИНСКИМ УЉЕМ
Лидија Р. Јеврић, Гордана Б. Копривица, Невена М. Мишљеновић, Братислав Ж.
Јовановић
Испитано је хроматографско понашање четири групе новосинтетисаних деривата с-триазина на танком слоју силика гела имрегнираног парафинским уљем. Ретенциони механизам је одређен употребом мобилних фаза: ацетон-вода, ацетонитрил-вода, диоксан-вода, тетрахидрофуран-вода, метанол-вода и етaнол-вода, варирањем запреминског удела модификатора у покретној фази. На танком слоју сили167
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ка гела импрегнираног парафинским уљем долази до обрнуто-фазног ретенционог
механизма. Добре корелације су постигнуте између ретенционе константе, RM0,
(ретенција растворка у чистој води), и нагиба, S, хроматографске једначине. Такође
су постигнуте задовољавајуће корелације између ретенционе константе и вредности logP, израчунатих коришћењем различитих рачунарских програма. То указује
да RM0 може бити алтернативни дескриптор у дефинисању липофилности испитиваних деривата с-триазина.
168
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REACTIVITY OF 17β-HYDROXY-17α-SUBSTITUTED ANDROSTANE
DERIVATIVES
Katarina M. Penov Gaši, Andrea R. Gaković, Jovana J. Ajduković,,
Maja Dj. Djurendić-Brenesel, Evgenija A. Djurendić, Marina P. Savić
and Marija N. Sakač

The work concerns the reactivity of 17β-hydroxy-17α-substituted androstanes 1-9.
Depending on the nature of the 17α-substituent, the presence of the neighboring 16-oximino group, and the reagent used, these compounds can undergo retroaddition (10-12)
and fragmentation-cyclization (14-16) reactions.
KEYWORDS: Androstane derivatives; Retroaddition; Fragmentation; Cyclization
reaction
INTRODUCTION
In our previous works (1-5) we synthesized a number of 17β-hydroxy-17α-substituted
androstane derivatives 1-9, which served as suitable precursors for the synthesis of some
new A-, B- and D-modified androstane derivatives with antiaromatase and cytotoxic activities (3-6).
Compounds 1-9 (Figure 1) showed the corresponding chemical reactivity under the
following reaction conditions. With boiling acetic anhydride, compounds 1, 3 and 4 gave
the corresponding 17-picolinylidene and 17-benzylidene derivatives (1-3). With potassium hydroxide in boiling ethylene glycol, compounds 5 and 6 afforded 16-amino-17-substituted-homo derivatives in the D-ring (4), while the same type of D-homo steroids
formed compounds 7 and 8 in the reaction with potassium tert-butoxide in boiling tertbutyl alcohol (4). On the other hand, 17β-hydroxy-16-oximino-17α-substituted derivatives 5-9, with p-toluenesulfonyl chloride in pyridine, acetic anhydride in pyridine, and
potassium tert-butoxide in tert-butyl alcohol, gave the corresponding 16,17-seco derivatives (4-6).
In view of all the above and our constant interest in 17β-hydroxy-17α-substituted
androstane derivatives, we studied in detail their chemical behavior in regard of the
nature of the C-17 substituent, first of all in the presence of the different bases. Special
attention was paid to the behavior of the 17β-hydroxy-17α-picolyl system under the conDr Katarina M. Penov Gaši, Prof.; M.Sc. Andrea R. Gaković, Assist.; M.Sc. Jovana J. Ajduković, Res. Assist.;
Dr Maja Dj. Djurendić-Brenesel, Sen. Res. Fell.; Dr Evgenija A. Djurendić, Prof.; M.Sc. Marina P. Savić, Res.
Assist.; Dr Marija N. Sakač, Prof., Department of Chemistry, Faculty of Sciences, Trg Dositeja Obradovića 3,
169
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ditions of N-carboxylation with ethyl esters of chloroacetic and 11-bromoundecanoic
acids. Also, the aim of the study was to establish the difference in reactivity of the 17βhydroxy-17α-substituted fragments in the groups of compounds 1-4 and 5-9, bearing in
mind that the latter group contains neighboring 16-oximino group. Another objective was
to establish whether there is an influence of the 4-en-3-one increment on reactivity of the
17β-hydroxy-17α-substituted androstanes.
Figure 1. 17β-Hydroxy-17α-substituted androstane derivatives
EXPERIMENTAL
General Procedure
Melting points were determined using a Büchi SMP 20 apparatus and are uncorrected. IR spectra were recorded on a Nexus 670 SP-IR spectrometer (wavenumbers in
cm-1). NMR spectra were taken on a Bruker AC 250E spectrometer operating at 250 MHz
(1H) and 62.5 MHz (13C) and are reported in ppm (-scale) downfield from the tetramethylsilane internal standard; coupling constants (J) are given in Hz. Mass spectra
(TOF) were recorded on a 6210 time-of-flight LC/MS Agilent Technologies (ESI+).
Chromatographic separations were performed on silica gel columns (Kieselgel 60, 0.0630.20 mm, Merck). All the reagents used were of analytical reagent grade. All solutions
were dried over anhydrous sodium sulfate.
Reaction of compounds 1-3 with ethyl chloroacetate
Compounds 1-3 (0.13 mmol) were dissolved in absolute ethanol (3 ml), ethyl chloroacetate (0.26 mmol) was added, and the reaction mixture was refluxed for 93 h (com170
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pounds 1 and 2) or for 48 h (compound 3). After cooling, the reaction mixture was poured into water (10 ml) and extracted with dichloromethane (4 × 5 ml). The extracts were
dried, the solvents evaporated, and crude products were purified by column chromatography (4 g silica gel, toluene-ethyl acetate 9:1 for compound 10; 12:1 for compound 11
and 15:1 for compound 12). Pure compound 10 (from 1, 51% yield; m.p. 151ºC, lit. (7)
m.p. 146-151ºC), compound 11 (from 2, 37% yield; m.p. 170ºC, lit. (7) m.p. 168-170ºC)
and compound 12 (from 3, 53% yield; m.p. 170-171ºC, lit. (7) m.p. 170-173 ºC).
Reaction of compound 1 with potassium tert-butoxide
Compound 1 (0.076 g, 0.2 mmol) was dissolved in 1 M potassium tert-butoxide in
tert-butyl alcohol (8 ml) and the reaction mixture was refluxed for 1 h. After cooling, the
reaction mixture was poured into water (15 ml), neutralized with HCl 1:1 and extracted
with diethyl ether (3 × 5 ml). The extract was dried and the solvent removed under reduced pressure, giving compound 10 (0.047 g, 82% yield; m.p. 151ºC).
Reaction of compound 1 with ethyl 11-bromoundecanoate
To compound 1 (0.190 g, 0.5 mmol), absolute ethanol (0.3 ml) and ethyl 11-bromoundecanoate (0.270 g, 1 mmol) were added and the reaction mixture was heated at the
boiling temperature for 80 h. After the reaction was complete, the ethanol was removed,
and benzene (5 ml) was added. The precipitate was recrystallized from acetone-ethyl
acetate affording a pure compound 13 (3) (0.075 g, 33%; m.p. 235-236ºC).
Reaction of compounds 6 and 7 with potassium tert-butoxide, or sodium ethoxide, or
potassium hydroxide
Compounds 6 and 7 (0.15 mmol) were dissolved in 1 M tert-BuOK in tert-butyl alcohol
(8 ml), or in 3 M EtONa in ethanol (5 ml), or in 3 M KOH in ethylene glycol (6 ml). The
reaction mixture was refluxed for 30 min (6) or 2 h (7) with tert-BuOK, 2.5 h with
EtONa, or 2 h with KOH. After the reactions were complete, the reaction mixtures were
diluted with water (15 ml), acidified to pH 2 with 2 M HCl and extracted with dichloromethane (4 × 5 ml). The combined extracts were dried and the solvent was evaporated.
The solid products were recrystallized from methanol, giving pure compounds 15 (61%
with tert-BuOK, 23% with EtONa and 84% with KOH; m.p. 318ºC, lit. (4) m.p. 317318ºC) and 16 (73% with tert-BuOK, 57% with EtONa and 31% with KOH; m.p. 249ºC,
lit. (4) m.p. 248-250ºC).
Compounds 1-4 were synthesized starting from dehydroepiandrosterone (10), applying known procedures (1-3). The addition of α-picolyllithium on the C-17 carbonyl group
of dehydroepiandrosterone yielded compound 1. Acetylation of compound 1 gave 3βacetoxy derivative 2, and the Oppenauer oxidation of compound 1 resulted in 4-en-3-one
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derivative 3. Scheme 1 presents chemical transformations of compounds 1-3 that were
performed in this work. In the reaction of compound 1 (17α-picolyl-17β-hydroxy system)
with potassium tert-butoxide in tert-butyl alcohol at the boiling temperature for 1h, elimination of α-picoline system was observed, which is the process opposite to the initial
reaction of addition of α-picolyllithium on the C-17 carbonyl group of dehydroepiandrosterone. The treatment of the reaction mixture gave dehydroepiandrosterone (10) in a
yield of 82%. Under the same conditions, compound 4 (17α-benzyl-17β-hydroxy system)
was stable and did not undergo retroaddition. When compound 1 was heated with sodium
ethoxide in ethanol at the boiling temperature, the reaction of retroaddition was much
slower, so that in 4 h compound 1 reacted only partly to give dehydroepiandrosterone
(10), which is in agreement with the lower basicity of the base used. However, by heating
compound 1 with potassium hydroxide in boiling ethylene glycol for 2 h the retroaddition
reaction proceeded to an end, so that the treatment of the reaction mixture and chromatographic purification afforded the retroaddition product 10 in a yield of 59%.
Quite unexpectedly, the retroaddition reaction was also observed in an attempt to prepare an N-carboxyalkyl derivative of compound 1 or its 3β-acetoxy derivative 2 with
ethyl chloroacetate in boiling ethanol in the course of 93 h. Apart from the unreacted
starting compounds 1 (27%) and 2 (34%) it was possible to isolate in a greater yield the
retroaddition products 10 (52%) and 11 (37%). Similarly, the retroaddition reaction was
confirmed with compound 3 under the same reaction conditions using ethyl chloroacetate
(as was the case with compounds 1 and 2), but in a shorter time (48 h), resulting in
androstenedione 12 in a yield of 53% (Scheme 1).
Scheme 1. (i) ClCH2COOEt, EtOH, reflux, 93 h (for 1 and 2) and 48 h (for 3); (ii) tBuOK, t-BuOH, reflux, 1 h, only for 1; (iii) cyclohexanone, (t-BuO)3Al, reflux, 3 h; (iv)
BrCH2(CH2)9COOEt, EtOH, reflux, 80 h, only for 1.
When 17α-picolyl derivative 1 was subjected to the Oppenauer oxidation, in addition
to the expected oxidation product 3 (40%), a retroaddition product, androst-4-ene-3,17dione (12), was also obtained in a small yield (6%) (3). With the aim to study the retroaddition conditions more thoroughly, compound 3 was subjected to the action of potassi172
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um tert-butoxide in tert-butyl alcohol and the reaction mixture was boiled for 1 h. However, no retroaddition took place, but the reaction yielded a very complex mixture.
Possible mechanisms of retroaddition for compounds 1 and 2 with potassium tertbutoxide and ethyl chloroacetate are shown in Scheme 2.
Scheme 2. Proposed mechanism of the retroaddition reaction
A different reaction product was obtained in the case of compound 1 using ethyl 11bromoundecanoate instead of ethyl chloroacetate. It can be assumed that no nucleophilic
attack on the N atom of the α-picolyl group of compounds 1 or 2 at C-11 took place, but,
most probably, a proton was abstracted from the C-10 of the ester, followed by elimination of HBr and formation of compound 13. Compound 13 was obtained previously by
the reaction of HBr in acetone with compound 1 (3).
On the other hand, on studying reactivity of the 17β-hydroxy-17α-substituted androstane in the presence of the neighboring 16-oximino group (compounds 5, 6 and 7) it was
concluded that it did not undergo retroaddition under alkaline conditions but a fragmentation-cyclization reaction in the D-homo ring, with the formation of compounds 14, 15
and 16 (Scheme 3). In our previous work (4), heating of oximino-alcohols 5 and 6 with
potassium hydroxide in boiling ethylene glycol gave the D-homo derivatives 14 and 15 in
yields 52% and 84%, respectively. In this work we found that lower yields of compound
15 were obtained irrespective of using stronger bases, but at lower reaction temperatures.
Using potassium tert-butoxide in tert-butyl alcohol at reflux during 30 min, compound 6
gave the 17-phenyl-D-homo product 15 in a yield of 61%, whereas heating with sodium
ethoxide in boiling ethanol for 2.5 h afforded compound 15 in a yield of only 23%.
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In the last two cases, the lower yield of compound 15 was probably influenced by the
bulkiness of the used bases, which hindered deprotonation of the D-seco intermediates
5a-7a and formation of the anions 5b-7b that undergo cyclization (Scheme 3).
Scheme 3. (i) t-BuOK, t-BuOH, reflux, 30 min (6) and 2 h (7); (ii) EtONa, EtOH, reflux,
2.5 h; (iii) KOH, ethylene glycol, reflux, 2 h.
In the case of a less bulky 17α-substituent (methyl group) in compound 7, the results
were almost opposite. We have previously found that the reaction with potassium tertbutoxide in tert-butyl alcohol at the boiling temperature resulted in the D-homo derivative 16 in a yield of 73% (4). In the present work we observed that the use of weaker
bases resulted in lower yields. Heating of compound 7 with sodium ethoxide in ethanol at
the boiling temperature for 2.5 h gave the D-homo derivative 16 in a yield of 57%,
whereas heating of the same compound with potassium hydroxide in boiling ethylene
glycol for 4 h resulted in the same compound 16 in a yield of only 31%. Most probably,
the crucial role in this case played the base strength, irrespectively of the bulkiness of its
molecule, especially because the anions were not additionally stabilized by the aromatic
ring, as was the case with the D-homo derivative 15.
In a previous work (6) we discussed the fact that 17α-substituents in the oximino alcohols 5-9 should have an H atom at the carbon that is directly bonded to the C-17, as a
precondition that, after fragmentation to the D-seco intermediate, they undergo cyclization to D-homo products. It was found that this was the reason why compound 9 in the reaction with potassium hydroxide in ethylene glycol did not undergo cyclization to yield
the corresponding 16-amino-17a-homo derivative, but the corresponding D-homo lactone
(6).
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Apart from the above reactions, the 17β-hydroxy-17α-substituted and 17α-unsubstituted androstane derivatives, in the reaction with the different reagents, undergo a fragmentation reaction and formation of only D-seco derivatives (4-6, 9).
CONCLUSION
It can be concluded that the 17β-hydroxy-17α- pycolyl, benzyl, methyl, ethyl and
phenyl androstane derivatives 1-9 can undergo different reactions: retroaddition and fragmentation-cyclization, which depends on the nature of the 17α-substituent, the structure
of the ring A, the presence of the neighboring 16-oximino group and the reagent used
(potassium tert-butoxide, sodium ethoxide, potassium hydroxide, ethyl chloroacetate and
ethyl 11-bromoundecanoate).
Acknowledgement
We would like to thank the Ministry of Science and Technological Development of
the Republic of Serbia for financial support (Grant No. 142052).
REFERENCES
1. D. Miljković, K. Gaši, M. Kindjer, S. Stanković, B. Ribár and G. Argay: Partial Synthesis of 21,27-Bisnordemissidine from Epiandrosterone and Dehydroepiandrosterone
Acetates. Crystal and Molecular Structure of 21,27-bisnordemissidine Hydrobromide.
Croat. Chem. Acta. 58 (1985) 721-736.
2. Lj. Medić Mijačević, D. Miljković, R. Kovačević, S. Stanković and K. Gaši: Synthesis and Antiandrogenic Activity of Some New 5-Аndrostene Derivatives. J. Serb.
Chem. Soc. 58 (1993) 615-619.
3. K. Penov Gaši, M. Djurendić-Brenesel, E. Djurendić, M. Sakač, J. Čanadi, J. Daljev,
T. Armbruster, S. Andrić, D. Sladić, T. Božić, I. Novaković and Z. Juranić: Synthesis
and Biological Evaluation of Some 17-Picolyl and 17-Picolynilidene Androst-5-ene
Derivatives. Steroids 72 (2007) 31-40.
4. K. Penov Gaši, D. Miljković, Lj. Medić-Mijačević, E. Djurendić, S. Stojanović, M.
Sakač, M. Djurendić, S. Stanković, D. Lazar, S. Andrić and R. Kovačević: Synthesis,
X-ray Crystal Structures and Biological Activity of 16-Amino-17-Substituted-DHomo Steroid Derivatives. Steroids 68 (2003) 667-676.
5. K. Penov Gaši, S. Stanković, J. Csanádi, E. Djurendić, M. Sakač, Lj. Medić-Mijačević, O. Arcson, S. Stojanović, S. Andrić, D. Molnar Gabor and R. Kovačević: New
Modified Androstane Derivatives as Aromatase Inhibitors. Steroids 66 (2001) 645653.
6. K. Penov Gaši, S. Stojanović, M. Sakač, E. Djurendić, J. Csanadi, D. Molnar Gabor,
D. Lazar and R. Kovačević: Synthesis and Biological Activity of Some 17a-Substituted Homolactones of Androst-5-ene Derivatives. Collect. Czech. Chem. Commun.
70 (2005) 1387-1396.
175
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DOI: 10.2298/APT1041169P
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7. www.chemblink.com/about.htm
8. K. Penov Gaši, S. Cvjetićanin, S. Stojanović, K. Kuhajda, Lj. Stupavský, J. Čanadi,
D. Molnar-Gabor, Lj. Medić-Mijačević, M. Sakač: The Chemical Transformation of
3β,17β-Dihydroxy-16-Oximino-5-Androstene. Acta Periodica Technologica 31
(2000) 675-683.
9. D. Miljković, J. Petrović, M. Stajić, M. Miljković: The Beckmann Fragmentation
Reaction of Some α-Hydroxy Ketoximes. J. Org. Chem. 38 (1973) 3585-3588.
РЕАКТИВНОСТ 17β-ХИДРОКСИ-17α-СУПСТИТУИСАНИХ
АНДРОСТАНСКИХ ДЕРИВАТА
Катарина М. Пенов Гаши, Андреа Р. Гаковић, Јована Ј. Ајдуковић, Маја Ђ.
Ђурендић-Бренесел, Евгенија А. Ђурендић, Марина П. Савић, Марија Н. Сакач
У овом раду је проучавана реактивност 17β-хидрокси-17α-супституисаних андростанских деривата 1-9, као прекурсора у синтези нових А-, B- и D-модификованих деривата са антиароматазном и цитотоксичном активношћу. У зависности од
природе 17α-супституента, присуства суседне 16-оксимино групе и употребљеног
реагенса, ови стероидни деривати показују одговарајућу хемијску реактивност у
присуству одабраних реагенаса као ацетанхидрида, калијум-хидроксида у кључалом етиленгликолу, калијум-t-бутоксида у кључалом t-бутанолу и/или етил-хлороацетату, при чему настају једињења 10-12 реакцијом ретроадиције, или фрагментационо-циклизационом реакцијом једињења 14-16. У раду су приказани могући механизми реакција ретроадиције, као и фрагментационо-циклизационе реакције при
чему настају D-секо и D-хомо деривати андрост-5-ена.
Received 10 February 2010
Accepted 6 April 2010
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CORRELATION BETWEEN THE LIPOPHILICITY AND ANTIFUNGAL
ACTIVITY OF SOME BENZOXAZOLE DERIVATIVES
Sanja O. Podunavac-Kuzmanović, Sonja D. Velimirović1
In the present work, a quantitative relationship between the lipophilicity and antifungal activity of some benzoxazole derivatives against Candida albicans was investigated
by using QSAR (quantitative structure-activity relationship) analyses. The descriptors
which describe numerically the lipophilicity, logP, were calculated using Chem-Office
Software version 7.0. The linear correlation between the minimal inhibitory concentration
(log1/cMIC) and lipophilicity descriptors was investigated. The best QSAR model predicting the antifungal activity of the investigated series of benzoxazole was developed. The
results are discussed on the basis of statistical data. High agreement between theoretical
and experimental inhibitory values was obtained. The results of this study indicate that the
lipophilicity parameter has a significant effect on antifungal activity of this class of compounds, which can be very useful in the design of new biologically active molecules.
KEY WORDS: benzoxazole derivatives, antifungal activity, lipophilicity, quantitative
structure-activity relationship.
INTRODUCTION
The benzoxazoles are a large chemical family used as antimicrobial agents against a
wide spectrum of microorganisms. The high therapeutic activity of the related drugs have
encouraged the medicinal chemists to synthesize a large number of novel chemotherapeutic agents. The incorporation of the benzoxazole nucleus is an important synthetic
strategy in drug discovery. This class of molecules have broaden the scope in remedying
various dispositions in clinical medicine. This heterocyclic system has different activities
as it can act as bacteriostat or bactericide, as well as fungicide and it is present in numerous antiviral drugs (1-12).
The resistance of some pathogens to standard antibiotic therapies is quickly becoming
a major public health problem all over the world. There is a real need for the discovery of
new antimicrobial agents, possibly acting through the mechanisms, which are distinct
from those of well-known classes of antimicrobial agents to which may clinically relevant pathogens may be now resistant.
Dr. Sanja O. Podunavac-Kuzmanović, Prof., Faculty of Technology, 21000 Novi Sad, Bul. Cara Lazara 1,
Serbia, M.Sc. Sonja D. Velimirović, Teacher, Primary School "Jovan Popović", 21000 Novi Sad, Ravanička 2,
Serbia
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A quantitative structure–activity relationship (QSAR) analysis was conducted to investigate the quantitative effect of structural properties of the compounds on their antimicrobial activity (13-18). The QSAR, as one of the most important methods in chemistry, gives information that is useful for drug design and medicinal chemistry. These
are mathematical models relating chemical structure to a wide variety of physical, chemical, technological and biological properties. The defined relationship between the
activity and molecular descriptors are used to estimate the property of molecules and/or
to find the parameters affecting the biological activity.
LogP is a frequently used molecular descriptor in QSAR analyses (19-23). It is a
quantitative descriptor of lipophilicity, one of the key determinants of pharmacokinetic
properties. The lipophilicity modifies the penetration of bioactive molecules through the
non-polar cell membranes. This property is usually characterized by the partition coefficient, which is essentially determined from distribution studies of the compound between
an immiscible polar and non-polar solvent pair. By knowing exact values for this parameter, it is possible to predict the inhibitory activity of a drug.
In view of above and in continuation of our studies on QSAR analyses (13-18, 22),
the goal of this investigation was to elucidate the quantitative effect of the lipophilicity on
antifungal activity of some benzoxazole derivatives against Candida albicans. The
objecttive was to develop a rapid and reliable method to predict antifungal activity of this
class of molecules.
EXPERIMENTAL
The structures of the benzoxazoles investigated in this study are presented in Table 1.
The results of antifungal activity against Candida albicans (MTCC 183) for all the benzoxazole derivatives were taken from the literature (24). 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.
Table 1. The structures of the compounds studied
O
R1
N
Compound
1
2
3
4
5
6
178
R1
H
C(CH3)3
NH2
NHCH3
C2H5
NHCOCH3
R2
R2
H
H
H
H
Cl
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Compound
7
8
9
10
11
12
13
14
15
16
17
18
19
R1
NH CH3
Cl
NO2
C2H5
F
N(CH3)2
CH3
C2H5
OCH3
F
NHCOCH3
NHCH3
N(CH3)2
R2
Cl
Cl
Cl
NH2
NH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Molecular Modeling and logP Calculations
Molecular modeling studies were performed by using CS Chem-Office Software
version 7.0 (Cambridge software) running on a P-III processor (25). All molecules were
constructed using Chem Draw Ultra 7.0 and saved as the template structure. For every
compound, the template structure was suitably changed considering its structural features,
copied to Chem 3D 7.0 to create a 3-D model and, finally, the model was cleaned up and
subjected to energy minimization using molecular mechanics (MM2). The minimization
was executed until the root mean square (RMS) gradient value reached a value smaller
than 0.1 kcal/molA. The Austin Model-1 (AM-1) method was used for re-optimization
until the RMS gradient attained a value smaller than 0.0001 kcal/molA using MOPAC.
The lowest energy structure was used for each molecule to calculate lipophilicity
parameters (Table 2).
Statistical Methods
The complete regression analysis was carried out by PASS 2005, GESS 2006, NCSS
Statistical Softwares (26).
The results of 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.
In this study, the log1/cMIC values were correlated against logP parameters calculated
using CS Chem-Office Software. The lipophilicity parameters are linearly related to the
inhibitory activity (MICs) (Eq. 1 and Fig. 1).
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Table 2. Data of the lipophilicity parameters used in this study and 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
Lipophilicity
logP
2.9522
3.5533
3.0524
3.2425
3.5071
3.8672
3.5871
3.5911
3.8465
3.2828
3.2823
3.6232
3.2328
3.3729
3.4927
3.2724
3.7629
3.2828
3.5232
log1/cMIC exper.
3.892
4.001
3.924
3.952
4.013
4.059
4.015
4.024
4.04
3.979
3.96
4.005
3.95
3.977
3.98
3.958
4.027
3.979
4.004
Antifungal activity
log1/cMIC predict.
3.909
4.003
3.924
3.955
3.996
4.053
4.010
4.009
4.050
3.960
3.960
4.014
3.953
3.976
3.995
3.960
4.038
3.960
3.999
Residuals
-0.017
-0.002
0.000
-0.003
0.017
-0.007
0.005
0.015
-0.011
0.019
0.000
0.009
-0.003
0.001
-0.015
-0.002
-0.011
0.019
0.005
1
log1/cMIC = 0.1589logP + 3.4398; r = 0.9643; s = 0.0119
4.06
r=0,9643
4.04
4.02
log1/cMICexp.
4.00
3.98
3.96
3.94
3.92
3.90
3.88
2.8
3.0
3.2
3.4
3.6
3.8
4.0
logP
Figure 1. Plot of the lipophilicity descriptor versus experimentally observed antifungal
activity against
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It is well known that there are three important components in any QSAR study:
development of models, validation of models and utility of developed models. Validation
is a crucial aspect of any QSAR analysis. The statistical quality of the resulting model, as
depicted in Eq. 1, is determined by the correlation coefficient (r) and standard error of
estimation (s).
Candida albicans
For testing the validity of the predictive power of derived model the cross-validation
statistical technique was used. This is the most common validation technique, where a
number of modified data sets are created by deleting, in each case, one or smaller group
of objects from the data in such a way that each object is taken away once and only once.
For each reduced data set, the model is calculated, and responses for the deleted objects
are predicted from the model. The simplest and most general cross-validation procedure
is the leave-one-out technique (LOO technique). This method uses cross-validated fewer
parameters: PRESS (predicted residual sum of squares), SSY (total sum of squares deviation), Q2 and r2adj. PRESS is an important cross-validation parameter as it is a good
approximation of the real predictive error of the models. Its value being less than SSY
points out that the model predicts better than chance, and can be considered statistically
significant. The present models have PRESS<<SSY. From the PRESS and SSY, Q2 can
be easily calculated:
Q2= 1 - PRESS/SSY
2
Table 3. Cross-validation parameters
Eq. 1
PRESS
SSY
PRESS/SSY
Q2
r2adj
0.0026
0.0306
0.0849
0.9150
0.9304
The high Q2 value observed for derived QSAR model is indicative of its reliability in
prediction of inhibitory activity.
However, 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, predicted log1/cMIC values of benzoxazole
investigated were calculated by using models (1) 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 0.019. Further, Fig. 2 shows the plot of the linear regression of the predicted versus
experimental values of the antifungal activity of benzoxazoles investigated.
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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 2. Plot of the predicted versus the experimentally observed antifungal activity
against Candida albicans
All the presented results suggest the dependence of the lipophilicity parameters on
antifungal behaviour of benzoxazoles investigated. By knowing exact values of these
parameters, we can accurately predict the inhibitory activity.
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 3. Plot of the residual values against the experimentally observed log1/cMIC values
To investigate the existence of a systematic error in developing the QSAR models,
the residuals of predicted values of the inhibitory activity were plotted against the
experimental values, as shown in Figure 3. The propagation of the residuals on both sides
of zero indicates that no systemic error exists, as suggested by Jalali-Heravi and Kyani
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(27). It indicates that these models can be successfully applied to predict the antifungal
activity of this class of molecules.
The results of this investigation indicate that the antifungal activity exhibited by the
tested compounds is governed by the partition coefficient, logP. Lipophilicity as a physicochemical parameter has an important effect on inhibitory activity, and this parameter
is usually related to the pharmacological activity. LogP is a measure of hydrophobicity,
which is important for the penetration and distribution of the drug, but also for the interaction of drug with the receptors. The derived relationship between the lipophilicity
parameters and inhibitory activity can be used to estimate the antifungal activity of other
benzoxazoles.
CONCLUSIONS
Molecular modeling and QSAR analysis were performed to find the quantitative
effects of the lipophilicity parameter, logP, of some benzoxazole derivatives on their
antifungal activity against Candida albicans. The logP values were calculated for each
molecule, and a high-quality mathematical model relating the inhibitory activity,
log1/cMIC, and logP was defined. The validity of the model has been established by the
determination of the appropriate statistical parameters. The established model was used to
predict the inhibitory activity of the benzoxazoles investigated and close agreement
between experimental and predicted values was obtained. The low residual activity and
high cross-validated r2 values (Q2) observed indicated the predictive ability of the developed QSAR model. It is concluded that the lipophilicity parameter is a convenient
quantity for modeling inhibition for the present set of benzoxazole derivatives.
Acknowledgement
These results are part of the project No. 142028, supported by the Ministry of Science and Technological Development of the Republic of Serbia.
REFERENCES
1. O. Temiz, I. Oren, E. Sener and I. Yalcin: Synthesis and microbiological activity of
some novel 5- or 6-methyl-2-(2,4-disubstituted phenyl) benzoxazole derivatives. Il
Farmaco 53 (1998) 337-341.
2. O. Temiz-Arpaci: QSARs of Some 5- or 6-Methyl-2-Substituted Benzoxazoles/Benzimidazoles against Candida albicans. Turk.J. Med. Sci. 31 (2001) 493-497.
3. Z. A. Kaplancikli, G. Turan- Zitouni, G. Revial and K. Guven: Synthesis and Study
of Antibacterial and Antifungal Activities of Novel 2-[[Benzoxazole/benzimidazole2-yl)sulfanyl]acetylamino]thiazoles. Arch. Pharm. Res. 27 (2004) 1081-1085.
4. M. Arisoy, O. Temiz-Arpaci, I. Yildiz, F. Kaynak-Onurdag, E. Aki, I. Yalçin and U.
Abbasoglu: Synthesis, Antimicrobial Activity and QSAR Studies of 2,5-Disubstituted
Benzoxazoles. SAR QSAR Environ Res. 19 (2008) 589-612.
183
APTEFF,41, 1-203 (2010)
DOI: 10.2298/APT1041177P
UDC: 547.7:54.062
BIBLID: 1450-7188 (2010) 41, 177-185
5. O. Temiz-Arpaci, I. Yildiz, S. Ozkan, F. Kaynak, E. Aki-Sener and I. Yalçin: Synthesis and Biological Activity of Some New Benzoxazoles. Eur. J. Med. Chem. 43
(2008) 1423-1431.
6. T. Ertan, I. Yildiz, B. Tekiner-Gulbas, K. Bolelli, O. Temiz-Arpaci, S. Ozkan, F.
Kaynak, I. Yalcin and E. Aki: Synthesis, Biological Evaluation and 2D-QSAR Analysis of Benzoxazoles as Antimicrobial Agents. Eur. J. Med. Chem. 44 (2009) 501510.
7. S. Alper-Hayta, M. Arisoy, O. Temiz-Arpaci, I. Yildiz, E. Aki, S. Ozkan and F. Kaynak: Synthesis, Antimicrobial Activity, Pharmacophore Analysis of Some New 2(Substitutedphenyl/benzyl)-5-[(2-benzofuryl)carboxamido] benzoxazoles. Eur. J.
Med. Chem. 43 (2008) 2568-2578.
8. E. Oksuzoglu, O. Temiz-Arpaci, B. Tekiner-Gulbas, H. Eroglu, G. Sen, S. Alper, I.
Yildiz, N. Diril, E. Aki-Sener and I. Yalcin: A Study on the Genotoxic Activities of
Some New Benzoxazoles. Med. Chem. Res. 16 (2007) 1–14.
9. E. Oksuzoglu, B. Tekiner-Gulbas, S. Alper, O. Temiz-Arpaci, T. Ertan, I. Yildiz, N.
Diril, E. Sener-Aki and I. Yalcin: Some Benzoxazoles and Benzimidazoles as DNA
Topoisomerase I and II Inhibitors. J. Enz. Inh. Med. Chem. 23 (2007) 37-42.
10. O. Temiz-Arpaci, A. Ozdemir, I. Yalçin, I. Yildiz, E. Aki-Sener and N. Altanlar: Synthesis and Antimicrobial Activity of Some 5-[2-(Morpholin-4-yl)acetamido] and/or 5[2-(4-Substituted piperazin-1-yl)acetamido]-2-(p-substituted phenyl)benzoxazoles.
Arch. Pharm. Chem. Life Sci. 338 (2005) 105-111.
11. I. Yildiz-Oren, B. Tekiner-Gulbas, O. Temiz-Arpaci, I. Yalcin and E. Aki-Sener:
Quantitative Structure-Activity Relationships using Comparative Molecular Field
Analysis Studies on 2-(p-Substitutedbenzyl)-5-(substituted carbonylamino)benzaoxazoles as Antibacterial Agents against Staphylococcus aureus. Asian J. Chem. 16
(2004) 1359-1366.
12. I. Yildiz-Oren, B. Tekiner-Gulbas, I. Yalcin, O. Temiz-Arpaci, E. Aki-Sener and N.
Altanlar: Synthesis and Antimicrobial Activity of New 2-[p-Substituted-benzyl]-5[substituted-carbonylamino]benzoxazoles, Arch. Pharm. Pharm. Med. Chem. 337
(2004) 402-410.
13. S.O. Podunavac-Kuzmanović, S.L. Markov and D.J. Barna: Relationship Between the
Lipophilicity and Antifungal Activity of Some Benzimidazole Derivatives. J. Theor.
Comp. Chem. 6 (2007) 687-698.
14. S.O. Podunavac-Kuzmanović, D.J. Barna and D.D. Cvetković: Quantitative Structure
Activity Relationship of Some 1-Benzylbenzimidazole Derivatives as Antifungal
Agents. Acta Periodica Technologica 38 (2007) 139-147.
15. N.U. Perišić-Janjić and S.O. Podunavac-Kuzmanović: RPTLC Study of QSRR and
QSAR for Some Benzimidazole Derivatives. J. Planar Chromatogr. 21 (2008) 135141.
16. N.U. Perišić-Janjić, S.O. Podunavac-Kuzmanović, J.S. Balaž and Đ. Vlaović: Chromatographic Behaviour and Lipophilicity of Some Benzimidazole Derivatives. J.
Planar Chromatogr. 13 (2000) 123-129.
17. S.O. Podunavac-Kuzmanović, D.D. Cvetković and D. J. Barna: The Effect of Lipophilicity on Antibacterial Activity of Some 1-Benzylbenzimidazole Derivatives. J.
Serb. Chem. Soc. 73 (2008) 967-978.
184
APTEFF,41, 1-203 (2010)
DOI: 10.2298/APT1041177P
UDC: 547.7:54.062
BIBLID: 1450-7188 (2010) 41, 177-185
18. S.O. Podunavac-Kuzmanović, D.D. Cvetković and D. J. Barna: QSAR Analysis of 2Amino or 2-Methyl-1-Substituted Benzimidazoles against Pseudomonas aeruginosa.
Int. J. Mol. Sci. 10 (2009) 1670-1682.
19. A. Leo, C. Hansch, and D. Elkins: Partition Coefficients and Their Uses. Chem. Rev.
71 (1971) 525-616.
20. ANasal, D. Siluk and R. Kaliszan: Chromatographic Retention Parameters in Medicinal Chemistry and Molecular Pharmacology. Curr. Med. Chem. 10 (2003) 381-426.
21. I. Tiperciuc and C. Sarbu: Prediction of the Chromatographic Retention (lipophilicity)
of Some New Methyl-Thiazole-Oxadiazoline Derivatives by Multivariate Regression
Methods. J. Liq. Chrom. Rel. Technol. 29 (2006) 2257-2270.
22. S.O. Podunavac-Kuzmanović, D.D. Cvetković and D.J. Barna: Correlations Between
the Lipophilicity and the Inhibitory Activity of Different Substituted Benzimidazoles.
CI&CEQ 15 (2009) 125-130.
23. T. Slawik and B. Paw: Lipophilicity of Some N-and O-Substituted Alkanoic Acids of
1, 2-Benzisothiazol-3 (2H)-one. J. Liq. Chromatogr. 27 (2004) 1043-1055.
24. O. Ursu, A. Costescu, M.V. Diudea and B. Parv: QSAR Modeling of Antifungal Activity of Some Heterocyclic Compounds. Croat. Chem. Acta 79 (2006) 483-488.
25. CS. Chem. Office, Version 7.0, Cambridge Soft Corporation, 100 Cambridge Park
Drive, Cambridge, MA 02140-2317, U.S.A. (2001).
26. www.ncss.com.
27. M. Jalali-Heravi and A. Kyani: Use of Computer-Assisted Methods for the Modeling
of the Retention Time of a Variety of Volatile Organic Compounds: A PCA-MLRANN Approach. J. Chem. Inf. Comput. Sci. 44 (2004) 1328-1335.
KОРЕЛАЦИЈA ИЗМЕЂУ ЛИПОФИЛНОСТИ И АНТИФУНГАЛНЕ
АКТИВНОСТИ НЕКИХ ДЕРИВАТА БЕНЗОКСАЗОЛА
Сања О. Подунавац-Кузмановић, Соња Д. Велимировић
Применом QSAR (quantitative structure-activity relationship) aнализе, у овом раду
испитанa је квантитативна зависност између липофилности и антифунгалне активности неких деривата бензоксазола према Candida albicans. Дескриптори који
нумерички описују липофилност, logP, израчунати су применом Chem-Office 7.0
програмског пакета. Испитана је линеарна зависност између минималне инхибиторне концентрације (log1/cMIC) и дескриптора липофилности. Добијен је QSAR модел
за предвиђање антифунгалне активности испитиване серије бензоксазола. Резултати су продискутовани на основу статистичких параметара. Добијено је веома
добро слагање између експерименталних и теоретски добијених података за инхибиторну активност. Резултати ових испитивања указују на то да параметар липофилности има значајан утицај на антифунгалну активност ове класе једињења и да
може бити веома користан при дизајнирању нових биолошки активних молекула.
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UTILISATION OF TOMATO WASTE AS A SOURCE OF POLYPHENOLIC
ANTIOXIDANTS
Sladjana M. Savatović, Gordana S. Ćetković, Jasna M. Čanadanović-Brunet
and Sonja M. Djilas
This study is concerned with the effects of two extraction procedures (using ultrasonic
bath and high performance homogenizer) on the extraction efficiency of polyphenolics
present in the tomato waste. The isolation of flavonoid fraction of obtained extracts was
performed by solid-phase extraction. The antioxidant activity of flavonoid fractions was
determined using different spectrophotometric tests, including reducing power and 2,2diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays. The content of total
polyphenolics and flavonoids in extract obtained employing homogenizer (E2) was
higher than in the extract obtained employing ultrasonic bath (E1), and it was 14.33
mg/g and 7.70 mg/g, respectively. The flavonoid fraction (EF2) of extract E2 showed
higher antioxidant activity than flavonoid fraction (EF1) of extract E1. The DPPH free
radical scavenging activity of fractions EF1 and EF2, expressed as EC50 value, were 0.78
mg/ml and 0.45 mg/ml, respectively. The obtained results show that tomato wastes can be
used as an easily accessible source of antioxidant polyphenolics.
KEYWORDS: Tomato wastes, polyphenolics, flavonoids, antioxidant activity
INRODUCTION
By-products of fruits and vegetables processing represent a major disposal problem for
the industry concerned, but they are also promising sources of compounds which may be
used for various purposes in the food, pharmaceutical and cosmetic industries (1).
Tomato (Lycopersicon esculentum) is, after potato, the second most consumed vegetable in the world and approximately 30% is consumed as processed products. Both
fresh and processed tomato possesses a high nutritional value, due to its content of different types of micronutrients: vitamins (C and E), folates, carotenoids (lycopene and β-carotene) and polyphenolic compounds (flavonoids - quercetin, kaempferol and narangenin,
and phenolic acid - caffeic, chlorogenic, ferulic and p-coumaric acids). The skin and seeds
of tomatoes have been found to be richer sources of polyphenolic compounds than the
pulp (2). George et al. (3) studied 12 genotypes of tomatoes, and found that the free
polyphenolic content (expressed as mg catechin/100 g, fresh weight) in pulps ranged from
Slađana M. Savatović, M.Sc., Dr. Gordana S. Ćetković, Prof., Dr. Jasna M. Čanadanović-Brunet, Prof., Dr. Sonja
M. Đilas, Prof., University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
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9.2 to 27.0 mg/100 g, compared to 10.4 to 40.0 mg/100 g in skin, and also that for each
genotype, the polyphenolic content in skin was higher than in pulp. A similar observation
has been made by Toor and Savage (4), who reported that the total polyphenolic content
(expressed as mg gallic acid equivalents/100 g) of skin and seeds of tomatoes were,
respectively, 29.1 and 22.0, compared to 12.7 mg/100 g in the pulp. However, when tomatoes are processed into products like ketchup, sauces or juice, 3-7% of their weight become waste (1, 5). Tomato waste, since it contains a significant amount of skin and seeds, is
a potential source of natural antioxidants.
The extraction and purification of phytochemicals from natural sources is desirable,
since these bioactive substances are often used in the preparation of dietary supplements,
nutraceuticals, functional food ingredients, food additives, pharmaceuticals and cosmetic
products (6). The purpose of the extraction of phytochemicals from their plant sources is
to liberate these compounds from the vacuolar structures where they are found, either
through rupturing plant tissue or through a process of diffusion (7). Extraction yield is dependent on the solvent and method of extraction (8). Water, aqueous mixtures of ethanol,
methanol and acetone are commonly used as solvents (9). The chosen extraction method
should enable complete extraction of the compounds of interest and avoid their chemical
transformation (10). The application of ultrasound as a laboratory-based technique for
assisting extraction from plant material is widely published (11). Among the several types
of sonicator systems currently available, bath and probe-type sonicators are used. The
homogenization is also a process which is commonly used for assisting extraction (12).
This study has been carried out with the aim to investigate the influence of applied
extraction procedures, ultrasonic bath and high performance homogenizer for solid-liquid
extraction on the contents of polyphenolics in the tomato waste extracts. The isolation of
flavonoid fraction of obtained extracts was performed using solid-phase extraction (SPE)
using CHROMABOND® PA column. The antioxidant activity of flavonoid fraction of
tomato waste extracts was determined by different tests, including the reducing power and
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays.
EXPERIMENTAL
Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, trichloroacetic acid,
chlorogenic acid and rutin were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). These chemicals were of analytical reagent grade. Other chemicals and solvents
used were of the highest analytical grade, obtained from „Zorka“ Šabac (Serbia).
Waste preparation
Tomato (genotype Zora) harvested in Serbia in 2008, was obtained from the Institute
of Field and Vegetable Crops, Novi Sad. Tomatoes (1 kg) were cleaned, cut in pieces and
tomato juice was prepared using the juice extractor Neo, SK-400. The obtained tomato
waste (yield 8.10 ± 0.39%) was used for the experiment.
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Extraction procedures
Sample of tomato waste (10 g) was extracted at room temperature, using an ultrasonic
bath Sonic 12GT (Vims elektrik, Loznica, Serbia). The extraction was performed three
times with different amounts of 80% ethanol: 160 ml in 30 min, 80 ml in 30 min, 80 ml in
15 min at room temperature. The total extraction time was 75 min. The three extracts were
combined and evaporated to dryness under reduced pressure. The weight of polyphenolic
extract obtained using a bath-type sonicator (E1) was m = 0.56 ± 0.02 g.
In the second procedure, sample of tomato waste (10 g) was extracted at room temperature, using a high performance homogenizer, Heidolph DIAX 900 (Heidolph Instruments GmbH, Kelheim, Germany). The extraction was performed with different amounts
of 80% ethanol: 160 ml in 30 min, 80 ml in 30 min, 80 ml in 15 min at room temperature.
The total extraction time was 75 min. The three extracts were combined and evaporated to
dryness under reduced pressure. The weight of polyphenolic extract obtained using the
high performance homogenizer (E2) was m = 0.58 ± 0.02 g.
SPE procedure
The isolation of flavonoid fraction was performed according to the method,
MACHEREY-NAGEL Appl. No. 300150 SPE (13). Tomato waste extract (0.4 g) was
dissolved in 4 ml of distilled water and passed under vacuum through conditioned (with 6
ml methanol, followed by 20 ml bidistilled water) CHROMABOND® PA column (500
mg; J.T. Baker, Phillipsburg, NJ, USA). For SPE, a vacuum manifold processor (system
spe-12G; J.T. Baker, Großgerau, Germany) was used. The column was washed with 8 ml
of distilled water and flavonoids were eluted with 6 ml of methanol. The flavonoid fraction was evapored to dryness under reduced pressure. The weights of flavonoid fractions
EF1 and EF2 obtained from extracts E1 and E2 were: EF1, m=0.0179 g and EF2,
m=0.0137 g.
Total polyphenolic content
The amount of total soluble polyphenolics in the tomato waste extracts E1, E2, EF1
and EF2 was determined spectrophotometrically by the Folin-Ciocalteu method (14). The
total polyphenolic content was expressed as mg of chlorogenic acid equivalents per g of
extract.
Total flavonoid content
Total flavonoids were measured in the tomato waste extracts E1, E2, EF1 and EF2 by
the aluminum chloride spectrophotometric assay (15). Total flavonoid content was expressed as mg of rutin equivalents per g of extract.
Free radical scavenging activity
The free radical scavenging activity of the flavonoid fractions EF1 and EF2 was determined spectrophotometrically using the DPPH method (16), modified for this assay.
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Briefly, a 0.5 ml of solution containing from 0.025 to 2 mg of EF1/EF2 in methanol or
0.5 ml of methanol (control) were mixed with 1.5 ml of 90 μM DPPH solution and 3 ml of
methanol. The mixture was vortexed and left at room temperature for 60 min, than the
absorbance was read against a blank at 515 nm using a UV-1800 spectrophotometer
(Shimadzu, Kyoto, Japan). The blank probe contained all components except the radicals.
The capability to scavenge the DPPH radicals, DPPH scavenging activity (SADPPH), was
calculated using the following equation:
SADPPH (%) = (AControl - ASample)/AControl × 100
where AControl is the absorbance of the control reaction (containing all reagents except the
extract) and ASample is the absorbance in the presence of the extract. The values of scavenging activity were calculated for the various concentrations of extract.
Reducing power
The reducing power of the flavonoid fractions EF1 and EF2 was determined by the
method of Oyaizu (17). For this puropse, suspension of EF1/EF2 (0.025 - 2.5 mg) in 1 ml
of distilled water or 1 ml of distilled water (control) 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, 1 ml of trichloroacetic acid (10%) was added and the
mixture was then centrifuged at 3000 rpm for 10 min. A 2 ml aliquot of the upper layer
was mixed with 2 ml of distilled water and 0.4 ml of 0.1% FeCl3 and the absorbance of the
mixture was measured at 700 nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto,
Japan). Increased absorbance of the reaction mixture indicates increased reduction capability.
Total Polyphenolic and Flavonoid Content
Two procedures were applied for the efficient extraction of polyphenolics from tomato
waste. The extractions were carried out using 80% aqueous ethanol solvent system. The
contents of total polyphenolics and flavonoids in the polyphenolic extracts (E1 and E2)
and in its flavonoid fractions (EF1 and EF2), as well as the ratio of total flavonoids/polyphenolics are presented in Table 1. The total polyphenolic contents in extracts and
flavonoid fractions were determined from the regression equation of chlorogenic acid calibration curve, and expressed as mg of chlorogenic acid equivalents per g of dry extract.
Similarly, total flavonoids in extracts and flavonoid fractions were determined from the
regression equation of rutin calibration curve, and expressed as mg of rutin equivalents per
g of dry extract. The content of total polyphenolics and flavonoids in E2 extract was higher than in E1 extract and it was 14.33 mg/g and 7.70 mg/g, respectively. Also, the content
of total polyphenolics and flavonoids in EF2 fraction was higher than in EF1 fraction, and
it was 73.22 mg/g and 69.82 mg/g, respectively. It is evident from Table 1 that the high
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performance homogenization (E2 and EF2) showed better results for the extraction of
polyphenolics and flavonoids than the ultrasonic assisted extraction (E1 and EF1).
Table 1. Total polyphenolics and flavonoids in tomato waste extracts and in its flavonoid
fractions
Extracts
E1
E2
EF1
EF2
Polyhenolics (mg/g)
11.41
14.33
71.03
73.22
Flavonids (mg/g) Flavonoids/polyphenolics
4.03
0.35
7.70
0.54
64.32
0.91
69.82
0.95
Based on spectrophotometric determination, it can be observed that the ratio total flavonoids/polyphenolics in flavonoid fractions obtained after SPE procedure, EF1 (0.91)
and EF2 (0.95), was very high (Table 1). The applied SPE showed good results for concentrating flavonoids from the extracts.
Antioxidant activity
The antioxidant activity of the EF1 and EF2 fractions of the tomato waste extracts was
determined using different tests, including reducing power and DPPH free radical scavenging assays. These extracts were chosen, since the content of flavonoids was higher
than in the extracts E1 and E2. The model of scavenging the stable DPPH radical is a
widely used method to evaluate antioxidant activity in a relatively short time compared
with other methods (18). The hydrogen atom or electron donation ability of the extract was
measured from the bleaching of a purple-coloured methanol solution of stable DPPH
radical. Figure 1 shows the dose response curve for the DPPH radical scavenging activity
(SADPPH) of the EF1 and EF2 fractions. The DPPH radical scavenging activities of the EF1
and EF2 fractions were concentration dependent. The high correlation coefficients
(R2>0.90), calculated from logarithmic regression analysis, indicated that there is a significant positive correlation between the concentration and DPPH radical scavenging activity.
100
SADPPH (%)
80
y = 17.679Ln(x) + 68.822
R2 = 0.9949
60
y = 17.143Ln(x) + 62.319
R2 = 0.9291
40
EFl1
EFl2
20
0
0.0
1.0
2.0
3.0
4.0
5.0
Concentration (mg/ml)
Figure 1. DPPH radical scavenging activity (SADPPH) of EF1 and EF2 fractions
The EC50 value, defined as the concentration of the extract required for 50% scavenging of DPPH radicals under experimental condition employed, is a parameter widely used
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to measure the free radical scavenging activity (19); a smaller EC50 value corresponds to a
higher antioxidant activity. The higher DPPH free radical scavenging activity, expressed
as EC50 value, showed the fraction EF2 (0.45 mg/ml) than the fraction EF1 (0.78 mg/ml).
For the measurements of the reducing power, the Fe3+ - Fe2+ transformation was
investigated in the presence of the EF1 and EF2 fractions using the method of Oyaizu
(17). In this assay, the yellow colour of the test solution changes to various shades of
green and blue, depending on the reducing power of the antioxidant in the extracts. The
capacity of the extracts to reduce the ferric-ferricyanide complex to the ferrous-ferricyanide complex of Prussian blue was determined by recording the absorbance at 700 nm
(20). Figure 2 shows the reducing powers of the EF1 and EF2 fractions. Like the DPPH
radical scavenging activity, the reducing power of the EF1 and EF2 fractions increased
with increasing concentration. All of the applied concentrations of EF2 flavonoid fraction
showed higher reducing powers than the EF1 fraction.
1,4
1,2
A700
1
0,8
EFl1
0,6
EFl2
0,4
0,2
0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Concentration (mg/ml)
Figure 2. Reducing power of different concentrations of EF1 and EF2 fractions
The obtained results show that the tomato waste should be regarded as a valuable product and has potential as a value-added ingredient which can be used because of their
favourable technological or nutritional properties for functional foods.
CONCLUSION
The analysis of the polyphenolic and flavonoid content in the tomato waste extracts
show that the high performance homogenization gave better results for the extraction of
polyphenolics than the bath-type sonication.
The ratio total flavonoids/polyphenolics in flavonoid fractions obtained after SPE
procedure was very high (>0.90), and this fact indicates that SPE is a good method for
concentrating flavonoids from polyphenolic extracts.
The antioxidant activity of the EF1 and EF2 flavonoid fractions increased with increasing concentration. The higher reducing power and DPPH free radical scavenging activity showed flavonoid fraction EF2 than the EF1. The EC50 value of the fraction EF2, determined based on DPPH radical scavenging activity, was 0.45 mg/ml.
Also, the obtained results show that the tomato waste can be used as an easily accessible source of antioxidant polyphenolics and flavonoids.
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Acknowledgement
These results are part of the project No. 23011, which is financially supported by the
Ministry of Science and Technological Development of the Republic of Serbia.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
A. Schieber, F.C. Stintzing and R. Carle: By-products of plant food processing as a
source of functional compounds – recent developments. Trends Food Sci. Tech. 12
(2001) 401-413.
I. Martínez-Valverde, M.J. Periago, G. Provan and A. Chesson: Phenolic compounds, lycopene and antioxidant activity in commercial varieties of tomato (Lycopersium esculentum). J. Sci. Food Agric. 82 (2002) 323-330.
B. George, C. Kaur, D.S. Khurdiya and H.C. Kapoor: Antioxidants in tomato (Lycopersium esculentum) as a function of genotype. Food Chem. 84 (2004) 45-51.
R.K. Toor and G.P. Savage: Antioxidant activity in different fractions of tomatoes.
Food Res. Int. 38 (2005) 487-494.
V. Oreopoulou and C. Tzia: Utilization of Plant By-Products for the Recovery of
Proteins, Dietary Fibers, Antioxidants, and Colorants in Utilization of By-Products
and Treatment of Waste in the Food Industry. Eds. V. Oreopoulou and W. Russ,
Springer, US (2007) pp. 209-232.
U.D. Chavan, F. Shahidi and M. Naczk: Extraction of condensed tannins from beach
pea (Luthyrus maritimus L.) as affected by different solvents. Food Chem. 75 (2001)
509-512.
J. Tabart, C. Kevers, A. Sipel, J. Pincemail, J.O. Defraigne and J. Dommes: Optimisation of extraction of phenolics and antioxidants from black currant leaves and buds
and of stability during storage. Food Chem. 105 (2007) 1268-1275.
A.H. Goli, M. Barzegar and M.A. Sahari: Antioxidant activity and total phenolic
compounds of pistachio (Pistachia vera) hull extracts. Food Chem. 92 (2005) 521525.
T. Sun and C. Ho: Antioxidant activities of buckwheat extracts. Food Chem. 90
(2005) 743-749.
Y. Zuo, H. Chen and Y. Deng: Simultaneous determination of catechins, caffeine
and gallic acids in green, oolong, black and pureh teas using HPLC with a photodiode array detector. Talanta 57 (2002) 307-316.
K. Vilkhu, R. Mawson, L. Simons and D. Bates: Applications and opportunities for
ultrasound assisted extraction in the food industry - A review. Innovat. Food Sci.
Emerg. Tech. 9 (2008) 161-169.
N. Castillo-Muñoz, M. Fernández-González, S. Gómez-Alonso, E. García-Romero
and I. Hermosín-Gutiérrez: Red-Color Related Phenolic Composition of Garnacha
Tintorera (Vitis vinifera L.) Grapes and Red Wines. J. Agric. Food Chem. 57 (2009)
7883-7891.
http://www.mn-net.com/DesktopModules/TabID/7647/defauld.aspx
193
APTEFF,41, 1-203 (2010)
DOI: 10.2298/APT1041187S
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BIBLID: 1450-7188 (2010) 40, 187-194
14. V.L. Singleton, R. Orthfer and R.M. Lamuela-Raventos: Analysis of total phenols
and others oxidation substrates and oxidant by means of Folin-Ciocalteu reagent.
Meth. Enzymol. 299 (1999) 152-178.
15. J. Zhishen, T. Mengcheng and W. Jianming: The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem.
64 (1999) 555-559.
16. W. Brand-Williams, M.E. Cuvelier and C. Berset: Use of a free radical method to
evaluate antioxidant activity. LWT-Food Science and Technology 28 (1995) 25-30.
17. M. Oyaizu: Studies on product of browning reaction prepared from glucose amine.
Japan. J. Nutr. 44 (1986) 307-315.
18. D. Villãno, M.S., Fernández-Pachón, M.L. Moyá, A.M. Troncoso and M.C. GarcíaParrilla: Radical scavenging ability of polyphenolic compounds towards DPPH free
radical. Talanta 71 (2007) 230-235.
19. M.E. Cuvelier, H. Richard and C. Berset: Comparison of the antioxidative activity
of some acid phenols: Structure-activity relationship. Biosci. Biotechnol. Biochem.
56 (1992) 324-325.
20. Y.C. Chung, C.T. Chang, W.W. Chao, C.F. Lin and S.T. Chou: Antioxidative activity and safety of the 50% ethanolic extract from red bean fermented by Bacillus
subtilis IMR-NK1. J. Agric. Food Chem. 50 (2002) 2454-2458.
ИСКОРИШЋЕЊЕ ОТПАТКА ПАРАДАЈЗА КАО ИЗВОРА
ПОЛИФЕНОЛНИХ АНТИОКСИДАНАТА
Слађаnа М. Саватовић, Гордана С. Ћетковић, Јасна М. Чанадановић-Брунет и
Соња М. Ђилас
У овом раду је испитан утицај ултразвучног купатила и хомогенизатора на
ефикасност екстракције полифенолних једињења из отпатка парадајза. Након екстракције, из добијених екстраката су издвојени флавоноиди екстракцијом на чврстој фази. Антиоксидативна активност фракција флавоноида испитана је спектрофотометријски 2,2-дифенил-1-пикрилхидразил (DPPH) тестом, а такође је испитана
и њихова редукциона способност. Садржај укупних полифенолних једињења и
флавоноида у екстракту добијеном применом хомогенизатора (E2) је већи у односу
на садржај у екстракту добијеном применом ултразвучног купатила (E1) и износи
14,33 mg/g, односно 7,70 mg/g. Фракција флавоноида (EF2) екстракта E2 показала
је већу антиоксидативну активност од фракције флавоноида (EF1) екстракта E1.
Скевинџер активност на DPPH радикале фракција EF1 и EF2 изражена као EC50 износи 0,78 mg/ml, односно 0,45 mg/ml. Добијени резултати показују да отпадак парадајза представља значајан извор полифенолних једињења.
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ANTIOXIDANT ACTIVITY OF MANDARIN (Citrus reticulata) PEEL
Vesna T. Tumbas, Gordana S. Ćetković, Sonja M. Djilas,
Jasna M. Čanadanović-Brunet, Jelena J. Vulić, Željko Knez and Mojca Škerget

Mandarin peel, a waste product coming from juice production, was extracted by conventional extraction with 70% acetone. Content of flavonoids in mandarin peel extract
(MPE) was determined by HPLC. Hesperidin was the most dominant flavonoid. Free
radical scavenging activity of MPE on stable DPPH radicals and reactive hydroxyl radicals was also evaluated. EC50 value determined in spectrophotometrical DPPH radical
assay was 0.179 mg/ml, while this value in ESR spin trapping hydroxyl radical assay was
0.415 mg/ml. Also, MPE showed protective effects in stabilising sunflower oil during
accelerated storage. The results indicated that mandarin peel can be a valuable source of
natural antioxidants.
KEYWORDS: Mandarin peel, DPPH radical, hydroxyl radical, TBARS
INTRODUCTION
Lipid peroxidation is a ubiquitous phenomenon, first associated with the development
of rancidity in food and implicated in the aging process as well as in several degenerative
diseases (1). A lot of scientific investigations have been performed to discover possible
functional properties of food compounds, antioxidant or otherwise, which could be efficient in preventing diseases like atherosclerosis, hyperglycemia, brain dysfunction and cancer. Most of the food additives and supplements present in modern food and pharmaceuticals are synthetic compounds, with well known harmful effects on human health.
Many epidemiological and experimental studies certify that natural antioxidants derived
from the diet can prevent the early onset of ROS (Reactive Oxygen Species) related
diseases.
Citrus is an important crop mainly used in food industries for fresh juice production
and peel is the main by-product of its processing. In general, fruit skin contains a higher
concentration of antioxidant substances than the flesh of the fruit (2). Citrus peel, which
represents roughly one half of the fruit mass, is a rich source of bioactive compounds
including natural antioxidants such as phenolic acids and flavonoids (3).
Dr. Vesna T. Tumbas, Assist., [email protected], Dr. Gordana Ćetković, Prof., Dr. Sonja M. Djilas, Prof., Dr.
Jasna M. Čanadanović-Brunet, Prof., Jelena J. Vulić, B.Sc., Faculty of Technology, University of Novi Sad,
Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia; Dr. Željko Knez, Prof., Dr. Mojca Škerget, Prof., Faculty of
Chemistry and Chemical Engineering, University of Maribor, Smetanova 17, 2000 Maribor, Slovenia
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Dried peels of mature mandarin Citrus reticulata, more familiar as chenpi in China, is
recorded in the Chinese Pharmacopeia as being appropriate for activation of vital energy
and circulation, elimination of phlegm, disperse physical stagnation, etc. The primary
active biological constituents of them are adrenergic amines (such as synephrine, octopamine, and tyramine) (4) and flavonoids (flavanones, flavones, and flavonols) and phenolic acids (5).
In this study we investigated the antioxidant activity of the extract of mandarin
(Citrus reticulata) peel. Mandarin peel extract (MPE) was prepared by conventional extraction with 70 % (v/v) aqueous solution of acetone. Contents of major flavonoids in
MPE were qualified and quantified by HPLC. Free radical scavenging activity of MPE
was determined on stable DPPH radicals by spectrophotometric method and on reactive
hydroxyl radicals by ESR spin-trapping method. Synthetic antioxidant butylated hydroxyanisole (BHA) was used for comparison. Furthermore, we studied the oxidative stabilisation of sunflower oil with MPE under accelerated conditions.
EXPERIMENTAL
Chemicals and reagents
Hesperidin and 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5,5-dimethyl-1-pyroline-Noxide (DMPO) were purchased from Fluka, narirutin was purchased from Extrasynthese
(France). All standards were HPLC grade. Trichloracetic acid (TCA), butylated hydroxyanisole (BHA), thiobarbituric acid (TBA) and 1,1,3,3-tetraetoxypropane (TEP) were obtained from Sigma Chemicals Co. (USA). Acetone, methanol, and anhydrous acetic acid
pro analysis grade purity were provided by Merck. Dimethylformamide (DMF), iron(II)
chloride tetrahydrate and hydrogen peroxide were obtained from J.T. Baker. Milli Q water produced by Milli-Q plus apparatus was used for HPLC analysis. Refined sunflower
oil was purchased from „Vital“, Vrbas (Serbia).
Preparation of MPE
Mandarin peels were collected from fruits bought at the local supermarket. Peels were
dried by hot air flow (40 - 50°C) and stored in dark and cool place. Dried peels were
ground before use and stored in dark at room temperature. For conventional extraction
the 70 % (v/v) aqueous solution of acetone was used. Extraction was performed in a glass
flask and the ratio of solvent volume and mass of material was 50 ml/g. After 2 hours of
mixing with magnetic stirrer at the temperature of 40°C, solvent was separated by vacuum water pump. After drying, the extract was weighed and stored in a cool place before analysis.
HPLC analysis of MPE
The content of hesperidin and narirutin in MPE was determined by HPLC method.
The standard solutions were prepared by dissolving hesperidin and narirutin in methanol.
MPE (10 mg) was dissolved in methanol (10 ml), sonicated and filtered before analysis.
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The HPLC system consisted of a Varian 9012 pump and Varian diode array detector
9065. Column Chomsep SS C-18 250  4.6 mm Microsorb 100 stationary phase with 5
μm particle size was used. The mobile phase consisted of two solvents: A: methanol, and
B: 2 % (v/v) acetic acid in Milli-Q water. The method started with linear gradient from
25 % A to 40 % A in 3 min, then changed to isocratic for 5 min at 40 % A, followed by
linear gradient from 40 % A to 70 % A for 30 min and 2 min isocratic at 70 % A. The
method continued with linear gradient from 70 % A to 80 % A in 10 min and finished
with isocratic for 5 min at 80 % A. The sample injection volume was 20 μl, and the injection was performed manually. The flow rate was 0.85 ml/min and detection was performed at 282 nm. The quantification was made with external standard.
DPPH free radical assay
The free radical scavenging activity of MPE was determined spectrophotometrically.
The hydrogen atom or electron donation abilities of the MPE was measured from the
bleaching of purple-colored methanol solution of stable 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH•). Briefly, 1 ml of solution of MPE in 95% methanol, in the final concentration range of 0.025-0.35 mg/ml, or 1 ml of methanol (control) were mixed with 3 ml of
90 μM DPPH solution (18 mg in 50 ml 95% methanol prepared daily) and 8 ml of 95%
methanol. The mixture was vortexed thoroughly for 1 min and left at room temperature
for 60 min, then the absorbance was read against control at 515 nm using UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The control probe contained all components
except for the radicals. The capability of MPE to scavenge DPPH radicals, DPPH• scavenging activity (SADPPH•), was calculated using the following equation: SADPPH• (%) =
100 × (AControl - ASample)/AControl, where AControl is the absorbance of the control reaction
(containing all reagents except the MPE) and ASample is the absorbance in the presence of
the MPE.
Hydroxyl radical assay
Hydroxyl radicals were obtained by the Fenton reaction and detected by spin trapping
in a system consisting of: 0.2 ml H2O2 (2 mM), 0.2 ml FeCl2 (0.3 mM), 0.2 ml of DMF
and 0.2 ml DMPO (112 mM) as spin trap (control sample). The influence of MPE on the
amounts of hydroxyl radicals trapped by DMPO was studied by adding the DMF solution
of the MPE to the reaction system in the concentration range of 0.05 – 2.0 mg/ml. ESR
spectra were recorded 2.5 min after mixing on an ESR spectrometer Bruker 300E (Rheinstetten, Germany), with the following spectrometer settings: field modulation 100 kHz,
modulation amplitude 0.512 G, receiver gain 1 × 104, time constant 81.92 ms, conversion
time 163.84 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64
GHz, power 20 mW, temperature 23°C. The SA•OH value of the MPE was defined as:
SA•OH (%)= 100 × (h0 – hx) / h0, where h0 and hx are the hight of the second peak in the
ESR spectrum of DMPO/•OH spin adduct of the samples without and with MPE, respectively.
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Assay of thiobarbituric acid-reactive substances (TBARS)
MPE was added to sunflower oil at a concentration of 200 mg/kg (legal limit for
synthetic antioxidants (6)). Control sample (without antioxidant) and sample with MPE
were stored in an incubator at a fixed temperature of 60°C for 24 days. Lipid oxidation of
sample was assessed by the 2-thiobarbituric acid (TBA) method. Analyses were conducted at 4-day intervals. In brief, an aliquot of 2 g of sample was homogenised with 10 ml
of TCA (5%) and 5 ml of BHA (0.8%) on an ultrasonic bath for 5 min and then centrifuged for 5 min at 3000 rpm. A volume of 2.5 ml of supernatant (water layer) was
added to 1.5 ml of TBA (0.8%) and heated in water bath (70°C) for 30 min for pink colour development. The tube was first cooled and then the absorbance was measured at
532 nm using UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). TBARS were calculated from a standard curve of malondialdehyde (MDA) freshly prepared by acidification of TEP in the range from 0.006 to 0.299 μg/ml and expressed as mg of MDA per
kg of sample.
Mandarin, as other citrus fruits, has nutritional importance due to its particular composition. Flavonoids, especially polymethoxyflavones and flavanones (hesperidin, narirutin and naringin) are identified in citrus pulp as well as in peel (7, 8). In the by-products
of citrus fruit after juice production, phenolic acids such as caffeic, p-coumaric, ferulic,
and sinapic acids have also been identified (9, 10).
In the present study, the presence of two flavonids, narirutin and hesperidin, was determined in MPE (Table 1 and Fig. 1). The amount of hesperidin detected in the MPE
was five times higher than that of narirutin.
Table 1. Results of HPLC analysis of MPE
Identified compound
Narirutin
Hesperidin
Content
mg/g extract
15.3
80.9
mg/g peel
5.93
31.42
Hesperidin exists largely in citrus peels and is considered as the main functional component. It is known that hesperidin is the most abundant flavanone in peel of mandarin
fruits (11, 8), and our result is accordance with these reports. However, Levaj et al. (12)
showed that the amounts of narirutin in mandarin’s peels were the highest (approximately
1.2 folds higher than the level of hesperidin) compared to the amounts of other flavanon
glycosides. Peels contained higher level of investigated flavanones than pulp. In mandarin peels, flavanon glycosides were present in a decreasing sequence, as follows: narirutin>hesperidin>naringin. Xu et al. (13) determined four citrus flavonoids: narirutin,
hesperidin, nobiletin and tangeretin, and seven phenolic acids, including four hydroxycinnamics (caffeic, p-coumaric, sinapic, and ferulic) and three hydroxybenzoics (protoca198
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techuic, p-hydroxybenzoic, and vanillic), in Satsuma mandarin peel extracted with hot
water, ferulic acid being the dominant one.
The model of scavenging the stable DPPH radical is a widely used method to evaluate
antioxidant activities in a relatively short time compared with other methods (14, 15).
Figure 1. HPLC chromatogram of MPE
Hydroxyl radical is physiologically important reactive oxygen species that reacts with
almost all substances (16). MPE showed prominent, concentration-dependent free-radical
scavenging activity on stable DPPH free radicals and reactive hydroxyl radicals (Fig. 2).
Figure 2. Free radical scavenging activity of MPE
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In the DPPH free radical assay MPE reached SADPPH• = 100% at the concentration of
0.35 mg/ml, while the same effect in the hydroxyl radical assay was achieved at the concentration of 2 mg/ml. Concentrations of MPE that remove 50% of DPPH• and hydroxyl
radicals were 0.179 mg/ml and 0.415 mg/ml, respectively. Hydroxyl radical scavenging
activity of investigated extract was higher than the activity of synthetic antioxidant BHA
(EC50•OH = 1.505 mg/ml), while DPPH free radical scavenging activity was lower than
BHA (EC50 DPPH• = 0.028 mg/ml).
Levaj et al. (12) also determined by the FRAP method higher antioxidant capacity of
peels than pulps of Satsuma and Clementine mandarins. They found good correlation
with total flavonoid, individual flavanones with antioxidant capacity, while Ma et al. (11)
found the correlation with total phenol content.
The TBARS value is an index of lipid oxidation, measuring malondialdehyde (MDA)
content. Hydroperoxides, the initial reaction product of polyunsaturated fatty acids, react
with oxygen to form MDA, which may contibute to off-flavour of oxidised oils. It can be
seen from Fig. 3 that MPE inhibited the formation of TBARS of sunflower oil during the
whole storage period. TBARS values of the experimental sunflower oil incresed gradually during storage period, with increased acceleration after the fourth day. Stabilisation
effect of MPE was strongest after eight days of storage, when TBARS value of the contol
was 1.65-fold higher than that of MPE and the inhibitory effect reached 39.27 %. However, the inhibiton of lipid peroxidation with MPE after 24 days of storage was weak
(5.25 %).
Figure 3. Effect of MPE on TBARS of sunflower oil under accelerated storage at 60°C
for 24 days
Magda et al. (17) found that mandarin peels inhibited lipid peroxidation in biscuits.
Addition of peels at the level of 10%, showing no adverse effects on the organoleptic properties of biscuits, improved the weight gain of rats, decreased the levels of serum total
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cholesterol, liver lipid and liver cholesterol and glucose blood. Authors suggested that
powdered citrus peels can be used as natural antioxidants in biscuits production.
Presence of polyphenolic compounds, namely hesperidin adn narirutin, good free radical scavenging activity and inhibition of lipid peroxidation of MPE, documented in this
paper, indicates that mandarin peel can be used as a safe food additive with antioxidant
activity.
CONCLUSION
Composition analysis showed that MPE contains hesperidin (80.9 mg/g extract) and
narirutin (15.3 mg/g extract).
MPE showed prominent free radical scavenging activity towards DPPH (EC50DPPH• =
0.179 mg/ml) and hydroxyl radicals (EC50•OH = 0.415 mg/ml). Hydroxyl radical scavenging activity was better than that of synthetic antioxidant BHA (EC50•OH = 1.505 mg/ml).
MPE showed protective effects against lipid peroxidation of sunflower oil during 24
days of storage at elevated temperature, with a strongest effect on TBARS value reduction after eight days of storage.
Based on the results presented here, we suggest that mandarin peel powders can be
used as substitutes for synthetic antioxidants, to increase the shelf life of food products
containing fats and oils, imparting helath benefits to the consumer.
Acknowledgement
This research is a part of the Project of Serbiа-Slovenia technology co-operation for
2010-2011, project No. 69-00-02/2009-01/10.
REFERENCES
1. C. Rice-Evans, R. Burdon: Free radical-lipid interactions and their pathological consequences. Prog. Lipid Res. 32 (1993) 71-110.
2. M.A. Awad, A. de Jagar, L.H. van der Plas, A.R. van der Krol: Flavonoid and chlorogenic acid changes in peel of ‘Elstar’ and ‘Jonagold’ apples during development
and ripening. Sci. Hortic. 90 (2001) 69–83.
3. B.B. Li, B. Smith, M.M. Hossain: Extraction of phenolics from citrus peels. I. Solvent
extraction method, Sep. Purif. Technol. 48 (2006) 182–188.
4. B. Avula, V.C. Joshi, A. Weerasooriya, I.A. Khan: Liquid chromatography for separation and quantitative determination of adrenergic amines and flavonoids from Poncirus trifoliatus Raf. fruits at different stages of growth. Chromatographia. 62(7-8)
(2005) 379-83.
5. G.H. Xu, X.Q. Ye, J.C. Chen, D.H. Liu: Effect of heat treatment on the phenolic
compounds and antioxidant capacity of citrus peel extract. J. Agric. Food Chem.
55(2) (2007) 330-5.
6. P.D. Duh, G.C. Yen: Antioxidant efficacy of methanolic extracts of peanut hulls in
soybean and peanut oils. JAOCS 74 (1997) 745-748.
201
APTEFF,41, 1-203 (2010)
DOI: 10.2298/APT1041195T
UDC: 634.322:543.645
BIBLID: 1450-7188 (2010) 40, 195-203
7. P. Mouly, E.M. Gaydou, A. Auffray: Simultaneous separation of flavanone glycosides and polymethoxylated flavones in citrus juices using liquid chromatography. J.
Chromatogr. 800 (1998) 171-179.
8. Y.-C. Wang, Y.-C. Chuang, H.-W. Hsu: The flavonoid, carotenoid and pectin content
in peels of citrus cultivated in Taiwan. Food Chem. 106 (2008) 277-284.
9. A. Bocco, M.E. Cuvelier, H. Richard, C. Berset: Antioxidant activity and phenolic
composition of citrus peel and seed extracts. J. Agric. Food Chem. 46 (1998) 21232129.
10. H. Peleg, M. Naim, R.L. Rouseff, U. Zehavi: Distribution of bound and free phenolic
acids in oranges (Citrus sinensis) and grapefruits (Citrus paradisi). J. Sci. Food Agric.
57 (1991) 417-26.
11. Y-Q. Ma, X-Q. Ye, Z-X. Fang, J-C. Chen, G-H. Xu, D-H. Liu: Phenolic Compounds
and Antioxidant Activity of Extracts from Ultrasonic Treatment of Satsuma Mandarin
(Citrus unshiu Marc.) Peels. J. Agric. Food. Chem. 56 (2008) 5682-5690.
12. B. Levaj, V. Dragović-Uzelac, D. Bursać Kovačević, N. Krasnići: Determination of
flavonoids in Pulp and Peel of Mandarin Fruits. ACS 74(3) (2009) 221-225.
13. G.H. Xu, J.C. Chen, D.H. Liu, Y.H. Zhang, P. Jiang, X.Q. Ye: Minerals, Phenolic
Compounds, and Antioxidant Capacity of Citrus Peel Extract by Hot Water. J. Food.
Sci. C: Food Chem. 73(1) (2008) C11-C18.
14. P.D. Duh, Y.Y. Tu, G.C. Yen: Antioxidant activity of water extract of Harng Jyur
(Chrysanthemum morifolium Ramat). Food Sci. Technol. 32 (1999) 269-77.
15. L.W. Chang, W.J. Yen, S.C. Huang, P.D. Duh: Antioxidant activity of sesame coat.
Food Chem. 78 (2002) 347-54.
16. M. Terashima, R. Watanabea, M. Uekia, S. Matsumura: Comprehensive evaluation of
antioxidant activity for various substances with 5-axe cobweb chart. Food Chem.
120(1) (2010) 150-155.
17. R.A. Magda, A.M. Awad, K.A. Selim: Evaluation of Mandarin and Navel Orange
Peels as Natural Sources of Antioxidant in Biscuits. Alex. J. Fd. Sci. & Technol.
Special Volume Conference (2008) 75-82.
202
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DOI: 10.2298/APT1041195T
UDC: 634.322:543.645
BIBLID: 1450-7188 (2010) 40, 195-203
АНТИОКСИДАТИВНА АКТИВНОСТ КОРЕ МАНДАРИНЕ
(Citrus reticulata)
Весна Т. Тумбас, Гордана С. Ћетковић, Соња М. Ђилас, Јасна М.
Чанадановић-Брунет, Јелена Ј. Вулић, Жељко Кнез и Мојца Шкергет
Kора мандарине, споредни производ који настаје током производње сока, екстрахована је конвенционалном екстракцијом 70% ацетоном. Садржај флавоноида у
екстракту коре мандарине одређен је течном хроматографијом високе ефикасности
(HPLC). Утврђено је да је најзаступљенији флавоноид хесперидин. Утврђене су и
антиоксидативне активности екстракта коре мандарине на стабилне DPPH и реактивне хидроксил радикале. EC50 вредност одређена у DPPH спектрофотометријском тесту износила је 0,179 мг/мл, док је вредност одређена ESR методом на хидроксил радикале била 0,415 мг/мл. Такође, екстракт коре мандарине показао је
заштитни ефекат током убрзане оксидације сунцокретовог уља изазване складиштењем. Добијени резултати указују на могућност употребе коре мандарине као значајног извора природних антиоксиданата.
203
INSTRUCTION FOR MANUSCRIPT PREPARATION
Acta Periodica Technologica, Novi Sad publishes reviews and scientific papers covering all branches of technology: food, chemical, biochemical, pharmaceutical, as well
as process engineering and related scientific fields.
Acta Periodica Technologica is published in English. The journal may include supplements from congresses, meetings or symposiums.
Submission of Papers. All correspondence, including submission of the manuscript,
notification of the Editor’s decision and requests for revision, takes place by e-mail:
[email protected] or [email protected].
Submission of paper implies that
- it has not been published previously (except in the form of an abstract or as a whole
in the proceedings of papers of a scientific meeting, or as part of a published
lecture or academic thesis),
- it is not under consideration for publication elsewhere, and
- it will not be published elsewhere in the same form, in English or in any other language, without the written consent of the publisher.
If the paper was given, wholly or in part, at a scientific meeting, this should be stated
in a footnote on the title page.
Preparation of manuscripts
Language: Manuscript should be written in English.
Typing: Manuscript must be written in Word with a font size 10 pt, double spaced
with wide margins (3 cm) on A4 pages (max. 10 pages for scientific papers). Number all
pages of the manuscript. Import tables and figures into the text.
General format. The manuscript should contain the following in this order: Title
page, ABSTRACT and KEY WORDS, INTRODUCTION, EXPERIMENTAL, RESULTS and DISCUSSION, CONCLUSION, ACKNOWLEDGEMENT and REFERENCES as well as ABSTRACT and KEY WORDS IN SERBIAN LANGUAGE.
Title page: On the first page should be the title without symbols, formulae or abbreviations (capital bold letters). Title should be concise and explanatory of the content of
the paper. Full name (name, initial and surname) of authors (without degrees, professional or official titles) should be given under the title, written in italic. Affilation of authors
should be given after the authors name. Indicate all affiliations with supscript letter immediately after authors name and in front of appropriate address. Provide the full postal
address of each affiliation, including the country name, and e-mail address of each
author.
Abstract of the paper (100-250 words, written in italic) should be given under the title and authors. Abstracts should contain the aim of investigated work, methods, results
and conclusion.
Keywords (normal letters, max. 5 keywords) should be listed afterwards.
Introduction should state previous relevant work with appropriate references, the
problem investigated and the aim of work.
Experimental. The methods and materials used should be stated clearly in sufficient
detail to permit the work to be repeated by others. Only new techniques should be described in detail; known methods must have adequately references.
Results and Discussion. Results should be presented concisely, with tables or illustrations for clarity. The significance of the findings should be discussed without repetition of the material in the Introduction. Adequate number of illustrations, graphs and
chemical formulae used must be kept on minimum.
Conclusion should indicate the significant contribution of the manuscript with its
applications.
Acknowledgements: These should be kept to a minimum.
References cited should be indicated in the text using Arabic numerals in brackets ( ),
in the order of appearing. All publications cited in the text should be presented in a list of
references given on a separate page. Abbreviations of journal titles should be given according to the International Codex for Abbreviations of Journal Titles (Chemical Abstracts). The list of references should be arranged according to their appearance in the text.
Give names of all authors (do not use „et.al.“), with their initials before respective surnames. Include article titles in journals. Journal titles should be abbreviated according to the
Chemical Abstracts Service Source Index, 2005 edition, and supplements. The abbreviated titles should be followed by the volume (in bold), number (if exists), year (in parentheses) and first and last page numbers.
Examples:
Journals: 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.
If pagination repeated in each number of one volume it is necessary to add this particular numeral.
Books: Banks, W. and Greenwood, C.T.: Starch and its Components, Edinburgh University Press, Edinburgh (1975) p.98.
Book with more chapters: 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.
Book of Abstracts: Noe, W., Howaldt, M., Ulber, R. and Scheper, T.: Immunobase
elution assay for process control, 8th European Congress on Biotechnology, Budapest, 1721 August 1997, Book of Abstracts WE 163, p. 246.
Thesis: Linstead, J.B.: Effects of adding natural antioxidants on colour stability of
paprika, Ph.D. (or M.Sc.) Thesis, University of Glasgow, 2006.
Patent: Miller, B.O.: U.S. Pat. 2542356 (1962), Dow Chemical Comp.: Abstr. 51
(1961) 2870.
Unpublished data: Should be cited with one of the following comments „in press“,
„unpublished work“ or „personal communication“.
Online citations: Should include the author, title, website and date of access (example: Wright, N.A.: The Standing of UK Histopathology Research 1997-2002.
http://pathsoc.org.uk (accessed 7 October 2004)).
Abstract and keywords in Serbian language should be given at the end of manuscript (after references), in extended form (max. length 1 page), printed in Cyrillic (normal letters) with the title (capital letters), full name(s) of each author(s) and affiliation(s)
(italic letters).
Chemical nomenclature and units. Authors are requested to use SI units and chemical nomenclature following the rules of Chemical Abstracts whenever possible.
Tables. Each Table is numbered with Arabic numeral, followed by the title (Table 1.
Result...).
Figures. Each drawing or figure should also be numbered with Arabic numerals followed by the title (Figure 1. Chromatogram of...). Graphs and charts must be prepared by
Microsoft Excel or Origin. Schemes must be prepared by Microsoft Visio or Corel Draw.
It is necessary to submit them in original extension (xls, vdr, cdr). Scanned black & white
schemes should be submitted in tif, wmf, or bpm form. Colour and black & white photographs should be submitted in jpg form.
Formulae and Equations. Type formulas and mathematical equations clearly, accurately placing superscripts and subscripts. Equations should be indicated in the text using
Arabic numerals in square brackets [ ]. Abbreviations and symbols-notation should be explained at first appearing, or on a separate list at the end of manuscript.
Additional information
Manuscript should be sent in one hard copy to the address: Faculty of Technology,
Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia, to the editor: Professor Dr. Sonja Djilas
(for Acta Periodica Technologica) and electronic form to the e-mail: [email protected]
or [email protected]. Authors are expected to propose the category of manuscript (review, original scientific paper).
Review process. All papers submitted to the journal will be reviewed by at least two
independent referees who will be asked to complete the refereeing job within 4-6 weeks.
Final decision on publication will be made by the Editorial Board. Manuscripts may be
sent back to authors for revision if necessary. Revised manuscript submissions should be
made as soon as possible (within 2 weeks) after the receipt of the referees comments.
Proofs. One set of page proofs will be sent by e-mail to the corresponding Author.
The author may list the corrections and return to the journal in an e-mail. Proofs must be
corrected and returned to the publishers within 48 hours of receipt.
Offprints. The corresponding author, at no cost, will be provided with a 10 free paper
offprints.
Author service. For inquiries relating to the submission of manuscript, please send
an a e-mail to the Editor.
THIS ISSUE OF ACTA PERIODICA TECHNOLOGICA
IS FINANCIALLY SUPPORTED BY:
Ministry of Science and Technological Development of Republic of Serbia,
Belgrade
Editorial:
Faculty of Technology, Bulevar cara Lazara 1,
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
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)

acta periodica technologica - Tehnoloski fakultet Novi Sad

Transcription

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