rezumatul - USAMV Cluj

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UNIVERSITATEA DE ŞTIINŢE
AGRICOLE ŞI MEDICINĂ
VETERINARĂ, CLUJ-NAPOCA
FACULTATEA DE ZOOTEHNIE ŞI
BIOTEHNOLOGII
DOMENIUL: BIOTEHNOLOGII
REZUMATUL
TEZEI DE DOCTORAT
SISTEME DE ÎNCAPSULARE A UNOR COMPUŞI
BIOACTIVI EXTRAŞI DIN ULEIURI VEGETALE
MONICA TRIF
Ing. Dipl. Biotehnolog
CONDUCĂTOR ŞTIINŢIFIC:
PROF. Dr. Dr. h.c. HORST A. DIEHL
2009
Sisteme de încapsulare a unor compuşi bioactivi extraşi din uleiuri vegetale
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CUPRINS
I. INTRODUCERE. SCOP ŞI OBIECTIVE............................................................................ III
PARTEA II. CONTRIBUŢII PROPRII (ORIGINALE) ......................................................... IX
CAPITOL II. CARACTERIZAREA ULEIURILOR FUNCŢIONALE UTILIZATE LA
BIOÎNCAPSULARE ............................................................................................................... IX
II.1. MATERIALE ŞI METODE ......................................................................................... IX
II.2. REZULTATE ŞI DISCUŢII.......................................................................................... X
II.3. CONCLUZII .............................................................................................................. XIV
CAPITOLUL III. BIOÎNCAPSULAREA ULEIURILOR: PROTOCOALE DE PREPARE A
CAPSULELOR ŞI CARACTERIZAREA LOR.................................................................... XV
III.1. MATERIALE ŞI METODE ...................................................................................... XV
III.2. REZULTATE ŞI DISCUŢII .................................................................................... XVI
III.3. CONCLUZII............................................................................................................XXII
CAPITOL IV. EFICIENŢA ÎNCAPSULĂRII ŞI STUDII DE ELIBERARE A ULEIURILOR
DIN CAPSULE ....................................................................................................................XXII
IV.1. MATERIALE ŞI METODE....................................................................................XXII
IV.2. REZULATTE ŞI DISCUŢII ................................................................................. XXIII
IV.3. CONCLUZII ......................................................................................................... XXVI
CAPITOL V. CARACTERIZAREA FTIR A OXIDĂRII ULEIURILOR .....................XXVII
V.1. MATERIALE ŞI METODE ..................................................................................XXVII
V.2. REZULTATE ŞI DISCUŢII..................................................................................XXVII
V.3. CONCLUZII.........................................................................................................XXVIII
CONCLUZII GENERALE ................................................................................................ XXIX
BIBLIOGRAFIE SELECTIVĂ ......................................................................................... XXXI
PUBLICAŢII PE DURATA STAGIULUI DOCTORAL SI PARTICIPARI LA
SIMPOZIOANE ŞI CONFERINŢE NATIONALE ŞI INTERNAŢIONALE............... XXXIV
II
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I. INTRODUCERE. SCOP ŞI OBIECTIVE
BIOÎNCAPSULAREA reprezintă o tehnologie nouă, bazată pe inserţia şi imobilizarea
moleculelor bioactive, în ‘’suporturi’’ specifice (matrici). Tehnologia încapsulării este bine
dezvoltată şi utilizată în industria farmaceutică, chimică, cosmetică, alimentară precum şi în
cea tipografică (Augustin et al., 2001; Heinzen, 2002). Potenţialul bioîncapsulării s-a
concretizat tot mai mult în domeniile biotehnologiei, mai ales în cele agricole şi alimentare. În
ultimele decenii, încapsularea compuşilor activi a devenit o tehnologie de mare interes şi
însemnătate, fiind adecvată atât pentru ingredienţii alimentari cât şi pentru cei chimici,
farmaceutici sau cosmetici.
Aplicarea acestei metode de success, de bioîncapsulare a compuşilor bioactivi extraşi
din uleiuri vegetale ar putea permite stabilirea combinaţilor şi a calitaţilor optime ale acestor
substanţe. Este de luat în considerare că o asemenea metodă şi anume bioîncapsularea,
aplicată în aria comercială, ar avea beneficii semnificative pentru industria farmaceutică,
alimentară şi cosmetică. În afară de aceasta este de consemnat faptul că, cercetarea şi
dezvoltarea în aceste domenii este semnificativă mai ales în ceea ce priveşte conservarea
compuşilor naturali bioactivi extraşi din plante.
Scopul acestei tezei constă în utilizarea diferitelor matrici naturale pentru
bioîncapsularea moleculelor bioactive prin metoda gelării ionice (‘’ionotropically crosslinked
gelation’’), precum şi în evaluarea diferenţelor de calitate şi a eficienţei parametrilor pentru
produşii încapsulaţi şi nu în ultimul rând a eliberării controlate a moleculelor bioactive din
matrici.
Structura tezei. Prima parte a acestei teze este reprezentată de un studiu de literatură, partea a
doua include rezultatele experimentale: materiale şi metode, rezultate şi discuţii, concluzii.
Prima parte (Studiul de literatură) este compusă din patru capitole (I-IV):
Capitolul I. Bioîncapsularea: definiţie, principii, aplicaţii, metode şi tehnici
Capitolul II. Uleiuri vegetale funcţionale: caracterizarea fizică, chimică şi autentificarea
Capitolul III: Încapsularea uleiurilor: matrici, metode şi tehnici de încapsulare, evaluarea
eficienţei şi a stabilităţii
Capitolul IV. Metode pentru caracterizarea capsulelor
Partea a doua a tezei (Contribuţiile proprii) include patru capitole, dupa cum urmează:
Capitolul V. Caracterizarea uleiurilor funcţionale utilizate pentru bioîncapsulare.
Această parte caracterizează patru uleiuri funcţionale (ulei de cânepa, ulei de dovleac, ulei
extra virgin de măsline şi ulei de cătină) analizate şi apoi încapsulate prin diferite tehnici:
spectroscopie de absorbţie în ultraviolet (UV), cromatografie de gaze (GC) cu detecţie prin
ionizare în flacără (FID) şi spectroscopie în infraroşu cu transformantă fourier echipată cu
reflectanţă atenuată orizontală (FTIR-ATR), determinările chimice fiind realizate în
conformitate cu metodele descrise în A.O.A.C. şi IOOC.
III
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Capitolul VI. Optimizarea protocoalelor de obţinere a caspsulelor utilizând matrici
naturale şi caracterizarea capsulelor ce conţin ulei încapsulat. Acest capitol descrie
protocoalele pentru: sinteza capsulelor goale de diferite mărimi şi concentraţii, sinteza
capsulelor de diferite mărimi şi concentraţii ce încorporează ulei, caracterizarea capsulelor
goale şi a celor ce conţin uleiuri (în funcţie de mărime şi morfologie), cuprinzând de
asemenea şi analiza FTIR-ATR şi termică a capsulelor.
Capitolul VII. Studierea eficienţei încapsulării şi a eliberării uleiurilor încapsulate. Acest
capitol conţine studii cu privire la eficienţa încapsulării uleiurilor funcţionale în diferite
matrici, determinarea ratei de eliberare a uleiurilor din capsule în timp şi în diferiţi solvenţi,
precum şi eliberarea in vitro a uleiurilor din capsule.
Capitolul VII. Caracterizarea FTIR-ATR a oxidării uleiurilor. Acest capitol include
analize comparative a uleiurilor libere şi încapsulate supuse oxidării în timp în condiţii UV.
Planul experimental se bazeaza pe urmatoarele obiective:
¾ Utilizarea diferitelor matrici naturale (precum alginatul, alginatul în complex cu kcaragenan şi gume: xantan şi guar, şi chitosan) în scopul încapsulării uleiurilor
funcţionale (ulei de dovleac, ulei extra virgin de măsline, ulei de cânepă şi ulei de
cătină)
¾ Îmbunătăţirea şi optimizarea metodelor de bioîncapsulare pentru uleiurile vegetale cu
proprietăţi funcţionale
¾ Investigarea morfologiei diferitelor capsule obţinute (microscopie electronică de
scanare), caracterizarea capsulelor (suprafaţă, diametru, perimetru, elongaţie,
sfericitate şi compactitate), analize FTIR
¾ Investigarea uleiurilor funcţionale bioîncapsulate: eficienţa şi stabilitatea încapsulării,
eliberarea controlată a uleiurilor încapsulate, materialul şi funcţionalitatea capsulelor
obţinute, caracterizarea FTIR a uleiurilor libere, a capsulelor obţinute şi oxidarea
uleiurilor libere şi încapsulate.
Cercetările prezentate au fost efectuate la Departamentul de Chimie şi Biochimie din
cadrul Universităţii de Ştiinţe Agricole şi Medicină Veterinară, Cluj-Napoca, în colaborare cu
Universitatea Tehnică Berlin (TU Berlin), Germania, Departamentul de Tehnologie a
Enzimelor, sub supravegherea Prof. Dr. rer. nat. Marion Ansorge-Schumacher. Aş dori de
asemenea să mulţumesc în mod special sponsorilor (Deutsche Bündestiftung Umwelt (DBU)
Germany şi EU COST 865) ce au facut aceste cercetări posibile, acordându-mi cele două
burse pentru studiile doctorale.
IV
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INTRODUCERE
Microîncapsularea este procesul de producere a capsulelor la o scară micrometrică sau
milimetrică, fiind cunoscute sub numele de capsule.
Bioîncapsularea beneficiază de principiile fundamentale ale încapsulării şi implică
învelirea efectivă a unei forme vii într-o membrană care este inertă, non-toxică pentru celulă,
şi stabilă la condiţiile interioare ale reacţiilor biochimice precum agitarea (Muralidhar R.V. et
al., 2001).
Microcapsula este o capsulă mică, iar procedura de preparare a acesteia este numită
microîncapsulare. Aceasta poate încorpora diferite tipuri de forme materiale pentru a
suplimeta funcţiile secundare şi/sau pentru a compensa în diferite condiţii de mediu.
Microcapsulele pot fi clasificate în trei categorii de bază în funcţie de morfologia
acestora: mononuleare, polinucleare sau de tip matrice.
Microcapsulele mononucleare conţin membrana care protejează compusul bioactiv; în
Fig.1. sunt prezentate câteva tipuri de capsule.
Fig. 1. Diferite tipuri de capsule utilizate (Birnbaum D.T. şi Brannon-Peppas L., 2003)
Capsulele polinucleare prezintă mai mulţi compuşi bioactivi încorporaţi în interiorul
unei membrane. Încapsularea de tip matrice conţine cmpusul bioactiv distribuit omogen pe
toată suprafaţa interioară.
Scopul microîncapsulării
În general există numeroase motive pentru care substanţele ar trebui încapsulate (Li S.P. şi
col., 1988; Finch C.A., 1985; Arshady, R., 1993):
•
•
•
•
•
•
•
Creştera stabilităţii pentru protejarea compuşilor activi de mediul extern
Pentru convertirea componenţilor lichizi activi într-un sistem solid uscat
Pentru separarea componenţilor incompatibili din punct de vedere funcţional
Pentru a masca proprietăţile nedorite a componenţilor activi
Pentru a proteja mediul extern al microcapsulelor de componenţi activi
Pentru a controla eliberarea compuşilr activi de procesel de eliberare întârziată sau
eliberarea susţinută
Separarea omponenţilor incompatibili
V
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•
•
•
Conversia lichidelor în solide
Mascarea mirosului, activităţii, etc.
În scop farmaceutic
Tehnologia de încapsulare este foarte bine dezvoltată fiind acceptată în multe industrii
precum: farmaceutică, chimică, cosmetică, alimentară (Augustin et al., 2001; Heinzen, 2002).
În industria alimetară, grăsimile şi uleiurile, compuşii aromatizanţi şi oleorezinele,
vitaminele, mineralele, coloranţii şi enzimele au fost deja încapsulate (Dziezak, 1988; Jackson
şi Lee, 1991; Shahidi şi Han, 1993).
Alegerea unei tehnici adecvate de bioîncapsulare depinde de utilizarea finală a
produsului şi de condiţiile de procesare implicate în obţinerea produsului final.
Bioîncapsularea îşi găseşte aplicaţie din ce mai multă aplicabilitate în domeniul
biotehnologiilor şi în special în alimentaţie şi agricultură. În ultimele decenii, încapsularea
compuşilor activi a devenit un process de mare interes şi însemnătate, fiind adecvat atât
pentru ingredienţii alimentari cât şi pentru cei chimici, farmaceutici sau cosmetici.
Pfutze S. (2003) consideră că tehnologiile de încapsulare pot fi divizate în două
categorii:
•
formarea matricea capsulelor: un ingredient activ şi protector formează granule
omogene. Produsul activ este uniform distribuit în granulă fiind înconjurat din
abundenţă de material protector, formând matricea activă.
•
formarea învelişului capsulelor: materialul activ este granulat şi acoperit de un strat
protector. Materialul activ şi protector este bine separat.
Obiectivul principal este construirea unei bariere între particulele componente şi
mediu. Această barieră reprezintă o protecţie împotriva oxigenului, apei, luminei; evitarea
contactului cu alte particule sau ingrediente; sau controlul eliberării lor în timp. Protecţia
compuşilor bioactivi pe parcursul procesării şi păstrării, precum şi eliberarea controlată în
tractusul gastrointestinal este o prioritate în exploatarea potenţialului benefic al multor
compuşi bioactivi.
Tehnicile utilizate la bioîncapsulare necesită un material drept înveliş şi o substanţă
protejată. Materialul utilizat trebuie aprobat de Administraţia Alimentaţiei şi Farmacie (US)
sau de Autoritatea Europeană pentru Securitatea Alimentelor (Europa) (Amrita şi col., 1999).
Coacervarea: încapsularea lichidelor
Coacervarea complexelor (sau faza de separare), este prima aplicaţie la scară largă a
tehnologiei de microîncapsulare. Coacervarea este un proces care are loc în soluţii coloidale şi
de multe ori privită ca metoda originală de încapsulare (Risch, 1995).
Aplicabilitate coacervării complexelor este enormă dar are şi limite datorită costurilor
ei ridicate, în unele aplicaţii. Aceasta include încapsularea:
aromelor
vitaminelor
cristaleor lichide pentru dispozitivele de display
sisteme de imprimare
VI
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ingredienţi activi pentru industria farmaceutică
bacteri şi celule
Matricile – materiale pentru încapsulare
Există diferite materiale ce pot fi utilizate pentru încapsulare precum: polielectroliţi
sintetici (Sukhorukov şi col., 1998; Donath şi col., 1998), polielectroliţi naturali (Shenoy şi
col., 2003), nanoparticule anorganice (Caruso şi col., 2001), grăsimi (Moya şi col., 2000),
coloranţi (Dai şi col., 2001), ioni polivalenţi (Radtchenk şi col., 2005), şi biomacromolecule
(Yang şi col., 2006).
În general trei clase de materiale au fost utilizate: materiale naturale derivate (colaen ş
alginat), matrici tisulare acelulare (submucoase intestinale) şi polimeri sintetici (acid
poliglicolic, etc.). Aceste clase de biomateriale au foste testate în concordanţă cu
biocomapatibilitatea lor (Pariente şi col., 2002).
Biopolimerii sunt polimeri care provin din surse naturale, sunt biodegradabili, şi
nontoxici. Pot fi produşi de sisteme biologice (ex: microorganisme, plante şi animale), sau
chimic sintetizate din materiale biologice (ex: amidon, grăsimi sau uleiuri, etc.).
Polimeri naturali şi derivaţi ai acestora: polimeri anionici: acid alginic, pectină,
caragenan; polimeri cationici:chitosan, polilizină; polimeri amfipatici: colagen (and gelatină),
chitină; polimeri neutri: dextran, agaroză, pululan.
Guma guar (E412, numită şi guaran) este extrasă din seminţele leguminoaselor din
familia Cyamopsis tetragonoloba. Guma guar prezintă vâscozitate scăzute dar este un bun
agent de întărire. Fiind un polimer non-ionic, nu este influenţat de pH, dar este influenţat de
temperaturi extreme la anumite pH-uri (ex: pH=3 la 50°C).
Alginatul (E400-E404) este produs extras din algele brune (Phaeophyceae, în special
Laminaria). Proprietăţile de gelifiere depind de interacţia cu unii ioni (Mg2+ << Ca2+ < Sr2+ <
Ba2+).
Caragenan (E407) este un nume colectiv atribuit polizaharidelor, obţinute prin
extracţia alcalină din algele roşii (Rhodophycae). Geluri puternice sunt formate de kcaragenan în prezenţa ionilor de K+ şi mai slab în prezenţa ionilor de Li+, Na+, Mg2+, Ca2+, sau
Sr2+.
Guma xantan (E415) este un polimer microbian preparat commercial prin fermentaţia
aerobică din Xanthomonas campestris. Guma xantan nu prezintă proprietăţi ridicate de
gelifiere, este hidratată uşor în apă rece, având aplicaţii ca şi emulgator, stabilizator.
Chitosanul este obţinut la scală industrială din carapacea crustaceelor (Yanga şi col.,
2000). În multe studii chitosanul este legat cu ajutorul aldehidelor, pecum glutaraldehida şi
formaldehida, pentru obţinerea lui sub o forma mai vâscoasă cu aplicaţii ca şi material de
încapsulat.
Mulţi componenţi naturali conţinuţi în uleiurile vegetale prezintă proprietăţi utile.
Uleiul de cânepă rezultă prin presarea seminţelor de cânepă (Cannabis sativa L).
Acidul oelic (Omega 9) conţinut în uleiul de cânepă menţine o bună funcţionalitate arterială.
În exces acidul oleic poate interfera cu acizii graşi esenţiali şi prostaglandinele.
VII
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Uleiul de măsline conţine trigliceroli şi cantităţi mici de acizi graşi liberi, glicerol,
pigmenţi, compuşi aromatizanţi, steroli, tocoferoli, fenoli, componenţi răşinoşi neidentificaţi,
etc.(Kiritsakis A., 1998).
Uleiul de dovleac este foarte sănătos, de caliate superioară, fiind în clasamentul
primelor 3 uleiuri nutritive. Seminţele de dovleac au un gust intens şi sunt bogate în acizi
graşi polinesaturaţi. Uleiul brun are un gust amărui. Conţinutul în tocoferoli ai uleiurilor
oscilează de la 27,1 la 75,1 μg/g de ulei pentru α-tocoferol, de la 74.9 la 492.8 μg/g pentru γtocoferol, şi de la 35.3 la 1109.7 μg/g pentru δ-tocopherol (Stevenson şi col., 2007)
Uleiurile de cătină conţin o cantitate ridicată de acizi esenţiali, linoleic şi alfa linoleic
(Chen şi col., 1990), care sunt precursori ai altor acizi graşi polinesaturaţi cum ar fi acidul
arahidonic sau eicosapentanoic. Este stocat în organitele extracitoplasmatice numite vezicule
de ulei, o formă naturală de încapsulare (Socaciu et all, 2007, 2008). Uleiul din pulpa frutelor
de cătină este bogat în acid palmitoleic şi acid oleic (Chen şi col., 1990).
Uleiurile conţin de asemenea flavonoizi (Chen şi col., 1991), carotenoizi, steroli liberi
şi esterificaţi, triterfenoli şi izoprenoli (Goncharova şi Glushenkova, 1996). Conţinutul în
carotenoizii variază de asemenea în funcţie de sursa de provenienţă a uleiului.
Proprietăţile fizice şi chimice ale uleiurilor funcţionale
Proprietăţile fizice şi chimice ale uleiurilor, incluzând indicele de iod, de saponificare
şi valorile de aciditate şi pentru peroxizi, indicele de refracţie, densitate şi materia
nesaponificabilă sunt determinate conform procedurilor standard. Indicele de iod măsoară
gradul de nesaturare al uleiurilor. Valoarea acestuia sub 100 demonstrază că uleiul prezintă un
grad redus de saturare (Pa Quart, 1979; Pearson, 1981). Indicele de saponificare este un
indicator al mediei masei moleculare a acizilor graşi prezenţi în ulei (AOAC, 1980; Pearson,
1981). Indicele de peroxid este frecvent utilizat pentru măsurarea stadiului de oxidare al
uleiului. Acesta indică oscilarea oxidativă a uleiului (deMan, 1992).
Tehnicile pentru caracterizarea şi autentificarea uleiurilor funcţionale
Există diferite tehnici pentru caracterizarea şi autentificare produselor alimetare.
Metodele de autentificare aplicate pentru uleiuri şi grăsimi pot fi clasificate ca şi chimice (de
separative) sau fizice (non-separative).
Spectrometrele de infraroşu cu transformantă fourier (FTIR) prezintă multe avantaje în
comparaţie cu instrumentele convenţionale de dispersie, printr-o excelentă reproductibilitate
şi acurateţe a lungimilor de undă, precisa manipulare spectrală şi utilizarea unor programe
chemometrice pentru calibrare. Accesoriile HATR au fost de asemenea larg utilizate în
dezvoltarea metodelor FTIR pentru analizarea uleiurilor şi a grăsimilor, deoarece acestea pot
oferi mijloace convenable şi simple pentru o manipulare uşoară (Sedman şi col., 1999).
Spectroscopia infraroşu de mijloc (MIR) poate fi utilizată pentru identificarea compuşilor
organici deoarece unele grupe de atomi prezintă proprietăţi ale frecvenţei de absorbţie a
vibraţiilor în regiunea infraroşie a spectrului electromagnetic. Reflectanţa orizontală totală
atenuată (HATR) este accesoriul cel mai des utilizat in metoda FTIR pentru analizele
uleiurilor şi a grăsimilor (Sedman şi col., 1999).
VIII
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O largă varietate de alimente utilizează pentru încapsulare aromatizanţi, acizi, baze,
îndulcitori artificiali, coloranţi, antioxidanţi, agenţi cu arome nedorite, mirosuri, etc. Aceştia
îşi păstreză bioactivitatea şi rămân accesibili agenţilor externi.
Fitosterolii, flavonoizii şi compusii organici cu sulf, reprezintă trei grupe de compuşi
caracteristici fructelor şi legumelor, care ar putea prezenta importanţă în reducerea riscului de
ateroscleorză. (Howard şi Kritchevsky, 1997). Unele substanţe fitochimice, cu ar fi acidul
ascorbic, carotenoizii, vitamina E, fitofenoli, izoflavoni şi fitosteroli, au fost evidenţiate ca
ingredienţi fiziologic activi ce îmbunătăţesc rezistenţa la anumite boli.
Încapsularea poate fi utilizată pentru condiţionarea uleiurilor în forme solide sau
solubile în apă, extinzând utilizarea lor în multe alte aplicaţii. Încapsularea uleiurilor include
ca metode şi tehnici: spray-drying, spray-chilling, fluid bed encapsulation, extrusion
encapsulation şi încapsularea prin coacervare.
Extrudarea este utilizată pentru încapsularea mineralelor şi vitaminelor în uleiuri
(grăsimi saturate) într-o matrice de tip polizaharidic (Van Lengerich şi Lakis, 2002). Protecţia
împotriva oxidării metil-linoleatului încapsulat cu gumă acacia prin metoda spray drying şi
freeze drying, depinde de umiditatea relativă a mediului (Minemoto şi col., 1997).
În majoritatea cazurilor matricile utilizate pentru încapsularea uleiurilor şi grăsimilor
sunt gume (acacia, arabică), proteine, carbohidraţi (cazeină/zaharuri), maltodextrină, betaciclodextrine, alginat de sodiu, gelatină.
PARTEA II. CONTRIBUŢII PROPRII (ORIGINALE)
CAPITOL II. CARACTERIZAREA ULEIURILOR FUNCŢIONALE UTILIZATE LA
BIOÎNCAPSULARE
Uleiurile extrase din plante (floarea-soarelui, dovleac, soia, rapita, etc.) sunt foarte
utilizate in domeniul alimentar, dar si in alte industrii (cosmetică, farmaceutică, etc.).
PrezintĂ o deosebita insemnatate datorita numerosilor componenţi benefici care intră în
alcătuirea lor. Calitatea şi autenticitatea acestor uleiuri se realizează prin diferite tehnici. Cele
mai utilizate trei tehnici în vederea caracterizării acestor uleiuri sunt: spectrometriA UV-Vis,
cromatografia gaz cu detecţie prin ionizare în flacără FID, şi spectroscopia infraroşu cu
transformantă fourier (FTIR).
II.1. MATERIALE ŞI METODE
Au fost alese în vederea încapsulării patru uleiuri de mare interes: cătină (SBO) extras
din fructele de catina, colectate din regiunea Clujului (Transilvania, nordul Romaniei), ulei de
măsline extra virgin (EVO) din Italia, cânepă (HP) şi dovleac (PK) din Romania.
Analizele chimice au fost determinate conform metodelor descrise de: A. O. A. C. şi
IOOC sau de Comisia Uniunii Europene (EU): aciditatea şi indicele de iod. Toate probele au
fost analizate în triplicat. Aciditatea a fost calculată luându-se în considerare conţinutul de
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acizi graşi liberi ale uleiurilor analizate, determinat prin titrare conform metodei oficiale Ca
5a-40. Indicele de iod a fost determinat prin metoda AOCS Cd 1c-85 (1997).
II.2. REZULTATE ŞI DISCUŢII
Determinarea acidităţii şi a indicelui de iod
Rezultatele analizelor chimice prezentate în Tabelul II.1. au demonstrat o bună
corelaţie a valorilor obţinute cu cele publicate în literatură.
Tabel II.1. Caracteristicile chimice şi fizice ale uleiurilor analizate în comparaţie cu literatura
Ulei de Cânepa
Ulei de măsline
extra virgin
Ulei de dovleac
Ulei de cătină
Caracteristicile fizice si chimice
Aciditate
1.93 / 4.0*
2.64 / 6.6*
1.32 / 4.0*
3.7 / 4.0*
(mg KOH/g ulei)
Indicele de iod**
162 / 145-166*
87 / 75-95*
130 / 116-133*
71 / 98-119*
**Indicele de Iod a fost calculat cu metoda AOCS Cd 1c-8
* Date din literatura
Aceste date demonstrează faptul că valorile acestor uleiuri corespund cu indicii de
calitate ai iodului din Codex, excepţie uleiul de cătină, a cărui valori în cazul acidităţii nu
corespund intervalelor acidităţii precizate în literatură.
Determinarea amprentei uleiurilor prin spectrometrie ultra violet/visibil (UV-Vis)
Caracterizarea spectrală (fingerprintul) specifică fiecărui ulei analizat prin UV-Vis
este prezentat in Fig. I.1. Diferenţele dintre un ulei autentic si un ulei falsificat a fost
demonstrate prin poziţia şi absorbanţa peakurilor caracteristice fiecărui ulei (Socaciu C. et al.,
2005).
Ulei de cânepă (Cannabis sativa L)
Amprenta spectrală UV-Vis caracteristică uleiului de cânepă în conformitate cu datele
precizate de OMLC, este dat de conţinutul ridicat în pigmenţi clorofilici, având absorbanţa
maximă la 411 nm (Fig.II.1.A.).
X
________________________________________________________________________________________
A.
C.
B
D.
Fig.II.1. Spectrele UV-Vis ale uleiurilor analizate (amprenta specifică în regiunea 350-600 nm
continând detalii referitoare la maximul absorbantei peakului specific: A. Ulei de cânepă; B.
Ulei de măsline extra virgin (EVO); C. Ulei de dovleac (PK); D. Ulei de cătină (SB)
Ulei de măsline extra virgin (Olea europaea )
Culoarea caracteristică uleiului de măsline depinde de majoritatea pigmenţilor
conţinuţi, în principiu acest ulei având un conţinut ridicat în carotenoide şi clorofile. Uleiul
provenit din măslinele ajunse la maturitate prezintă o culoare galbenă datorită continutului în
pigmenţi carotenoidici galbeni. În general culoarea acestui ulei variază şi este datorată
combinaţiei şi diferetelor proporţii de pigmenţi. Exista o simplă ecuaţie: Culoarea= clorofilă
(verde) + carotenoide (galben) + alţi pigmenţi.
Conţinutul în pigmenţi clorofilici se diminueaza odată cu atingerea maturităţii
fructelor. Fingărprintul specific uleiului de măsline analizat este atribuit ‘’ecuaţiei culorii’’
menţionată anterior (Fig.II.1.D.).
Ulei de dovleac (Cucurbita pepo)
Amprenta spectrală (fingerprintul) a acestui ulei este acceptat ca avand doua umere,
unul la 418 nm cu absorbanţă mai mică, şi unul la 435 nm cu absorbanţă mai mare
XI
________________________________________________________________________________________
(Fig.II.1.C.), în cazul falsificării sau oxidării, acest ulei prezintă absorbanţele celor două
umere schimbate (Lankmayr şi col., 2004).
Uleiul de cătină (Hippophae rhamnoides)
Spectrului acestui ulei demonstrează ca fingărprintul este dat de cele trei umere care
dau spectrul, care sunt caracteristice carotenoidelor, mai exact beta-carotenului, intre 400 şi
500 nm (Fig.II.1.A.), acesta fiind compusul principal al acestui ulei (Lichtenthaler şi
Buschmann, 2001).
Analiza uleiurilor prin spectroscopia infrarosu cu furie transformata (FTIR)
Studiile FTIR ale uleiurilor analizate au demonstrate existenta relatiei intre fecventele
si absorbantele benzilor specifice si compozitia acestora. Aceste frecvenţe şi valoarea
absorbanţei lor, au fost utilizate în continuare pentru evaluarea oxidarii uleiurilor (Guillen, M.
D. şi Cabo, N, 1997, 1998, 1999, 2000, 2002).
În conformitate cu aceste spectre au fost identificate principalele benzi şi frecvente în
domeniul infraroşu ale uleiurilor analizate ( Tabel II.2.).
Tabel.II.2. Benzile infraroşu relevante ale uleiurilor investigate
Nr.
banda
HP
EOV
PK
SB
(cm-1)
(cm-1)
(cm-1)
(cm-1)
Grupul functional
Modul de vibratie
1
3008
3005
3008
3006
=C-H (cis-)
de întindere
2
2956
2956
2956
2956
-C-H (CH3)
de întindere (asimetrică)
3
2923
2923
2923
2922
-C-H (CH2)
4
2853
2853
2854
2853
-C-H (CH2)
5
1742
1742
1742
1742
-C=O (ester)
de întindere
6
1654
1653
1653
1653
-C=C- (cis-)
de întindere
7
1463
1464
1464
1464
-C-H (CH2, CH3)
8
1456
1456
9
1418
1417
1418
1417
10
1396
1402
1398
1402
11
1377
1377
1377
1377
1317
1319
1238
1238
12
13
1236
1456
=C-H (cis-)
de deformare
de deformare
-C-H (CH3)
de deformare (simetrică)
de deformare
1238
-C-O, -CH2-
de întindere, de deformare
XII
________________________________________________________________________________________
14
1155
1159
1157
1161
-C-O, -CH2-
15
1120
1118
1120
1116
-C-O
de întindere
16
1097
1097
1099
1095
-C-O
de întindere
17
1028
1028
1029
1033
-C-O
de întindere
958
962
968
-HC=CH- (trans-)
de deformare înafara
planului
-HC=CH- (cis-)
de deformare înafara
planului
18
19
914
20
721
914
721
721
721
-(CH2)n-, -HC=CH(cis-)
de întindere, de deformare
de deformare ( rocking)
Spectrele uleiurilor analizate par a fi in principiu similare, însă diferenţele în
intensitatea benzilor ca de altfel şi a frecvenţelor fac posibilă diferenţierea foarte clară a
compoziţiei acestor uleiuri (see Fig. II.2.).
Fig.II.2. Spectrul FTIR-ATR al zonei de fingerprint (1700-800 cm-1) a uleiurilor analizate
HP= cânepă, EVO (EOV) = măsline extra virgin; PK= dovleac; SB= cătină
XIII
________________________________________________________________________________________
Profilul acizilor graşi prin cromatografia gaz
Compoziţia acizilor grasi analizaţi prin GC-FID in acest studio sunt evidentiati in
Tabelul II.3. Profilul acizilor graşi a fost comparat cu al uleiurilor din literature.
Tabel.II.3. Compoziţia procentuală a acilor graşi din uleiurile analizate
Acizi grasi %
Ulei de Canepa
Ulei de masline
extra virgin
Ulei de dovleac
Ulei de catină
Palmitic (16:0)
7.48
7.28
6.29
7.76
Stearic (18:0)
1.66
2.67
3.64
0.3
Arachidic (20:0)
1.06
-
-
0.11
Σ saturati %
10.02
9.95
9.93
8.17
-
-
-
5.4
14.94
36.81
42.44
6.3
72.6
43.14
46.71
-
-
0.93
0.92
0.8
Eicosadienoic
(C20:2)
0.55
-
-
-
Σ nesaturati %
87.54
80.88
90.07
12.5
C18:1/C18:2
0.21
0.85
0.91
6.3
-
0.022
0.02
-
Palmitoleic
(C16:1)
Oleic
(C18:1)
Linoleic
(C18:2)
Linolenic
(18:3n3)
omega 3 : omega
6 acizi grasi
II.3. CONCLUZII
Prin GC-FID, s-a determinat compoziţia în acizi graşi a uleiurilor analizate şi s-a facut
comparaţia cu datele din literatură. În urma acestei analize s-au concluzionat urmatoarele:
•
•
compoziţia uleiului de cânepă nu corespunde cu valorile precizate în literatură pentru
acizii graşi, acesta avand un conţinut mai scăzut. Acidul oleic se incadrează in
intervalul prevăzut in literatură
principalii acizi graşi în uleiul de măsline extra virgin sunt acidul oleic şi linoleic, şi de
asemenea în cantitate mai mică acidul lonoleic
XIV
________________________________________________________________________________________
•
•
în cazul uleiului de dovleac, compoziţia în acizi graşi corespunde cu valorile precizate
în litaratura, excepţie facând acizii palmitic şi stearic care sunt prezenţi in concentrţii
mai mici
profilul acizilor graşi a uleiului de cătină a demonstrat faptul că acest ulei provine din
pulpa/pielea fructelor şi nu din seminţe, fiind foarte bogat in acidul palmitic şi oleic
CAPITOLUL III. BIOÎNCAPSULAREA ULEIURILOR:
PREPARE A CAPSULELOR ŞI CARACTERIZAREA LOR
PROTOCOALE
DE
III.1. MATERIALE ŞI METODE
În vederea realizării parţii experimentale din acest capitol s-au utilizat urmatoarele:
•
•
•
matrici pentru încapsulare: alginat, k-caragenan, chitosan, gumă xantan şi
gumă guar, procurate de la Sigma Aldrich
solvenţii si reactanţii necesari de asemenea de la Sigma Aldrich
uleiurile utilizate la încapsulare au fost prezentate in capitolul anterior
Protocol pentru sintetizarea capsulelor goale de diferite mărimi şi concentraţii
Diferite concentratii de alginat (1%, 1.5%, 2% w/v), amestec de: alginat si caragenan,
alginat si guma xantha, alginat si guma guar au fost dizolvate in apa deionizata pentru ~ 30
minute. Diferite concentratii de chitosan (1%, 1.5%, 2% w/v) au fost dizolvate 0.7% v/v acid
acetic glacial.
Alginatul şi amestecul de alginat au fost pipetate într-o solutie de 2% CaCl2 în apa (ca şi
baie de întărire), utilizând o pompa peristaltica cu un injector de 0.4 x 20mm, iar capsulele au
fost formate instantaneu.
Chitosanul a fost pipetat in 5% (w/v) solutie de NaTPP in apa (ca si baie de intarire),
utilizând pipeta pentru control.
Dupa ~ 1h, capsulele au fost separate din baia de intarire şi transferate în placi “Petri”
pentru protecţie si conservare.
Protocol pentru sintetizarea capsulelor de diferite mărimi şi concentraţii cu uleiri
încorporate
S-au luat în considerare doar concentraţiile de matrici care au prezentat emulsiile cele mai
stabile. De asemenea vâscozitatea soluţiilor a fost considerat un factor principal în vederea
alegerii concentraţiilor de matrici. Protocolul pentru obţinerea capsulelor a fost descris
anterior.
XV
________________________________________________________________________________________
Evaluarea microscopica a emulsiilor înaintea încapsulării
Evaluarea microscopică a emulsiilor înaintea încapsulării a fost determinată utilizând
un microscop Olimpus optical microscope BX51M, echipat cu camera digitală.
Caracterizarea capsulelor: dimensiuni şi morfologie, analize FTIR şi termice
Parametri luaţi in considerare în vederea caracterizării capsulelor, precum dimensiune,
arie, perimetru, elongaţie si compactitate, au fost determinaţi utilizând UTHSCSA ImageTool
ca şi software.
Morfologia suprafeţei capsulelor a fost determinată utilizându-se microspia electronică
scanată (Hitachi S-2700, iMOXS, cu detector BSE). Capsulele analizate au fost suflate şi
învelite în aur înaintea supunerii analizelor microscopice.
III.2. REZULTATE ŞI DISCUŢII
Evaluarea microscopica a emulsiilor înaintea încapsulării
Stabilitatea emulsiilor este un factor cheie în evaluarea în condiţii de temperatura în
vederea păstrării timp mai îndelungat a produselor pe baze de emulsii.
Mărimea picăturilor de ulei dispersate in structura matricilor dizolvate care au fost
comparate in vederea evaluarii stabilitatii emulsiilor obtinute. Emuliile cu cea mai bună
stabilitate în timp au fost utilizate mai departe pentru încapsulare. Mărimea picăturilor de
uleiuri au fost dispersate uniform in matrici, în funcţie de stabilitatea matricei, uniformitatea
crescând odată creşterea concentraţiei matricilor (Fig.III.1.).
Caracterizarea capsulelor
Dupa obtinerea emulsiilor, si pipetarea lor in baile de intarire, datorita interactiilor cu
ionii de legare in vederea formarii gelurilor.
Capsulele continand uleiuri au avut o forma aproximativ sferica, culoare variind intre
alb-galbui si portocaliu.
Luându-se in considerare toate caracteristicile capsulelor obtinute, si facand o
comparatie intre aceste caracteristici ale capsulelor goale şi a celor continand uleiuri, s-a
constatat ca incorporarea uleiurilor în capsule modifica aceste caracteristici (Fig.III.2.).
Compararea capsulelor între ele continand uleiuri, a demonstrat ca sfericitatea şi
compactitatea nu au fost prea mult afectate de incorporarea uleiurilor. Insa in cazul
diametrului, ariei, elongatia, au fost clar determinate diferente foarte mari.
XVI
________________________________________________________________________________________
A.
B.
C.
D.
Parameter values/Valoarea parametrilor
A
G
C
A
G
A
-C R
(
A
R 0.5
A
(0 :0
G
-X .75 .5)
:0
G
.7
(0
5)
.7
A
G
5
:
0
X
A
.7
G G
5)
-G (0
.5
G
:
0
(0
A
.7 .5)
G
-G 5:0
.7
G
5)
(0
.5
:0
.5
A
)
G
oi
A
G l2%
oi
l1
.5
%
A
G
oi
l1
%
C
H
oi
l
C
H 2%
oi
l1
.5
%
C
H
oi
l1
%
Fig. III.1. Imagini microscopice ale diferitelor emulsii: A. alginat 2%; B. alginat 1%; C. complex alginat-gumă
guar; D. complex alginat-gumă xantan. Scala reprezintă 5 μm.
9
8
7
Area / Aria (cm2)
6
5
4
3
2
1
Perimeter / Perimetru
Elongation (axes ratio)/
Elongatia (raportul axelor)
Roundness (up to 1) /
Sfericitatea val. max. 1
Diameter / Diametrul (cm)
0
Compactness (up to 1)/
Compactitatea (val. max. 1)
Samples/Probele
Fig.III.2. Reprezentarea grafică comparată a caracteristicilor capsulelor din complexul alginat cu k-caragenan,
gume xantan şi guar, alginat şi chitosan continând ulei: AG-CAR (0.5:0.5) = complex alginat-k-carrageenan
(raport 0.5:0.5) continând ulei; : AG-CAR (0.75:0.75) = complex alginat-k-carrageenan (raport 0.75:0.75)
continând ulei; AG-XG (0.75:0.75) = complex alginate-guma xantan (raport 0.75:0.75) continând ulei; AG-XG
(0.5:0.5) = complex alginate-guma xantan (raport 0.5:0.5) continând ulei;AG -GG (0.75:0.75) = complex
alginate-guma guar (raport 0.75:0.75) continând ulei; AG -GG (0.5:0.5) = complex alginate-guma guar (raport
0.5:0.5) continând ulei; AGoil2% = capsule alginat 2% continând ulei; AGoil1.5% = capsule alginat 1.5%
continând ulei; AGoil1% = capsule alginat 1% continând ulei; ; CHoil2% = capsule chitosan 2% continând
ulei; CHoil1.5% = capsule chitosan 1.5% continând ulei; CHoil1% = capsule chitosan 1% continând ulei
XVII
________________________________________________________________________________________
Microscopia electronică scanată (SEM)
Scopul acestor analize a fost de a evalua si caracteriza topologia capsulelor obtinute
continand uleiuri. Suprafata capsulelor a fost non-regulara, aceasta datorita picaturilor de ulei
prezente, exceptand chitosanul care prezinta o suprafata mult mai mata (Fig.III.3.A).
Fotografiile SEM ale capsulelor nu prezintă porozitate (Fig.III.3.).
B.
A.
Fig.III.3. Morfologia suprafetei diferitelor capsule obtinute continand uleiuri utilizand microscopia electronica
de scanare: A. alginat-caragnan complex; B. chitosan. Bara indicand scala este reprezentata in fiecare poza.
Magnificatia 70x.
Analizele FTIR
Caracterizarea FTIR a matricilor
În urma analizelor FTIR-ATR s-a realizat caracterizarea matricilor utilizate la
încapsulare, realizându-se astfel o comparaţie între matrici (AG, CAR, CH, GG, XG).
Principalele frecvenţe caracteristice matricilor în vederea identificării individuale sunt: 32443302 cm-1 (O-H stretch), 1400-1474 cm-1 (CH2 bending), 1000-1200 cm-1 (C-O şi C-C
stretch), 924-1000 cm-1 ( poly OH şi CH2 twist), 776-892 cm-1(glycoside).
Vibraţiile şi grupurile
funcţionale
AG
CAR
GG
XG
CH
O–H intindere
3244
3514
3299
3302
3289
grupul poliOH
O-H +
N-H de
întindere
XVIII
________________________________________________________________________________________
C–H întinderea grupului CH2
2926
2953, 2911, 2894
2884
-
2935
C-O de întindere ( COOH)
1597
-
1636
-
1651
deformarea grupării CH2
1408
1474, 1400
1408
1400
1428
O-H deformare
-
1223 ( S=O vibraţia
de întindere a sulfet
esterului)
1350
1247
-
C-O şi C-C întindere
1200-1000
-
1145
1150
1151
–CH2OH modul de întindere
1054
1063
1054
Gruparea C–OH alcolică
1024
1024
-
1025
1024
948, 902,
924, 910
1016
-
-
Provenite de la
acizii: guluronic
şi maluronic
Grupările
polihidroxi
809
842
866,777
785
892,
1061
(C-O întinderea zaharidelor)
–CH2 vibraţie
legaturile glicozidice
Sulfatul galactozic, (1,4; 1,6) legatura
C-H de
legatura glicozidică galactozei şi
deformare
manozei
C-C întindere
776
FTIR characterization of different beads containing oils
Spectrele matricelor, ale capsulelor goale, capsulelor continand uleiuri au fost
analizate. Concentratia matricelor nu a influentat caracteristicile capsulelor prin FTIR. Un
exemplu concludent este reprezentat in Fig.III.4., spectrele uleiului de cătină (SB) şi ale
capsulelor din alginat 2% conţinand ulei SB.
În urma încapsulării uleiului de SB în capsule de alginate, se produce o creştere a
intensităţii absorbanţei la 3400 cm-1 (care este direct proporţională cu creşterea concentraţiei
de alginate utilizată la încapsulare) precum şi o shiftare a unor peakuri spre valori şi frecvenţe
mai scăzute în regiunea 1000-1500 cm-1, regiune specifică uleiului de SB
Spectrele amestecului de uleiuri si capsule au demonstrat prezenta peakurilor specifice
uleiurilor in doua zone distuncte (2800-2900 cm-1 şi 1700-900 cm-1), confirmându-se astfel
prezenţa uleiurilor în capsule.
XIX
________________________________________________________________________________________
Fig.III.4. Spectru FTIR-ATR înregistrat pentru: A. Capsule de alginat 2% conţinând SB; B. pudră alginat; C.
ulei SB; D. capsule goale de alginat 2%
Analize termice
Analize DSC
Termogramele DSC ale uleiurilor libere si ale diferitelor capsulelor continand uleiuri,
au fost masurate.
Cateva dintre peakurile endotermice, ca si exemplu ale uleiului de catina, si ale unor
capsule continand ulei de catina, sunt prezentate in reprezentarea grafica din Fig.III.5.;
temperature peakurilor cerste direct proportional cu cresterea temperaturii, fiecare peak fiind
characteristic fiecărui tip de capsula obţinută.
Analize termogravimetrice
Termogramele TGA ale uleiurilor libere si ale diferitelor capsulelor continand uleiuri,
au fost masurate.
Cateva rezultate referitoare la pierderea in masa a diferitelor tipuri de capsule
obtinute, este prezentata in graficul din Fig.III.6.. Pierderea în greutate, reprezentata in figura
anterioara, demonstreaza ca aceasta se datoreaza continutului ridicat in apa a unor capsule.
Uleiurile nu influenteaza foarte mult pierderea in greutate, la un ulei liber aceasta fiind de
99.44%. Dar in timpul procesului de oxidare aceste uleiuri pierd din greutate, datorita
reactiilor oxidarii.
XX
________________________________________________________________________________________
200
180
160
Temperature (°C)
140
120
100
80
60
40
20
0
AG 2%
AG
1.5%
Alginate AG-CAR
1%
(0.75%)
CH 2%
CH 1%
AG-GG
AG-KG
SB
Fig. III.5. Reprezentarea grafica a peakurilor endotermice ale unor tipuri de capsule
DSC si TGA au fost in ultimul timp foarte mult utilizate in monitorizarea stabilitatii, a
comportamentului termic, a parametrilor de cinetica in diferite uleiuri (Jayadas et al., 2006;
Milovanovic et al., 2006; Bahruddin et al., 2008). În acest studiu analizele termice au fost
efectuate pana la temperatura de 300°C. Diferenţele nu foarte mari între probe se datoreaza
tocmai acestei temperature, deoarece conform cu literatura, oxidarea uleiuriloe prin metode
termice se poate determina la expunerea probelor la o temperatura mai mare de 300°C, iar
pierderea in greutate poate fi pana la 10%, aceasta depinzând de natura uleiului (Jayadas et
al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008).
120
Restmass %
100
80
60
40
20
0
AG 2%
AG 1.5%
AG-CAR
(0.75%)
AG-GG
AG-KG
SB
Fig.III.6. Reprezentarea grafică a pierderii de masă % a probelor analizate TGA
Scopul acestor analize termice a fost acela de a analiza şi a determina stabilitatea
termică a capsulelor obţinute continând diferite uleiuri, în vederea viitoarelor aplicaţii ale
acestora în domeniul alimentar şi cosmetic. În astfel de aplicaţii se cunoaşte necesitatea
XXI
________________________________________________________________________________________
sterilizării probelor sau expunerea la presiuni înalte în vederea evitării biohazardului sau
contaminării, aceste tratamente fiind facute în timpul proceselor tehnologice.
III.3. CONCLUZII
Studiile experimentale realizate, cu scopul bioîncapsulării unor uleiuri funcţionale în
matrici naturale, utilizând ca şi metodă ‘’ionotropically crosslinked gelation’’, au demonstrate
posibilitatea utilizării acestei tehnici în vederea bioîncapsulării unor compuşi naturali şi
eliberarea lor condiţionată.
Cele mai bune matrici în vederea bioîncapsulării uleiurilor s-au dovedit a fi: alginatul
şi chitosanul în concentrţii de 2%, 1.5% şi 1%, complexele alginatului cu k-caragenan, şi
gume guar şi xantan în raport de concentraţie 0.75:0.75.
Rezultatele au arătat faptul că bioîncapsularea uleiului a afectat diametrul capsulelor,
acesta crescând direct proporţional cu cantitatea de ulei utilizată pentru încapsulare. De
asemenea şi ceilalţi parametri masuraţi în vederea caracterizării capsulelor au fost influenţaţi
şi de cantitatea de ulei utilizată.
Prin analizele FTIR-ATR, diferitele capsule conţinând uleiuri au prezentat peakuri
care sunt atribuite atat uleiurilor cât şi capsulelor goale (regiunile dintre 2800-2900 cm-1 şi
1700-900 cm-1). Astfel se demonstrează prezenţa uleiurilor în capsule, deci încapsularea
acestora.
CAPITOL IV. EFICIENŢA ÎNCAPSULĂRII ŞI STUDII DE ELIBERARE A
ULEIURILOR DIN CAPSULE
IV.1. MATERIALE ŞI METODE
Eficinţa încapsulării e uleiurilor în capsule
Încapsularea uleiurilor a fost determinată calculând cantitatea de beta-caroten sau
cantitatea de carotenoid care este principalul component major al fiecărui ulei analizat înainte
şi după încapsulare. Această determinare a fost realizată spectrofotometric, iar eficinţa
încapsulării (EE%) a fost calculată conform ecuaţiei:
EE% = C1/C2 x 100,
C1= concentraţia carotenoid din ulei iniţială
C2= concentraţia carotenoid din ulei după încapsulare
XXII
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Măsurarea ratei de eliberare a uleiurilor din capsule
Eliberarea controlată a uleiurilor din capsule a fost determinata de asemenea
spectrofotometric, utilizând un spectrofotometru CarWin 50 UV-VIS. Măsurătorile au fost
facute în triplicat la temperatura camerei, utilizându-se cuvete de cuarţ de 2 mm.
Eliberarea in vitro a uleiurilor din capsule
Stimularea fluidului gastric a fost realizată conform urmatorul protocol:
•
timp de o ora la pH 1.2 intr-o solutie de 0.1N HCl cu⁄si 5 ml Sanzyme (sirop de
enzime continand 80 mg papaina, 40 mg pepsina and 10 mg sanzyme 2000)
•
in urmatoarele 2-3 ore capsulele au fost transferate in intr-o solutie in vederea
stimulatii fluidului intestinal pH 4.5 tot asa cu⁄si fara enzyme
•
urmatoare ore au fost transferate in solutie stimuland fluidul intestinal la pH 7.4,
aceasta fiind formata din KH2PO4 1.074g in 30 ml de 0.2N NaOH, si pancreatina 275
mg (utilizand “Triferment”)
•
toate testările s-au efectuat la 37ºC cu barbotare continuă de CO2
IV.2. REZULATTE ŞI DISCUŢII
Eficinţa încapsulării e uleiurilor în capsule
Eficienţa încapsulării este reprezentata în graficul din Fig.IV.1. pentru diferite tipuri de
capsule. Valorile prezentate sunt ale uleiului de cătină, însa pentru toata uleiurile analizate
aceasta eficienţă a prezentat aceleaşi valori.
După cum se poate observa şi în graficul prezentat, eficienţa încapsulării este direct
proporţionala cu creşterea concentraţiei matricilor. Dintre toate matricile şi complexele de
matrici utilizate, după cum se poate observa şi in graficul din Fig.IV.1., s-a obţinut cea mai
bună eficienţa a încapsulării utilizând ca şi matrici: alginatul în concentraţie de 2%, chitosanul
in aceeaşi concentraţie, urmate de concentraţiile de 1.5%, şi de alginatul în complex cu kcaragenan şi gume în raport de 0.75:0.75%.
XXIII
________________________________________________________________________________________
Fig.IV.1. Reprezentarea grafica comparata a eficientei incapsularii a uleiurilor in capsule din complexul alginat
cu k-caragenan, gume xantan si guar, alginat si chitosan : AG2% = capsule alginat 2% ; CH2% = capsule
chitosan 2% ; CH1.5% = capsule chitosan 1.5% ; AG1.5% = capsule alginat 1.5% ; AG-CAR (0.75:0.75) =
complex alginat-k-carrageenan (raport 0.75:0.75) ; AG-XG (0.75:0.75) = complex alginate-guma xantan (raport
0.75:0.75); CH1% = capsule chitosan 1%; AG -GG (0.75:0.75) = complex alginate-guma guar (raport
0.75:0.75) ; AG1% = capsule alginat 1% ; AG -CAR (0.5:0.5) = complex alginat-k-carrageenan (raport
0.5:0.5); AG -GG (0.5:0.5) = complex alginate-guma guar (raport 0.5:0.5); AG-XG (0.5:0.5) = complex
alginate-guma xantan (raport 0.5:0.5)
Măsurarea ratei de eliberare a uleiurilor din capsule
Ca şi sisteme de eliberare s-au luat in considerare 3 solvenţi: tetrahidrofuran (THF),
metanol si hexan. În toate cazuri s-a evidentiat o rapida eliberare in THF ca si solvent din
toate capsule obtinute, si o mai lenta eliberare in cazul metanolului si o foarte slaba in cazul
hexanului (vezi Fig.IV.2., graficele fiind parte din lucrarea publicata in revista Chemické
Listy Journal (IF=0.683)). THF este considerat şi în litaratura ca fiind solventul cel mai
eficient în extracţia carotenoidelor, lucru dovedit şi în acest studiu. Rata de eliberare a
uleiurilor din capsule depinde de difuzitatea şi solubilitatea uleiului în matrice.
Eliberarea uleiurilor din capsule a demonstrat faptul ca alginatul, complexul dintre
alginat cu k-caragenan şi gume, precum şi chitosanul, sunt matrici pretabile la încapsularea
uleiurilor vegetale.
XXIV
________________________________________________________________________________________
0.4
Absorbance (u.a.)
0.35
Alginate-carrageenan
complex in hexane
0.3
0.25
Alginate-carrageenan
complex in methanol
Alginate 2% in methanol
0.2
0.15
Alginate 2% in hexane
0.1
0.05
0
0
100
200
300
400
Wavelenght (nm)
A.
0.7
Absorbance (a.u.)
0.6
Chitosan 1.5% in methanol
0.5
Chitosan 1% in methanol
0.4
Chitosan 1% in hexane
Chitosan 1.5% in hexane
0.3
Alginate 2% in methanol
0.2
Alginate 2% in hexane
0.1
0
0
100
200
300
400
Wavelenght (nm)
B.
Fig.IV.2. Reprezentarea grafica a eliberarii in timp din diferitele capsule in metanol si hexan, ca si solventi
Eliberarea in vitro a uleiurilor din capsule
În cazul mimării mediului digestiv s-a demosntrat stabilitatea capsulelor la pH 1.2 si pH
4.5, dizolvarea acestora si eliberarea continutului realizandu-se la pH 7.4, atat in cazul
solutiilor continand enzime cat si a celor fara continut enzimatic în cazul capsulelor din
alginat şi alginat în complex cu k-caragenan şi gume (Fig.IV.3.), ceea ce demonstreaza
aplicabilitatea viitoare a acestor capsule continand ulei de catina ca si nutraceutice sau in
industria farmaceutica. Capsulele de chitosan nu s-au dizolvat la nici unul dintre pH-urile
testate.
XXV
________________________________________________________________________________________
A.
B.
C.
D.
Fig.IV.3. Eliberarea ”in vitro” a uleiului de catina din capsulele de alginate 2% de la stanga la dreapta in fiecare
poza fluidele stimulatoare fara enzyme si cu adios de enzyme (Sanzyme): A. capsulele obtinute; B. dupa 1 ora in
stimulatul fluid gastric la pH 1.2; C. dupa 3 ore in mixul dintre fluidul gastric si fluidul intestinal la pH 4.5; D. in
stimulatul fluid intestinal pH 7.4 dupa 30 minute
IV.3. CONCLUZII
Studiile referitoare la eficienţa încapsulării şi stabilitatea capsulelor conţinând uleiuri au
demonstrat:
1. Creşterea concentraţiei matricilor sau a complexului de matrici determină obţinerea
unei mai bune eficienţe la încapsulare. s-a obţinut cea mai bună eficienţa a încapsulării
utilizând ca şi matrici: alginatul în concentraţie de 2%, chitosanul in aceeaşi
concentraţie, urmate de concentraţiile de 1.5%, şi de alginatul în complex cu kcaragenan şi gume în raport de 0.75:0.75%.
2. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea şi solubilitatea uleiului
în matrice. Eliberarea a fost mai lentă in cazul hexanului, mai ridicată în cazul
metanolului şi cea mai buna eliberare fiind in THF, indiferent de matricea sau
concentraţia utilizată la încapsulare.
3. Stabilitatea capsulelor la pH 1.2 si pH 4.5, dizolvarea acestora şi eliberarea
continutului realizandu-se la pH 7.4.
XXVI
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CAPITOL V. CARACTERIZAREA FTIR A OXIDĂRII ULEIURILOR
V.1. MATERIALE ŞI METODE
Analiza spectrala in domeniul IR s-a utilizat spectrofotometru FT-IR Shimadzu
Prestige-21, echipat cu Reflectanta Totala Atenuata Orizontala (HATR), cu accesoriu de
ZnSe. Masuratorile au fost efectuate in domeniul infrarosu 650-4000 cm-1, 100 scanari fiecare
proba la rezolutia 2 cm-1. Dupa fiecare proba accesoriul a fost spalat cu acetona.
Uleiurile libere si capsulele cu uleiuri au fost supuse in vederea oxidarii la temperatura
de 105ºC, si la iradiere UV (254µm) timp de o ora, 4 ore si 6 ore.
S-au inregistrat spectrele FTIR-ATR dupa fiecare oxidare, atat la uleiul liber cat si
extras din capsule. In cazul analizelor FTIR-ATR au fost posibile inregistrarea spectrelor
capsulelor cu ulei, nefiind necesara extracţia uleiului din capsule.
V.2. REZULTATE ŞI DISCUŢII
În cazul analizelor FTIR-ATR a fost stabilit stadiul oxidării, calculandu-se raportul
între intensităţile principalele peakuri considerate markeri ai oxidarii conform literaturi
(Guillén and Cabo, 1999, 2000, 2002): A2853/A3005, A1746/A3006, A1474/A3006, A1377/A3006 and
A1163/A3006, înainte şi după tratamentul UV.
S-a constat ca uleiul liber avea un stagiu mai avansat al oxidarii 2 sau 3, in timp ce
uleiul incapsulat se afla in stagiu 1 al oxidarii. S-a demosntrat astfel ca uleiul de catina
incapsulat in diferitele capsule obtinute din matricile utilizate a fost mult mai protejat
impotriva oxidarii in urma diferitelor tratamente, comparativ cu uleiul liber (ex: la uleiul de
cătină, Fig.V.1.).
Cea mai bună protecţie împotriva oxidării a fost asigurată de urmatoarele capsule
formate din matrcile şi concentraţiile urmatoare: alginat 1%, chitosan 1.5%, complexele
alginate-gumă gum şi alginat-gum xantan în raport 0.5:0.5, şi alginat-k-caragenan complex în
raport 0.75:0.75.
XXVII
Ratio values/Valoarea raportelor
________________________________________________________________________________________
8
7
Oil free/Ulei liber
6
5
Oil from AG 1%/Ulei din AG 1%
4
3
Oil from AG 1.5%/Ulei din AG 1.5%
2
1
0
A
B C
D
E
A
B C
D
E
A
B C D
E
After 1h UV/Dupa 1h After 4h UV/Dupa 4h After 6h UV/Dupa 6h
UV
UV
UV
Types of ratios on time/Tipul rapoartelor in timp
Fig. V.1. Reprezentarea grafica a uleiului de canepa liber si incapsulate (in diferite capsule
de alginate) in timpul oxidarii
(A= A2853/A3005-3008, B= A1744/ A3005-3008, C= A1464/ A3005-3008, D= A1377/ A3005-3008, E= A1160/
A3005-3008)
V.3. CONCLUZII
Uleiurile încapsulate prezintă o mai buna stabilitate împotriva oxidării provocate de
diferite conditii comparativ cu uleiurile libere.
Cea mai bună protecţie împotriva oxidării a fost asigurată de urmatoarele capsule
formate din matrcile şi concentraţiile urmatoare: alginat 1%, chitosan 1.5%, complexele
alginate-gumă gum şi alginat-gum xantan în raport 0.5:0.5, şi alginat-k-caragenan complex în
raport 0.75:0.75.
Spectroscopia FTIR este considerată o foarte buna tehnică de monitorizare a oxidării
uleiurilor libere şi încapsulate, dovedindu-se totodată a fi o tehnică rapidă, acurată şi simplă.
XXVIII
________________________________________________________________________________________
CONCLUZII GENERALE
În conformitate cu scopul si obiectivele acestei teze de doctorat, s-a realizat
bioîncapsularea a patru uleiuri funcţionale din plante în diferite matrici naturale, precum şi
evaluarea eficienţei încapsulării, stabilitatea şi eliberarea uleiurilor din capsulele obtinute.
S-a realizat o evaluare comparativă si sistematică a calităţii celor patru uleiuri
funcţionale din plante înaintea încapsulării lor: uleiul de cânepă (HP), uleiul extra virgin de
măsline (EVO), uleiul de dovleac (PK) şi uleiul de cătină (SB) (de provenienţă din România
şi Italia).
Având în vedere obiectivele propuse s-a realizat:
I. S-au identificat caracteristicile uleiurilor înainte de încapsulare, stabilindu-se
markeri de calitate si autenticitate:
1. Majoritatea uleiurilor analizate prezintă indicele de iod în conformiatte cu
specificaţiile din CODEX 210, excepţie uleiul de cătină care prezintă a valoare mai
scazută.
2. Spectrele UV-Vis ale uleiurilor au relevant peakurile specifice, ca şi markeri ai
autenticităâii.
3. Studiile FTIR-ATR au demonstrat relaţia dintre benzile aparute în spectre şi
compoziţia specifică fiecarui ulei, putându-se astfel stabili fingerprintul specific
uleiurilor studiate.
4. Analizele GC-FID au demonstrate faptul rpofilul acizilor din compoziţia uleiurilor
analizate este în conformitate cu datele din literatură.
II. Studiile experimentale utilizând ca şi metodă gelarea ionicăde
numită:‘’ionotropically crosslinked gelation’’, în vederea bioîncapsulării uleiurilor
funcţionale în matrici naturale, au demonstrat stabilitatea şi eliberarea controlată a
uleiurilor bioîncapsulate
1.
S-a reusit obţinerea diferitelor tipuri de capsule utilizănd matrici naturale şi
complexe dintre acestea, fiind incorporate uleiuri
2.
Caracteristicile capsulelor obţinute (aria, perimetrul, compactitatea, sfericitatea şi
elongaţia), în special mărimea lor, au fost influenţate de conţinutul de uleiuri
încapsulate.
3.
Cele mai bune matrici pentru bioîncapsularea uleiurilor au fost: alginat 2%,
chitosan 2%, şi alginate în complex cu k-caragenan, gumă guar şi gumă xantan în
raport de 0.75:0.75.
III. Caracterizarea capsulelor a fost realizată prin diferite metode: SEM, FTIR,
analize DSC şi TGA
1.
Suprafata capsulelor analizată prin SEM, a fost non-regulara, aceasta datorita
picaturilor de ulei prezente, exceptand chitosanul care prezinta o suprafata mult
mai mata
XXIX
________________________________________________________________________________________
2.
Prin analizele FTIR-ATR, diferitele capsule conţinând uleiuri au prezentat peakuri
care sunt atribuite atat uleiurilor cât şi capsulelor goale (regiunile dintre 2800-2900
cm-1 şi 1700-900 cm-1).
3.
Termogramele DSC au arătat faptul că temperature peakurilor creşte direct
proportional cu cresterea temperaturii, fiecare peak fiind characteristic fiecărui tip
de capsula obţinută.
4.
Analizele TGA au demonstrate ca pierderea în greutate se datoreaza continutului
ridicat in apa a unor capsule. Încapsularea uleiurilor nu afectează pierderea în
greutate a capsulelor.
IV. Evaluarea eficienţei încapsulării
1. Creşterea concentraţiei matricilor sau a complexului de matrici determină
obţinerea unei mai bune eficienţe la încapsulare. s-a obţinut cea mai bună eficienţa
a încapsulării utilizând ca şi matrici: alginatul în concentraţie de 2%, chitosanul in
aceeaşi concentraţie, urmate de concentraţiile de 1.5%, şi de alginatul în complex
cu k-caragenan şi gume în raport de 0.75:0.75%.
2. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea şi solubilitatea
uleiului în matrice. Eliberarea a fost mai lentă in cazul hexanului, mai ridicată în
cazul metanolului şi cea mai buna eliberare fiind in THF, indiferent de matricea
sau concentraţia utilizată la încapsulare.
3. Stabilitatea capsulelor la pH 1.2 si pH 4.5, dizolvarea acestora şi eliberarea
continutului realizandu-se la pH 7.4.
V. Protecţia bioîncapsulării a uleiurilor împotriva
1. Uleiurile încapsulate prezintă o mai buna stabilitate împotriva oxidării provocate
de diferite conditii comparativ cu uleiurile libere.
2. Cea mai bună protecţie împotriva oxidării a fost asigurată de urmatoarele capsule
formate din matrcile şi concentraţiile urmatoare: alginat 1%, chitosan 1.5%,
complexele alginate-gumă gum şi alginat-gum xantan în raport 0.5:0.5, şi alginatk-caragenan complex în raport 0.75:0.75.
XXX
________________________________________________________________________________________
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35.
SOCACIU C., C., MIHIS, A., NOKE, 2008, Oleosome Fractions Separated From
Sea Buckthorn Berries: Yield And Stability Studies. in : Seabuckthorn, A
Multipurpose Wonder Plant, ed. V.Singh, vol.III, Indus International, India, ISBN:
978-81-7035-520-525, 322-326,.
36. SOCACIU C., C.MIHIS, M.TRIF, H.A.DIEHL, 2007, Seabuckthorn fruit oleosomes
as natural, micro-encapsulated oilbodies:separation, characterization, stability
evaluation, Proc.15th Int. Symposium on Bioencapsulation, 6-8 Sept, Univ. Viena,
Austria, P3-19, 1-3.
37. SOCACIU C., RANGA F., DIEHL, H., 2005, UV-VIS spectrometry applied for the
quality and authenticity evaluation of edible oils from Romania, Buletin USAMV-CN,
62, 1454-2382.
38. TRIF M., M.ANSORGE-SCHUMACHER, C.SOCACIU, H.A.DIEHL, 2007,
Application of FTIR spectroscopy to evaluate the oxidation of encapsulated
seabuckthron oil, 15th Int. Symposium on Bioencapsulation, Universitatea din Viena,
Austria, P3-07, 1-3
39. TRIF M., M.ANSORGE-SCHUMACHER, CHEDEA, V., SOCACIU, C., 2007,
Release rates measurement of encapsulated castor oil using alginate as
microencapsulation matrix, Proc.Int.Symp., Nanotech Insight, 10-17 martie, Luxor,
Egipt, 157-159.
40. TRIF, M., 2007, Determination of encapsulated seabuckthorn oil oxidation usiing
FTIR-ATR spectroscopy, 63-64, Buletin USAMV-CN, 06-51, 1-3.
41. ZELLER, B.L., SALEEB, F.Z., LUDESCHER, R.D., 1999, Trends in Development
of Porous Carbohydrate Food Ingredients for Use in Flavor Encapsulation. Trends in
Food Science & Technology, 9, 389-394
XXXIII
________________________________________________________________________________________
PUBLICAŢII PE DURATA STAGIULUI DOCTORAL SI PARTICIPARI LA
SIMPOZIOANE ŞI CONFERINŢE NATIONALE ŞI INTERNAŢIONALE
2009
1. Socaciu C., Trif. M, A. Baciu, T. Nicula, A. Nicula, ‘’ Encapsulation of plant oleosomes
and oleoresins in mixed carbohydrate matrices’’, COST865, Spring
Meeting
"Microcapsule property assesment", Luxemburg 2009, Proceeding
2008
1. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. „Bioencapsulated
seabuckthorn oil: controlled release rates in different solvents”, Bull. USAMV-CN,
65/2008, ISSN 1454-2382, Romania
2. Pece Aurelia, D. Vodnar, Monica Trif, C. Coroian, Camelia Raducu, G. Muresan,
“Study of the physico-chemical parameters from buffalo raw milk during different
lactations”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania
3. Pece Aurelia, D. Vodnar, Monica Trif, “Corelation between microbiological and
physico-chemical parameters from buffalo raw milk during different lactations”, Bull.
USAMV-CN, 65/2008, ISSN 1454-2382, Romania
4. Carmen Socaciu, Baciu A., Trif M., “Oleosome-rich pectin network as a new, natural
bioencapsulation matrix”, XVI International Conference on Bioencapsulation Dublin,
Ireland ; September 2008, Proceeding
5. Monica Trif, Carmen Socaciu, Andreea Stanila, “The evaluation of encapsulated
Seabuckthorn oil properties usind FTIR”, CIGR - International Conference of
Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola,
Processing Conference - 4th CIGR Section VI International Symposium On Food And
Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797
6. Andreea Stanila and Monica Trif, “Antioxidant activity of carotenoide extracts from
HIPPOPHAE RHAMNOIDES”, CIGR - International Conference of Agricultural
Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola, Processing
Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess
Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797
7. Monica Trif, Carmen Socaciu and Horst Diehl, “Evaluation of effiency, release and
oxidation stability of seabuckthorn encapsulated oil using FTIR spectroscopy”, 7th Joint
Meeting of AFERP, ASP, GA, PSE & SIF, August 2008, Athens, Greece, Book of
Abstracts, pg.39
8. Monica Trif and Carmen Socaciu, “Evaluation of effiency, release and oxidation
stability of Seabuckthorn microencapsulated oil using Fourier Transformed Infrared
Spectroscopy”, 4th Meeting on Chemistry and Life, and accepted to be published in
Chemické Listy Journal (current IF=0.683)
XXXIV
________________________________________________________________________________________
2007
1. Monica Trif, Marion Ansorge-Schumacher, Veronica S. Chedea, Carmen Socaciu,
‘’Release rates measurement of encapsulated castor oil using alginate as
microencapsulation matrix’’, The International Conference on Nanotechnology: Science
and Application (NanoTech Insight), Luxor, 10-17 March 2007, Egipt
2. Chedea V.S., Kefalas P., Trif M. and Socaciu C. ‘’Stability studies of encapsulated
carotenoid extract from orange waste using pullulan as microencapsulation matrice’’,
Nano Tech Insight, Luxor, 10-17 March 2007, Egipt
3. Monica Trif, Marion Ansorge-Schumacher, Carmen Socaciu, ‘’Application of FTIR
Spectroscopy for determination of oxidation of encapsulated sea buckthorn oil’’,
Proc.XV International workshop on Bioencapsulation and COST865 Meeting, 2007,
Wien, Austria, published in extenso
4. Carmen Socaciu, Cristina Mihis, Monica Trif, Horst A. Diehl, ‘’Seabuckthorn fruit
oleosomes as natural, microencapsulated oilbodies: separation, characterization, stability
evaluation oil’’, Proc. XV International workshop on Bioencapsulation and COST865
Meeting, 2007, Wien, Austria, published in extenso
5. Socaciu C., Trif M., Ranga F., Fetea F., Bunea A., Dulf F., Bele C. and Echim C.
‘’Quality and authenticity of seabuckthorn oils using succesive UV-Vis, FT-IR, NMR
spectroscopy and HPLC-, GC- chromatography fingerprints’’, 3rd Conf. Int.
Seabuckthorn Assoc., 2007, Quebec, Canada
6. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. ‘’Determination of
encapsulated Sea buckthorn oil oxidation using FTIR-ATR spectroscopy’’, Bull.
USAMV-CN, 63-64/2007, ISSN 1843-5262, Romania
2006
1. Monica Trif, “Seabuckthorn oleosomes as stabilized bioactive nanostrustures with
applications in microencapsulation nutraceuticals”, Symposium IRC Transylvania
“Innovations in Agriculture, Biotechnologies, Animal Breeds and Veterinary Medicine”,
2006, USAMV Cluj-Napoca, Romania
2004
1. Veerle Minne, Monica Trif, J.M.C. Geuns, Corina Catana, “Steviozide and steviol
determination in callus culture of Stevia rebaudiana Bertoni”, Bull. USAMV-CN,
61/2004, ISSN 1454-2382, Romania
XXXV
UNIVERSITY OF AGRICULTURAL
SCIENCES AND VETERINARY
MEDICINE, CLUJ-NAPOCA
FACULTY OF ANIMAL BREEDS
AND BIOTECHNOLOGY
BIOTECHNOLOGY FIELD
PHD THESIS
BIOENCAPSULATION SYSTEMS OF BIOACTIVE
COMPOUNDS EXTRACTED FROM PLANT OILS
(SUMMARY)
MONICA TRIF
Dipl. Eng. Biotechnologist
SCIENTIFIC SUREVISOR:
PROF. Dr. Dr. h.c. HORST A. DIEHL
2009
Bioencapsulation systems of bioactive compounds extracted from plants oils
________________________________________________________________________________________
TABLE OF CONTENTS
I. INTRODUCTION. AIMS AND OBJECTIVES .................................................................. III
PART II. ORIGINAL CONTRIBUTIONS .............................................................................. X
CHAPTER II. CHARACTERIZATION OF FUNCTIONAL OILS USED FOR
BIOENCAPSULATION........................................................................................................... X
II.1. MATERIALS AND METHODS................................................................................... X
II.2. RESULTS AND DISCUSSIONS.................................................................................. X
II.3. CONCLUSIONS......................................................................................................... XV
CHAPTER III. BIOENCAPSULATED OILS: BEADS PREPARATION PROTOCOLS AND
CHARACTERIZATION……. ............................................................................................. XVI
III.1. MATERIALS AND METHODS ............................................................................. XVI
III.2. RESULTS AND DISCUSSIONS ...........................................................................XVII
III.3. CONCLUSIONS ................................................................................................... XXIII
CHAPTER IV. ENCAPSULATION EFFICIENCY AND RELEASE STUDIES............ XXIII
IV.1. MATERIALS AND METHODS .......................................................................... XXIII
IV.2. RESULTS AND DISSCUSIONS ......................................................................... XXIV
IV.3. CONCLUSIONS ..................................................................................................XXVII
CHAPTER V. FTIR CHARACTERIZATION OF OIL OXIDATION ..........................XXVIII
V.1. MATERIALS AND METHODS .........................................................................XXVIII
V.2. RESULTS AND DISCUSSIONS.........................................................................XXVIII
V.3. CONCLUSIONS .................................................................................................... XXIX
GENERAL CONCLUSIONS............................................................................................. XXX
SELECTED BIBLIOGRAPHY ........................................................................................XXXII
PUBLICATIONS RELEASED DURING PhD..................................................... .........XXXVI
II
________________________________________________________________________________________
I.
INTRODUCTION. AIMS AND OBJECTIVES
BIOENCAPSULATION is a novel technology which use bioactive molecules to be
inserted , immobilized on specific supports ( matrices). Encapsulation technology is now well
developed and accepted within the pharmaceutical, chemical, cosmetic, foods and printing
industries (Augustin et al., 2001; Heinzen, 2002). It appears that bioencapsulation has a strong
potential in most biotechnology fields and especially in agriculture and food. The
encapsulation of active components has become a very attractive process in the last decades,
being adequate for food ingredients as well as for chemicals, drugs or cosmetics.
The application of a successful method to bioencapsulate bioactive compounds
extracted from plant oils could enable the optimum combinations and qualities of these
substances to be established. It is envisaged that such a combination be bioencapsulated into a
commercial field would have significant benefits for the pharmaceutical, food and
cosmeceutical industry. Furthermore, research and development in these fields are of
significant benefits for the preservation of natural bioactive compounds extracted from plants.
The aim of this thesis was to use different natural matrices to bioencapsulate of
bioactive molecules (plant oils) using as method ionotropically crosslinked gelation, and to
evaluate different quality and efficiency parameters for the bioencapsulated products, as well
the controlled release of bioactive molecules from the matrix.
Thesis structure. The first part of the thesis is a bibliographic report and the second part
contains the experimental procedures: material and methods, results and discussions, and
conclusions.
The first part (Literature studies) includes four chapters (I-IV):
Chapter I. Bioencapsulation: definition, principles, applications, methods and techniques
Chapter II. Functional plant oils: physical and chemical characterization and authentification
Chapter III. Oil encapsulation: matrices, encapsulation methods and techniques, efficiency
and stability evaluation
Chapter IV. Methods for beads characterization
Part two (Original Contribution) is included in four chapters as follows:
Chapter V. Characterization of functional oils used for bioencapsulation. This part
characterize the functional fourth oils (hemp oil, pumpkin oil, extra virgin olive oil and
seabuckthorn oil) analyzed and encapsulated by different techniques: ultraviolet (UV)
spectrometry, Gas-Chromatography (GC) with Flame Ionization Detection (FID) and Fourier
transformed Infrared spectroscopy equipped with horizontal attenuated total reflectance
(FTIR-ATR), and chemical determinations were carried out according to the methods
described in the A. O. A. C. and IOOC.
Chapter VI. Bioencapsulated oils: beads preparation protocols and characterization. This
chapter describes the protocols: for synthesis of empty beads of different sizes and
concentrations and for synthesis of beads of different sizes and concentrations incorporating
small oil droplets, characterizes the beads empty and containing oils (by sizes, morphology)
and analyses the beads by FTIR and thermal (differential scanning calorimetry and
termogravimetric).
III
________________________________________________________________________________________
Chapter VII. Encapsulation efficiency and release studies. This chapter includes the studies
regarding encapsulation efficiency of functional oils encapsulated in different matrices,
release rate measurements of oils from beads on time and in different solvents, and in vitro
release oils from the beads.
Chapter VIII. FTIR characterization of oil oxidation. This chapter includes the comparative
analysis of oil free and encapsulated oxidized on time under UV conditions.
The experimental work is focused on following objectives:
¾ Use of different natural matrices (such as alginate, alginate in complex with kcarrageenan and gums: xanthan and guar, chitosan) to encapsulate functional oils
(pumpkin oil, extra virgin olive oil, hemp oil and seabuckthorn oil)
¾ Improvement and optimization of bioencapsulation methods for vegetable oils with
functional properties
¾ Investigations of different obtained beads: morphology (scanning electron
microscopy), characterization of beads (area, diameter, perimeter, elongation,
compactness), Fourier transform infrared spectroscopy (FTIR) analysis
¾ Investigations of bioencapsulated functional oils: encapsulation efficiency and
stability, control release of oils encapsulated, material and functionality of the beads
obtained , FTIR characterization of: free oils, obtained beads and oxidation of free and
encapsulated oils
The work presented was carried out in the Department of Chemistry and Biochemistry
at the University of Agricultural Sciences and Veterinary Medicine (USAMV), Cluj-Napoca,
Romania, in collaboration with the Technical University Berlin (TU Berlin), Germany,
Department of Enzyme Technology, under supervision of Prof. Dr. rer. nat. Marion AnsorgeSchumacher. I would like to thank the sponsors who made this work possible providing
scholarships to pursue doctoral studies: Deutsche Bündestiftung Umwelt (DBU) Germany and
EU COST 865.
IV
________________________________________________________________________________________
INTRODUCTION
Microencapsulation is a process to produce capsules in the micrometer to millimeter
range known as microcapsules.
A microcapsule is a tiny capsule and its preparation procedure, called
microencapsulation, can endow various traits to the core material in order to add secondary
functions and/or compensate for shortcomings.
Microcapsules can be classified in three basic categories according to their
morphology as mono-cored (mononuclear), poly-cored (polynuclear), and matrix types.
The schematic presentation of different types of microcapsules is shown in the
following figure Fig.1.:
Fig. 1. Variations on microcapsules formulation
(Birnbaum D.T. and Brannon-Peppas L., 2003)
Mono-cored (mononuclear) microcapsules contain the shell around the core. Polycored (polynuclear) capsules have many cores enclosed within the shell. In matrix
encapsulation, the core material is distributed homogeneously into the shell material.
Purposes of microencapsulation
Generally, there are a numbers of reasons why substances should be encapsulated (Li S.P. et
a.l, 1988; Finch C.A., 1985; Arshady, R., 1993):
•
•
•
•
•
•
•
•
•
•
•
Increasing stability to protect reactive substances from the environment.
To convert liquid active components into a dry solid system.
To separate incompatible components for functional reasons.
To mask undesired properties of the active components.
To protect the immediate environment of the microcapsules from the active
components.
To control release of the active components for delayed (timed) release or long-acting
(sustained) release.
Separation of incompatible components.
Conversion of liquids to free-flowing solids.
Masking of odor, activity, etc.
Protection of immediate environment.
Targeting of drugs.
V
________________________________________________________________________________________
Encapsulation technology is now well developed and accepted within the
pharmaceutical, chemical, cosmetic, foods and printing industries (Augustin et al., 2001;
Heinzen, 2002).
It appears that bioencapsulation has a strong potential in most biotechnology fields
and especially in agriculture and food. The encapsulation of active components has become a
very attractive process in the last decades, being adequate for food ingredients as well as for
chemicals, drugs or cosmetics.
The main objective is to build a barrier between the component in the particle and the
environment. This barrier may protect against oxygen, water, light; avoid contact with other
ingredients, e.g. a heavy meal; or control diffusion. The preservation of bioactive food
ingredients through product processing and storage, and their controlled release in the
gastrointestinal tract is yet a major obstacle for the full exploitation of the health potential of
many food bioactive components. Challenges facing introduction of bioactive compounds into
foods are not limited solely to their inclusion in free flowing powder or solution.
In food products, fats and oils, aroma compounds and oleoresins, vitamins, minerals,
colorants, and enzymes have been encapsulated (Dziezak, 1988; Jackson and Lee, 1991;
Shahidi and Han, 1993).
The choice of appropriate bioencapsulation technique depends upon the end use of the
product and the processing conditions involved in the manufacturing product.
All bioencapsulation techniques require a core material and an enveloping solution.
The material has to be approved by the Food and Drug Administration (US) or European
Food Safety Authority (Europe) (Amrita et al., 1999).
Pfutze S. (2003) considers that the technologies to accomplish encapsulation can be
divided into two groups:
•
formation of matrix capsules : an active and protective ingredient form homogeneous
granules. The active is well distributed within the granule and is enclosed by the
abundance of the protective material, forming a matrix for the active.
•
formation of defined shell capsules : the active material is granules and coated with a
protective layer. Active and protective material is clearly separated.
Coacervation: encapsulation of liquids
Complex coacervation, (or phase separation), is the first large application of a
microencapsulation technology. Coacervation, which is a phenomenon occurring in colloidal
solutions, is often regarded as the original method of encapsulation (Risch, 1995).
The applicability of complex coacervation is enormous but has been limited due to its
relatively high costs. It includes the encapsulation of:
Flavors
Vitamins
Fragrances (scratch and sniff)
Liquid Crystals for display devices
Ink systems for carbonless copy paper
VI
________________________________________________________________________________________
Active ingredients for drug delivery
Bacteria and cells
Matrices – materials for encapsulation
Enormous range of different materials can be used for encapsulation, such as synthetic
polyelectrolytes (Sukhorukov G.B. et al., 1998; Donath E. et al., 1998), natural
polyelectrolytes (Shenoy D.B. et al., 2003) inorganic nanoparticles (Caruso F. et al., 2001),
lipids (Moya S. et al., 2000), dye (Dai Z. et al., 2001), multivalent ion (Radtchenko I.L. et al.,
2005), and biomacromolecules (Yang H. et al., 2006).
Biopolymers are polymers that are generated from renewable natural sources, are
often biodegradable, and not toxic to produce. They can be produced by biological systems
(i.e. micro-organisms, plants and animals), or chemically synthesized from biological starting
materials (e.g. sugars, starch, natural fats or oils, etc.).
Natural polymers and their derivatives: anionic polymers: HA, alginic acid, pectin,
carrageenan, chondroitin sulfate, dextran sulfat; cationic polymers: chitosan, polylysine;
amphipathic polymers: collagen (and gelatin), carboxymethyl chitin, fibrin; neutral polymers:
dextran, agarose, pullulan.
The ability of carbohydrates, such as starches, maltodextrins, corn syrup solids and
gums, to bind flavours is complemented by their diversity, low cost, and widespread use in
foods and makes them the preferred choice for encapsulation.
Guar gum (E412, also called guaran) is extracted from the seed of the leguminous
shrub Cyamopsis tetragonoloba, where it acts as a food and water store. Guar gum shows
high low-shear viscosity but is strongly shear-thinning. Being non-ionic, it is not affected by
ionic strength or pH but will degrade at pH extremes at temperature (for example, pH 3 at
50°C).
Alginates (E400-E404) are produced by brown seaweeds (Phaeophyceae, mainly
Laminaria). Gelling properties depends on the ion binding (Mg2+ << Ca2+ < Sr2+ < Ba2+) with
the control of the di-cation addition being important for the production of homogeneous gels.
Carrageenan (E407) is a collective term for polysaccharides prepared by alkaline
extraction (and modification) from red seaweed (Rhodophycae). The strongest gels of κcarrageenan are formed with K+ rather than Li+, Na+, Mg2+, Ca2+, or Sr2+.
Xanthan gum (E415) is a microbial desiccation-resistant polymer prepared
commercially by aerobic submerged fermentation from Xanthomonas campestris. Xanthan
gum is mainly considered to be non-gelling and used for the control of viscosity due to the
tenuous associations endowing it with weak-gel shear-thinning properties. It hydrates rapidly
in cold water without lumping to give a reliable viscosity, encouraging its use as thickener,
stabilizer, emulsifier and foaming agent.
Chitin is obtained in industrial scale from shrimps and crustaceans in general (Yanga
et al., 2000). In many studies, chitosan has been crosslinked with aldehydes, such as
glutaraldehyde and formaldehyde, to make it a more rigid polymer for use as a core material
in research on controlled release. However, biological acceptance of these cross-linked
products depends upon the amount of cross-linking agent in the final products and the toxicity
of aldehydes has been enormously limited the utilization of the cross-linked chitosan
microparticles in the pharmaceutical field.
VII
________________________________________________________________________________________
Many components naturally present in vegetable oils have been shown to have beneficial
properties.
Hempseed oil is pressed from the seed of the hemp plant (i.e., non-drug varieties of
Cannabis sativa L). The Oleic Acid (Omega 9) contained in Hemp Seed Oil helps keep
arteries supple because of its fluidity. In excess Oleic acid can interfere with EFA's and
prostaglandin's.
Olive oil contains triacylglycerols and small quantities of free fatty acits, glycerol,
pigments, aroma compounds, sterols, tocopherols, phenols, unidentified resinous components
and others (Kiritsakis A., 1998). Among these constituents the usaponifiable fraction , which
covers a small percentage (0,5-15%) plays a significant role on human health (Waterman and
Lockwood, 2007). Olive oil is considerably rich in monounsaturated fats, most notably oleic
acid.
Pumpkin oil is a healthy, high quality, specialty oil, ranked in the top 3 most nutritious.
Pumpkin seed oil has an intense nutty taste and is rich in polyunsaturated fatty acids. Brown
oil has a bitter taste. The tocopherol content of the oils is ranging from 27.1 to 75.1 μg/g of
oil for α-tocopherol, from 74.9 to 492.8 μg/g for γ-tocopherol, and from 35.3 to 1109.7 μg/g
for δ-tocopherol (Stevenson D.G. et al., 2007).
Most often seabuckthorn oil is called “Nature's anti-oxidant cocktail”, because it has a
unique composition, combining a cocktail of components usually only found separately. The
seabuckthorn oil is stored in extra-chromoplastic organelle, named oil bodies, a natural form
of encapsulation (Socaciu et al., 2007, 2008). Seabuckthorn seed oil contains a high content of
the two essential fatty acids, linoleic acid and α-linolenic acid (Chen et al., 1990), which are
precursors of other polyunsaturated fatty acids such as arachidonic and eicosapentaenoic
acids. The oil from the pulp/peel of seabuckthorn berries is rich in palmitoleic acid and oleic
acid (Chen et al. 1990).
Oils include also flavonoids (Chen et al., 1991), carotenoids, free and esterified
sterols, triterphenols, and isoprenols (Goncharova and Glushenkova, 1996). Carotenoids also
vary depending upon the source of the oil.
The physical and chemical properties of functional oils
The physical and chemical properties of oils, including iodine, saponification, acid and
peroxide values, refractive index, density and unsaponifiable matter are determined according
to standard procedures. Iodine value measures the unsaturation of oil. The fact that the iodine
value is lower than 100 shows that the oil is of lower degree of saturation (Pa Quart, 1979;
Pearson, 1981). The saponification value is an indication of the average molecular mass of
fatty acids present in oil. The acid value has been shown to be a general indication of the
edibility of oils (AOAC, 1980; Pearson, 1981). The peroxide value is frequently used to
measure the progress of oxidation of oil. It indicates the oxidative rancidity of oil. (deMan,
1992).
The techniques to characterize and authentify of functional oils
Several techniques to characterize and authentify the food products have been
proposed. The authentication methods applied to oils and fats can be classified as chemical (=
VIII
________________________________________________________________________________________
separative) or physical (= non-separative). The most widely used and accepted physical
technique for oil and fat authentication is ultraviolet (UV) spectrometry. Other promising
physical techniques which have been investigated for oil and fat characterization and
authentication include mass spectrometry, pyrolysis mass spectrometry, GC-electron
ionisation mass spectrometry, nuclear magnetic resonance and infrared spectrometry (IR).
Fourier transform infrared (FTIR) spectrometers have many advantages over
conventional dispersive instruments, with more energy throughput, excellent wavenumber
reproducibility and accuracy, extensive and precise spectral manipulation capabilities
(rationing, subtraction, derivative spectra and deconvolution) and advanced chemometric
software to handle calibration development. FTIR spectroscopy can provide much more
information on the characteristics, composition and/or chemical changes taking place in fats
and oils than can be obtained from conventional dispersive IR instruments. Furthermore from
a practical viewpoint, FTIR quantitative analysis methods are generally rapid (1-2 min), can
be automated and reduce the need for solvents and toxic reagents associated with wet
chemical methods for fats and oils analyses, making the development of FTIR methods timely
in view of present efforts to eliminate toxic solvents
Horizontal attenuated total reflectance (HATR) accessories also have been widely
used in the development of FTIR methods for the analysis of fats and oils, because they
provide a simple and convenient means of sample handling (Sedman et al., 1999).
Mid infrared (MIR) spectroscopy can be used to identify organic compounds because
some groups of atoms display characteristic vibrational absorption frequencies in this infrared
region of the electromagnetic spectrum. Edible fats and oils in their neat form are ideal
candidates for FTIR analysis, in either the attenuated total reflectance or the transmission
mode.
A wide variety of foods is encapsulated- flavoring agents, acids, bases, artificial
sweeteners, colourants, leavening agents, antioxidants, agents with undesirable flavors, odors
and nutrients, among others. They retain their bioactivity and remain accessible to external
reagents.
Phytosterols, flavonoids and sulphur containing compounds represent three groups of
compounds found in fruits and vegetables, which may be important in reducing the risk of
atherosclerosis (Howard and Kritchevsky, 1997). Some phytochemicals such as ascorbic acid,
carotenoids, vitamin E, polyphenols, isoflavone and phytosterols have been highlighted as
physiologically-active ingredients that help fight certain diseases.
Natural products such as phytochemicals and herbal extracts are being widely used by
consumers as alternatives to prescription drugs for allergic diseases. Many of the compounds
found in plants have useful applications in the pharmaceutical, food processing and various
other industries.
Encapsulation also masks some objectionable flavors, e.g. fish oil and some bitter
antibiotics. Encapsulation can be used to convert oils into solid and water soluble forms and
extend their use in many product applications. The encapsulation of oils, include as methods
and techniques: spray-drying, spray-chilling, fluid bed encapsulation, extrusion encapsulation,
and encapsulation by complex coacervation. Oils high in omega-3 fatty acids may be spraydried and oil encapsulated in a dry matrix with very low exposure to surface oxidation.
In most of the cases the matrices used to encapsulated oils and fats are gums (acacia,
arabic), proteins, carbohydrates (casein/sugar), maltodextrin, beta-cyclodextrin, sodium
alginate, gelatin.
IX
________________________________________________________________________________________
PART II. ORIGINAL CONTRIBUTIONS
CHAPTER II. CHARACTERIZATION OF FUNCTIONAL OILS USED FOR
BIOENCAPSULATION
Edible oils extracted from plant sources (sunflower, pumpkin, soybean, rapeseed,
olive, etc.) are important in foods and in various industries (e. g. cosmetics, pharmaceuticals,
lubricants). They are key components of the diet and also provide characteristic flavours and
textures to foods. To check their quality and safety, the oils analysis is made by different
techniques. Three techniques are generally applied to characterize such oils: ultraviolet (UV)
spectrometry, Gas-Chromatography (GC) with Flame Ionization Detection (FID) and Fourier
transformed Infrared spectroscopy equipped with horizontal attenuated total reflectance
(FTIR-ATR).
II.1. MATERIALS AND METHODS
Samples of four different oils were examined: seabuckthorn oil (SBO) extracted from
seabuckthorn fruits, collected from Cluj county (Transylvania, North of Romania), extra
virgin olive oil (EVO) purchased on the Italien market, hemp oil (HP) and pumpkin oil (PK)
were purchased on Romanian market.
The following chemical determinations were carried out according to the methods
described in the A. O. A. C. and IOOC or by the Commission of the European Union (EU):
acid value and iodine number. All tests were performed in triplicate. Acid value was
calculated from the free fatty acid content of the analyzed oils, determined by titration
according to the modified official method Ca 5a-40. The iodine value has been determined by
the AOCS method Cd 1c-85 (1997).
II.2. RESULTS AND DISCUSSIONS
Determination of acid and iodine value
The results of chemical analysis are presented in Table 1. indicating that oil
characteristics are in good agreement with current published values.
These data indicate that the oils investigated correspond to Codex quality indicators
for iodine values, except SB oil, and do not correspond for the acid values.
X
________________________________________________________________________________________
Table 1. Chemical and physical characteristics of analyzed oils compared with literature
Hemp
Oil
Extra Virgin Olive
Oil
Pumpkin
Sea buckthorn
Oil
Oil
Ulei de Măsline
Extra Virgin
Ulei de Dovleac
Ulei de Cătina
Ulei de Canepa
Chemical and physical characteristics
Caracteristicile chimice si fizice
Acid value+
(mg KOH/g Oil)
Aciditatea+
4.0
6.6
4.0
4.0
145-166**
75-94**
116-133**
98-119‡
(mg KOH/g ulei)
Iodine value
Indicele de iod
+
CODEX 210/CODEX STAN 33;**Firestone D., 1999; ‡Albulescu M. et al., 2006
Determination of ultra violet/visible (UV-Vis) oils fingerprint
A spectral characterization (fingerprint) of the oil samples by UV-Vis is presented in
Fig. II.1. The difference between a typical authentic (accepted) and not authentic (rejected) oil
has been determined based on peaks’ position and intensities (Socaciu C. et al., 2005).
Hemp (Cannabis sativa L) oil
The fingerprint spectral characterization of hemp oil according with data from
OMLC, is given by the content of chlorophyll with the maximum absorbance at 411 nm
(Fig.II.1.A.).
Virgin Olive (Olea europaea ) oil
The color of extra virgin olive oil is dependent on the pigments, usually having high
carotenoid and chlorophyll content. Rippen olives give a yellow oil because of the carotenoid
(yellow red) pigments. The color of the oil is influenced by the exact combination and
proportions of pigments. A simple equation : Color = Chlorophyll (Green) + Carotenoids
(Yellow red) + other pigments (“color equation”).
XI
________________________________________________________________________________________
A.
B
D.
C.
Fig.II.1.UV-Vis spectra of investigated oil - fingerprint for regions 3X0-600 nm with specific
of maximum absorbance peak: A. hemp oil (HP); B. extra virgin olive oil (EVO); C. pumpkin
oil (PK); D. seabuckthorn oil (SB)
The chlorophyll content decreases as the fruit matures so olives picked green produce
a greener oil with a "grassy" flavor. The fingerprint of the extra virgin olive oil we attributed
to the “color equation” mentioned previously (Fig.II.1.B.).
Pumpkin (Cucurbita pepo) oil
The representative fingerprint of this oil accepted have the peak at 418 nm lower and
the peak at 435 nm higher (Fig.II.1.C.) compared to the not accepted oils which have a high
peak at 418 nm and a low one at 435 nm (Lankmayr et al., 2004).
Seabuckthorn (Hippophae rhamnoides) oil
The absorption maxima from seabuckthorn oil spectrum shows that the fingerprint of
this oil has a broad absorption with the three maxima or shoulders in the blue spectral range
between 400 and 500 nm, corresponding to the carotenoids (Fig.II.1.D.). The main nutrient in
seabuckthorn oil is beta-carotene. According with literature and compared to the three
maxima in the spectra of seabuckthorn oil, is it obviously that the fingerprint of this oil is
given by beta-carotene (Lichtenthaler and Buschmann, 2001).
XII
________________________________________________________________________________________
Fourier Transform Infrared Spectroscopy (FTIR) analysis of oils
FTIR studies of edible oils have proved the existence of relationships between
frequency and absorbance values of certain bands of the oil FTIR and the oil composition as
well as between some of these spectroscopic parameters and the oil oxidation level (Guillen,
M. D. and Cabo, N, 1997, 1998, 1999, 2000, 2002).
According to these spectra, we identified the relevant infrared frequencies (bands)
useful to and assign the specificity of the oils investigated ( Table 2.).
Table 2. Relevant infrared bands and assignments of the oils investigated
No.
Bands
Nr.
banda
HP
EVO
PK
SB
Functional group
Mode of vibration
HP
EVO
PK
SB
Grupul functional
Modul de vibratie
(cm-1)
(cm-1)
(cm-1)
(cm-1)
1
3008
3005
3008
3006
=C-H (cis-)
stretching
2
2956
2956
2956
2956
-C-H (CH3)
stretching (asymetric)
3
2923
2923
2923
2922
-C-H (CH2)
stretching (asymetric)
4
2853
2853
2854
2853
-C-H (CH2)
stretching (symetric)
5
1742
1742
1742
1742
-C=O (ester)
stretching
6
1654
1653
1653
1653
-C=C- (cis-)
stretching
7
1463
1464
1464
1464
-C-H (CH2, CH3)
8
1456
1456
9
1418
1417
1418
1417
10
1396
1402
1398
1402
11
1377
1377
1377
1377
1317
1319
12
1456
=C-H (cis-)
bending (rocking)
bending
-C-H (CH3)
bending (symmetric)
bending
13
1236
1238
1238
1238
-C-O, -CH2-
stretching, bending
14
1155
1159
1157
1161
-C-O, -CH2-
stretching, bending
15
1120
1118
1120
1116
-C-O
stretching
16
1097
1097
1099
1095
-C-O
stretching
17
1028
1028
1029
1033
-C-O
stretching
958
962
968
-HC=CH- (trans-)
bending out of plane
-HC=CH- (cis-)
bending out of plane
-(CH2)n-, -HC=CH(cis-)
bending (rocking)
18
19
914
20
721
914
721
721
721
XIII
________________________________________________________________________________________
Although oil spectra seem to be similar, they show differences in the intensity of their
bands as well as in the exact frequency at which the maximum absorbance is produced in each
case, due to the different nature and composition of the oil under study (see Fig. II.2.).
Fig.II.2. FTIR-ATR fingerprint spectra (1700-800 cm-1) of analyzed oils: Fingerprint oils:
HP= hemp, EVO (EOV) = extra virgin olive; PK= pumpkin; SB= seabuckthorn
Gas-Chromatography Determination of fatty acid profile
The composition of fatty acids analyzed by GC-FID in this study is shown in Table 3.
The fatty acid composition of the analyzed oils has been compared with the composition of
genuine oils reported in the literature or by the direct analysis of the genuine oils (Table 3.).
Table 3. Fatty acid composition (percentage) of the investigated vegetable
Fatty acid %
Hemp Oil
Extra virgin
Olive oil
Pumpkin oil
Seabuckthorn oil
Ulei de Canepă
Ulei Extra Virgin
de Măsline
Ulei de Dovleac
Ulei de Cătină
7.48
7.28
6.29
7.76
Acizi graşi %
Palmitic acid
(16:0)
XIV
________________________________________________________________________________________
Stearic acid (18:0)
1.66
2.67
3.64
0.3
Arachidic acid
(20:0)
1.06
-
-
0.11
Σ saturated %
10.02
9.95
9.93
8.17
-
-
-
5.4
Oleic acid
14.94
36.81
42.44
6.3
(C18:1)
Linoleic acid
(C18:2)
72.6
43.14
46.71
-
-
0.93
0.92
0.8
0.55
-
-
-
Σ unsaturated %
87.54
80.88
90.07
12.5
C18:1/C18:2
0.21
0.85
0.91
6.3
-
0.022
0.02
-
Palmitoleic
(C16:1)
Linolenic
(18:3n3)
Eicosadienoic acid
(C20:2)
omega 3 : omega
6 fatty acids
II.3. CONCLUSIONS
By GC-FID, the fatty acid composition of the analyzed oils has been determined and
compared with the composition of genuine oils reported in the literature or by the direct
analysis of the genuine oils. Regarding the content in fatty acids, GC-FID analysis revealed
that:
•
•
•
•
hemp oil composition does not agree with the literature for most of the fatty acids,
hemp oil contains lower values as the value reported. Oleic acid at least ranged
between the values mentioned.
primary fatty acids of extra virgin olive oil are oleic and linoleic acid with a small
amount of linolenic acid.
for pumpkin seed oil, the fatty acid composition is in good agreement with the profile
for most of the fatty acids, excepting palmitic and stearic acid found in lower
concentrations.
the fatty acids composition of seabuckthorn oil demonstrated that this oil is from
pulp/peel (whole) berries, being rich in palmitoleic acid and oleic acid.
XV
________________________________________________________________________________________
CHAPTER III. BIOENCAPSULATED OILS: BEADS PREPARATION PROTOCOLS
AND CHARACTERIZATION
III.1. MATERIALS AND METHODS
The following chemicals were used:
•
•
•
as matrices for encapsulation: alginate, k-carrageenan, chitosan, xanthan gum
and guar gum from Sigma Aldrich
the others solvents and reactants also from Sigma Aldrich
the oils used were purchased as we mentioned before.
Protocol for synthesis of empty beads of different sizes and concentrations
Different concentrations of alginate (1%, 1.5%, 2% w/v), mixture of: alginate and
carrageenan, alginate and xanthan gum, alginate and guar gum were dissolved in de-ionized
water stirred for ~ 30 minutes, different concentrations of chitosan (1%, 1.5%, 2% w/v) was
dissolved in acetic acid 0.7% v/v, than were dropped into a stirred hardening bath, using a
peristaltic pump with injector 0.4 x 20mm, and the beds were formed instantaneously.
After ~ 1h, the beads were separated from this hardening bath and were put on Petri dishes
for ‘’protection’’ and ‘’conservation’’.
Protocol for synthesis of beads of different sizes and concentrations incorporating small oil
droplets
Different solutions containing matrices obtained were used to prepare the mixtures
(emulsions) with oils; the mixtures were continuously stirred to maintain the emulsions. The
emulsions formed were dropped into the hardening bath, using a pipette for controlled
injection.
Taking into consideration the viscosity of the solutions obtained, were chosen the
combinations between this two matrices having not so high viscosity. First the emulsions
obtained, were evaluated microscopically and than were dropped into the hardening bath.
After ~ 1h, the beads were separated from this hardening bath and were put on Petri dishes
for ‘’protection’’ and ‘’conservation’’.
Microscopic evaluation of emulsions before encapsulation
Microscopic evaluation of emulsions before encapsulation was imaged using an
Olimpus optical microscope BXX1M equipped with a digital camera.
Beads Characterization: sizes and morphology, FTIR and thermal analysis
The obtained bead sizes, areas, perimeters, elongation and compactness were
measured using the UTHSCSA ImageTool software.
XVI
________________________________________________________________________________________
The surface morphology of freeze- and air-dried hydrogels was determined using a
scanning electron microscope (Hitachi S-2700, iMOXS, with BSE detector). Beads samples
were sputtered with gold and scanned at an accelerating voltage of 15 kV.
III.2. RESULTS AND DISCUSSIONS
Microscopic evaluation of emulsions before encapsulation
Stability of emulsions (including the composition and microstructure) is a key element
for evaluation of the lifetime and temperature conditions for the storage and use of emulsion
based products. The oil droplets sizes and shapes dispersed in the structure of matrices
dissolved were compared in order to evaluate the stability of the emulsions.
The drop size distributions of emulsions were determined by optical microscopy
associated to an image analysis technique. It was observed that oils droplets in emulsion
coalescence after a few minutes when the matrices concentrations increased (because no
emulsificator was used to help the emulsion formation), being necessary to drop it
immediately into the hardening bath. Different concentrations of matrices were used to
encapsulate the oils. The first evaluation of the solution of matrices dissolved was done.
The oils droplets were homogenized uniformly, they are smaller with the increasing of
matrix (Fig.III.1.). This demonstrated that the good oils encapsulation increased with the
increasing of matrix concentration.
A.
B.
C.
D.
Fig. III.1. Microscopic images with different emulsions using as matrices: A. alginate 2%; B.
alginate 1%; C. alginate-guar gum complex; D. alginate-xanthan gum complex. The scale bar
represents 5 μm.
XVII
________________________________________________________________________________________
Beads Characterization
After the emulsion was formed, it was extruded into the hardening bath and the gel
formed by the action of cross-linking agents. The beads containing functional oils were
almost spherical, and slightly yellowish, whereas those containing extra virgin olive oil,
pumpkin oil, hemp oil and were less transparent and light yellowish, and the beads containing
seabuckthorn oil were orange in color. This result was owing to original color presented in an
oil phase.
Parameter values/Valoarea parametrilor
AG
AG -C
-C AR
A
(
AG R ( 0.5
-X 0.7 :0.5
G 5: )
AG (0. 0.75
7
AG -X 5:0 )
-G G (0 .75
G
.5 )
AG (0. :0.
-G 75: 5)
G 0.7
(0 5)
.5
:
AG 0.5
)
AG oil2
oi %
l1
AG .5%
oi
C l1%
H
C oil2
Ho %
il1
C .5%
Ho
il1
%
Comparating all the matrices and concentration of matrices used to obtain beads with
oils encapsulated (Fig.III.2.), and taking into consideration all the characteristics of the
different beads containing different types of oils, most specially roundness and compactness,
which are two important characteristics in cosmetic and nutraceutical applications, and not to
forget elongation coefficient, the most suitable for oils encapsulation are: alginate 2%,
chitosan 2%, and alginate in complex with k-carrageenan, xanthan and guar gums in ratio
0.75:0.75.
9
8
7
Area / Aria (cm2)
6
5
4
3
2
1
Perimeter / Perimetru
Elongation (axes ratio)/
Elongatia (raportul axelor)
Roundness (up to 1) /
Sfericitatea val. max. 1
Diameter / Diametrul (cm)
0
Compactness (up to 1)/
Compactitatea (val. max. 1)
Samples/Probele
Fig.III.2. Comparative graphic representation of characteristics of alginate complex with kcarrageenan, xanthan and guar gums, alginate and chitosan beads obtained containing oil:
AG-CAR (0.5:0.5) = alginate-k-carrageenan (ratio 0.5:0.5) complex beads containing oil;
AG-CAR (0.75:0.75) = alginate-k-carrageenan (ratio 0.5:0.5) complex beads containing oil;
AG-XG (0.75:0.75) = alginate-xanthan gum (ratio 0.75:0.75) complex beads containing oil;
AG-XG (0.5:0.5) = alginate-xanthan gum (ratio 0.5:0.5) complex beads containing oil; AGGG (0.75:0.75) = alginate-guar gum (ratio 0.75:0.75) complex beads containing oil; AG-GG
(0.5:0.5) = alginate-guar gum (ratio 0.5:0.5) complex beads containing oil; AGoil2% =
alginate 2% beads containing oil; AGoil1.5% = alginate 1.5% beads containing oil; AGoil1%
= alginate 1% beads containing oil; CHoil2% = chitosan 2% beads containing oil; CHoil1.5%
= chitosan 1.5% beads containing oil; CHoil1% = chitosan 1% beads containing oil
XVIII
________________________________________________________________________________________
Scanning electron microscopy
The purpose of the scanning electron microscopy study was to obtain a topographical
characterization of beads.
The surface of beads obtained is non regular due to the oil droplets dispersed all over
the internal structure, except the chitosan beads which do not present such an irregular surface
(Fig.III.3.A and B.). The SEM pictures of beads revealed that the surfaces were found to be
non porous.
B.
A.
Fig.III.3. Scanning electron micrographs of external structure of different beads containing
oils: A. alginate-carrageenan complex; B. chitosan. The scale bars are shown on the
individual photographs. Magnification 70x.
FTIR analysis
FTIR Characterization of matrices
By FTIR-ATR spectra we were able not only to identify the main wave numbers
specific to free matrices (AG, CAR, CH, GG, XG) and to discriminate later the differences
when oils were free or incorporated. The wave numbers useful for matrices discriminations
were identified at 3244-3302 cm-1 (O-H stretch), 1400-1474 cm-1 (CH2 bending), 1000-1200 1
(C-O and C-C stretch), 924-1000 cm-1 ( poly OH and CH2 twist), 776-892 cm-1(glycoside
links).
To summarize, FTIR spectroscopy can discriminate between the different matrices:
Functional group and
vibration
AG
CAR
GG
XG
CH
O–H stretching vibration
3244
3514
3299
3302
3289
PolyOH groups
O-H +
N-H strech
C–H stretching of CH2 group
2926
2953, 2911, 2894
2884
-
2935
C-O stretching ( COOH)
1597
-
1636
-
1651
XIX
________________________________________________________________________________________
Deformations of CH2 group (
bending)
1408
1474, 1400
1408
1400
1428
O-H bending
-
1223 ( S=O strech
sulphate ester
1350
1247
-
C-O and C-C ring stretching
1200-1000
-
1145
1150
1151
–CH2OH stretching mode
1054
1063
1054
C–OH alcoholic
1024
1024
-
1025
1024
948, 902,
924, 910
1016
-
-
Gululonic &
mannuronic
Polyhydroxy groups
809
842
866,777
892,
Galactose sulphate,
glycosidic link
(1,4; 1,6) link
galactose
785 C-H
rocking,
bending
1061
(C-O stretching saccharide)
–CH2 twisting vibration
Glycosidic links
and mannose
776
C-C stretching
FTIR characterization of different beads containing oils
The spectra of empty beads obtained, beads containing oils and free oils were
recorded. Matrices concentrations did not the affected the FTIR-ATR characteristics peak
intensities. As example in Fig.III.4. are shown FTIR-ATR spectra of SB oil and alginate 2%
beads containing SB oil.
The encapsulation of SB oil in alginate induces the decrease of absorbance intensity at
3400 cm-1(which was proportional with the increase of alginate percentage) and shifts of
absorbance peaks to lower-wavenumbers in the region 1000-1500 cm-1 specific to the
encapsulated SB oil comparing with the free SB oil.
By FTIR-ATR analysis, mixture of oils and different blank beads showed the peaks
attributable to both oils and empty beads. This confirms the oils entrapment into the beads at
the molecular level, the oil specific double peaks (regions between 2800-2900 cm-1 and 1700900 cm-1) which are present also in the free oils.
XX
________________________________________________________________________________________
Fig.III.4. FTIR-ATR spectra of: A. alginate 2% beads containing SB oil; B. alginate powder;
C. SB oil; D. alginate 2% beads empty
Thermal analysis
DSC measurements
The DSC thermograms of the free functional oils as well as alginate beads containing
oils, alginate/k-carrageenan, alginate-guar gum and alginate-xanthan complex beads, and
chitosan beads containing oils were measured.
Some endothermal peaks of seabuckthorn oil and beads containing seabuckthorn oil
are shown in Fig.III.5.; the peaks temperature increased with the increasing of matrices
concentration, and for each matrice is a characteristic endothermal peak.
Thermogravimetric analysis
The TGA thermograms of the free functional oils as well as alginate beads containing
oils, alginate/k-carrageenan, alginate-guar gum and alginate-xanthan complex beads, and
chitosan beads containing oils were measured.
As is shown in Fig.III.6., which is the graphic representation of restmass% of some
samples, the peaks temperature increased with the increasing of matrices concentration, this is
due to the high content of the beads water. Oils do not influence so much the restmass% of the
capsules.
XXI
________________________________________________________________________________________
200
180
160
Temperature (°C)
140
120
100
80
60
40
20
0
AG 2%
AG
1.5%
Alginate AG-CAR CH 2%
1%
(0.75%)
CH 1%
AG-GG
AG-KG
SB
Fig. III.5. Graphic representation of DSC endothermic peaks of some samples
DSC and TGA has been widely applied in the monitoring of oxidative stability,
thermal behavior, kinetic parameters in various oil samples (Jayadas et al., 2006; Milovanovic
et al., 2006; Bahruddin et al., 2008). The oxidative decomposition of saturated fatty acids
according with literature showed weight loss before 380°C (Bahruddin et al., 2008). Because
on this study the highest temperature of thermal analysis measurements has been 300°C, is
not taking into consideration this aspect regarding monitoring oxidative stability. This should
be an explanation why the analyzed oils did not loss so much weight during thermal
measurements, according with literature weight loss % should be more than 10% depending
on the oil sample (Jayadas et al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008).
120
Restmass %
100
80
60
40
20
0
AG 2%
AG 1.5%
AG-CAR
(0.75%)
AG-GG
AG-KG
SB
Fig.III.6. Graphic representation of restmass% of some samples of TGA analysis
XXII
________________________________________________________________________________________
The aim of this study regarding the thermal measurements was to analyze the thermal
behavior and to check the stability of beads containing different functional oils obtained in the
context of their further applications on food or cosmetic. For this purpose it is know that in
most of the cases especially on food field the products are sterilize or are expose to high
pressures treatments in order to avoid the biohazard or the contamination, these treatments
being done during technological process.
III.3. CONCLUSIONS
Our experimental studies using the ionotropically crosslinked gelation to
microencapsulate functional oils into natural matrices demonstrates which the best
technological conditions are in order to assure stable beads and controlled conditions of
bioactive molecules release.
Are considered to be the best concentrations from all tested as suitable for oils
encapsulation: alginate and chitosan 2%, 1.5% and 1%, complexes of alginate with kcarrageenan, xanthan and guar gums in ratio concentrations of 0.75:0.75.
The results show that the amount of oil encapsulated in different matrices affected the
mean diameter of the beads. The size of the gel beads increased with the amount of oil
encapsulated. Also the other characteristics of capsules (area, perimeter, roundness and
elongation) chanced after oil encapsulation.
By FTIR-ATR analysis, mixture of oils and different blank beads showed the peaks
attributable to both oils and empty beads. This confirms the oils entrapment into the beads at
the molecular level, the oil specific double peaks (regions between 2800-2900 cm-1 and 1700900 cm-1) which are present also in the free oils.
CHAPTER IV. ENCAPSULATION EFFICIENCY AND RELEASE STUDIES
IV.1. MATERIALS AND METHODS
Encapsulation efficiency of the beads
The oils encapsulation was determined calculating the amount of β-carotene or total
carotenoids content of each oil analyzed before and after encapsulation. The samples were
assayed for β-carotene or total carotenoids content of each oil according previous analysis
when was identified the UV-Vis fingerprint, spectrophotometrically.
Encapsulation efficiency (EE%) was calculated by using formulae:
EE% = C1/C2 x XL0,
C1= carotenoid concentration in the oil
C2= carotenoid concentration after release from beads
Also from the hardening baths, after encapsulation process, were extracted the
carotenoids with THF for a better efficiency calculation.
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Release rate measurements of oil from beads
Control release of carotenoids contents in the oils from beads were measurements
spectrophotometrically. The absorption spectra were obtained in a CarWin X0 UV-VIS
spectrometer. All measurements were performed with the substances inside a 2 mm long
quartz glass cuvette. All spectra were recorded at room temperature and the results are the
average of 3 runs.
In vitro release oils from the beads
The scheme of using the artificial simulated fluids at different pH was as follows:
• 1st hour: simulated gastric fluid of pH 1.2
• 2nd to 3rd hour: mixture of simulated gastric and intestinal fluid of pH 4.5
• 4th to 7th hour: simulated intestinal fluid of pH 7.4
In vitro oil release studies were performed as per scheme in different simulated fluids.
Simulation of gastrointestinal (GI) transit conditions was achieved by using different
dissolution media.
Simulated gastric fluid (SGF) pH 1.2 consisted of 0.1N HCl and X ml Sanzyme (enzyme
syrup containing 80 mg papain, 40 mg pepsin and XL mg sanzyme 2000); pH adjusted to 1.2
±0.1.
Simulated intestinal fluid (SIF) pH 4.5 was prepared by mixing SGF pH 1.2 and SIF pH
XX.4 in a ratio 3XX:61; pH adjusted to 4.X ±0.1.
Simulated intestinal fluid (SIF) pH 7.4 consisted of KH2PO4 1.0XX4g in 30 ml of 0.2N
NaOH, and pancreatin 2XXX mg (using “Triferment”); pH adjusted to XX.4 ±0.1.
The experiment was performed into an incubator with a continuous supply of carbon
dioxide at 37ºC.
IV.2. RESULTS AND DISSCUSIONS
Encapsulation efficiency of the beads
UV-Vis analysis of the extracts from hardening baths, did not show significant values. In
the cases of low encapsulation efficiency the absorbance values were ranging from 0.0001 to
0.0003, we can say that the efficiency encapsulation is enough to be calculated using formulae
mentioned before.
According with formulae described on Material and Methods, the encapsulation efficiency
is presented on the following table (Fig.IV.1.) for the different types of beads, and related to
each oil. The dates presented represent the average of values for the same beads and different
oils.
XXIV
________________________________________________________________________________________
Increasing the concentration of matrices or complex matrices the better encapsulation
efficienciens were obtained.
The best concentrations, of all matrices and complex of matrices used, as is shown in
the graphical comparation in Fig.IV.1. to get the bet encapsulation efficiency were using
alginate in concentration 2%, chitosan in concentration 2%, following concentration of 1.5%
from these matrices, and alginate in complex with k-carrageenan and gums in ratio
0.75:0.75%.
Fig.IV.1. Comparative graphic representation of encanspuation efficiency of oils in alginate
complex with k-carrageenan, xanthan and guar gums, alginate and chitosan beads obtained
AG2% = alginate 2% beads; CH2% = chitosan 2% beads ; CH1.5% = chitosan 1.5% beads ;
AG1.5% = alginate 1.5% beads; AG-CAR (0.75:0.75) = alginate-k-carrageenan; AG-XG
(0.75:0.75) = alginate-xanthan gum (ratio 0.75:0.75) complex beads ; CH1% = chitosan 1%
beads; AG-GG (0.75:0.75) = alginate-guar gum (ratio 0.75:0.75) complex beads; AG1% =
alginate 1% beads; AG-CAR (0.5:0.5) = alginate-k-carrageenan (ratio 0.5:0.5) complex
beads; AG-GG (0.5:0.5) = alginate-guar gum (ratio 0.5:0.5) complex beads; AG-XG (0.5:0.5)
= alginate-xanthan gum (ratio 0.5:0.5) complex beads
Release rate measurements of oil from beads in organic solvents
As an example, the influence of matrix concentration on release rate and the same
swelling property of the alginate-carrageenan complex (ratio 0.75:0.75) beads containing SB
oil in methanol, hexane and THF are shown in the graphic representation of Fig.IV.2. The
best release of the oil was obtained from the alginate beads or alginate complexes with kcarrageenan and gums, comparing with a slower release of the oil from chitoan beads.
Under these conditions the release rate was substantially slower in hexane than in the
case of the methanol and the best release was obtained into THF for the all different type of
XXV
________________________________________________________________________________________
beads obtained. THF was demonstrated to be one of the best solvent to extract carotenoides,
and this example confirmed the same expectations, but because is considerated a very toxic
solvent, is impossible to use it in cosmetic field. The release rate depends of the diffusivity
and solubility of the oil in the matrix, and the swelling collapse transition in the gel.
3
Absorbance (a.u.)
2.5
2
Methanol
Hexane
1.5
THF
1
0.5
0
0
50
100
150
200
250
Time (minutes)
300
350
400
Fig.IV.2. Graphic representation of the absorbance values of seabuckthorn oil release in time
at 445 (methanol and hexane) and 454 nm (THF) from different from alginate-carrageenan
complex (ratio 0.75:0.75) fresh beads into: methanol, hexane and THF
Release rate of oil from the beads showed that the alginate, alginate-k-carrageenan
complexe and comples with gums, and chitosan are suitable microencapsulation matrices for
oils.
In vitro artificial simulated release oils from the beads
The swelling volumes of the alginate and alginate complex beads with guar gum and
xanthan gum increased at higher pH. The swelling volume at pH 7.4 was higher than at pH
1.2 or pH 4.X. Higher swelling at higher pH condition suggest that the calcium alginate ionic
interaction was reduced at high pH, Na+ ions will displace Ca++ ions leading to lowering the
concentration of Ca++ ions in the beads.
Therefore, at high pH condition the swelling volumes increased, and the beads dissolved
in media with/without enzyme. Chitosan beads did not increase in volume or dissolve like
alginate or alginate complex beads with guar gum and xanthan gum, suggesting higher
strenghtness under tested conditions (Fig.IV.3.).
XXVI
________________________________________________________________________________________
A.
B.
C.
D.
Fig.IV.3. In vitro seabuckthorn oil release from alginate 2% beads from left to right in
each picture the stimulated fluids without enzymes and containing enzymes: A. fresh
beads; B. after 1st hour in simulated gastric fluid of pH 1.2; C. after 3rd hours in mixture of
simulated gastric and intestinal fluid of pH 4.5; D. in simulated intestinal fluid of pH 7.4
after 30 minutes
IV.3. CONCLUSIONS
The studies regarding encapsulation efficiency and stability of oils containing beads show:
1. Increasing the concentration of matrices or complex matrices improved the
encapsulation efficiency was obtained. The best concentrations, of all matrices and
complex of matrices used, to get the best encapsulation efficiency, were using alginate
in concentration 2%, chitosan in concentration 2%, following concentration of 1.5%
from these matrices, and alginate in complex with k-carrageenan and gums in ratio
0.75:0.75%.
2. The release rate depends of the diffusivity and solubility of the oil in the matrix, and
the swelling collapse transition in the gel.
3. The release rate was substantially slower into hexane than into methanol and the best
release was obtained into THF for the type of beads obtained.
4. In vitro oil release studies shown that capsules from alginate, and alginate in complex
with carrageenan and gums are completely dissolved at pH 7.4, chitosan beads being
not.
XXVII
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CHAPTER V. FTIR CHARACTERIZATION OF OIL OXIDATION
V.1. MATERIALS AND METHODS
The FTIR spectra were obtained with a Fourier transform spectrometer Spectrum One
(PerkinElmer), equipped with the universal ATR as an internal reflection accessory which
have Composite Zinc Selenide (ZnSe) and Diamond crystals. Each spectrum was from 4000
to 6X0 cm-1. Between measurements the crystal was cleaned with acetone.
The oxidation process under UV light on time (after 1h, 4h and 6h) was done using an
UV lamp (2X4 μm), each oil an all obtained beads containing oils were exposed under these
conditions.
V.2. RESULTS AND DISCUSSIONS
The oxidation process under UV light (2X4 μm) on time (after 1h, 4h and 6h) was
monitored calculating the ratios between absorbance of some bands of the spectra of free oil,
according with literature (Guillén and Cabo, 1999, 2000, 2002) and encapsulated oil in
different type of beads obtained: A2853/A3005, A1746/A3006, A1474/A3006, A1377/A3006 and
A1163/A3006, before and after treatment under UV. The values are given for these ratios could
be considered as indicative parameters of the oxidation level of different kinds of oils.
All oils free obtained values showed SS or TS stage oxidation, comparing with the
values of oils encapsulated in FS stage of oxidation (see as an example Fig.V.1., the oxidation
on time of HP oil free and encapsulated).
The best protection from all this concentrations used against UV treatment was found
to be alginate 1%, chitosan 1.5%, alginate-guar gum and alginate-xanthan gum complexs in
ratio 0.5:0.5, and alginate-k-carrageenan complex in ratio 0.75:0.75.
XXVIII
Ratio values/Valoarea raportelor
________________________________________________________________________________________
8
7
Oil free/Ulei liber
6
5
4
3
Oil from AG 1.5%/Ulei din AG 1.5%
2
1
0
A
B C
D
E
A
B C
D
E
A
B C D
E
After 1h UV/Dupa 1h After 4h UV/Dupa 4h After 6h UV/Dupa 6h
UV
UV
UV
Types of ratios on time/Tipul rapoartelor in timp
Fig. V.1. Graphic representation of the hemp oil free and encapsulated (in different alginate
concentrations beads) under oxidation changes
(A= A2853/A3005-3008, B= A1744/ A3005-3008, C= A1464/ A3005-3008, D= A1377/ A3005-3008, E= A1160/
A3005-3008)
V.3. CONCLUSIONS
The usefulness of absorbance ratios and frequency data to measure the oxidative
stability and oxidation degree of encapsulated oils directly into the beads was studied.
All free oils show SS or TS stage oxidation, compared with the values of encapsulated
oils in FS stage of oxidation.
The best protection against UV treatment was found to be alginate 1%, chitosan 1.5%,
alginate-guar gum and alginate-xanthan gum complexs in ratio 0.5:0.5, and alginate-kcarrageenan complex in ratio 0.75:0.75.
FTIR spectroscopy has been found to be a versatile technique for evaluating the
oxidative stability of oils free and encapsulated, and for providing information on the
oxidation degree of an oil sample in a simple, fast and accurate way.
XXIX
________________________________________________________________________________________
GENERAL CONCLUSIONS
According to the aims and objectives of this PhD thesis, we succeeded to
bioencapsulate four different oils extracted from plants using different natural matrices, and to
evaluate the encapsulation efficiency, stability and release of these from the beads obtained.
We analyzed four different oils, hemp oil (HP), extra virgin olive oil (EVO), pumpkin
oil (PK) and seabuckthorn oil (SB) (provided from Romanian industry or from Italy). Before
being encapsulated, these oils were analyzed and then.
In agreement with the objectives proposed, our results can be summarized as
follows (conclusions I-V):
I. We identified the oil characteristics, before to be encapsulated, establishing
their quality and authenticity markers :
1. Majority of analysed oils had similar iodine values as specified in CODEX 210,
except the seabuckthorn oil which had a lower iodine value compared with the
specification.
2. The UV-Vis spectra of the oil samples showed their specific peak position and
intensity, as markers of authenticity.
3. The FTIR-ATR studies of analyzed oils proved the relationships existing between
frequency and absorbance values of certain absorption bands and the oil composition,
establishing their fingerprint.
4. The GC-FID analysis revealed that composition of genuine oils reported in the
literature or by the direct analysis of the genuine oils.
II. Our experimental studies using the ionotropically crosslinked gelation to
bioencapsulate functional oils into natural matrices demonstrates which are the best
technological conditions in order to assure stable beads and controlled conditions of
bioactive molecules release.
1. We succeeded to obtain different beads using matrices as alginate and different
complexes between alginate and k-carrageenan and different gums, including the four oils by
the gellation mechanism.
2. The size of the gel beads increased as the amount of oil used.
3. The other characteristics of capsules analyzed show changes after oil encapsulation
(area, diameter, perimeter, elongation, compactness, roundness). Especially roundness and
compactness, are the two important bead characteristics for cosmetic and nutraceutical
applications,
4. The best concentrations of matrices to encapsulate oils encapsulation alginate 2%,
chitosan 2%, and alginate in complex with k-carrageenan, xanthan and guar gums in ratio
0.75:0.75.
XXX
________________________________________________________________________________________
III. Characterization of microcapsules was made by different and complementary
methods: SEM, FTIR, DSC, TGA analysis
1. The surface of beads obtained by SEM is non regular due to the oil droplets dispersed
all over the internal structure, except the chitosan beads which do not present such an
irregular surface
2. By FTIR-ATR analysis, mixture of oils and different blank beads showed the
differences in fingerprinting empty and oil-containing beads.
3. The DSC thermograms of the free functional oils as well as oil-containing beads
showed that the phase transition temperature increases with the matrix concentration into the
bead, and each matrix has characteristic endothermal peak.
4. TGA analysis showed that the restmass % of the samples and the peaks temperature
increased with the increase of matrix concentration, due to the high content of the beads
water. Oils do not influence so much the restmass% of the capsules.
IV. Evaluation of encapsulation efficiency
1. The best concentration of matrix into capsules proved to be 2% , either using alginate or
chitosan, better than 1,5% and alginate in complex with k-carrageenan and gums in ratio
0.75:0.75%.
2. The release rate depends on the diffusivity and solubility of the oil in the matrix, and
the swelling collapse transition in the gel. The release rate was substantially slower in hexane
than into methanol and the best release was obtained into THF for the all different type of
beads obtained.
3. In vitro oil release studies shown that capsules from alginate, and alginate in complex
with carrageenan and gums are completely dissolved at pH 7.4, excepting chitosan beads.
V. Protective action of bioencapsulation against oil oxidation by UV
1. Ratios between absorbance of different bands of the FTIR spectra were indicators of
oils oxidation, and of stages of the oxidation. The best protection against UV treatment was
found to be alginate 1%, chitosan 1.5%, alginate-guar gum and alginate-xanthan gum
complexs in ratio 0.5:0.5, and alginate-k-carrageenan complex in ratio 0.75:0.75.
XXXI
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XXXIV
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PUBLICATIONS RELEASED DURING PhD
2009
1. Socaciu C., Trif. M, A. Baciu, T. Nicula, A. Nicula, ‘’ Encapsulation of plant oleosomes
and oleoresins in mixed carbohydrate matrices’’, COST865, Spring
Meeting
"Microcapsule property assesment", Luxemburg 2009, Proceeding
2008
1. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. „Bioencapsulated
seabuckthorn oil: controlled release rates in different solvents”, Bull. USAMV-CN,
65/2008, ISSN 1454-2382, Romania
2. Pece Aurelia, D. Vodnar, Monica Trif, C. Coroian, Camelia Raducu, G. Muresan,
“Study of the physico-chemical parameters from buffalo raw milk during different
lactations”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania
3. Pece Aurelia, D. Vodnar, Monica Trif, “Corelation between microbiological and
physico-chemical parameters from buffalo raw milk during different lactations”, Bull.
USAMV-CN, 65/2008, ISSN 1454-2382, Romania
4. Carmen Socaciu, Baciu A., Trif M., “Oleosome-rich pectin network as a new, natural
bioencapsulation matrix”, XVI International Conference on Bioencapsulation Dublin,
Ireland ; September 2008, Proceeding
5. Monica Trif, Carmen Socaciu, Andreea Stanila, “The evaluation of encapsulated
Seabuckthorn oil properties usind FTIR”, CIGR - International Conference of
Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola,
Processing Conference - 4th CIGR Section VI International Symposium On Food And
Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797
6. Andreea Stanila and Monica Trif, “Antioxidant activity of carotenoide extracts from
HIPPOPHAE RHAMNOIDES”, CIGR - International Conference of Agricultural
Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola, Processing
Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess
Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797
7. Monica Trif, Carmen Socaciu and Horst Diehl, “Evaluation of effiency, release and
oxidation stability of seabuckthorn encapsulated oil using FTIR spectroscopy”, 7th Joint
Meeting of AFERP, ASP, GA, PSE & SIF, August 2008, Athens, Greece, Book of
Abstracts, pg.39
8. Monica Trif and Carmen Socaciu, “Evaluation of effiency, release and oxidation
stability of Seabuckthorn microencapsulated oil using Fourier Transformed Infrared
Spectroscopy”, 4th Meeting on Chemistry and Life, and accepted to be published in
Chemické Listy Journal (current IF=0.683)
XXXV
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2007
1. Monica Trif, Marion Ansorge-Schumacher, Veronica S. Chedea, Carmen Socaciu,
‘’Release rates measurement of encapsulated castor oil using alginate as
microencapsulation matrix’’, The International Conference on Nanotechnology: Science
and Application (NanoTech Insight), Luxor, 10-17 March 2007, Egipt
2. Chedea V.S., Kefalas P., Trif M. and Socaciu C. ‘’Stability studies of encapsulated
carotenoid extract from orange waste using pullulan as microencapsulation matrice’’,
Nano Tech Insight, Luxor, 10-17 March 2007, Egipt
3. Monica Trif, Marion Ansorge-Schumacher, Carmen Socaciu, ‘’Application of FTIR
Spectroscopy for determination of oxidation of encapsulated sea buckthorn oil’’,
Proc.XV International workshop on Bioencapsulation and COST865 Meeting, 2007,
Wien, Austria, published in extenso
4. Carmen Socaciu, Cristina Mihis, Monica Trif, Horst A. Diehl, ‘’Seabuckthorn fruit
oleosomes as natural, microencapsulated oilbodies: separation, characterization, stability
evaluation oil’’, Proc. XV International workshop on Bioencapsulation and COST865
Meeting, 2007, Wien, Austria, published in extenso
5. Socaciu C., Trif M., Ranga F., Fetea F., Bunea A., Dulf F., Bele C. and Echim C.
‘’Quality and authenticity of seabuckthorn oils using succesive UV-Vis, FT-IR, NMR
spectroscopy and HPLC-, GC- chromatography fingerprints’’, 3rd Conf. Int.
Seabuckthorn Assoc., 2007, Quebec, Canada
6. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. ‘’Determination of
encapsulated Sea buckthorn oil oxidation using FTIR-ATR spectroscopy’’, Bull.
USAMV-CN, 63-64/2007, ISSN 1843-5262, Romania
2006
1. Monica Trif, “Seabuckthorn oleosomes as stabilized bioactive nanostrustures with
applications in microencapsulation nutraceuticals”, Symposium IRC Transylvania
“Innovations in Agriculture, Biotechnologies, Animal Breeds and Veterinary Medicine”,
2006, USAMV Cluj-Napoca, Romania
2004
1. Veerle Minne, Monica Trif, J.M.C. Geuns, Corina Catana, “Steviozide and steviol
determination in callus culture of Stevia rebaudiana Bertoni”, Bull. USAMV-CN,
61/2004, ISSN 1454-2382, Romania
XXXVI

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