REZUMATUL - USAMV Cluj-Napoca

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 

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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. 

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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: 

Utilizarea diferitelor matrici naturale (precum alginatul, alginatul în complex cu k- 

caragenan 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) 

Îmbunătăţirea şi optimizarea metodelor de bioîncapsulare pentru uleiurile vegetale cu 

proprietăţi funcţionale 

Investigarea morfologiei diferitelor capsule obţinute (microscopie electronică de 

scanare), caracterizarea capsulelor (suprafaţă, diametru, perimetru, elongaţie, 

sfericitate şi compactitate), analize FTIR 

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. 

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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 

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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 

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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 (Mg 2+


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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). 

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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 

Aciditate 

(mg KOH/g ulei) 

Ulei de Cânepa Ulei de măsline 

extra virgin 

Caracteristicile fizice si chimice 

Ulei de dovleac Ulei de cătină 

1.93 / 4.0* 2.64 / 6.6* 1.32 / 4.0* 3.7 / 4.0* 

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. B 

C. 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.). 

Nr. 

banda 

HP 

(cm -1 ) 

EOV 

(cm -1 ) 

Tabel.II.2. Benzile infraroşu relevante ale uleiurilor investigate 

PK 

(cm -1 ) 

SB 

(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) 



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 1456 

9 1418 1417 1418 1417 =C-H (cis-) de deformare 

10 1396 1402 1398 1402 de deformare 

11 1377 1377 1377 1377 -C-H (CH3) de deformare (simetrică) 

12 1317 1319 de deformare 

13 1236 1238 1238 1238 -C-O, -CH2- de întindere, de deformare 

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14 1155 1159 1157 1161 -C-O, -CH2- de întindere, de deformare 

15 1120 1118 1120 1116 -C-O de întindere 



18 958 962 968 -HC=CH- (trans-) de deformare înafara 

planului 

19 914 914 -HC=CH- (cis-) de deformare înafara 

planului 

20 721 721 721 721 -(CH2)n-, -HC=CH- 

(cis-) 

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


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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. 

Acizi grasi % 

Tabel.II.3. Compoziţia procentuală a acilor graşi din uleiurile analizate 

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 

Palmitoleic 

(C16:1) 

Oleic 

(C18:1) 

Linoleic 

(C18:2) 

Linolenic 

(18:3n3) 

Eicosadienoic 

(C20:2) 

- 

14.94 

72.6 

- 

0.55 

- 

36.81 

43.14 

0.93 

- 

- 

42.44 

46.71 

Σ nesaturati % 87.54 80.88 90.07 12.5 

C18:1/C18:2 0.21 0.85 0.91 6.3 

omega 3 : omega 

6 acizi grasi 

II.3. CONCLUZII 

0.92 

- 0.022 0.02 - 

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 

- 

5.4 

6.3 

- 

0.8 

- 

XIV


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• 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: PROTOCOALE DE 

PREPARE A CAPSULELOR ŞI CARACTERIZAREA LOR 

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. 

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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


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A. B. 

C. D. 

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. 

Parameter values/Valoarea parametrilor 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

AG-CAR (0.5:0.5) 

AG-XG (0.75:0.75) 

AG-CAR (0.75:0.75) 

AG-XG (0.5:0.5) 

AG-GG (0.75:0.75) 

AG-GG (0.5:0.5) 

Samples/Probele 

AGoil2% 

AGoil1.5% 

AGoil1% 

CHoil2% 

CHoil1.5% 

CHoil1% 

Area / Aria (cm2) 

Perimeter / Perimetru 

Elongation (axes ratio)/ 

Elongatia (raportul axelor) 

Roundness (up to 1) / 

Sfericitatea val. max. 1 

Diameter / Diametrul (cm) 

Compactness (up to 1)/ 

Compactitatea (val. max. 1) 

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 

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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.). 

A. B. 

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: 3244- 

3302 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 

O–H intindere 3244 3514 

AG CAR GG XG CH 

grupul poliOH 

3299 3302 3289 

O-H + 

N-H de 

întindere 

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________________________________________________________________________________________ 

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 1061 

Gruparea C–OH alcolică 

(C-O întinderea zaharidelor) 

–CH2 vibraţie 948, 902, 

1024 1024 - 1025 1024 

Provenite de la 

acizii: guluronic 

şi maluronic 

924, 910 

Grupările 

polihidroxi 

legaturile glicozidice 809 842 

Sulfatul galactozic, 

legatura glicozidică 

FTIR characterization of different beads containing oils 

1016 - - 

866,777 

(1,4; 1,6) legatura 

galactozei şi 

manozei 

785 

C-H de 

deformare 

C-C întindere 

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. 

892, 

776 

XIX


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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. 

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Temperature (°C) 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

AG 2% AG 

1.5% 

Alginate 

1% 

AG-CAR 

(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). 

Restmass % 

120 

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 

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________________________________________________________________________________________ 

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 

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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


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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


________________________________________________________________________________________ 

A. 

B. 

Absorbance (u.a.) 

Absorbance (a.u.) 

0.4 

0.35 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

0 

0 100 200 

Wavelenght (nm) 

300 400 

0 100 200 

Wavelenght (nm) 

300 400 

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 

Alginate-carrageenan 

complex in hexane 

Alginate-carrageenan 

complex in methanol 

Alginate 2% in methanol 

Alginate 2% in hexane 

Chitosan 1.5% in methanol 

Chitosan 1% in methanol 

Chitosan 1% in hexane 

Chitosan 1.5% in hexane 

Alginate 2% in methanol 

Alginate 2% in hexane 

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


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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


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Ratio values/Valoarea raportelor 

8 

7 

6 

5 

4 

3 

2 

1 

0 

A B C D E A B C D E A B C D E 

After 1h UV/Dupa 1h 

UV 


UV 


UV 

Types of ratios on time/Tipul rapoartelor in timp 

Oil free/Ulei liber 

Oil from AG 1%/Ulei din AG 1% 

Oil from AG 1.5%/Ulei din AG 1.5% 


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


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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 

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XXXIII


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2009 

PUBLICAŢII PE DURATA STAGIULUI DOCTORAL SI PARTICIPARI LA 

SIMPOZIOANE ŞI CONFERINŢE NATIONALE ŞI INTERNAŢIONALE 

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 - 4 th 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 - 4 th 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”, 7 th 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’’, 3 rd 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: 

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) 

Improvement and optimization of bioencapsulation methods for vegetable oils with 

functional properties 

Investigations of different obtained beads: morphology (scanning electron 

microscopy), characterization of beads (area, diameter, perimeter, elongation, 

compactness), Fourier transform infrared spectroscopy (FTIR) analysis 

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 Ansorge- 

Schumacher. 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 (Mg 2+


________________________________________________________________________________________ 

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 

Acid value + 

(mg KOH/g Oil) 

Aciditatea + 

(mg KOH/g ulei) 

Iodine value 

Indicele de iod 

Hemp 

Oil 

Ulei de Canepa 

Extra Virgin Olive 

Oil 

Ulei de Măsline 

Extra Virgin 

Chemical and physical characteristics 

Caracteristicile chimice si fizice 

Pumpkin 

Oil 

Ulei de Dovleac 

Sea buckthorn 

Oil 

Ulei de Cătina 

4.0 6.6 4.0 4.0 

145-166** 75-94** 116-133** 98-119‡ 

+ 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 

C. D. 

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


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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.). 

No. 

Bands 

Nr. 

banda 

HP 

HP 

(cm -1 ) 

Table 2. Relevant infrared bands and assignments of the oils investigated 

EVO 

EVO 

(cm -1 ) 

PK 

PK 

(cm -1 ) 

SB 

SB 

(cm -1 ) 

Functional group 

Grupul functional 

1 3008 3005 3008 3006 =C-H (cis-) stretching 

Mode of vibration 

Modul de vibratie 

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 1456 

9 1418 1417 1418 1417 =C-H (cis-) bending (rocking) 

10 1396 1402 1398 1402 bending 

11 1377 1377 1377 1377 -C-H (CH3) bending (symmetric) 

12 1317 1319 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 

18 958 962 968 -HC=CH- (trans-) bending out of plane 

19 914 914 -HC=CH- (cis-) bending out of plane 

20 721 721 721 721 -(CH2)n-, -HC=CH- 

(cis-) 

bending (rocking) 

XIII


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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.). 

Fatty acid % 

Acizi graşi % 

Palmitic acid 

(16:0) 

Table 3. Fatty acid composition (percentage) of the investigated vegetable 

Hemp Oil 

Ulei de Canepă 

Extra virgin 

Olive oil 

Ulei Extra Virgin 

de Măsline 

Pumpkin oil 

Ulei de Dovleac 

Seabuckthorn oil 

Ulei de Cătină 

7.48 7.28 6.29 7.76 

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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 

Palmitoleic 

(C16:1) 

Oleic acid 

(C18:1) 

Linoleic acid 

(C18:2) 

Linolenic 

(18:3n3) 

Eicosadienoic acid 

(C20:2) 

- 

14.94 

72.6 

- 

0.55 

- 

36.81 

43.14 

0.93 

- 

- 

42.44 

46.71 

Σ unsaturated % 87.54 80.88 90.07 12.5 

C18:1/C18:2 0.21 0.85 0.91 6.3 

omega 3 : omega 

6 fatty acids 

II.3. CONCLUSIONS 

0.92 

- 0.022 0.02 - 

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. 

- 

5.4 

6.3 

- 

0.8 

- 

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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. 

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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. 

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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. 

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. 

Parameter values/Valoarea parametrilor 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

AG-CAR (0.5:0.5) 

AG-CAR (0.75:0.75) 

AG-XG (0.75:0.75) 

AG-XG (0.5:0.5) 

AG-GG (0.75:0.75) 

AG-GG (0.5:0.5) 

AGoil2% 

Samples/Probele 

AGoil1.5% 

AGoil1% 

CHoil2% 

CHoil1.5% 

CHoil1% 

Area / Aria (cm2) 

Perimeter / Perimetru 

Elongation (axes ratio)/ 

Elongatia (raportul axelor) 

Roundness (up to 1) / 

Sfericitatea val. max. 1 

Diameter / Diametrul (cm) 

Compactness (up to 1)/ 

Compactitatea (val. max. 1) 

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; AG- 

GG (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 

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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. 

A. B. 

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 

O–H stretching vibration 3244 3514 

AG CAR GG XG CH 

PolyOH groups 

3299 3302 3289 

O-H + 

N-H strech 

C–H stretching of CH2 group 2926 2953, 2911, 2894 2884 - 2935 

C-O stretching ( COOH) 1597 - 1636 - 1651 

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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 1061 

C–OH alcoholic 

(C-O stretching saccharide) 

–CH2 twisting vibration 948, 902, 

1024 1024 - 1025 1024 

Gululonic & 

mannuronic 

924, 910 

Polyhydroxy groups 

Glycosidic links 809 842 

Galactose sulphate, 

glycosidic link 

FTIR characterization of different beads containing oils 

1016 - - 

866,777 

(1,4; 1,6) link 

galactose 

and mannose 

785 C-H 

rocking, 

bending 

C-C stretching 

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 1700- 

900 cm -1 ) which are present also in the free oils. 

892, 

776 

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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. 

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Temperature (°C) 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

AG 2% AG 

1.5% 

Alginate 

1% 

AG-CAR 

(0.75%) 

CH 2% 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). 

Restmass % 

120 

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 

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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 1700- 

900 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: 

• 1 st hour: simulated gastric fluid of pH 1.2 

• 2 nd to 3 rd hour: mixture of simulated gastric and intestinal fluid of pH 4.5 

• 4 th to 7 th 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. 

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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 

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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. 

Absorbance (a.u.) 

3 

2.5 

2 

1.5 

1 

0.5 

0 

0 50 100 150 200 250 300 350 400 

Time (minutes) 

Methanol 

Hexane 

THF 

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.). 

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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 1 st hour in simulated gastric fluid of pH 1.2; C. after 3 rd 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. 

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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. 

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________________________________________________________________________________________ 

Ratio values/Valoarea raportelor 

8 

7 

6 

5 

4 

3 

2 

1 

0 

A B C D E A B C D E A B C D E 


UV 


UV 


UV 

Types of ratios on time/Tipul rapoartelor in timp 

Oil free/Ulei liber 


Oil from AG 1.5%/Ulei din AG 1.5% 


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. 

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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. 

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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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35. SOCACIU C., C., MIHIS, A., NOKE, 2008, Oleosome Fractions Separated From 

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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 - 4 th 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 - 4 th 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”, 7 th 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’’, 3 rd 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

REZUMATUL - USAMV Cluj-Napoca

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