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UNIVERSITATEA DE TIINE AGRICOLE I MEDICIN VETERINAR, CLUJ-NAPOCA FACULTATEA DE ZOOTEHNIE I BIOTEHNOLOGII DOMENIUL: BIOTEHNOLOGII

REZUMATUL
TEZEI DE DOCTORAT

SISTEME DE NCAPSULARE A UNOR COMPUI BIOACTIVI EXTRAI DIN ULEIURI VEGETALE

MONICA TRIF Ing. Dipl. Biotehnolog

CONDUCTOR TIINIFIC: PROF. Dr. Dr. h.c. HORST A. DIEHL

2009

Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

CUPRINS

I. INTRODUCERE. SCOP I OBIECTIVE............................................................................ III PARTEA II. CONTRIBUII PROPRII (ORIGINALE) ......................................................... IX CAPITOL II. CARACTERIZAREA ULEIURILOR FUNCIONALE UTILIZATE LA BIONCAPSULARE ............................................................................................................... IX II.1. MATERIALE I METODE ......................................................................................... IX II.2. REZULTATE I DISCUII.......................................................................................... X II.3. CONCLUZII .............................................................................................................. XIV CAPITOLUL III. BIONCAPSULAREA ULEIURILOR: PROTOCOALE DE PREPARE A CAPSULELOR I CARACTERIZAREA LOR.................................................................... XV III.1. MATERIALE I METODE ...................................................................................... XV III.2. REZULTATE I DISCUII .................................................................................... XVI III.3. CONCLUZII............................................................................................................XXII CAPITOL IV. EFICIENA NCAPSULRII I STUDII DE ELIBERARE A ULEIURILOR DIN CAPSULE ....................................................................................................................XXII IV.1. MATERIALE I METODE....................................................................................XXII IV.2. REZULATTE I DISCUII ................................................................................. XXIII IV.3. CONCLUZII ......................................................................................................... XXVI CAPITOL V. CARACTERIZAREA FTIR A OXIDRII ULEIURILOR .....................XXVII V.1. MATERIALE I METODE ..................................................................................XXVII V.2. REZULTATE I DISCUII..................................................................................XXVII V.3. CONCLUZII.........................................................................................................XXVIII CONCLUZII GENERALE ................................................................................................ XXIX BIBLIOGRAFIE SELECTIV ......................................................................................... XXXI PUBLICAII PE DURATA STAGIULUI DOCTORAL SI PARTICIPARI LA SIMPOZIOANE I CONFERINE NATIONALE I INTERNAIONALE............... XXXIV

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I. INTRODUCERE. SCOP I OBIECTIVE BIONCAPSULAREA reprezint o tehnologie nou, bazat pe inseria i imobilizarea moleculelor bioactive, n suporturi specifice (matrici). Tehnologia ncapsulrii este bine dezvoltat i utilizat n industria farmaceutic, chimic, cosmetic, alimentar precum i n cea tipografic (Augustin et al., 2001; Heinzen, 2002). Potenialul bioncapsulrii s-a concretizat tot mai mult n domeniile biotehnologiei, mai ales n cele agricole i alimentare. n ultimele decenii, ncapsularea compuilor activi a devenit o tehnologie de mare interes i nsemntate, fiind adecvat att pentru ingredienii alimentari ct i pentru cei chimici, farmaceutici sau cosmetici. Aplicarea acestei metode de success, de bioncapsulare a compuilor bioactivi extrai din uleiuri vegetale ar putea permite stabilirea combinailor i a calitailor optime ale acestor substane. Este de luat n considerare c o asemenea metod i anume bioncapsularea, aplicat n aria comercial, ar avea beneficii semnificative pentru industria farmaceutic, alimentar i cosmetic. n afar de aceasta este de consemnat faptul c, cercetarea i dezvoltarea n aceste domenii este semnificativ mai ales n ceea ce privete conservarea compuilor naturali bioactivi extrai din plante. Scopul acestei tezei const n utilizarea diferitelor matrici naturale pentru bioncapsularea moleculelor bioactive prin metoda gelrii ionice (ionotropically crosslinked gelation), precum i n evaluarea diferenelor de calitate i a eficienei parametrilor pentru produii ncapsulai i nu n ultimul rnd a eliberrii controlate a moleculelor bioactive din matrici.

Structura tezei. Prima parte a acestei teze este reprezentat de un studiu de literatur, partea a doua include rezultatele experimentale: materiale i metode, rezultate i discuii, concluzii. Prima parte (Studiul de literatur) este compus din patru capitole (I-IV): Capitolul I. Bioncapsularea: definiie, principii, aplicaii, metode i tehnici Capitolul II. Uleiuri vegetale funcionale: caracterizarea fizic, chimic i autentificarea Capitolul III: ncapsularea uleiurilor: matrici, metode i tehnici de ncapsulare, evaluarea eficienei i a stabilitii Capitolul IV. Metode pentru caracterizarea capsulelor Partea a doua a tezei (Contribuiile proprii) include patru capitole, dupa cum urmeaz: Capitolul V. Caracterizarea uleiurilor funcionale utilizate pentru bioncapsulare. Aceast parte caracterizeaz patru uleiuri funcionale (ulei de cnepa, ulei de dovleac, ulei extra virgin de msline i ulei de ctin) analizate i apoi ncapsulate prin diferite tehnici: spectroscopie de absorbie n ultraviolet (UV), cromatografie de gaze (GC) cu detecie prin ionizare n flacr (FID) i spectroscopie n infrarou cu transformant fourier echipat cu reflectan atenuat orizontal (FTIR-ATR), determinrile chimice fiind realizate n conformitate cu metodele descrise n A.O.A.C. i IOOC.

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Capitolul VI. Optimizarea protocoalelor de obinere a caspsulelor utiliznd matrici naturale i caracterizarea capsulelor ce conin ulei ncapsulat. Acest capitol descrie protocoalele pentru: sinteza capsulelor goale de diferite mrimi i concentraii, sinteza capsulelor de diferite mrimi i concentraii ce ncorporeaz ulei, caracterizarea capsulelor goale i a celor ce conin uleiuri (n funcie de mrime i morfologie), cuprinznd de asemenea i analiza FTIR-ATR i termic a capsulelor. Capitolul VII. Studierea eficienei ncapsulrii i a eliberrii uleiurilor ncapsulate. Acest capitol conine studii cu privire la eficiena ncapsulrii uleiurilor funcionale n diferite matrici, determinarea ratei de eliberare a uleiurilor din capsule n timp i n diferii solveni, precum i eliberarea in vitro a uleiurilor din capsule. Capitolul VII. Caracterizarea FTIR-ATR a oxidrii uleiurilor. Acest capitol include analize comparative a uleiurilor libere i ncapsulate supuse oxidrii n timp n condiii UV. Planul experimental se bazeaza pe urmatoarele obiective: Utilizarea diferitelor matrici naturale (precum alginatul, alginatul n complex cu kcaragenan i gume: xantan i guar, i chitosan) n scopul ncapsulrii uleiurilor funcionale (ulei de dovleac, ulei extra virgin de msline, ulei de cnep i ulei de ctin) mbuntirea i optimizarea metodelor de bioncapsulare pentru uleiurile vegetale cu proprieti funcionale Investigarea morfologiei diferitelor capsule obinute (microscopie electronic de scanare), caracterizarea capsulelor (suprafa, diametru, perimetru, elongaie, sfericitate i compactitate), analize FTIR Investigarea uleiurilor funcionale bioncapsulate: eficiena i stabilitatea ncapsulrii, eliberarea controlat a uleiurilor ncapsulate, materialul i funcionalitatea capsulelor obinute, caracterizarea FTIR a uleiurilor libere, a capsulelor obinute i oxidarea uleiurilor libere i ncapsulate. Cercetrile prezentate au fost efectuate la Departamentul de Chimie i Biochimie din cadrul Universitii de tiine Agricole i Medicin Veterinar, Cluj-Napoca, n colaborare cu Universitatea Tehnic Berlin (TU Berlin), Germania, Departamentul de Tehnologie a Enzimelor, sub supravegherea Prof. Dr. rer. nat. Marion Ansorge-Schumacher. A dori de asemenea s mulumesc n mod special sponsorilor (Deutsche Bndestiftung Umwelt (DBU) Germany i EU COST 865) ce au facut aceste cercetri posibile, acordndu-mi cele dou burse pentru studiile doctorale.

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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

INTRODUCERE

Microncapsularea este procesul de producere a capsulelor la o scar micrometric sau milimetric, fiind cunoscute sub numele de capsule. Bioncapsularea beneficiaz de principiile fundamentale ale ncapsulrii i implic nvelirea efectiv a unei forme vii ntr-o membran care este inert, non-toxic pentru celul, i stabil la condiiile interioare ale reaciilor biochimice precum agitarea (Muralidhar R.V. et al., 2001). Microcapsula este o capsul mic, iar procedura de preparare a acesteia este numit microncapsulare. Aceasta poate ncorpora diferite tipuri de forme materiale pentru a suplimeta funciile secundare i/sau pentru a compensa n diferite condiii de mediu. Microcapsulele pot fi clasificate n trei categorii de baz n funcie de morfologia acestora: mononuleare, polinucleare sau de tip matrice. Microcapsulele mononucleare conin membrana care protejeaz compusul bioactiv; n Fig.1. sunt prezentate cteva tipuri de capsule.

Fig. 1. Diferite tipuri de capsule utilizate (Birnbaum D.T. i Brannon-Peppas L., 2003) Capsulele polinucleare prezint mai muli compui bioactivi ncorporai n interiorul unei membrane. ncapsularea de tip matrice conine cmpusul bioactiv distribuit omogen pe toat suprafaa interioar. Scopul microncapsulrii

n general exist numeroase motive pentru care substanele ar trebui ncapsulate (Li S.P. i col., 1988; Finch C.A., 1985; Arshady, R., 1993): Cretera stabilitii pentru protejarea compuilor activi de mediul extern Pentru convertirea componenilor lichizi activi ntr-un sistem solid uscat Pentru separarea componenilor incompatibili din punct de vedere funcional Pentru a masca proprietile nedorite a componenilor activi Pentru a proteja mediul extern al microcapsulelor de componeni activi Pentru a controla eliberarea compuilr activi de procesel de eliberare ntrziat sau eliberarea susinut Separarea omponenilor incompatibili
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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

Conversia lichidelor n solide Mascarea mirosului, activitii, etc. n scop farmaceutic

Tehnologia de ncapsulare este foarte bine dezvoltat fiind acceptat n multe industrii precum: farmaceutic, chimic, cosmetic, alimentar (Augustin et al., 2001; Heinzen, 2002). n industria alimetar, grsimile i uleiurile, compuii aromatizani i oleorezinele, vitaminele, mineralele, coloranii i enzimele au fost deja ncapsulate (Dziezak, 1988; Jackson i Lee, 1991; Shahidi i Han, 1993). Alegerea unei tehnici adecvate de bioncapsulare depinde de utilizarea final a produsului i de condiiile de procesare implicate n obinerea produsului final. Bioncapsularea i gsete aplicaie din ce mai mult aplicabilitate n domeniul biotehnologiilor i n special n alimentaie i agricultur. n ultimele decenii, ncapsularea compuilor activi a devenit un process de mare interes i nsemntate, fiind adecvat att pentru ingredienii alimentari ct i pentru cei chimici, farmaceutici sau cosmetici. Pfutze S. (2003) consider c tehnologiile de ncapsulare pot fi divizate n dou categorii: formarea matricea capsulelor: un ingredient activ i protector formeaz granule omogene. Produsul activ este uniform distribuit n granul fiind nconjurat din abunden de material protector, formnd matricea activ. formarea nveliului capsulelor: materialul activ este granulat i acoperit de un strat protector. Materialul activ i protector este bine separat.

Obiectivul principal este construirea unei bariere ntre particulele componente i mediu. Aceast barier reprezint o protecie mpotriva oxigenului, apei, luminei; evitarea contactului cu alte particule sau ingrediente; sau controlul eliberrii lor n timp. Protecia compuilor bioactivi pe parcursul procesrii i pstrrii, precum i eliberarea controlat n tractusul gastrointestinal este o prioritate n exploatarea potenialului benefic al multor compui bioactivi. Tehnicile utilizate la bioncapsulare necesit un material drept nveli i o substan protejat. Materialul utilizat trebuie aprobat de Administraia Alimentaiei i Farmacie (US) sau de Autoritatea European pentru Securitatea Alimentelor (Europa) (Amrita i col., 1999). Coacervarea: ncapsularea lichidelor Coacervarea complexelor (sau faza de separare), este prima aplicaie la scar larg a tehnologiei de microncapsulare. Coacervarea este un proces care are loc n soluii coloidale i de multe ori privit ca metoda original de ncapsulare (Risch, 1995). Aplicabilitate coacervrii complexelor este enorm dar are i limite datorit costurilor ei ridicate, n unele aplicaii. Aceasta include ncapsularea: aromelor vitaminelor cristaleor lichide pentru dispozitivele de display sisteme de imprimare
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ingredieni activi pentru industria farmaceutic bacteri i celule Matricile materiale pentru ncapsulare Exist diferite materiale ce pot fi utilizate pentru ncapsulare precum: polielectrolii sintetici (Sukhorukov i col., 1998; Donath i col., 1998), polielectrolii naturali (Shenoy i col., 2003), nanoparticule anorganice (Caruso i col., 2001), grsimi (Moya i col., 2000), colorani (Dai i col., 2001), ioni polivaleni (Radtchenk i col., 2005), i biomacromolecule (Yang i col., 2006). n general trei clase de materiale au fost utilizate: materiale naturale derivate (colaen alginat), matrici tisulare acelulare (submucoase intestinale) i polimeri sintetici (acid poliglicolic, etc.). Aceste clase de biomateriale au foste testate n concordan cu biocomapatibilitatea lor (Pariente i col., 2002). Biopolimerii sunt polimeri care provin din surse naturale, sunt biodegradabili, i nontoxici. Pot fi produi de sisteme biologice (ex: microorganisme, plante i animale), sau chimic sintetizate din materiale biologice (ex: amidon, grsimi sau uleiuri, etc.). Polimeri naturali i derivai ai acestora: polimeri anionici: acid alginic, pectin, caragenan; polimeri cationici:chitosan, polilizin; polimeri amfipatici: colagen (and gelatin), chitin; polimeri neutri: dextran, agaroz, pululan. Guma guar (E412, numit i guaran) este extras din seminele leguminoaselor din familia Cyamopsis tetragonoloba. Guma guar prezint vscozitate sczute dar este un bun agent de ntrire. Fiind un polimer non-ionic, nu este influenat de pH, dar este influenat de temperaturi extreme la anumite pH-uri (ex: pH=3 la 50C). Alginatul (E400-E404) este produs extras din algele brune (Phaeophyceae, n special Laminaria). Proprietile de gelifiere depind de interacia cu unii ioni (Mg2+ << Ca2+ < Sr2+ < Ba2+). Caragenan (E407) este un nume colectiv atribuit polizaharidelor, obinute prin extracia alcalin din algele roii (Rhodophycae). Geluri puternice sunt formate de kcaragenan n prezena ionilor de K+ i mai slab n prezena ionilor de Li+, Na+, Mg2+, Ca2+, sau Sr2+. Guma xantan (E415) este un polimer microbian preparat commercial prin fermentaia aerobic din Xanthomonas campestris. Guma xantan nu prezint proprieti ridicate de gelifiere, este hidratat uor n ap rece, avnd aplicaii ca i emulgator, stabilizator. Chitosanul este obinut la scal industrial din carapacea crustaceelor (Yanga i col., 2000). n multe studii chitosanul este legat cu ajutorul aldehidelor, pecum glutaraldehida i formaldehida, pentru obinerea lui sub o forma mai vscoas cu aplicaii ca i material de ncapsulat. Muli componeni naturali coninui n uleiurile vegetale prezint proprieti utile. Uleiul de cnep rezult prin presarea seminelor de cnep (Cannabis sativa L). Acidul oelic (Omega 9) coninut n uleiul de cnep menine o bun funcionalitate arterial. n exces acidul oleic poate interfera cu acizii grai eseniali i prostaglandinele.
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Uleiul de msline conine trigliceroli i cantiti mici de acizi grai liberi, glicerol, pigmeni, compui aromatizani, steroli, tocoferoli, fenoli, componeni rinoi neidentificai, etc.(Kiritsakis A., 1998). Uleiul de dovleac este foarte sntos, de caliate superioar, fiind n clasamentul primelor 3 uleiuri nutritive. Seminele de dovleac au un gust intens i sunt bogate n acizi grai polinesaturai. Uleiul brun are un gust amrui. Coninutul n tocoferoli ai uleiurilor oscileaz de la 27,1 la 75,1 g/g de ulei pentru -tocoferol, de la 74.9 la 492.8 g/g pentru tocoferol, i de la 35.3 la 1109.7 g/g pentru -tocopherol (Stevenson i col., 2007) Uleiurile de ctin conin o cantitate ridicat de acizi eseniali, linoleic i alfa linoleic (Chen i col., 1990), care sunt precursori ai altor acizi grai polinesaturai cum ar fi acidul arahidonic sau eicosapentanoic. Este stocat n organitele extracitoplasmatice numite vezicule de ulei, o form natural de ncapsulare (Socaciu et all, 2007, 2008). Uleiul din pulpa frutelor de ctin este bogat n acid palmitoleic i acid oleic (Chen i col., 1990). Uleiurile conin de asemenea flavonoizi (Chen i col., 1991), carotenoizi, steroli liberi i esterificai, triterfenoli i izoprenoli (Goncharova i Glushenkova, 1996). Coninutul n carotenoizii variaz de asemenea n funcie de sursa de provenien a uleiului.

Proprietile fizice i chimice ale uleiurilor funcionale Proprietile fizice i chimice ale uleiurilor, incluznd indicele de iod, de saponificare i valorile de aciditate i pentru peroxizi, indicele de refracie, densitate i materia nesaponificabil sunt determinate conform procedurilor standard. Indicele de iod msoar gradul de nesaturare al uleiurilor. Valoarea acestuia sub 100 demonstraz c uleiul prezint un grad redus de saturare (Pa Quart, 1979; Pearson, 1981). Indicele de saponificare este un indicator al mediei masei moleculare a acizilor grai prezeni n ulei (AOAC, 1980; Pearson, 1981). Indicele de peroxid este frecvent utilizat pentru msurarea stadiului de oxidare al uleiului. Acesta indic oscilarea oxidativ a uleiului (deMan, 1992).

Tehnicile pentru caracterizarea i autentificarea uleiurilor funcionale Exist diferite tehnici pentru caracterizarea i autentificare produselor alimetare. Metodele de autentificare aplicate pentru uleiuri i grsimi pot fi clasificate ca i chimice (de separative) sau fizice (non-separative). Spectrometrele de infrarou cu transformant fourier (FTIR) prezint multe avantaje n comparaie cu instrumentele convenionale de dispersie, printr-o excelent reproductibilitate i acuratee a lungimilor de und, precisa manipulare spectral i utilizarea unor programe chemometrice pentru calibrare. Accesoriile HATR au fost de asemenea larg utilizate n dezvoltarea metodelor FTIR pentru analizarea uleiurilor i a grsimilor, deoarece acestea pot oferi mijloace convenable i simple pentru o manipulare uoar (Sedman i col., 1999). Spectroscopia infrarou de mijloc (MIR) poate fi utilizat pentru identificarea compuilor organici deoarece unele grupe de atomi prezint proprieti ale frecvenei de absorbie a vibraiilor n regiunea infraroie a spectrului electromagnetic. Reflectana orizontal total atenuat (HATR) este accesoriul cel mai des utilizat in metoda FTIR pentru analizele uleiurilor i a grsimilor (Sedman i col., 1999).
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O larg varietate de alimente utilizeaz pentru ncapsulare aromatizani, acizi, baze, ndulcitori artificiali, colorani, antioxidani, ageni cu arome nedorite, mirosuri, etc. Acetia i pstrez bioactivitatea i rmn accesibili agenilor externi. Fitosterolii, flavonoizii i compusii organici cu sulf, reprezint trei grupe de compui caracteristici fructelor i legumelor, care ar putea prezenta importan n reducerea riscului de ateroscleorz. (Howard i Kritchevsky, 1997). Unele substane fitochimice, cu ar fi acidul ascorbic, carotenoizii, vitamina E, fitofenoli, izoflavoni i fitosteroli, au fost evideniate ca ingredieni fiziologic activi ce mbuntesc rezistena la anumite boli. ncapsularea poate fi utilizat pentru condiionarea uleiurilor n forme solide sau solubile n ap, extinznd utilizarea lor n multe alte aplicaii. ncapsularea uleiurilor include ca metode i tehnici: spray-drying, spray-chilling, fluid bed encapsulation, extrusion encapsulation i ncapsularea prin coacervare. Extrudarea este utilizat pentru ncapsularea mineralelor i vitaminelor n uleiuri (grsimi saturate) ntr-o matrice de tip polizaharidic (Van Lengerich i Lakis, 2002). Protecia mpotriva oxidrii metil-linoleatului ncapsulat cu gum acacia prin metoda spray drying i freeze drying, depinde de umiditatea relativ a mediului (Minemoto i col., 1997). n majoritatea cazurilor matricile utilizate pentru ncapsularea uleiurilor i grsimilor sunt gume (acacia, arabic), proteine, carbohidrai (cazein/zaharuri), maltodextrin, betaciclodextrine, alginat de sodiu, gelatin.

PARTEA II. CONTRIBUII PROPRII (ORIGINALE)


CAPITOL II. CARACTERIZAREA ULEIURILOR FUNCIONALE UTILIZATE LA BIONCAPSULARE Uleiurile extrase din plante (floarea-soarelui, dovleac, soia, rapita, etc.) sunt foarte utilizate in domeniul alimentar, dar si in alte industrii (cosmetic, farmaceutic, etc.). Prezint o deosebita insemnatate datorita numerosilor componeni benefici care intr n alctuirea lor. Calitatea i autenticitatea acestor uleiuri se realizeaz prin diferite tehnici. Cele mai utilizate trei tehnici n vederea caracterizrii acestor uleiuri sunt: spectrometriA UV-Vis, cromatografia gaz cu detecie prin ionizare n flacr FID, i spectroscopia infrarou cu transformant fourier (FTIR).

II.1. MATERIALE I METODE

Au fost alese n vederea ncapsulrii patru uleiuri de mare interes: ctin (SBO) extras din fructele de catina, colectate din regiunea Clujului (Transilvania, nordul Romaniei), ulei de msline extra virgin (EVO) din Italia, cnep (HP) i dovleac (PK) din Romania. Analizele chimice au fost determinate conform metodelor descrise de: A. O. A. C. i IOOC sau de Comisia Uniunii Europene (EU): aciditatea i indicele de iod. Toate probele au fost analizate n triplicat. Aciditatea a fost calculat lundu-se n considerare coninutul de
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acizi grai 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 I DISCUII

Determinarea aciditii i a indicelui de iod Rezultatele analizelor chimice prezentate n Tabelul II.1. au demonstrat o bun corelaie a valorilor obinute cu cele publicate n literatur. Tabel II.1. Caracteristicile chimice i fizice ale uleiurilor analizate n comparaie cu literatura
Ulei de Cnepa Ulei de msline extra virgin Ulei de dovleac Ulei de ctin

Caracteristicile fizice si chimice Aciditate (mg KOH/g ulei) Indicele de iod** 162 / 145-166* 87 / 75-95* 130 / 116-133* 71 / 98-119* 1.93 / 4.0* 2.64 / 6.6* 1.32 / 4.0* 3.7 / 4.0*

**Indicele de Iod a fost calculat cu metoda AOCS Cd 1c-8 * Date din literatura

Aceste date demonstreaz faptul c valorile acestor uleiuri corespund cu indicii de calitate ai iodului din Codex, excepie uleiul de ctin, a crui valori n cazul aciditii nu corespund intervalelor aciditii precizate n literatur.

Determinarea amprentei uleiurilor prin spectrometrie ultra violet/visibil (UV-Vis) Caracterizarea spectral (fingerprintul) specific fiecrui ulei analizat prin UV-Vis este prezentat in Fig. I.1. Diferenele dintre un ulei autentic si un ulei falsificat a fost demonstrate prin poziia i absorbana peakurilor caracteristice fiecrui ulei (Socaciu C. et al., 2005).

Ulei de cnep (Cannabis sativa L) Amprenta spectral UV-Vis caracteristic uleiului de cnep n conformitate cu datele precizate de OMLC, este dat de coninutul ridicat n pigmeni clorofilici, avnd absorbana maxim la 411 nm (Fig.II.1.A.).

Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

A.

C.

D.

Fig.II.1. Spectrele UV-Vis ale uleiurilor analizate (amprenta specific n regiunea 350-600 nm continnd detalii referitoare la maximul absorbantei peakului specific: A. Ulei de cnep; B. Ulei de msline extra virgin (EVO); C. Ulei de dovleac (PK); D. Ulei de ctin (SB)

Ulei de msline extra virgin (Olea europaea ) Culoarea caracteristic uleiului de msline depinde de majoritatea pigmenilor coninui, n principiu acest ulei avnd un coninut ridicat n carotenoide i clorofile. Uleiul provenit din mslinele ajunse la maturitate prezint o culoare galben datorit continutului n pigmeni carotenoidici galbeni. n general culoarea acestui ulei variaz i este datorat combinaiei i diferetelor proporii de pigmeni. Exista o simpl ecuaie: Culoarea= clorofil (verde) + carotenoide (galben) + ali pigmeni. Coninutul n pigmeni clorofilici se diminueaza odat cu atingerea maturitii fructelor. Fingrprintul specific uleiului de msline analizat este atribuit ecuaiei culorii menionat anterior (Fig.II.1.D.).

Ulei de dovleac (Cucurbita pepo) Amprenta spectral (fingerprintul) a acestui ulei este acceptat ca avand doua umere, unul la 418 nm cu absorban mai mic, i unul la 435 nm cu absorban mai mare
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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

(Fig.II.1.C.), n cazul falsificrii sau oxidrii, acest ulei prezint absorbanele celor dou umere schimbate (Lankmayr i col., 2004).

Uleiul de ctin (Hippophae rhamnoides) Spectrului acestui ulei demonstreaz ca fingrprintul este dat de cele trei umere care dau spectrul, care sunt caracteristice carotenoidelor, mai exact beta-carotenului, intre 400 i 500 nm (Fig.II.1.A.), acesta fiind compusul principal al acestui ulei (Lichtenthaler i 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 frecvene i valoarea absorbanei lor, au fost utilizate n continuare pentru evaluarea oxidarii uleiurilor (Guillen, M. D. i Cabo, N, 1997, 1998, 1999, 2000, 2002). n conformitate cu aceste spectre au fost identificate principalele benzi i frecvente n domeniul infrarou ale uleiurilor analizate ( Tabel II.2.).

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


Nr. banda 1 2 3 4 5 6 7 8 9 10 11 12 13 1236 HP (cm-1) 3008 2956 2923 2853 1742 1654 1463 1456 1418 1396 1377 EOV (cm-1) 3005 2956 2923 2853 1742 1653 1464 1456 1417 1402 1377 1317 1238 1418 1398 1377 1319 1238 1238 -C-O, -CH2PK (cm-1) 3008 2956 2923 2854 1742 1653 1464 SB (cm-1) 3006 2956 2922 2853 1742 1653 1464 1456 1417 1402 1377 -C-H (CH3) =C-H (cis-) de deformare de deformare de deformare (simetric) de deformare de ntindere, de deformare =C-H (cis-) -C-H (CH3) -C-H (CH2) -C-H (CH2) -C=O (ester) -C=C- (cis-) -C-H (CH2, CH3) de ntindere de ntindere (asimetric) de ntindere (asimetric) de ntindere (asimetric) de ntindere de ntindere Grupul functional Modul de vibratie

XII

Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________ 14 15 16 17 18 19 20 914 721 721 1155 1120 1097 1028 1159 1118 1097 1028 958 1157 1120 1099 1029 962 914 721 721 1161 1116 1095 1033 968 -C-O, -CH2-C-O -C-O -C-O -HC=CH- (trans-) -HC=CH- (cis-) -(CH2)n-, -HC=CH(cis-) de ntindere, de deformare de ntindere de ntindere de ntindere de deformare nafara planului de deformare nafara planului de deformare ( rocking)

Spectrele uleiurilor analizate par a fi in principiu similare, ns diferenele n intensitatea benzilor ca de altfel i a frecvenelor fac posibil diferenierea foarte clar a compoziiei acestor uleiuri (see Fig. II.2.).

Fig.II.2. Spectrul FTIR-ATR al zonei de fingerprint (1700-800 cm-1) a uleiurilor analizate HP= cnep, EVO (EOV) = msline extra virgin; PK= dovleac; SB= ctin

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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

Profilul acizilor grai prin cromatografia gaz Compoziia acizilor grasi analizai prin GC-FID in acest studio sunt evidentiati in Tabelul II.3. Profilul acizilor grai a fost comparat cu al uleiurilor din literature.

Tabel.II.3. Compoziia procentual a acilor grai din uleiurile analizate


Acizi grasi % Ulei de Canepa Ulei de masline extra virgin 7.28 2.67 9.95 Ulei de dovleac Ulei de catin

Palmitic (16:0) Stearic (18:0) Arachidic (20:0) saturati % Palmitoleic (C16:1) Oleic (C18:1) Linoleic (C18:2) Linolenic (18:3n3) Eicosadienoic (C20:2) nesaturati % C18:1/C18:2 omega 3 : omega 6 acizi grasi

7.48 1.66 1.06 10.02 -

6.29 3.64 9.93 -

7.76 0.3 0.11 8.17 5.4

14.94

36.81

42.44

6.3

72.6

43.14

46.71

0.93

0.92

0.8

0.55 87.54 0.21 -

80.88 0.85 0.022

90.07 0.91 0.02

12.5 6.3 -

II.3. CONCLUZII

Prin GC-FID, s-a determinat compoziia n acizi grai a uleiurilor analizate i s-a facut comparaia cu datele din literatur. n urma acestei analize s-au concluzionat urmatoarele: compoziia uleiului de cnep nu corespunde cu valorile precizate n literatur pentru acizii grai, acesta avand un coninut mai sczut. Acidul oleic se incadreaz in intervalul prevzut in literatur principalii acizi grai n uleiul de msline extra virgin sunt acidul oleic i linoleic, i de asemenea n cantitate mai mic acidul lonoleic
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n cazul uleiului de dovleac, compoziia n acizi grai corespunde cu valorile precizate n litaratura, excepie facnd acizii palmitic i stearic care sunt prezeni in concentrii mai mici profilul acizilor grai a uleiului de ctin a demonstrat faptul c acest ulei provine din pulpa/pielea fructelor i nu din semine, fiind foarte bogat in acidul palmitic i oleic

CAPITOLUL III. BIONCAPSULAREA ULEIURILOR: PREPARE A CAPSULELOR I CARACTERIZAREA LOR III.1. MATERIALE I METODE

PROTOCOALE

DE

n vederea realizrii parii experimentale din acest capitol s-au utilizat urmatoarele: matrici pentru ncapsulare: alginat, k-caragenan, chitosan, gum xantan i gum guar, procurate de la Sigma Aldrich solvenii si reactanii necesari de asemenea de la Sigma Aldrich uleiurile utilizate la ncapsulare au fost prezentate in capitolul anterior

Protocol pentru sintetizarea capsulelor goale de diferite mrimi i concentraii 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 i amestecul de alginat au fost pipetate ntr-o solutie de 2% CaCl2 n apa (ca i baie de ntrire), utiliznd 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), utiliznd pipeta pentru control. Dupa ~ 1h, capsulele au fost separate din baia de intarire i transferate n placi Petri pentru protecie si conservare.

Protocol pentru sintetizarea capsulelor de diferite mrimi i concentraii cu uleiri ncorporate S-au luat n considerare doar concentraiile de matrici care au prezentat emulsiile cele mai stabile. De asemenea vscozitatea soluiilor a fost considerat un factor principal n vederea alegerii concentraiilor de matrici. Protocolul pentru obinerea capsulelor a fost descris anterior.

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Evaluarea microscopica a emulsiilor naintea ncapsulrii Evaluarea microscopic a emulsiilor naintea ncapsulrii a fost determinat utiliznd un microscop Olimpus optical microscope BX51M, echipat cu camera digital.

Caracterizarea capsulelor: dimensiuni i morfologie, analize FTIR i termice

Parametri luai in considerare n vederea caracterizrii capsulelor, precum dimensiune, arie, perimetru, elongaie si compactitate, au fost determinai utiliznd UTHSCSA ImageTool ca i software. Morfologia suprafeei capsulelor a fost determinat utilizndu-se microspia electronic scanat (Hitachi S-2700, iMOXS, cu detector BSE). Capsulele analizate au fost suflate i nvelite n aur naintea supunerii analizelor microscopice.

III.2. REZULTATE I DISCUII

Evaluarea microscopica a emulsiilor naintea ncapsulrii Stabilitatea emulsiilor este un factor cheie n evaluarea n condiii de temperatura n vederea pstrrii timp mai ndelungat a produselor pe baze de emulsii. Mrimea picturilor de ulei dispersate in structura matricilor dizolvate care au fost comparate in vederea evaluarii stabilitatii emulsiilor obtinute. Emuliile cu cea mai bun stabilitate n timp au fost utilizate mai departe pentru ncapsulare. Mrimea picturilor de uleiuri au fost dispersate uniform in matrici, n funcie de stabilitatea matricei, uniformitatea crescnd odat creterea concentraiei 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. Lundu-se in considerare toate caracteristicile capsulelor obtinute, si facand o comparatie intre aceste caracteristici ale capsulelor goale i a celor continand uleiuri, s-a constatat ca incorporarea uleiurilor n capsule modifica aceste caracteristici (Fig.III.2.). Compararea capsulelor ntre ele continand uleiuri, a demonstrat ca sfericitatea i compactitatea nu au fost prea mult afectate de incorporarea uleiurilor. Insa in cazul diametrului, ariei, elongatia, au fost clar determinate diferente foarte mari.
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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

A.

B.

C.

D.

Fig. III.1. Imagini microscopice ale diferitelor emulsii: A. alginat 2%; B. alginat 1%; C. complex alginat-gum guar; D. complex alginat-gum xantan. Scala reprezint 5 m.
Param eter values/Valoarea param etrilor A G C A G A -C R ( A R 0.5 A (0 :0 G -X .75 .5) :0 G .7 (0 5) .7 A G 5 : 0 X A .7 G G 5) -G (0 .5 G : 0 (0 A .7 .5) G -G 5:0 .7 G 5) (0 .5 :0 .5 A ) G oi A G l2% oi l1 .5 % A G oi l1 % C H oi l C H 2% oi l1 .5 % C H oi l1 % 9 8 7 6 5 4 3 2 1 0

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)

Samples/Probele

Fig.III.2. Reprezentarea grafic comparat a caracteristicilor capsulelor din complexul alginat cu k-caragenan, gume xantan i guar, alginat i chitosan continnd ulei: AG-CAR (0.5:0.5) = complex alginat-k-carrageenan (raport 0.5:0.5) continnd ulei; : AG-CAR (0.75:0.75) = complex alginat-k-carrageenan (raport 0.75:0.75) continnd ulei; AG-XG (0.75:0.75) = complex alginate-guma xantan (raport 0.75:0.75) continnd ulei; AG-XG (0.5:0.5) = complex alginate-guma xantan (raport 0.5:0.5) continnd ulei;AG -GG (0.75:0.75) = complex alginate-guma guar (raport 0.75:0.75) continnd ulei; AG -GG (0.5:0.5) = complex alginate-guma guar (raport 0.5:0.5) continnd ulei; AGoil2% = capsule alginat 2% continnd ulei; AGoil1.5% = capsule alginat 1.5% continnd ulei; AGoil1% = capsule alginat 1% continnd ulei; ; CHoil2% = capsule chitosan 2% continnd ulei; CHoil1.5% = capsule chitosan 1.5% continnd ulei; CHoil1% = capsule chitosan 1% continnd ulei

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Microscopia electronic scanat (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 prezint 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 n urma analizelor FTIR-ATR s-a realizat caracterizarea matricilor utilizate la ncapsulare, realizndu-se astfel o comparaie ntre matrici (AG, CAR, CH, GG, XG). Principalele frecvene caracteristice matricilor n vederea identificrii individuale sunt: 32443302 cm-1 (O-H stretch), 1400-1474 cm-1 (CH2 bending), 1000-1200 cm-1 (C-O i C-C stretch), 924-1000 cm-1 ( poly OH i CH2 twist), 776-892 cm-1(glycoside).

Vibraiile i grupurile funcionale OH intindere

AG 3244

CAR 3514 grupul poliOH

GG 3299

XG 3302

CH 3289 O-H + N-H de ntindere

XVIII

Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________ CH ntinderea grupului CH2 C-O de ntindere ( COOH) deformarea gruprii CH2 O-H deformare 2926 1597 1408 2953, 2911, 2894 1474, 1400 1223 ( S=O vibraia de ntindere a sulfet esterului) 1063 1024 2884 1636 1408 1350 1400 1247 2935 1651 1428 -

C-O i C-C ntindere CH2OH modul de ntindere Gruparea COH alcolic (C-O ntinderea zaharidelor) CH2 vibraie

1200-1000 1054 1024

1145 1054 -

1150

1151 1061

1025

1024

948, 902, Provenite de la acizii: guluronic i maluronic

924, 910 Gruprile polihidroxi 842

1016

legaturile glicozidice

809

866,777

785

892, 776

Sulfatul galactozic, (1,4; 1,6) legatura C-H de legatura glicozidic galactozei i deformare manozei C-C ntindere

FTIR characterization of different beads containing oils

Spectrele matricelor, ale capsulelor goale, capsulelor continand uleiuri au fost analizate. Concentratia matricelor nu a influentat caracteristicile capsulelor prin FTIR. Un exemplu concludent este reprezentat in Fig.III.4., spectrele uleiului de ctin (SB) i ale capsulelor din alginat 2% coninand ulei SB. n urma ncapsulrii uleiului de SB n capsule de alginate, se produce o cretere a intensitii absorbanei la 3400 cm-1 (care este direct proporional cu creterea concentraiei de alginate utilizat la ncapsulare) precum i o shiftare a unor peakuri spre valori i frecvene mai sczute n regiunea 1000-1500 cm-1, regiune specific uleiului de SB Spectrele amestecului de uleiuri si capsule au demonstrat prezenta peakurilor specifice uleiurilor in doua zone distuncte (2800-2900 cm-1 i 1700-900 cm-1), confirmndu-se astfel prezena uleiurilor n capsule.

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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

Fig.III.4. Spectru FTIR-ATR nregistrat pentru: A. Capsule de alginat 2% coninnd SB; B. pudr 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 fiecrui tip de capsula obinut.

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 n 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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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________
200 180 160 140 Temperature (C) 120 100 80 60 40 20 0 AG 2% AG 1.5% Alginate AG-CAR 1% (0.75%) CH 2% CH 1% AG-GG AG-KG SB

Fig. III.5. Reprezentarea grafica a peakurilor endotermice ale unor tipuri de capsule

DSC si TGA au fost in ultimul timp foarte mult utilizate in monitorizarea stabilitatii, a comportamentului termic, a parametrilor de cinetica in diferite uleiuri (Jayadas et al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008). n acest studiu analizele termice au fost efectuate pana la temperatura de 300C. Diferenele nu foarte mari ntre 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 300C, iar pierderea in greutate poate fi pana la 10%, aceasta depinznd de natura uleiului (Jayadas et al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008).
120 100 Restmass % 80 60 40 20 0 AG 2% AG 1.5% AG-CAR (0.75%) AG-GG AG-KG SB

Fig.III.6. Reprezentarea grafic a pierderii de mas % a probelor analizate TGA

Scopul acestor analize termice a fost acela de a analiza i a determina stabilitatea termic a capsulelor obinute continnd diferite uleiuri, n vederea viitoarelor aplicaii ale acestora n domeniul alimentar i cosmetic. n astfel de aplicaii se cunoate necesitatea
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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

sterilizrii probelor sau expunerea la presiuni nalte n vederea evitrii biohazardului sau contaminrii, aceste tratamente fiind facute n timpul proceselor tehnologice.

III.3. CONCLUZII Studiile experimentale realizate, cu scopul bioncapsulrii unor uleiuri funcionale n matrici naturale, utiliznd ca i metod ionotropically crosslinked gelation, au demonstrate posibilitatea utilizrii acestei tehnici n vederea bioncapsulrii unor compui naturali i eliberarea lor condiionat. Cele mai bune matrici n vederea bioncapsulrii uleiurilor s-au dovedit a fi: alginatul i chitosanul n concentrii de 2%, 1.5% i 1%, complexele alginatului cu k-caragenan, i gume guar i xantan n raport de concentraie 0.75:0.75. Rezultatele au artat faptul c bioncapsularea uleiului a afectat diametrul capsulelor, acesta crescnd direct proporional cu cantitatea de ulei utilizat pentru ncapsulare. De asemenea i ceilali parametri masurai n vederea caracterizrii capsulelor au fost influenai i de cantitatea de ulei utilizat. Prin analizele FTIR-ATR, diferitele capsule coninnd uleiuri au prezentat peakuri care sunt atribuite atat uleiurilor ct i capsulelor goale (regiunile dintre 2800-2900 cm-1 i 1700-900 cm-1). Astfel se demonstreaz prezena uleiurilor n capsule, deci ncapsularea acestora.

CAPITOL IV. EFICIENA NCAPSULRII I STUDII DE ELIBERARE A ULEIURILOR DIN CAPSULE IV.1. MATERIALE I METODE Eficina ncapsulrii e uleiurilor n capsule ncapsularea uleiurilor a fost determinat calculnd cantitatea de beta-caroten sau cantitatea de carotenoid care este principalul component major al fiecrui ulei analizat nainte i dup ncapsulare. Aceast determinare a fost realizat spectrofotometric, iar eficina ncapsulrii (EE%) a fost calculat conform ecuaiei:

EE% = C1/C2 x 100,

C1= concentraia carotenoid din ulei iniial C2= concentraia carotenoid din ulei dup ncapsulare

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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

Msurarea ratei de eliberare a uleiurilor din capsule Eliberarea controlat a uleiurilor din capsule a fost determinata de asemenea spectrofotometric, utiliznd un spectrofotometru CarWin 50 UV-VIS. Msurtorile au fost facute n triplicat la temperatura camerei, utilizndu-se cuvete de cuar de 2 mm.

Eliberarea in vitro a uleiurilor din capsule Stimularea fluidului gastric a fost realizat conform urmatorul protocol: timp de o ora la pH 1.2 intr-o solutie de 0.1N HCl cusi 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 cusi 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 testrile s-au efectuat la 37C cu barbotare continu de CO2

IV.2. REZULATTE I DISCUII

Eficina ncapsulrii e uleiurilor n capsule Eficiena ncapsulrii este reprezentata n graficul din Fig.IV.1. pentru diferite tipuri de capsule. Valorile prezentate sunt ale uleiului de ctin, nsa pentru toata uleiurile analizate aceasta eficien a prezentat aceleai valori. Dup cum se poate observa i n graficul prezentat, eficiena ncapsulrii este direct proporionala cu creterea concentraiei matricilor. Dintre toate matricile i complexele de matrici utilizate, dup cum se poate observa i in graficul din Fig.IV.1., s-a obinut cea mai bun eficiena a ncapsulrii utiliznd ca i matrici: alginatul n concentraie de 2%, chitosanul in aceeai concentraie, urmate de concentraiile de 1.5%, i de alginatul n complex cu kcaragenan i gume n raport de 0.75:0.75%.

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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

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)

Msurarea ratei de eliberare a uleiurilor din capsule Ca i sisteme de eliberare s-au luat in considerare 3 solveni: tetrahidrofuran (THF), metanol si hexan. n 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 Chemick Listy Journal (IF=0.683)). THF este considerat i n litaratura ca fiind solventul cel mai eficient n extracia carotenoidelor, lucru dovedit i n acest studiu. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea i solubilitatea uleiului n matrice. Eliberarea uleiurilor din capsule a demonstrat faptul ca alginatul, complexul dintre alginat cu k-caragenan i gume, precum i chitosanul, sunt matrici pretabile la ncapsularea uleiurilor vegetale.

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Sisteme de ncapsulare a unor compui bioactivi extrai din uleiuri vegetale ________________________________________________________________________________________

0.4 0.35 Absorbance (u.a.) 0.3 0.25 0.2 0.15 Alginate 2% in hexane 0.1 0.05 0 0 100 200 Wavelenght (nm) 300 400 Alginate-carrageenan complex in hexane Alginate-carrageenan complex in methanol Alginate 2% in methanol

A.
0.7 0.6 Absorbance (a.u.) 0.5 0.4 0.3 0.2 0.1 0 0 100 200 Wavelenght (nm) 300 400 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

B.
Fig.IV.2. Reprezentarea grafica a eliberarii in timp din diferitele capsule in metanol si hexan, ca si solventi

Eliberarea in vitro a uleiurilor din capsule n cazul mimrii 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 n cazul capsulelor din alginat i alginat n complex cu k-caragenan i 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.

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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 eficiena ncapsulrii i stabilitatea capsulelor coninnd uleiuri au demonstrat:

1. Creterea concentraiei matricilor sau a complexului de matrici determin obinerea unei mai bune eficiene la ncapsulare. s-a obinut cea mai bun eficiena a ncapsulrii utiliznd ca i matrici: alginatul n concentraie de 2%, chitosanul in aceeai concentraie, urmate de concentraiile de 1.5%, i de alginatul n complex cu kcaragenan i gume n raport de 0.75:0.75%. 2. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea i solubilitatea uleiului n matrice. Eliberarea a fost mai lent in cazul hexanului, mai ridicat n cazul metanolului i cea mai buna eliberare fiind in THF, indiferent de matricea sau concentraia utilizat la ncapsulare. 3. Stabilitatea capsulelor la pH 1.2 si pH 4.5, dizolvarea acestora i eliberarea continutului realizandu-se la pH 7.4.
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CAPITOL V. CARACTERIZAREA FTIR A OXIDRII ULEIURILOR V.1. MATERIALE I 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 105C, si la iradiere UV (254m) 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 extracia uleiului din capsule.

V.2. REZULTATE I DISCUII

n cazul analizelor FTIR-ATR a fost stabilit stadiul oxidrii, calculandu-se raportul ntre intensitile principalele peakuri considerate markeri ai oxidarii conform literaturi (Guilln and Cabo, 1999, 2000, 2002): A2853/A3005, A1746/A3006, A1474/A3006, A1377/A3006 and A1163/A3006, nainte i dup 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 ctin, Fig.V.1.). Cea mai bun protecie mpotriva oxidrii a fost asigurat de urmatoarele capsule formate din matrcile i concentraiile urmatoare: alginat 1%, chitosan 1.5%, complexele alginate-gum gum i alginat-gum xantan n raport 0.5:0.5, i alginat-k-caragenan complex n raport 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 After 1h UV/Dupa 1h After 4h UV/Dupa 4h After 6h UV/Dupa 6h UV UV UV


Oil from AG 2%/Ulei din AG 2% Oil from AG 1.5%/Ulei din AG 1.5% Oil free/Ulei liber

Oil from AG 1%/Ulei din AG 1%

Types of ratios on time/Tipul rapoartelor in timp

Fig. V.1. Reprezentarea grafica a uleiului de canepa liber si incapsulate (in diferite capsule de alginate) in timpul oxidarii (A= A2853/A3005-3008, B= A1744/ A3005-3008, C= A1464/ A3005-3008, D= A1377/ A3005-3008, E= A1160/ A3005-3008)

V.3. CONCLUZII Uleiurile ncapsulate prezint o mai buna stabilitate mpotriva oxidrii provocate de diferite conditii comparativ cu uleiurile libere. Cea mai bun protecie mpotriva oxidrii a fost asigurat de urmatoarele capsule formate din matrcile i concentraiile urmatoare: alginat 1%, chitosan 1.5%, complexele alginate-gum gum i alginat-gum xantan n raport 0.5:0.5, i alginat-k-caragenan complex n raport 0.75:0.75. Spectroscopia FTIR este considerat o foarte buna tehnic de monitorizare a oxidrii uleiurilor libere i ncapsulate, dovedindu-se totodat a fi o tehnic rapid, acurat i simpl.

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CONCLUZII GENERALE n conformitate cu scopul si obiectivele acestei teze de doctorat, s-a realizat bioncapsularea a patru uleiuri funcionale din plante n diferite matrici naturale, precum i evaluarea eficienei ncapsulrii, stabilitatea i eliberarea uleiurilor din capsulele obtinute. S-a realizat o evaluare comparativ si sistematic a calitii celor patru uleiuri funcionale din plante naintea ncapsulrii lor: uleiul de cnep (HP), uleiul extra virgin de msline (EVO), uleiul de dovleac (PK) i uleiul de ctin (SB) (de provenien din Romnia i Italia). Avnd n vedere obiectivele propuse s-a realizat:

I. S-au identificat caracteristicile uleiurilor nainte de ncapsulare, stabilindu-se markeri de calitate si autenticitate: 1. Majoritatea uleiurilor analizate prezint indicele de iod n conformiatte cu specificaiile din CODEX 210, excepie uleiul de ctin care prezint a valoare mai scazut. 2. Spectrele UV-Vis ale uleiurilor au relevant peakurile specifice, ca i markeri ai autenticitii. 3. Studiile FTIR-ATR au demonstrat relaia dintre benzile aparute n spectre i compoziia specific fiecarui ulei, putndu-se astfel stabili fingerprintul specific uleiurilor studiate. 4. Analizele GC-FID au demonstrate faptul rpofilul acizilor din compoziia uleiurilor analizate este n conformitate cu datele din literatur. II. Studiile experimentale utiliznd ca i metod gelarea ionicde numit:ionotropically crosslinked gelation, n vederea bioncapsulrii uleiurilor funcionale n matrici naturale, au demonstrat stabilitatea i eliberarea controlat a uleiurilor bioncapsulate 1. 2. S-a reusit obinerea diferitelor tipuri de capsule utiliznd matrici naturale i complexe dintre acestea, fiind incorporate uleiuri Caracteristicile capsulelor obinute (aria, perimetrul, compactitatea, sfericitatea i elongaia), n special mrimea lor, au fost influenate de coninutul de uleiuri ncapsulate. Cele mai bune matrici pentru bioncapsularea uleiurilor au fost: alginat 2%, chitosan 2%, i alginate n complex cu k-caragenan, gum guar i gum xantan n raport de 0.75:0.75.

3.

III. Caracterizarea capsulelor a fost realizat prin diferite metode: SEM, FTIR, analize DSC i TGA 1. Suprafata capsulelor analizat prin SEM, a fost non-regulara, aceasta datorita picaturilor de ulei prezente, exceptand chitosanul care prezinta o suprafata mult mai mata
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2.

Prin analizele FTIR-ATR, diferitele capsule coninnd uleiuri au prezentat peakuri care sunt atribuite atat uleiurilor ct i capsulelor goale (regiunile dintre 2800-2900 cm-1 i 1700-900 cm-1). Termogramele DSC au artat faptul c temperature peakurilor crete direct proportional cu cresterea temperaturii, fiecare peak fiind characteristic fiecrui tip de capsula obinut. Analizele TGA au demonstrate ca pierderea n greutate se datoreaza continutului ridicat in apa a unor capsule. ncapsularea uleiurilor nu afecteaz pierderea n greutate a capsulelor.

3.

4.

IV. Evaluarea eficienei ncapsulrii 1. Creterea concentraiei matricilor sau a complexului de matrici determin obinerea unei mai bune eficiene la ncapsulare. s-a obinut cea mai bun eficiena a ncapsulrii utiliznd ca i matrici: alginatul n concentraie de 2%, chitosanul in aceeai concentraie, urmate de concentraiile de 1.5%, i de alginatul n complex cu k-caragenan i gume n raport de 0.75:0.75%. 2. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea i solubilitatea uleiului n matrice. Eliberarea a fost mai lent in cazul hexanului, mai ridicat n cazul metanolului i cea mai buna eliberare fiind in THF, indiferent de matricea sau concentraia utilizat la ncapsulare. 3. Stabilitatea capsulelor la pH 1.2 si pH 4.5, dizolvarea acestora i eliberarea continutului realizandu-se la pH 7.4. V. Protecia bioncapsulrii a uleiurilor mpotriva 1. Uleiurile ncapsulate prezint o mai buna stabilitate mpotriva oxidrii provocate de diferite conditii comparativ cu uleiurile libere. 2. Cea mai bun protecie mpotriva oxidrii a fost asigurat de urmatoarele capsule formate din matrcile i concentraiile urmatoare: alginat 1%, chitosan 1.5%, complexele alginate-gum gum i alginat-gum xantan n raport 0.5:0.5, i alginatk-caragenan complex n raport 0.75:0.75.

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BIBLIOGRAFIE SELECTIV AOAC, 1980, Official Methods of Analysis of the Association of Official Analytical Chemistry. 13th Ed., AOAC, Washington DC 2. AOAC, 1984, Iodine absorption number of oils and fats. In AOAC official methods of analysis (14th ed.). Washington, DC, 506 3. BAETEN, V., AND APARICIO, R., 2000, Edible oils and fats authentication by Fourier transform Raman spectrometry, Biotechnol. Agron. Soc. Environ. 4 (4), 196 203. 4. BAETEN, V., AND PIERNA, J. A. F., 2005, Detection of the presence of hazelnut oil in olive oil by FTRaman and FT-MIR spectroscopy, Journal of Agricultural and Food Chemistry, 53, 6201-6206. 5. BENITA S., 2006, Microencapsulation-Methods and Industrial Applications, 2nd edition, Taylor&Francis, CRC Press, New York. 6. BIRNBAUM, D.T., AND BRANNON-PEPPAS, L., 2003, Molecular weight distribution changes during degradation and release of PLGA nanoparticles containing epirubicin HCl, Journal of Biomaterials Science, Polymer Edition, 14 (1), 87-102. 7. CODEX ALIMENTARIUS, CODEX STANDARD FOR OLIVE OIL, VIRGIN AND REFINED, AND FOR REFINED OLIVE-POMACE OIL CODEX STAN 331981 (Rev. 1-1989), 25-39. 8. CODEX-STAN 210, CODEX STANDARD FOR NAMED VEGETABLE OILS, (Amended 2003, 2005), 1-13. 9. CODEX-STAN 210, Other quality and composition factors Commission Regulation (EEC) no. 2568/91, J. Eur. Commun., No. L, 248, 5.9.91, CODEX STANDARD FOR OLIVE OIL, VIRGIN AND REFINED, AND FOR REFINED OLIVE-POMACE OIL CODEX STAN 33-1981 (Rev. 1-1989). 10. DAI, Z., VOIGT, A., DONATH, E., MHWALD, H., 2001, Novel Encapsulated Functional Dye Particles Based on Alternately Adsorbed Multilayers of Active Oppositely Charged Macromolecular Species, Macromolecular Rapid Communications, 22 (10), 756 762. 11. DE MAN J.M., 1992, Chemical and physical properties of fatty acids, In: Chow CK (ed) Fatty Acids in Foods and Their Health Implications. Marcel Dekker Inc. New York, 18 46. 12. DHANIKULA AB, AND PANCHAGNULA R., 2004, Development and Characterization of Biodegradable Chitosan Films for Local Delivery of Paclitaxel, The AAPS Journal, 6 (3), Article 27. 13. DULIEU, C., PONCELET, D., NEUFELD, R., 1999, Encapsulation and immobilization techniques, In: Cell Encapsulation Technology and Therapeutics, W.M. Khtreiber, R.P. Lanza and W.L. Chick, eds., Birkhuser, Boston, 3-17 14. DZIEEZAK, J.D., 1988, Microencapsulation and encapsulated ingredients, Food Technol. 45(4), 136. 15. GUILLEN, M. D. AND CABO, N., 1997, Characterization of edible oils and lard by Fourier transform infrared spectroscopy. Relationships between composition and frequency of concrete bands in the fingerprint region, J. Am. Oil Chem. Soc., 74 (10), 12811286. 16. GUILLEN, M. D. AND CABO, N., 1997, Infrared Spectroscopy in the Study of Edible Oils and Fats, J Sci Food Agric., 75, 1-11. 17. GUILLEN, M. D. AND CABO, N., 1998, Relationships between the composition of edible oils and lard and the ratio of the absorbance of specific bands of their Fourier
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transform infrared spectra. Role of some bands of the fingerprint region, J. Agric. Food Chem., 46 (5), 17881793. 18. GUILLEN, M. D. AND CABO, N., 1999, Usefulness of the frequencies of some Fourier transform infrared spectroscopic bands for evaluating the composition of edible oil mixtures. Fett-Lipid, 101 (2), 71 76. 19. GUILLEN, M. D. AND CABO, N., 1999, Usefulness of the frequency data of the Fourier transform infrared spectra to evaluate the degree of oxidation of edible oils, J. Agric. Food Chem., 47 (2), 709 719. 20. GUILLEN, M. D. AND CABO, N., 2000, Some of the most significant changes in the Fourier transform infrared spectra of edible oils under oxidative conditions, J. Sci. Food Agric., 80 (14), 2028 2036. 21. GUILLEN, M. D. AND CABO, N., 2002, Fourier transform infrared spectra data versus peroxide and anisidine values to determine oxidative stability of edible oils, Food Chem., 77 (4), 503510. 22. GUILLEN, M.D., CARTON, I., GOICOECHEA, E. AND URIARTE, P.S., 2008, Characterization of Cod Liver Oil by Spectroscopic Techniques. New Approaches for the Determination of Compositional Parameters, Acyl Groups, and Cholesterol from 1H Nuclear Magnetic Resonance and Fourier Transform Infrared Spectral Data, J. Agric. Food Chem., 56, 90729079. 23. HEINZEN, C., 2002, Microencapsulation solve time dependent problems for foodmakers. European Food and Drink Review, 3, 2730. 24. KRAJEWSKA, B., 2005, Membrane-based Processes Performed with use of Chitin/Chitosan Materials, Separation & Purification Technology, 41, 305312. 25. LAPITSKY, Y. AND KALER, E. W., 2006, Surfactant and polyelectrolyte gel particles for encapsulation and release of aromatic oils, Soft Matter, 2, 779-784. 26. LICHTENTHALER H.K., BUSCHMANN C., 2001, Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. Curr. Prot. Food Anal. Chem. F4.3.1 F 4.3.8. 27. MADDUR NAGARAJU SATHEESH KUMAR, SIDDARAMAIA, 2007, Thermogravimetric Analysis and Morphological Behavior of Castor Oil Based PolyurethanePolyester Nonwoven Fabric Composites, Journal of Applied Polymer Science, 106, 35213528. 28. MATEA, C.T., NEGREA, O., HAS, I., IFRIM, S., BELE, C., 2008, Tocopherol and fatty acids contents of selected Romanian cereals grains, Chem. Listy, 99, 1234-2345. 29. OZEN, B. F. AND MAUER, L. J., 2002, Detection of Hazelnut Oil Adulteration Using FT-IR Spectroscopy, J. Agric. Food Chem., 50 (14), 38983901. 30. OZEN, B. F., WEISS, I., et al., 2003, Dietary supplement oil classification and detection of adulteration using Fourier transform infrared spectroscopy, Journal of Agricultural and Food Chemistry, 51, 5871-5876. 31. PARTANEN, R., YOSHII, H., KALLIO, H., YANG, B. AND FORSSELL, P., 2002, Encapsulation of sea buckthorn kernel oil in modified starches. Journal of the American Oil Chemists' Society (JAOCS), 79 (3), 219-223. 32. PEREIRA, L., SOUSA, A., COELHO, H., AMADO, A.M., RIBEIRO-CLARO, P.J.A., 2003, Use of FTIR, FT-Raman and 13C-NMR spectroscopy for identification of some seaweed phycocolloids, Biomolecular Engineering, 20, 223-228. 33. PFUTZE, S., 2003, Encapsulatation and granulation, XI International Workshop on Bioencapsulation, 3-6. 34. PONCELET D., 2006, Microencapsulation: Fundamentals, methods and applications, in Surface Chemistry in Biomedical and Environmental Science ( Brlitz J. and Gunko K. eds), NATO Science Series, Springer verlag, 23-34.
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35.

SOCACIU C., C., MIHIS, A., NOKE, 2008, Oleosome Fractions Separated From Sea Buckthorn Berries: Yield And Stability Studies. in : Seabuckthorn, A Multipurpose Wonder Plant, ed. V.Singh, vol.III, Indus International, India, ISBN: 978-81-7035-520-525, 322-326,. 36. SOCACIU C., C.MIHIS, M.TRIF, H.A.DIEHL, 2007, Seabuckthorn fruit oleosomes as natural, micro-encapsulated oilbodies:separation, characterization, stability evaluation, Proc.15th Int. Symposium on Bioencapsulation, 6-8 Sept, Univ. Viena, Austria, P3-19, 1-3. 37. SOCACIU C., RANGA F., DIEHL, H., 2005, UV-VIS spectrometry applied for the quality and authenticity evaluation of edible oils from Romania, Buletin USAMV-CN, 62, 1454-2382. 38. TRIF M., M.ANSORGE-SCHUMACHER, C.SOCACIU, H.A.DIEHL, 2007, Application of FTIR spectroscopy to evaluate the oxidation of encapsulated seabuckthron oil, 15th Int. Symposium on Bioencapsulation, Universitatea din Viena, Austria, P3-07, 1-3 39. TRIF M., M.ANSORGE-SCHUMACHER, CHEDEA, V., SOCACIU, C., 2007, Release rates measurement of encapsulated castor oil using alginate as microencapsulation matrix, Proc.Int.Symp., Nanotech Insight, 10-17 martie, Luxor, Egipt, 157-159. 40. TRIF, M., 2007, Determination of encapsulated seabuckthorn oil oxidation usiing FTIR-ATR spectroscopy, 63-64, Buletin USAMV-CN, 06-51, 1-3. 41. ZELLER, B.L., SALEEB, F.Z., LUDESCHER, R.D., 1999, Trends in Development of Porous Carbohydrate Food Ingredients for Use in Flavor Encapsulation. Trends in Food Science & Technology, 9, 389-394

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PUBLICAII PE DURATA STAGIULUI DOCTORAL SI PARTICIPARI LA SIMPOZIOANE I CONFERINE NATIONALE I INTERNAIONALE 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 Agrcola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797 6. Andreea Stanila and Monica Trif, Antioxidant activity of carotenoide extracts from HIPPOPHAE RHAMNOIDES, CIGR - International Conference of Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrcola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797 7. Monica Trif, Carmen Socaciu and Horst Diehl, Evaluation of effiency, release and oxidation stability of seabuckthorn encapsulated oil using FTIR spectroscopy, 7th Joint Meeting of AFERP, ASP, GA, PSE & SIF, August 2008, Athens, Greece, Book of Abstracts, pg.39 8. Monica Trif and Carmen Socaciu, Evaluation of effiency, release and oxidation stability of Seabuckthorn microencapsulated oil using Fourier Transformed Infrared Spectroscopy, 4th Meeting on Chemistry and Life, and accepted to be published in Chemick Listy Journal (current IF=0.683)

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2007

1. Monica Trif, Marion Ansorge-Schumacher, Veronica S. Chedea, Carmen Socaciu, Release rates measurement of encapsulated castor oil using alginate as microencapsulation matrix, The International Conference on Nanotechnology: Science and Application (NanoTech Insight), Luxor, 10-17 March 2007, Egipt 2. Chedea V.S., Kefalas P., Trif M. and Socaciu C. Stability studies of encapsulated carotenoid extract from orange waste using pullulan as microencapsulation matrice, Nano Tech Insight, Luxor, 10-17 March 2007, Egipt 3. Monica Trif, Marion Ansorge-Schumacher, Carmen Socaciu, Application of FTIR Spectroscopy for determination of oxidation of encapsulated sea buckthorn oil, Proc.XV International workshop on Bioencapsulation and COST865 Meeting, 2007, Wien, Austria, published in extenso 4. Carmen Socaciu, Cristina Mihis, Monica Trif, Horst A. Diehl, Seabuckthorn fruit oleosomes as natural, microencapsulated oilbodies: separation, characterization, stability evaluation oil, Proc. XV International workshop on Bioencapsulation and COST865 Meeting, 2007, Wien, Austria, published in extenso 5. Socaciu C., Trif M., Ranga F., Fetea F., Bunea A., Dulf F., Bele C. and Echim C. Quality and authenticity of seabuckthorn oils using succesive UV-Vis, FT-IR, NMR spectroscopy and HPLC-, GC- chromatography fingerprints, 3rd Conf. Int. Seabuckthorn Assoc., 2007, Quebec, Canada 6. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. Determination of encapsulated Sea buckthorn oil oxidation using FTIR-ATR spectroscopy, Bull. USAMV-CN, 63-64/2007, ISSN 1843-5262, Romania

2006

1. Monica Trif, Seabuckthorn oleosomes as stabilized bioactive nanostrustures with applications in microencapsulation nutraceuticals, Symposium IRC Transylvania Innovations in Agriculture, Biotechnologies, Animal Breeds and Veterinary Medicine, 2006, USAMV Cluj-Napoca, Romania 2004

1. Veerle Minne, Monica Trif, J.M.C. Geuns, Corina Catana, Steviozide and steviol determination in callus culture of Stevia rebaudiana Bertoni, Bull. USAMV-CN, 61/2004, ISSN 1454-2382, Romania

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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

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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).
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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 AnsorgeSchumacher. I would like to thank the sponsors who made this work possible providing scholarships to pursue doctoral studies: Deutsche Bndestiftung Umwelt (DBU) Germany and EU COST 865.

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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.
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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
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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 50C). Alginates (E400-E404) are produced by brown seaweeds (Phaeophyceae, mainly Laminaria). Gelling properties depends on the ion binding (Mg2+ << Ca2+ < Sr2+ < Ba2+) with the control of the di-cation addition being important for the production of homogeneous gels. Carrageenan (E407) is a collective term for polysaccharides prepared by alkaline extraction (and modification) from red seaweed (Rhodophycae). The strongest gels of carrageenan are formed with K+ rather than Li+, Na+, Mg2+, Ca2+, or Sr2+. Xanthan gum (E415) is a microbial desiccation-resistant polymer prepared commercially by aerobic submerged fermentation from Xanthomonas campestris. Xanthan gum is mainly considered to be non-gelling and used for the control of viscosity due to the tenuous associations endowing it with weak-gel shear-thinning properties. It hydrates rapidly in cold water without lumping to give a reliable viscosity, encouraging its use as thickener, stabilizer, emulsifier and foaming agent. Chitin is obtained in industrial scale from shrimps and crustaceans in general (Yanga et al., 2000). In many studies, chitosan has been crosslinked with aldehydes, such as glutaraldehyde and formaldehyde, to make it a more rigid polymer for use as a core material in research on controlled release. However, biological acceptance of these cross-linked products depends upon the amount of cross-linking agent in the final products and the toxicity of aldehydes has been enormously limited the utilization of the cross-linked chitosan microparticles in the pharmaceutical field.
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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 g/g of oil for -tocopherol, from 74.9 to 492.8 g/g for -tocopherol, and from 35.3 to 1109.7 g/g for -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 -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 (=
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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.
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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.

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Table 1. Chemical and physical characteristics of analyzed oils compared with literature
Hemp Oil Ulei de Canepa Extra Virgin Olive Oil Ulei de Msline Extra Virgin Pumpkin Oil Ulei de Dovleac Sea buckthorn Oil Ulei de Ctina

Chemical and physical characteristics Caracteristicile chimice si fizice Acid value+ (mg KOH/g Oil) Aciditatea+ (mg KOH/g ulei) Iodine value Indicele de iod
+

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).

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A.

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).

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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.).

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


No. Bands Nr. banda 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 914 721 721 1236 1155 1120 1097 1028 HP HP (cm-1) 3008 2956 2923 2853 1742 1654 1463 1456 1418 1396 1377 EVO EVO (cm-1) 3005 2956 2923 2853 1742 1653 1464 1456 1417 1402 1377 1317 1238 1159 1118 1097 1028 958 1418 1398 1377 1319 1238 1157 1120 1099 1029 962 914 721 721 1238 1161 1116 1095 1033 968 -C-O, -CH2-C-O, -CH2-C-O -C-O -C-O -HC=CH- (trans-) -HC=CH- (cis-) -(CH2)n-, -HC=CH(cis-) PK PK (cm-1) 3008 2956 2923 2854 1742 1653 1464 SB SB (cm-1) 3006 2956 2922 2853 1742 1653 1464 1456 1417 1402 1377 -C-H (CH3) =C-H (cis-) bending (rocking) bending bending (symmetric) bending stretching, bending stretching, bending stretching stretching stretching bending out of plane bending out of plane bending (rocking) =C-H (cis-) -C-H (CH3) -C-H (CH2) -C-H (CH2) -C=O (ester) -C=C- (cis-) -C-H (CH2, CH3) stretching stretching (asymetric) stretching (asymetric) stretching (symetric) stretching stretching Functional group Grupul functional Mode of vibration Modul de vibratie

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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.). Table 3. Fatty acid composition (percentage) of the investigated vegetable
Fatty acid % Hemp Oil Extra virgin Olive oil Ulei Extra Virgin de Msline 7.28 Pumpkin oil Seabuckthorn oil

Acizi grai % Ulei de Canep Palmitic acid (16:0) 7.48

Ulei de Dovleac 6.29

Ulei de Ctin 7.76

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Bioencapsulation systems of bioactive compounds extracted from plants oils ________________________________________________________________________________________ Stearic acid (18:0) Arachidic acid (20:0) saturated % Palmitoleic (C16:1) Oleic acid (C18:1) Linoleic acid (C18:2) Linolenic (18:3n3) Eicosadienoic acid (C20:2) unsaturated % C18:1/C18:2 omega 3 : omega 6 fatty acids 0.93 0.92 0.8 1.66 1.06 10.02 2.67 9.95 3.64 9.93 0.3 0.11 8.17 5.4

14.94

36.81

42.44

6.3

72.6

43.14

46.71

0.55 87.54 0.21 -

80.88 0.85 0.022

90.07 0.91 0.02

12.5 6.3 -

II.3. CONCLUSIONS

By GC-FID, the fatty acid composition of the analyzed oils has been determined and compared with the composition of genuine oils reported in the literature or by the direct analysis of the genuine oils. Regarding the content in fatty acids, GC-FID analysis revealed that: hemp oil composition does not agree with the literature for most of the fatty acids, hemp oil contains lower values as the value reported. Oleic acid at least ranged between the values mentioned. primary fatty acids of extra virgin olive oil are oleic and linoleic acid with a small amount of linolenic acid. for pumpkin seed oil, the fatty acid composition is in good agreement with the profile for most of the fatty acids, excepting palmitic and stearic acid found in lower concentrations. the fatty acids composition of seabuckthorn oil demonstrated that this oil is from pulp/peel (whole) berries, being rich in palmitoleic acid and oleic acid.

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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 m.
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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 AG AG -C -C AR A ( AG R ( 0.5 -X 0.7 :0.5 G 5: ) AG (0. 0.75 7 AG -X 5:0 ) -G G (0 .75 G .5 ) AG (0. :0. -G 75: 5) G 0.7 (0 5) .5 : AG 0.5 ) AG oil2 oi % l1 AG .5% oi C l1% H C oil2 Ho % il1 C .5% Ho il1 % 9 8 7 6 5 4 3 2 1 0

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)

Samples/Probele

Fig.III.2. Comparative graphic representation of characteristics of alginate complex with kcarrageenan, xanthan and guar gums, alginate and chitosan beads obtained containing oil: AG-CAR (0.5:0.5) = alginate-k-carrageenan (ratio 0.5:0.5) complex beads containing oil; AG-CAR (0.75:0.75) = alginate-k-carrageenan (ratio 0.5:0.5) complex beads containing oil; AG-XG (0.75:0.75) = alginate-xanthan gum (ratio 0.75:0.75) complex beads containing oil; AG-XG (0.5:0.5) = alginate-xanthan gum (ratio 0.5:0.5) complex beads containing oil; AGGG (0.75:0.75) = alginate-guar gum (ratio 0.75:0.75) complex beads containing oil; AG-GG (0.5:0.5) = alginate-guar gum (ratio 0.5:0.5) complex beads containing oil; AGoil2% = alginate 2% beads containing oil; AGoil1.5% = alginate 1.5% beads containing oil; AGoil1% = alginate 1% beads containing oil; CHoil2% = chitosan 2% beads containing oil; CHoil1.5% = chitosan 1.5% beads containing oil; CHoil1% = chitosan 1% beads containing oil

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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 OH stretching vibration AG 3244 CAR 3514 PolyOH groups GG 3299 XG 3302 CH 3289 O-H + N-H strech CH stretching of CH2 group C-O stretching ( COOH) 2926 1597 2953, 2911, 2894 2884 1636 2935 1651

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Bioencapsulation systems of bioactive compounds extracted from plants oils ________________________________________________________________________________________ Deformations of CH2 group ( bending) O-H bending C-O and C-C ring stretching CH2OH stretching mode COH alcoholic (C-O stretching saccharide) CH2 twisting vibration 948, 902, Gululonic & mannuronic Glycosidic links 809 924, 910 Polyhydroxy groups 842 Galactose sulphate, glycosidic link 866,777 (1,4; 1,6) link galactose and mannose 785 C-H rocking, bending C-C stretching 892, 776 1016 1408 1200-1000 1054 1024 1474, 1400 1223 ( S=O strech sulphate ester 1063 1024 1408 1350 1145 1054 1025 1400 1247 1150 1428 1151 1061 1024

FTIR characterization of different beads containing oils

The spectra of empty beads obtained, beads containing oils and free oils were recorded. Matrices concentrations did not the affected the FTIR-ATR characteristics peak intensities. As example in Fig.III.4. are shown FTIR-ATR spectra of SB oil and alginate 2% beads containing SB oil. The encapsulation of SB oil in alginate induces the decrease of absorbance intensity at 3400 cm-1(which was proportional with the increase of alginate percentage) and shifts of absorbance peaks to lower-wavenumbers in the region 1000-1500 cm-1 specific to the encapsulated SB oil comparing with the free SB oil. By FTIR-ATR analysis, mixture of oils and different blank beads showed the peaks attributable to both oils and empty beads. This confirms the oils entrapment into the beads at the molecular level, the oil specific double peaks (regions between 2800-2900 cm-1 and 1700900 cm-1) which are present also in the free oils.

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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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200 180 160 140 Temperature (C) 120 100 80 60 40 20 0 AG 2% AG 1.5% Alginate AG-CAR CH 2% 1% (0.75%) CH 1% AG-GG AG-KG SB

Fig. III.5. Graphic representation of DSC endothermic peaks of some samples

DSC and TGA has been widely applied in the monitoring of oxidative stability, thermal behavior, kinetic parameters in various oil samples (Jayadas et al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008). The oxidative decomposition of saturated fatty acids according with literature showed weight loss before 380C (Bahruddin et al., 2008). Because on this study the highest temperature of thermal analysis measurements has been 300C, is not taking into consideration this aspect regarding monitoring oxidative stability. This should be an explanation why the analyzed oils did not loss so much weight during thermal measurements, according with literature weight loss % should be more than 10% depending on the oil sample (Jayadas et al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008).
120 100 Restmass % 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 1700900 cm-1) which are present also in the free oils.

CHAPTER IV. ENCAPSULATION EFFICIENCY AND RELEASE STUDIES IV.1. MATERIALS AND METHODS Encapsulation efficiency of the beads The oils encapsulation was determined calculating the amount of -carotene or total carotenoids content of each oil analyzed before and after encapsulation. The samples were assayed for -carotene or total carotenoids content of each oil according previous analysis when was identified the UV-Vis fingerprint, spectrophotometrically. Encapsulation efficiency (EE%) was calculated by using formulae: EE% = C1/C2 x XL0, C1= carotenoid concentration in the oil C2= carotenoid concentration after release from beads Also from the hardening baths, after encapsulation process, were extracted the carotenoids with THF for a better efficiency calculation.
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Release rate measurements of oil from beads


Control release of carotenoids contents in the oils from beads were measurements spectrophotometrically. The absorption spectra were obtained in a CarWin X0 UV-VIS spectrometer. All measurements were performed with the substances inside a 2 mm long quartz glass cuvette. All spectra were recorded at room temperature and the results are the average of 3 runs.

In vitro release oils from the beads The scheme of using the artificial simulated fluids at different pH was as follows: 1st hour: simulated gastric fluid of pH 1.2 2nd to 3rd hour: mixture of simulated gastric and intestinal fluid of pH 4.5 4th to 7th hour: simulated intestinal fluid of pH 7.4 In vitro oil release studies were performed as per scheme in different simulated fluids. Simulation of gastrointestinal (GI) transit conditions was achieved by using different dissolution media. Simulated gastric fluid (SGF) pH 1.2 consisted of 0.1N HCl and X ml Sanzyme (enzyme syrup containing 80 mg papain, 40 mg pepsin and XL mg sanzyme 2000); pH adjusted to 1.2 0.1. Simulated intestinal fluid (SIF) pH 4.5 was prepared by mixing SGF pH 1.2 and SIF pH XX.4 in a ratio 3XX:61; pH adjusted to 4.X 0.1. Simulated intestinal fluid (SIF) pH 7.4 consisted of KH2PO4 1.0XX4g in 30 ml of 0.2N NaOH, and pancreatin 2XXX mg (using Triferment); pH adjusted to XX.4 0.1. The experiment was performed into an incubator with a continuous supply of carbon dioxide at 37C.

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.

3 2.5 Absorbance (a.u.) 2 1.5 1 0.5 0 0 50 100 150 200 250 Time (minutes) 300 350 400
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 1st hour in simulated gastric fluid of pH 1.2; C. after 3rd hours in mixture of simulated gastric and intestinal fluid of pH 4.5; D. in simulated intestinal fluid of pH 7.4 after 30 minutes

IV.3. CONCLUSIONS The studies regarding encapsulation efficiency and stability of oils containing beads show:

1. Increasing the concentration of matrices or complex matrices improved the encapsulation efficiency was obtained. The best concentrations, of all matrices and complex of matrices used, to get the best encapsulation efficiency, were using alginate in concentration 2%, chitosan in concentration 2%, following concentration of 1.5% from these matrices, and alginate in complex with k-carrageenan and gums in ratio 0.75:0.75%. 2. The release rate depends of the diffusivity and solubility of the oil in the matrix, and the swelling collapse transition in the gel. 3. The release rate was substantially slower into hexane than into methanol and the best release was obtained into THF for the type of beads obtained. 4. In vitro oil release studies shown that capsules from alginate, and alginate in complex with carrageenan and gums are completely dissolved at pH 7.4, chitosan beads being not.
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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 m), 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 m) 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 (Guilln 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 After 1h UV/Dupa 1h After 4h UV/Dupa 4h After 6h UV/Dupa 6h UV UV UV


Oil from AG 2%/Ulei din AG 2% Oil from AG 1.5%/Ulei din AG 1.5% Oil free/Ulei liber

Oil from AG 1%/Ulei din AG 1%

Types of ratios on time/Tipul rapoartelor in timp

Fig. V.1. Graphic representation of the hemp oil free and encapsulated (in different alginate concentrations beads) under oxidation changes (A= A2853/A3005-3008, B= A1744/ A3005-3008, C= A1464/ A3005-3008, D= A1377/ A3005-3008, E= A1160/ A3005-3008)

V.3. CONCLUSIONS The usefulness of absorbance ratios and frequency data to measure the oxidative stability and oxidation degree of encapsulated oils directly into the beads was studied. All free oils show SS or TS stage oxidation, compared with the values of encapsulated oils in FS stage of oxidation. The best protection against UV treatment was found to be alginate 1%, chitosan 1.5%, alginate-guar gum and alginate-xanthan gum complexs in ratio 0.5:0.5, and alginate-kcarrageenan complex in ratio 0.75:0.75. FTIR spectroscopy has been found to be a versatile technique for evaluating the oxidative stability of oils free and encapsulated, and for providing information on the oxidation degree of an oil sample in a simple, fast and accurate way.

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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.

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SELECTED BIBLIOGRAPHY AOAC, 1980, Official Methods of Analysis of the Association of Official Analytical Chemistry. 13th Ed., AOAC, Washington DC 2. AOAC, 1984, Iodine absorption number of oils and fats. In AOAC official methods of analysis (14th ed.). Washington, DC, 506 3. BAETEN, V., AND APARICIO, R., 2000, Edible oils and fats authentication by Fourier transform Raman spectrometry, Biotechnol. Agron. Soc. Environ. 4 (4), 196 203. 4. BAETEN, V., AND PIERNA, J. A. F., 2005, Detection of the presence of hazelnut oil in olive oil by FTRaman and FT-MIR spectroscopy, Journal of Agricultural and Food Chemistry, 53, 6201-6206. 5. BENITA S., 2006, Microencapsulation-Methods and Industrial Applications, 2nd edition, Taylor&Francis, CRC Press, New York. 6. BIRNBAUM, D.T., AND BRANNON-PEPPAS, L., 2003, Molecular weight distribution changes during degradation and release of PLGA nanoparticles containing epirubicin HCl, Journal of Biomaterials Science, Polymer Edition, 14 (1), 87-102. 7. CODEX ALIMENTARIUS, CODEX STANDARD FOR OLIVE OIL, VIRGIN AND REFINED, AND FOR REFINED OLIVE-POMACE OIL CODEX STAN 331981 (Rev. 1-1989), 25-39. 8. CODEX-STAN 210, CODEX STANDARD FOR NAMED VEGETABLE OILS, (Amended 2003, 2005), 1-13. 9. CODEX-STAN 210, Other quality and composition factors Commission Regulation (EEC) no. 2568/91, J. Eur. Commun., No. L, 248, 5.9.91, CODEX STANDARD FOR OLIVE OIL, VIRGIN AND REFINED, AND FOR REFINED OLIVE-POMACE OIL CODEX STAN 33-1981 (Rev. 1-1989). 10. DAI, Z., VOIGT, A., DONATH, E., MHWALD, H., 2001, Novel Encapsulated Functional Dye Particles Based on Alternately Adsorbed Multilayers of Active Oppositely Charged Macromolecular Species, Macromolecular Rapid Communications, 22 (10), 756 762. 11. DE MAN J.M., 1992, Chemical and physical properties of fatty acids, In: Chow CK (ed) Fatty Acids in Foods and Their Health Implications. Marcel Dekker Inc. New York, 18 46. 12. DHANIKULA AB, AND PANCHAGNULA R., 2004, Development and Characterization of Biodegradable Chitosan Films for Local Delivery of Paclitaxel, The AAPS Journal, 6 (3), Article 27. 13. DULIEU, C., PONCELET, D., NEUFELD, R., 1999, Encapsulation and immobilization techniques, In: Cell Encapsulation Technology and Therapeutics, W.M. Khtreiber, R.P. Lanza and W.L. Chick, eds., Birkhuser, Boston, 3-17 14. DZIEEZAK, J.D., 1988, Microencapsulation and encapsulated ingredients, Food Technol. 45(4), 136. 15. GUILLEN, M. D. AND CABO, N., 1997, Characterization of edible oils and lard by Fourier transform infrared spectroscopy. Relationships between composition and frequency of concrete bands in the fingerprint region, J. Am. Oil Chem. Soc., 74 (10), 12811286. 16. GUILLEN, M. D. AND CABO, N., 1997, Infrared Spectroscopy in the Study of Edible Oils and Fats, J Sci Food Agric., 75, 1-11. 17. GUILLEN, M. D. AND CABO, N., 1998, Relationships between the composition of edible oils and lard and the ratio of the absorbance of specific bands of their Fourier
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transform infrared spectra. Role of some bands of the fingerprint region, J. Agric. Food Chem., 46 (5), 17881793. 18. GUILLEN, M. D. AND CABO, N., 1999, Usefulness of the frequencies of some Fourier transform infrared spectroscopic bands for evaluating the composition of edible oil mixtures. Fett-Lipid, 101 (2), 71 76. 19. GUILLEN, M. D. AND CABO, N., 1999, Usefulness of the frequency data of the Fourier transform infrared spectra to evaluate the degree of oxidation of edible oils, J. Agric. Food Chem., 47 (2), 709 719. 20. GUILLEN, M. D. AND CABO, N., 2000, Some of the most significant changes in the Fourier transform infrared spectra of edible oils under oxidative conditions, J. Sci. Food Agric., 80 (14), 2028 2036. 21. GUILLEN, M. D. AND CABO, N., 2002, Fourier transform infrared spectra data versus peroxide and anisidine values to determine oxidative stability of edible oils, Food Chem., 77 (4), 503510. 22. GUILLEN, M.D., CARTON, I., GOICOECHEA, E. AND URIARTE, P.S., 2008, Characterization of Cod Liver Oil by Spectroscopic Techniques. New Approaches for the Determination of Compositional Parameters, Acyl Groups, and Cholesterol from 1H Nuclear Magnetic Resonance and Fourier Transform Infrared Spectral Data, J. Agric. Food Chem., 56, 90729079. 23. HEINZEN, C., 2002, Microencapsulation solve time dependent problems for foodmakers. European Food and Drink Review, 3, 2730. 24. KRAJEWSKA, B., 2005, Membrane-based Processes Performed with use of Chitin/Chitosan Materials, Separation & Purification Technology, 41, 305312. 25. LAPITSKY, Y. AND KALER, E. W., 2006, Surfactant and polyelectrolyte gel particles for encapsulation and release of aromatic oils, Soft Matter, 2, 779-784. 26. LICHTENTHALER H.K., BUSCHMANN C., 2001, Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. Curr. Prot. Food Anal. Chem. F4.3.1 F 4.3.8. 27. MADDUR NAGARAJU SATHEESH KUMAR, SIDDARAMAIA, 2007, Thermogravimetric Analysis and Morphological Behavior of Castor Oil Based PolyurethanePolyester Nonwoven Fabric Composites, Journal of Applied Polymer Science, 106, 35213528. 28. MATEA, C.T., NEGREA, O., HAS, I., IFRIM, S., BELE, C., 2008, Tocopherol and fatty acids contents of selected Romanian cereals grains, Chem. Listy, 99, 1234-2345. 29. OZEN, B. F. AND MAUER, L. J., 2002, Detection of Hazelnut Oil Adulteration Using FT-IR Spectroscopy, J. Agric. Food Chem., 50 (14), 38983901. 30. OZEN, B. F., WEISS, I., et al., 2003, Dietary supplement oil classification and detection of adulteration using Fourier transform infrared spectroscopy, Journal of Agricultural and Food Chemistry, 51, 5871-5876. 31. PARTANEN, R., YOSHII, H., KALLIO, H., YANG, B. AND FORSSELL, P., 2002, Encapsulation of sea buckthorn kernel oil in modified starches. Journal of the American Oil Chemists' Society (JAOCS), 79 (3), 219-223. 32. PEREIRA, L., SOUSA, A., COELHO, H., AMADO, A.M., RIBEIRO-CLARO, P.J.A., 2003, Use of FTIR, FT-Raman and 13C-NMR spectroscopy for identification of some seaweed phycocolloids, Biomolecular Engineering, 20, 223-228. 33. PFUTZE, S., 2003, Encapsulatation and granulation, XI International Workshop on Bioencapsulation, 3-6. 34. PONCELET D., 2006, Microencapsulation: Fundamentals, methods and applications, in Surface Chemistry in Biomedical and Environmental Science ( Brlitz J. and Gunko K. eds), NATO Science Series, Springer verlag, 23-34.
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35.

SOCACIU C., C., MIHIS, A., NOKE, 2008, Oleosome Fractions Separated From Sea Buckthorn Berries: Yield And Stability Studies. in : Seabuckthorn, A Multipurpose Wonder Plant, ed. V.Singh, vol.III, Indus International, India, ISBN: 978-81-7035-520-525, 322-326,. 36. SOCACIU C., C.MIHIS, M.TRIF, H.A.DIEHL, 2007, Seabuckthorn fruit oleosomes as natural, micro-encapsulated oilbodies:separation, characterization, stability evaluation, Proc.15th Int. Symposium on Bioencapsulation, 6-8 Sept, Univ. Viena, Austria, P3-19, 1-3. 37. SOCACIU C., RANGA F., DIEHL, H., 2005, UV-VIS spectrometry applied for the quality and authenticity evaluation of edible oils from Romania, Buletin USAMV-CN, 62, 1454-2382. 38. TRIF M., M.ANSORGE-SCHUMACHER, C.SOCACIU, H.A.DIEHL, 2007, Application of FTIR spectroscopy to evaluate the oxidation of encapsulated seabuckthron oil, 15th Int. Symposium on Bioencapsulation, Universitatea din Viena, Austria, P3-07, 1-3 39. TRIF M., M.ANSORGE-SCHUMACHER, CHEDEA, V., SOCACIU, C., 2007, Release rates measurement of encapsulated castor oil using alginate as microencapsulation matrix, Proc.Int.Symp., Nanotech Insight, 10-17 martie, Luxor, Egipt, 157-159. 40. TRIF, M., 2007, Determination of encapsulated seabuckthorn oil oxidation usiing FTIR-ATR spectroscopy, 63-64, Buletin USAMV-CN, 06-51, 1-3. 41. ZELLER, B.L., SALEEB, F.Z., LUDESCHER, R.D., 1999, Trends in Development of Porous Carbohydrate Food Ingredients for Use in Flavor Encapsulation. Trends in Food Science & Technology, 9, 389-394

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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 Agrcola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797 6. Andreea Stanila and Monica Trif, Antioxidant activity of carotenoide extracts from HIPPOPHAE RHAMNOIDES, CIGR - International Conference of Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrcola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797 7. Monica Trif, Carmen Socaciu and Horst Diehl, Evaluation of effiency, release and oxidation stability of seabuckthorn encapsulated oil using FTIR spectroscopy, 7th Joint Meeting of AFERP, ASP, GA, PSE & SIF, August 2008, Athens, Greece, Book of Abstracts, pg.39 8. Monica Trif and Carmen Socaciu, Evaluation of effiency, release and oxidation stability of Seabuckthorn microencapsulated oil using Fourier Transformed Infrared Spectroscopy, 4th Meeting on Chemistry and Life, and accepted to be published in Chemick Listy Journal (current IF=0.683)

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2007

1. Monica Trif, Marion Ansorge-Schumacher, Veronica S. Chedea, Carmen Socaciu, Release rates measurement of encapsulated castor oil using alginate as microencapsulation matrix, The International Conference on Nanotechnology: Science and Application (NanoTech Insight), Luxor, 10-17 March 2007, Egipt 2. Chedea V.S., Kefalas P., Trif M. and Socaciu C. Stability studies of encapsulated carotenoid extract from orange waste using pullulan as microencapsulation matrice, Nano Tech Insight, Luxor, 10-17 March 2007, Egipt 3. Monica Trif, Marion Ansorge-Schumacher, Carmen Socaciu, Application of FTIR Spectroscopy for determination of oxidation of encapsulated sea buckthorn oil, Proc.XV International workshop on Bioencapsulation and COST865 Meeting, 2007, Wien, Austria, published in extenso 4. Carmen Socaciu, Cristina Mihis, Monica Trif, Horst A. Diehl, Seabuckthorn fruit oleosomes as natural, microencapsulated oilbodies: separation, characterization, stability evaluation oil, Proc. XV International workshop on Bioencapsulation and COST865 Meeting, 2007, Wien, Austria, published in extenso 5. Socaciu C., Trif M., Ranga F., Fetea F., Bunea A., Dulf F., Bele C. and Echim C. Quality and authenticity of seabuckthorn oils using succesive UV-Vis, FT-IR, NMR spectroscopy and HPLC-, GC- chromatography fingerprints, 3rd Conf. Int. Seabuckthorn Assoc., 2007, Quebec, Canada 6. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. Determination of encapsulated Sea buckthorn oil oxidation using FTIR-ATR spectroscopy, Bull. USAMV-CN, 63-64/2007, ISSN 1843-5262, Romania

2006

1. Monica Trif, Seabuckthorn oleosomes as stabilized bioactive nanostrustures with applications in microencapsulation nutraceuticals, Symposium IRC Transylvania Innovations in Agriculture, Biotechnologies, Animal Breeds and Veterinary Medicine, 2006, USAMV Cluj-Napoca, Romania 2004

1. Veerle Minne, Monica Trif, J.M.C. Geuns, Corina Catana, Steviozide and steviol determination in callus culture of Stevia rebaudiana Bertoni, Bull. USAMV-CN, 61/2004, ISSN 1454-2382, Romania

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