rezumatul - usamvcluj.ro · pot fi produşi de sisteme biologice (ex: microorganisme, plante şi...

UNIVERSITATEA DE ŞTIINŢE

AGRICOLE ŞI MEDICINĂ VETERINARĂ, CLUJ-NAPOCA

FACULTATEA DE ZOOTEHNIE ŞI

BIOTEHNOLOGII

DOMENIUL: BIOTEHNOLOGII

REZUMATUL

TEZEI DE DOCTORAT

SISTEME DE ÎNCAPSULARE A UNOR COMPUŞI BIOACTIVI EXTRAŞI DIN ULEIURI VEGETALE

MONICA TRIF

Ing. Dipl. Biotehnolog

CONDUCĂTOR ŞTIINŢIFIC:

PROF. Dr. Dr. h.c. HORST A. DIEHL

2009

Sisteme de încapsulare a unor compuşi bioactivi extraşi din uleiuri vegetale ________________________________________________________________________________________

II

CUPRINS

I. INTRODUCERE. SCOP ŞI OBIECTIVE............................................................................ III

PARTEA II. CONTRIBUŢII PROPRII (ORIGINALE) ......................................................... IX

CAPITOL II. CARACTERIZAREA ULEIURILOR FUNCŢIONALE UTILIZATE LA BIOÎNCAPSULARE ............................................................................................................... IX

II.1. MATERIALE ŞI METODE ......................................................................................... IX

II.2. REZULTATE ŞI DISCUŢII.......................................................................................... X

II.3. CONCLUZII .............................................................................................................. XIV

CAPITOLUL III. BIOÎNCAPSULAREA ULEIURILOR: PROTOCOALE DE PREPARE A CAPSULELOR ŞI CARACTERIZAREA LOR.................................................................... XV

III.1. MATERIALE ŞI METODE...................................................................................... XV

III.2. REZULTATE ŞI DISCUŢII .................................................................................... XVI

III.3. CONCLUZII............................................................................................................XXII

CAPITOL IV. EFICIENŢA ÎNCAPSULĂRII ŞI STUDII DE ELIBERARE A ULEIURILOR DIN CAPSULE....................................................................................................................XXII

IV.1. MATERIALE ŞI METODE....................................................................................XXII

IV.2. REZULATTE ŞI DISCUŢII ................................................................................. XXIII

IV.3. CONCLUZII ......................................................................................................... XXVI

CAPITOL V. CARACTERIZAREA FTIR A OXIDĂRII ULEIURILOR .....................XXVII

V.1. MATERIALE ŞI METODE ..................................................................................XXVII

V.2. REZULTATE ŞI DISCUŢII..................................................................................XXVII

V.3. CONCLUZII.........................................................................................................XXVIII

CONCLUZII GENERALE ................................................................................................ XXIX

BIBLIOGRAFIE SELECTIVĂ ......................................................................................... XXXI

PUBLICAŢII PE DURATA STAGIULUI DOCTORAL SI PARTICIPARI LA SIMPOZIOANE ŞI CONFERINŢE NATIONALE ŞI INTERNAŢIONALE............... XXXIV


III

I. INTRODUCERE. SCOP ŞI OBIECTIVE

BIOÎNCAPSULAREA reprezintă o tehnologie nouă, bazată pe inserţia şi imobilizarea moleculelor bioactive, în ‘’suporturi’’ specifice (matrici). Tehnologia încapsulării este bine dezvoltată şi utilizată în industria farmaceutică, chimică, cosmetică, alimentară precum şi în cea tipografică (Augustin et al., 2001; Heinzen, 2002). Potenţialul bioîncapsulării s-a concretizat tot mai mult în domeniile biotehnologiei, mai ales în cele agricole şi alimentare. În ultimele decenii, încapsularea compuşilor activi a devenit o tehnologie de mare interes şi însemnătate, fiind adecvată atât pentru ingredienţii alimentari cât şi pentru cei chimici, farmaceutici sau cosmetici.

Aplicarea acestei metode de success, de bioîncapsulare a compuşilor bioactivi extraşi din uleiuri vegetale ar putea permite stabilirea combinaţilor şi a calitaţilor optime ale acestor substanţe. Este de luat în considerare că o asemenea metodă şi anume bioîncapsularea, aplicată în aria comercială, ar avea beneficii semnificative pentru industria farmaceutică, alimentară şi cosmetică. În afară de aceasta este de consemnat faptul că, cercetarea şi dezvoltarea în aceste domenii este semnificativă mai ales în ceea ce priveşte conservarea compuşilor naturali bioactivi extraşi din plante.

Scopul acestei tezei constă în utilizarea diferitelor matrici naturale pentru bioîncapsularea moleculelor bioactive prin metoda gelării ionice (‘’ionotropically crosslinked gelation’’), precum şi în evaluarea diferenţelor de calitate şi a eficienţei parametrilor pentru produşii încapsulaţi şi nu în ultimul rând a eliberării controlate a moleculelor bioactive din matrici.

Structura tezei. Prima parte a acestei teze este reprezentată de un studiu de literatură, partea a doua include rezultatele experimentale: materiale şi metode, rezultate şi discuţii, concluzii.

Prima parte (Studiul de literatură) este compusă din patru capitole (I-IV):

Capitolul I. Bioîncapsularea: definiţie, principii, aplicaţii, metode şi tehnici

Capitolul II. Uleiuri vegetale funcţionale: caracterizarea fizică, chimică şi autentificarea

Capitolul III: Încapsularea uleiurilor: matrici, metode şi tehnici de încapsulare, evaluarea eficienţei şi a stabilităţii

Capitolul IV. Metode pentru caracterizarea capsulelor

Partea a doua a tezei (Contribuţiile proprii) include patru capitole, dupa cum urmează:

Capitolul V. Caracterizarea uleiurilor funcţionale utilizate pentru bioîncapsulare. Această parte caracterizează patru uleiuri funcţionale (ulei de cânepa, ulei de dovleac, ulei extra virgin de măsline şi ulei de cătină) analizate şi apoi încapsulate prin diferite tehnici: spectroscopie de absorbţie în ultraviolet (UV), cromatografie de gaze (GC) cu detecţie prin ionizare în flacără (FID) şi spectroscopie în infraroşu cu transformantă fourier echipată cu reflectanţă atenuată orizontală (FTIR-ATR), determinările chimice fiind realizate în conformitate cu metodele descrise în A.O.A.C. şi IOOC.


IV

Capitolul VI. Optimizarea protocoalelor de obţinere a caspsulelor utilizând matrici naturale şi caracterizarea capsulelor ce conţin ulei încapsulat. Acest capitol descrie protocoalele pentru: sinteza capsulelor goale de diferite mărimi şi concentraţii, sinteza capsulelor de diferite mărimi şi concentraţii ce încorporează ulei, caracterizarea capsulelor goale şi a celor ce conţin uleiuri (în funcţie de mărime şi morfologie), cuprinzând de asemenea şi analiza FTIR-ATR şi termică a capsulelor.

Capitolul VII. Studierea eficienţei încapsulării şi a eliberării uleiurilor încapsulate. Acest capitol conţine studii cu privire la eficienţa încapsulării uleiurilor funcţionale în diferite matrici, determinarea ratei de eliberare a uleiurilor din capsule în timp şi în diferiţi solvenţi, precum şi eliberarea in vitro a uleiurilor din capsule.

Capitolul VII. Caracterizarea FTIR-ATR a oxidării uleiurilor. Acest capitol include analize comparative a uleiurilor libere şi încapsulate supuse oxidării în timp în condiţii UV.

Planul experimental se bazeaza pe urmatoarele obiective:

Utilizarea diferitelor matrici naturale (precum alginatul, alginatul în complex cu k- caragenan şi gume: xantan şi guar, şi chitosan) în scopul încapsulării uleiurilor funcţionale (ulei de dovleac, ulei extra virgin de măsline, ulei de cânepă şi ulei de cătină)

Îmbunătăţirea şi optimizarea metodelor de bioîncapsulare pentru uleiurile vegetale cu proprietăţi funcţionale

Investigarea morfologiei diferitelor capsule obţinute (microscopie electronică de scanare), caracterizarea capsulelor (suprafaţă, diametru, perimetru, elongaţie, sfericitate şi compactitate), analize FTIR

Investigarea uleiurilor funcţionale bioîncapsulate: eficienţa şi stabilitatea încapsulării, eliberarea controlată a uleiurilor încapsulate, materialul şi funcţionalitatea capsulelor obţinute, caracterizarea FTIR a uleiurilor libere, a capsulelor obţinute şi oxidarea uleiurilor libere şi încapsulate.

Cercetările prezentate au fost efectuate la Departamentul de Chimie şi Biochimie din cadrul Universităţii de Ştiinţe Agricole şi Medicină Veterinară, Cluj-Napoca, în colaborare cu Universitatea Tehnică Berlin (TU Berlin), Germania, Departamentul de Tehnologie a Enzimelor, sub supravegherea Prof. Dr. rer. nat. Marion Ansorge-Schumacher. Aş dori de asemenea să mulţumesc în mod special sponsorilor (Deutsche Bündestiftung Umwelt (DBU) Germany şi EU COST 865) ce au facut aceste cercetări posibile, acordându-mi cele două burse pentru studiile doctorale.


V

INTRODUCERE

Microîncapsularea este procesul de producere a capsulelor la o scară micrometrică sau milimetrică, fiind cunoscute sub numele de capsule.

Bioîncapsularea beneficiază de principiile fundamentale ale încapsulării şi implică învelirea efectivă a unei forme vii într-o membrană care este inertă, non-toxică pentru celulă, şi stabilă la condiţiile interioare ale reacţiilor biochimice precum agitarea (Muralidhar R.V. et al., 2001).

Microcapsula este o capsulă mică, iar procedura de preparare a acesteia este numită microîncapsulare. Aceasta poate încorpora diferite tipuri de forme materiale pentru a suplimeta funcţiile secundare şi/sau pentru a compensa în diferite condiţii de mediu.

Microcapsulele pot fi clasificate în trei categorii de bază în funcţie de morfologia acestora: mononuleare, polinucleare sau de tip matrice.

Microcapsulele mononucleare conţin membrana care protejează compusul bioactiv; în Fig.1. sunt prezentate câteva tipuri de capsule.

Fig. 1. Diferite tipuri de capsule utilizate (Birnbaum D.T. şi Brannon-Peppas L., 2003)

Capsulele polinucleare prezintă mai mulţi compuşi bioactivi încorporaţi în interiorul

unei membrane. Încapsularea de tip matrice conţine cmpusul bioactiv distribuit omogen pe toată suprafaţa interioară.

Scopul microîncapsulării

În general există numeroase motive pentru care substanţele ar trebui încapsulate (Li S.P. şi col., 1988; Finch C.A., 1985; Arshady, R., 1993):

• Creştera stabilităţii pentru protejarea compuşilor activi de mediul extern • Pentru convertirea componenţilor lichizi activi într-un sistem solid uscat • Pentru separarea componenţilor incompatibili din punct de vedere funcţional • Pentru a masca proprietăţile nedorite a componenţilor activi • Pentru a proteja mediul extern al microcapsulelor de componenţi activi • Pentru a controla eliberarea compuşilr activi de procesel de eliberare întârziată sau

eliberarea susţinută • Separarea omponenţilor incompatibili


VI

• Conversia lichidelor în solide • Mascarea mirosului, activităţii, etc. • În scop farmaceutic

Tehnologia de încapsulare este foarte bine dezvoltată fiind acceptată în multe industrii precum: farmaceutică, chimică, cosmetică, alimentară (Augustin et al., 2001; Heinzen, 2002).

În industria alimetară, grăsimile şi uleiurile, compuşii aromatizanţi şi oleorezinele, vitaminele, mineralele, coloranţii şi enzimele au fost deja încapsulate (Dziezak, 1988; Jackson şi Lee, 1991; Shahidi şi Han, 1993).

Alegerea unei tehnici adecvate de bioîncapsulare depinde de utilizarea finală a produsului şi de condiţiile de procesare implicate în obţinerea produsului final. Bioîncapsularea îşi găseşte aplicaţie din ce mai multă aplicabilitate în domeniul biotehnologiilor şi în special în alimentaţie şi agricultură. În ultimele decenii, încapsularea compuşilor activi a devenit un process de mare interes şi însemnătate, fiind adecvat atât pentru ingredienţii alimentari cât şi pentru cei chimici, farmaceutici sau cosmetici.

Pfutze S. (2003) consideră că tehnologiile de încapsulare pot fi divizate în două categorii: • formarea matricea capsulelor: un ingredient activ şi protector formează granule

omogene. Produsul activ este uniform distribuit în granulă fiind înconjurat din abundenţă de material protector, formând matricea activă.

• formarea învelişului capsulelor: materialul activ este granulat şi acoperit de un strat protector. Materialul activ şi protector este bine separat. Obiectivul principal este construirea unei bariere între particulele componente şi

mediu. Această barieră reprezintă o protecţie împotriva oxigenului, apei, luminei; evitarea contactului cu alte particule sau ingrediente; sau controlul eliberării lor în timp. Protecţia compuşilor bioactivi pe parcursul procesării şi păstrării, precum şi eliberarea controlată în tractusul gastrointestinal este o prioritate în exploatarea potenţialului benefic al multor compuşi bioactivi.

Tehnicile utilizate la bioîncapsulare necesită un material drept înveliş şi o substanţă protejată. Materialul utilizat trebuie aprobat de Administraţia Alimentaţiei şi Farmacie (US) sau de Autoritatea Europeană pentru Securitatea Alimentelor (Europa) (Amrita şi col., 1999).

Coacervarea: încapsularea lichidelor

Coacervarea complexelor (sau faza de separare), este prima aplicaţie la scară largă a tehnologiei de microîncapsulare. Coacervarea este un proces care are loc în soluţii coloidale şi de multe ori privită ca metoda originală de încapsulare (Risch, 1995).

Aplicabilitate coacervării complexelor este enormă dar are şi limite datorită costurilor ei ridicate, în unele aplicaţii. Aceasta include încapsularea:

aromelor vitaminelor cristaleor lichide pentru dispozitivele de display sisteme de imprimare


VII

ingredienţi activi pentru industria farmaceutică bacteri şi celule

Matricile – materiale pentru încapsulare

Există diferite materiale ce pot fi utilizate pentru încapsulare precum: polielectroliţi sintetici (Sukhorukov şi col., 1998; Donath şi col., 1998), polielectroliţi naturali (Shenoy şi col., 2003), nanoparticule anorganice (Caruso şi col., 2001), grăsimi (Moya şi col., 2000), coloranţi (Dai şi col., 2001), ioni polivalenţi (Radtchenk şi col., 2005), şi biomacromolecule (Yang şi col., 2006).

În general trei clase de materiale au fost utilizate: materiale naturale derivate (colaen ş alginat), matrici tisulare acelulare (submucoase intestinale) şi polimeri sintetici (acid poliglicolic, etc.). Aceste clase de biomateriale au foste testate în concordanţă cu biocomapatibilitatea lor (Pariente şi col., 2002).

Biopolimerii sunt polimeri care provin din surse naturale, sunt biodegradabili, şi nontoxici. Pot fi produşi de sisteme biologice (ex: microorganisme, plante şi animale), sau chimic sintetizate din materiale biologice (ex: amidon, grăsimi sau uleiuri, etc.).

Polimeri naturali şi derivaţi ai acestora: polimeri anionici: acid alginic, pectină, caragenan; polimeri cationici:chitosan, polilizină; polimeri amfipatici: colagen (and gelatină), chitină; polimeri neutri: dextran, agaroză, pululan.

Guma guar (E412, numită şi guaran) este extrasă din seminţele leguminoaselor din familia Cyamopsis tetragonoloba. Guma guar prezintă vâscozitate scăzute dar este un bun agent de întărire. Fiind un polimer non-ionic, nu este influenţat de pH, dar este influenţat de temperaturi extreme la anumite pH-uri (ex: pH=3 la 50°C).

Alginatul (E400-E404) este produs extras din algele brune (Phaeophyceae, în special Laminaria). Proprietăţile de gelifiere depind de interacţia cu unii ioni (Mg2+ << Ca2+ < Sr2+ < Ba2+).

Caragenan (E407) este un nume colectiv atribuit polizaharidelor, obţinute prin extracţia alcalină din algele roşii (Rhodophycae). Geluri puternice sunt formate de k-caragenan în prezenţa ionilor de K+ şi mai slab în prezenţa ionilor de Li+, Na+, Mg2+, Ca2+, sau Sr2+.

Guma xantan (E415) este un polimer microbian preparat commercial prin fermentaţia aerobică din Xanthomonas campestris. Guma xantan nu prezintă proprietăţi ridicate de gelifiere, este hidratată uşor în apă rece, având aplicaţii ca şi emulgator, stabilizator.

Chitosanul este obţinut la scală industrială din carapacea crustaceelor (Yanga şi col., 2000). În multe studii chitosanul este legat cu ajutorul aldehidelor, pecum glutaraldehida şi formaldehida, pentru obţinerea lui sub o forma mai vâscoasă cu aplicaţii ca şi material de încapsulat.

Mulţi componenţi naturali conţinuţi în uleiurile vegetale prezintă proprietăţi utile.

Uleiul de cânepă rezultă prin presarea seminţelor de cânepă (Cannabis sativa L). Acidul oelic (Omega 9) conţinut în uleiul de cânepă menţine o bună funcţionalitate arterială. În exces acidul oleic poate interfera cu acizii graşi esenţiali şi prostaglandinele.


VIII

Uleiul de măsline conţine trigliceroli şi cantităţi mici de acizi graşi liberi, glicerol, pigmenţi, compuşi aromatizanţi, steroli, tocoferoli, fenoli, componenţi răşinoşi neidentificaţi, etc.(Kiritsakis A., 1998).

Uleiul de dovleac este foarte sănătos, de caliate superioară, fiind în clasamentul primelor 3 uleiuri nutritive. Seminţele de dovleac au un gust intens şi sunt bogate în acizi graşi polinesaturaţi. Uleiul brun are un gust amărui. Conţinutul în tocoferoli ai uleiurilor oscilează de la 27,1 la 75,1 μg/g de ulei pentru α-tocoferol, de la 74.9 la 492.8 μg/g pentru γ-tocoferol, şi de la 35.3 la 1109.7 μg/g pentru δ-tocopherol (Stevenson şi col., 2007)

Uleiurile de cătină conţin o cantitate ridicată de acizi esenţiali, linoleic şi alfa linoleic (Chen şi col., 1990), care sunt precursori ai altor acizi graşi polinesaturaţi cum ar fi acidul arahidonic sau eicosapentanoic. Este stocat în organitele extracitoplasmatice numite vezicule de ulei, o formă naturală de încapsulare (Socaciu et all, 2007, 2008). Uleiul din pulpa frutelor de cătină este bogat în acid palmitoleic şi acid oleic (Chen şi col., 1990).

Uleiurile conţin de asemenea flavonoizi (Chen şi col., 1991), carotenoizi, steroli liberi şi esterificaţi, triterfenoli şi izoprenoli (Goncharova şi Glushenkova, 1996). Conţinutul în carotenoizii variază de asemenea în funcţie de sursa de provenienţă a uleiului.

Proprietăţile fizice şi chimice ale uleiurilor funcţionale

Proprietăţile fizice şi chimice ale uleiurilor, incluzând indicele de iod, de saponificare şi valorile de aciditate şi pentru peroxizi, indicele de refracţie, densitate şi materia nesaponificabilă sunt determinate conform procedurilor standard. Indicele de iod măsoară gradul de nesaturare al uleiurilor. Valoarea acestuia sub 100 demonstrază că uleiul prezintă un grad redus de saturare (Pa Quart, 1979; Pearson, 1981). Indicele de saponificare este un indicator al mediei masei moleculare a acizilor graşi prezenţi în ulei (AOAC, 1980; Pearson, 1981). Indicele de peroxid este frecvent utilizat pentru măsurarea stadiului de oxidare al uleiului. Acesta indică oscilarea oxidativă a uleiului (deMan, 1992).

Tehnicile pentru caracterizarea şi autentificarea uleiurilor funcţionale

Există diferite tehnici pentru caracterizarea şi autentificare produselor alimetare. Metodele de autentificare aplicate pentru uleiuri şi grăsimi pot fi clasificate ca şi chimice (de separative) sau fizice (non-separative).

Spectrometrele de infraroşu cu transformantă fourier (FTIR) prezintă multe avantaje în comparaţie cu instrumentele convenţionale de dispersie, printr-o excelentă reproductibilitate şi acurateţe a lungimilor de undă, precisa manipulare spectrală şi utilizarea unor programe chemometrice pentru calibrare. Accesoriile HATR au fost de asemenea larg utilizate în dezvoltarea metodelor FTIR pentru analizarea uleiurilor şi a grăsimilor, deoarece acestea pot oferi mijloace convenable şi simple pentru o manipulare uşoară (Sedman şi col., 1999). Spectroscopia infraroşu de mijloc (MIR) poate fi utilizată pentru identificarea compuşilor organici deoarece unele grupe de atomi prezintă proprietăţi ale frecvenţei de absorbţie a vibraţiilor în regiunea infraroşie a spectrului electromagnetic. Reflectanţa orizontală totală atenuată (HATR) este accesoriul cel mai des utilizat in metoda FTIR pentru analizele uleiurilor şi a grăsimilor (Sedman şi col., 1999).


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O largă varietate de alimente utilizează pentru încapsulare aromatizanţi, acizi, baze, îndulcitori artificiali, coloranţi, antioxidanţi, agenţi cu arome nedorite, mirosuri, etc. Aceştia îşi păstreză bioactivitatea şi rămân accesibili agenţilor externi.

Fitosterolii, flavonoizii şi compusii organici cu sulf, reprezintă trei grupe de compuşi caracteristici fructelor şi legumelor, care ar putea prezenta importanţă în reducerea riscului de ateroscleorză. (Howard şi Kritchevsky, 1997). Unele substanţe fitochimice, cu ar fi acidul ascorbic, carotenoizii, vitamina E, fitofenoli, izoflavoni şi fitosteroli, au fost evidenţiate ca ingredienţi fiziologic activi ce îmbunătăţesc rezistenţa la anumite boli.

Încapsularea poate fi utilizată pentru condiţionarea uleiurilor în forme solide sau solubile în apă, extinzând utilizarea lor în multe alte aplicaţii. Încapsularea uleiurilor include ca metode şi tehnici: spray-drying, spray-chilling, fluid bed encapsulation, extrusion encapsulation şi încapsularea prin coacervare.

Extrudarea este utilizată pentru încapsularea mineralelor şi vitaminelor în uleiuri (grăsimi saturate) într-o matrice de tip polizaharidic (Van Lengerich şi Lakis, 2002). Protecţia împotriva oxidării metil-linoleatului încapsulat cu gumă acacia prin metoda spray drying şi freeze drying, depinde de umiditatea relativă a mediului (Minemoto şi col., 1997).

În majoritatea cazurilor matricile utilizate pentru încapsularea uleiurilor şi grăsimilor sunt gume (acacia, arabică), proteine, carbohidraţi (cazeină/zaharuri), maltodextrină, beta-ciclodextrine, alginat de sodiu, gelatină.

PARTEA II. CONTRIBUŢII PROPRII (ORIGINALE)

CAPITOL II. CARACTERIZAREA ULEIURILOR FUNCŢIONALE UTILIZATE LA BIOÎNCAPSULARE

Uleiurile extrase din plante (floarea-soarelui, dovleac, soia, rapita, etc.) sunt foarte utilizate in domeniul alimentar, dar si in alte industrii (cosmetică, farmaceutică, etc.). PrezintĂ o deosebita insemnatate datorita numerosilor componenţi benefici care intră în alcătuirea lor. Calitatea şi autenticitatea acestor uleiuri se realizează prin diferite tehnici. Cele mai utilizate trei tehnici în vederea caracterizării acestor uleiuri sunt: spectrometriA UV-Vis, cromatografia gaz cu detecţie prin ionizare în flacără FID, şi spectroscopia infraroşu cu transformantă fourier (FTIR).

II.1. MATERIALE ŞI METODE

Au fost alese în vederea încapsulării patru uleiuri de mare interes: cătină (SBO) extras din fructele de catina, colectate din regiunea Clujului (Transilvania, nordul Romaniei), ulei de măsline extra virgin (EVO) din Italia, cânepă (HP) şi dovleac (PK) din Romania.

Analizele chimice au fost determinate conform metodelor descrise de: A. O. A. C. şi IOOC sau de Comisia Uniunii Europene (EU): aciditatea şi indicele de iod. Toate probele au fost analizate în triplicat. Aciditatea a fost calculată luându-se în considerare conţinutul de


X

acizi graşi liberi ale uleiurilor analizate, determinat prin titrare conform metodei oficiale Ca 5a-40. Indicele de iod a fost determinat prin metoda AOCS Cd 1c-85 (1997).

II.2. REZULTATE ŞI DISCUŢII

Determinarea acidităţii şi a indicelui de iod

Rezultatele analizelor chimice prezentate în Tabelul II.1. au demonstrat o bună corelaţie a valorilor obţinute cu cele publicate în literatură.

Tabel II.1. Caracteristicile chimice şi fizice ale uleiurilor analizate în comparaţie cu literatura

Ulei de Cânepa Ulei de măsline extra virgin

Ulei de dovleac Ulei de cătină

Caracteristicile fizice si chimice

Aciditate

(mg KOH/g ulei)

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

Indicele de iod** 162 / 145-166* 87 / 75-95* 130 / 116-133* 71 / 98-119*

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

* Date din literatura

Aceste date demonstrează faptul că valorile acestor uleiuri corespund cu indicii de calitate ai iodului din Codex, excepţie uleiul de cătină, a cărui valori în cazul acidităţii nu corespund intervalelor acidităţii precizate în literatură.

Determinarea amprentei uleiurilor prin spectrometrie ultra violet/visibil (UV-Vis)

Caracterizarea spectrală (fingerprintul) specifică fiecărui ulei analizat prin UV-Vis este prezentat in Fig. I.1. Diferenţele dintre un ulei autentic si un ulei falsificat a fost demonstrate prin poziţia şi absorbanţa peakurilor caracteristice fiecărui ulei (Socaciu C. et al., 2005).

Ulei de cânepă (Cannabis sativa L)

Amprenta spectrală UV-Vis caracteristică uleiului de cânepă în conformitate cu datele precizate de OMLC, este dat de conţinutul ridicat în pigmenţi clorofilici, având absorbanţa maximă la 411 nm (Fig.II.1.A.).


XI

A. B

C. D.

Fig.II.1. Spectrele UV-Vis ale uleiurilor analizate (amprenta specifică în regiunea 350-600 nm continând detalii referitoare la maximul absorbantei peakului specific: A. Ulei de cânepă; B.

Ulei de măsline extra virgin (EVO); C. Ulei de dovleac (PK); D. Ulei de cătină (SB)

Ulei de măsline extra virgin (Olea europaea )

Culoarea caracteristică uleiului de măsline depinde de majoritatea pigmenţilor conţinuţi, în principiu acest ulei având un conţinut ridicat în carotenoide şi clorofile. Uleiul provenit din măslinele ajunse la maturitate prezintă o culoare galbenă datorită continutului în pigmenţi carotenoidici galbeni. În general culoarea acestui ulei variază şi este datorată combinaţiei şi diferetelor proporţii de pigmenţi. Exista o simplă ecuaţie: Culoarea= clorofilă (verde) + carotenoide (galben) + alţi pigmenţi.

Conţinutul în pigmenţi clorofilici se diminueaza odată cu atingerea maturităţii fructelor. Fingărprintul specific uleiului de măsline analizat este atribuit ‘’ecuaţiei culorii’’ menţionată anterior (Fig.II.1.D.).

Ulei de dovleac (Cucurbita pepo)

Amprenta spectrală (fingerprintul) a acestui ulei este acceptat ca avand doua umere, unul la 418 nm cu absorbanţă mai mică, şi unul la 435 nm cu absorbanţă mai mare


XII

(Fig.II.1.C.), în cazul falsificării sau oxidării, acest ulei prezintă absorbanţele celor două umere schimbate (Lankmayr şi col., 2004).

Uleiul de cătină (Hippophae rhamnoides)

Spectrului acestui ulei demonstrează ca fingărprintul este dat de cele trei umere care dau spectrul, care sunt caracteristice carotenoidelor, mai exact beta-carotenului, intre 400 şi 500 nm (Fig.II.1.A.), acesta fiind compusul principal al acestui ulei (Lichtenthaler şi Buschmann, 2001).

Analiza uleiurilor prin spectroscopia infrarosu cu furie transformata (FTIR)

Studiile FTIR ale uleiurilor analizate au demonstrate existenta relatiei intre fecventele si absorbantele benzilor specifice si compozitia acestora. Aceste frecvenţe şi valoarea absorbanţei lor, au fost utilizate în continuare pentru evaluarea oxidarii uleiurilor (Guillen, M. D. şi Cabo, N, 1997, 1998, 1999, 2000, 2002).

În conformitate cu aceste spectre au fost identificate principalele benzi şi frecvente în domeniul infraroşu ale uleiurilor analizate ( Tabel II.2.).

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

Nr. banda

HP

(cm-1)

EOV

(cm-1)

PK

(cm-1)

SB

(cm-1)

Grupul functional Modul de vibratie

1 3008 3005 3008 3006 =C-H (cis-) de întindere

2 2956 2956 2956 2956 -C-H (CH3) de întindere (asimetrică)



5 1742 1742 1742 1742 -C=O (ester) de întindere

6 1654 1653 1653 1653 -C=C- (cis-) de întindere

7 1463 1464 1464 1464 -C-H (CH2, CH3)

8 1456 1456 1456

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

10 1396 1402 1398 1402 de deformare

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

12 1317 1319 de deformare

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


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

15 1120 1118 1120 1116 -C-O de întindere

16 1097 1097 1099 1095 -C-O de întindere

17 1028 1028 1029 1033 -C-O de întindere

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

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

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

de deformare ( rocking)

Spectrele uleiurilor analizate par a fi in principiu similare, însă diferenţele în intensitatea benzilor ca de altfel şi a frecvenţelor fac posibilă diferenţierea foarte clară a compoziţiei acestor uleiuri (see Fig. II.2.).

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


XIV

Profilul acizilor graşi prin cromatografia gaz

Compoziţia acizilor grasi analizaţi prin GC-FID in acest studio sunt evidentiati in Tabelul II.3. Profilul acizilor graşi a fost comparat cu al uleiurilor din literature.

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

Acizi grasi % Ulei de Canepa Ulei de masline extra virgin

Ulei de dovleac Ulei de catină

Palmitic (16:0) 7.48 7.28 6.29 7.76

Stearic (18:0) 1.66 2.67 3.64 0.3

Arachidic (20:0) 1.06 - - 0.11

Σ saturati % 10.02 9.95 9.93 8.17

Palmitoleic (C16:1)

Oleic

(C18:1) Linoleic

(C18:2)

Linolenic

(18:3n3)

Eicosadienoic (C20:2)

-

14.94

72.6

-

0.55

-

36.81

43.14

0.93

-

-

42.44

46.71

0.92

-

5.4

6.3

-

0.8

-

Σ nesaturati % 87.54 80.88 90.07 12.5

C18:1/C18:2 0.21 0.85 0.91 6.3

omega 3 : omega 6 acizi grasi - 0.022 0.02 -

II.3. CONCLUZII

Prin GC-FID, s-a determinat compoziţia în acizi graşi a uleiurilor analizate şi s-a facut comparaţia cu datele din literatură. În urma acestei analize s-au concluzionat urmatoarele:

• compoziţia uleiului de cânepă nu corespunde cu valorile precizate în literatură pentru acizii graşi, acesta avand un conţinut mai scăzut. Acidul oleic se incadrează in intervalul prevăzut in literatură

• principalii acizi graşi în uleiul de măsline extra virgin sunt acidul oleic şi linoleic, şi de asemenea în cantitate mai mică acidul lonoleic


XV

• în cazul uleiului de dovleac, compoziţia în acizi graşi corespunde cu valorile precizate în litaratura, excepţie facând acizii palmitic şi stearic care sunt prezenţi in concentrţii mai mici

• profilul acizilor graşi a uleiului de cătină a demonstrat faptul că acest ulei provine din pulpa/pielea fructelor şi nu din seminţe, fiind foarte bogat in acidul palmitic şi oleic

CAPITOLUL III. BIOÎNCAPSULAREA ULEIURILOR: PROTOCOALE DE PREPARE A CAPSULELOR ŞI CARACTERIZAREA LOR

III.1. MATERIALE ŞI METODE În vederea realizării parţii experimentale din acest capitol s-au utilizat urmatoarele:

• matrici pentru încapsulare: alginat, k-caragenan, chitosan, gumă xantan şi gumă guar, procurate de la Sigma Aldrich

• solvenţii si reactanţii necesari de asemenea de la Sigma Aldrich • uleiurile utilizate la încapsulare au fost prezentate in capitolul anterior

Protocol pentru sintetizarea capsulelor goale de diferite mărimi şi concentraţii

Diferite concentratii de alginat (1%, 1.5%, 2% w/v), amestec de: alginat si caragenan, alginat si guma xantha, alginat si guma guar au fost dizolvate in apa deionizata pentru ~ 30 minute. Diferite concentratii de chitosan (1%, 1.5%, 2% w/v) au fost dizolvate 0.7% v/v acid acetic glacial.

Alginatul şi amestecul de alginat au fost pipetate într-o solutie de 2% CaCl2 în apa (ca şi baie de întărire), utilizând o pompa peristaltica cu un injector de 0.4 x 20mm, iar capsulele au fost formate instantaneu.

Chitosanul a fost pipetat in 5% (w/v) solutie de NaTPP in apa (ca si baie de intarire), utilizând pipeta pentru control.

Dupa ~ 1h, capsulele au fost separate din baia de intarire şi transferate în placi “Petri” pentru protecţie si conservare.

Protocol pentru sintetizarea capsulelor de diferite mărimi şi concentraţii cu uleiri încorporate

S-au luat în considerare doar concentraţiile de matrici care au prezentat emulsiile cele mai stabile. De asemenea vâscozitatea soluţiilor a fost considerat un factor principal în vederea alegerii concentraţiilor de matrici. Protocolul pentru obţinerea capsulelor a fost descris anterior.


XVI

Evaluarea microscopica a emulsiilor înaintea încapsulării

Evaluarea microscopică a emulsiilor înaintea încapsulării a fost determinată utilizând un microscop Olimpus optical microscope BX51M, echipat cu camera digitală.

Caracterizarea capsulelor: dimensiuni şi morfologie, analize FTIR şi termice

Parametri luaţi in considerare în vederea caracterizării capsulelor, precum dimensiune, arie, perimetru, elongaţie si compactitate, au fost determinaţi utilizând UTHSCSA ImageTool ca şi software.

Morfologia suprafeţei capsulelor a fost determinată utilizându-se microspia electronică scanată (Hitachi S-2700, iMOXS, cu detector BSE). Capsulele analizate au fost suflate şi învelite în aur înaintea supunerii analizelor microscopice.

III.2. REZULTATE ŞI DISCUŢII

Evaluarea microscopica a emulsiilor înaintea încapsulării

Stabilitatea emulsiilor este un factor cheie în evaluarea în condiţii de temperatura în vederea păstrării timp mai îndelungat a produselor pe baze de emulsii.

Mărimea picăturilor de ulei dispersate in structura matricilor dizolvate care au fost comparate in vederea evaluarii stabilitatii emulsiilor obtinute. Emuliile cu cea mai bună stabilitate în timp au fost utilizate mai departe pentru încapsulare. Mărimea picăturilor de uleiuri au fost dispersate uniform in matrici, în funcţie de stabilitatea matricei, uniformitatea crescând odată creşterea concentraţiei matricilor (Fig.III.1.).

Caracterizarea capsulelor

Dupa obtinerea emulsiilor, si pipetarea lor in baile de intarire, datorita interactiilor cu ionii de legare in vederea formarii gelurilor.

Capsulele continand uleiuri au avut o forma aproximativ sferica, culoare variind intre alb-galbui si portocaliu.

Luându-se in considerare toate caracteristicile capsulelor obtinute, si facand o comparatie intre aceste caracteristici ale capsulelor goale şi a celor continand uleiuri, s-a constatat ca incorporarea uleiurilor în capsule modifica aceste caracteristici (Fig.III.2.). Compararea capsulelor între ele continand uleiuri, a demonstrat ca sfericitatea şi compactitatea nu au fost prea mult afectate de incorporarea uleiurilor. Insa in cazul diametrului, ariei, elongatia, au fost clar determinate diferente foarte mari.


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A. B.

C. D.

Fig. III.1. Imagini microscopice ale diferitelor emulsii: A. alginat 2%; B. alginat 1%; C. complex alginat-gumă guar; D. complex alginat-gumă xantan. Scala reprezintă 5 μm.

0

1

2

3

4

5

6

7

8

9

AG-CAR (0

.5:0.5

)

AG-CAR (0

.75:0.

75)

AG-XG (0

.75:0

.75)

AG-XG (0

.5:0.

5)

AG-GG (0

.75:0.

75)

AG-GG (0

.5:0.5

)

AGoil2%

AGoil1.5

%

AGoil1%

CHoil2%

CHoil1.5

%

CHoil1%

Samples/Probele

Para

met

er v

alue

s/Va

loar

ea p

aram

etril

or

Area / Aria (cm2)

Perimeter / Perimetru

Elongation (axes ratio)/Elongatia (raportul axelor)Roundness (up to 1) /Sfericitatea val. max. 1 Diameter / Diametrul (cm)

Compactness (up to 1)/Compactitatea (val. max. 1)

Fig.III.2. Reprezentarea grafică comparată a caracteristicilor capsulelor din complexul alginat cu k-caragenan,

gume xantan şi guar, alginat şi chitosan continând ulei: AG-CAR (0.5:0.5) = complex alginat-k-carrageenan (raport 0.5:0.5) continând ulei; : AG-CAR (0.75:0.75) = complex alginat-k-carrageenan (raport 0.75:0.75)

continând ulei; AG-XG (0.75:0.75) = complex alginate-guma xantan (raport 0.75:0.75) continând ulei; AG-XG (0.5:0.5) = complex alginate-guma xantan (raport 0.5:0.5) continând ulei;AG -GG (0.75:0.75) = complex

alginate-guma guar (raport 0.75:0.75) continând ulei; AG -GG (0.5:0.5) = complex alginate-guma guar (raport 0.5:0.5) continând ulei; AGoil2% = capsule alginat 2% continând ulei; AGoil1.5% = capsule alginat 1.5%

continând ulei; AGoil1% = capsule alginat 1% continând ulei; ; CHoil2% = capsule chitosan 2% continând ulei; CHoil1.5% = capsule chitosan 1.5% continând ulei; CHoil1% = capsule chitosan 1% continând ulei


XVIII

Microscopia electronică scanată (SEM)

Scopul acestor analize a fost de a evalua si caracteriza topologia capsulelor obtinute continand uleiuri. Suprafata capsulelor a fost non-regulara, aceasta datorita picaturilor de ulei prezente, exceptand chitosanul care prezinta o suprafata mult mai mata (Fig.III.3.A).

Fotografiile SEM ale capsulelor nu prezintă porozitate (Fig.III.3.).

A. B.

Fig.III.3. Morfologia suprafetei diferitelor capsule obtinute continand uleiuri utilizand microscopia electronica de scanare: A. alginat-caragnan complex; B. chitosan. Bara indicand scala este reprezentata in fiecare poza.

Magnificatia 70x.

Analizele FTIR

Caracterizarea FTIR a matricilor

În urma analizelor FTIR-ATR s-a realizat caracterizarea matricilor utilizate la încapsulare, realizându-se astfel o comparaţie între matrici (AG, CAR, CH, GG, XG). Principalele frecvenţe caracteristice matricilor în vederea identificării individuale sunt: 3244-3302 cm-1 (O-H stretch), 1400-1474 cm-1 (CH2 bending), 1000-1200 cm-1 (C-O şi C-C stretch), 924-1000 cm-1 ( poly OH şi CH2 twist), 776-892 cm-1(glycoside).

Vibraţiile şi grupurile funcţionale

AG CAR GG XG CH

O–H intindere 3244 3514

grupul poliOH

3299 3302 3289

O-H +

N-H de întindere


XIX

C–H întinderea grupului CH2 2926 2953, 2911, 2894 2884 - 2935

C-O de întindere ( COOH) 1597 - 1636 - 1651

deformarea grupării CH2 1408 1474, 1400 1408 1400 1428

O-H deformare - 1223 ( S=O vibraţia de întindere a sulfet

esterului)

1350 1247 -

C-O şi C-C întindere 1200-1000 - 1145 1150 1151

–CH2OH modul de întindere 1054 1063 1054 1061

Gruparea C–OH alcolică

(C-O întinderea zaharidelor)

1024 1024 - 1025 1024

–CH2 vibraţie 948, 902,

Provenite de la acizii: guluronic şi maluronic

924, 910

Grupările polihidroxi

1016 - -

legaturile glicozidice 809 842

Sulfatul galactozic, legatura glicozidică

866,777

(1,4; 1,6) legatura galactozei şi

manozei

785

C-H de deformare

C-C întindere

892,

776

FTIR characterization of different beads containing oils

Spectrele matricelor, ale capsulelor goale, capsulelor continand uleiuri au fost analizate. Concentratia matricelor nu a influentat caracteristicile capsulelor prin FTIR. Un exemplu concludent este reprezentat in Fig.III.4., spectrele uleiului de cătină (SB) şi ale capsulelor din alginat 2% conţinand ulei SB.

În urma încapsulării uleiului de SB în capsule de alginate, se produce o creştere a intensităţii absorbanţei la 3400 cm-1 (care este direct proporţională cu creşterea concentraţiei de alginate utilizată la încapsulare) precum şi o shiftare a unor peakuri spre valori şi frecvenţe mai scăzute în regiunea 1000-1500 cm-1, regiune specifică uleiului de SB

Spectrele amestecului de uleiuri si capsule au demonstrat prezenta peakurilor specifice uleiurilor in doua zone distuncte (2800-2900 cm-1 şi 1700-900 cm-1), confirmându-se astfel prezenţa uleiurilor în capsule.


XX

Fig.III.4. Spectru FTIR-ATR înregistrat pentru: A. Capsule de alginat 2% conţinând SB; B. pudră alginat; C.

ulei SB; D. capsule goale de alginat 2%

Analize termice

Analize DSC

Termogramele DSC ale uleiurilor libere si ale diferitelor capsulelor continand uleiuri, au fost masurate.

Cateva dintre peakurile endotermice, ca si exemplu ale uleiului de catina, si ale unor capsule continand ulei de catina, sunt prezentate in reprezentarea grafica din Fig.III.5.; temperature peakurilor cerste direct proportional cu cresterea temperaturii, fiecare peak fiind characteristic fiecărui tip de capsula obţinută.

Analize termogravimetrice

Termogramele TGA ale uleiurilor libere si ale diferitelor capsulelor continand uleiuri, au fost masurate.

Cateva rezultate referitoare la pierderea in masa a diferitelor tipuri de capsule obtinute, este prezentata in graficul din Fig.III.6.. Pierderea în greutate, reprezentata in figura anterioara, demonstreaza ca aceasta se datoreaza continutului ridicat in apa a unor capsule. Uleiurile nu influenteaza foarte mult pierderea in greutate, la un ulei liber aceasta fiind de 99.44%. Dar in timpul procesului de oxidare aceste uleiuri pierd din greutate, datorita reactiilor oxidarii.


XXI

0

20

40

60

80

100

120

140

160

180

200

AG 2% AG 1.5%

Alginate 1%

AG-CAR(0.75%)

CH 2% CH 1% AG-GG AG-KG SB

Tem

pera

ture

(°C

)

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

DSC si TGA au fost in ultimul timp foarte mult utilizate in monitorizarea stabilitatii, a comportamentului termic, a parametrilor de cinetica in diferite uleiuri (Jayadas et al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008). În acest studiu analizele termice au fost efectuate pana la temperatura de 300°C. Diferenţele nu foarte mari între probe se datoreaza tocmai acestei temperature, deoarece conform cu literatura, oxidarea uleiuriloe prin metode termice se poate determina la expunerea probelor la o temperatura mai mare de 300°C, iar pierderea in greutate poate fi pana la 10%, aceasta depinzând de natura uleiului (Jayadas et al., 2006; Milovanovic et al., 2006; Bahruddin et al., 2008).

0

20

40

60

80

100

120

AG 2% AG 1.5% AG-CAR(0.75%)

AG-GG AG-KG SB

Rest

mas

s %

Fig.III.6. Reprezentarea grafică a pierderii de masă % a probelor analizate TGA

Scopul acestor analize termice a fost acela de a analiza şi a determina stabilitatea termică a capsulelor obţinute continând diferite uleiuri, în vederea viitoarelor aplicaţii ale acestora în domeniul alimentar şi cosmetic. În astfel de aplicaţii se cunoaşte necesitatea


XXII

sterilizării probelor sau expunerea la presiuni înalte în vederea evitării biohazardului sau contaminării, aceste tratamente fiind facute în timpul proceselor tehnologice.

III.3. CONCLUZII

Studiile experimentale realizate, cu scopul bioîncapsulării unor uleiuri funcţionale în matrici naturale, utilizând ca şi metodă ‘’ionotropically crosslinked gelation’’, au demonstrate posibilitatea utilizării acestei tehnici în vederea bioîncapsulării unor compuşi naturali şi eliberarea lor condiţionată.

Cele mai bune matrici în vederea bioîncapsulării uleiurilor s-au dovedit a fi: alginatul şi chitosanul în concentrţii de 2%, 1.5% şi 1%, complexele alginatului cu k-caragenan, şi gume guar şi xantan în raport de concentraţie 0.75:0.75.

Rezultatele au arătat faptul că bioîncapsularea uleiului a afectat diametrul capsulelor, acesta crescând direct proporţional cu cantitatea de ulei utilizată pentru încapsulare. De asemenea şi ceilalţi parametri masuraţi în vederea caracterizării capsulelor au fost influenţaţi şi de cantitatea de ulei utilizată.

Prin analizele FTIR-ATR, diferitele capsule conţinând uleiuri au prezentat peakuri care sunt atribuite atat uleiurilor cât şi capsulelor goale (regiunile dintre 2800-2900 cm-1 şi 1700-900 cm-1). Astfel se demonstrează prezenţa uleiurilor în capsule, deci încapsularea acestora.

CAPITOL IV. EFICIENŢA ÎNCAPSULĂRII ŞI STUDII DE ELIBERARE A ULEIURILOR DIN CAPSULE

IV.1. MATERIALE ŞI METODE

Eficinţa încapsulării e uleiurilor în capsule

Încapsularea uleiurilor a fost determinată calculând cantitatea de beta-caroten sau cantitatea de carotenoid care este principalul component major al fiecărui ulei analizat înainte şi după încapsulare. Această determinare a fost realizată spectrofotometric, iar eficinţa încapsulării (EE%) a fost calculată conform ecuaţiei:

EE% = C1/C2 x 100, C1= concentraţia carotenoid din ulei iniţială

C2= concentraţia carotenoid din ulei după încapsulare


XXIII

Măsurarea ratei de eliberare a uleiurilor din capsule

Eliberarea controlată a uleiurilor din capsule a fost determinata de asemenea spectrofotometric, utilizând un spectrofotometru CarWin 50 UV-VIS. Măsurătorile au fost facute în triplicat la temperatura camerei, utilizându-se cuvete de cuarţ de 2 mm.

Eliberarea in vitro a uleiurilor din capsule

Stimularea fluidului gastric a fost realizată conform urmatorul protocol:

• timp de o ora la pH 1.2 intr-o solutie de 0.1N HCl cu⁄si 5 ml Sanzyme (sirop de enzime continand 80 mg papaina, 40 mg pepsina and 10 mg sanzyme 2000)

• in urmatoarele 2-3 ore capsulele au fost transferate in intr-o solutie in vederea stimulatii fluidului intestinal pH 4.5 tot asa cu⁄si fara enzyme

• urmatoare ore au fost transferate in solutie stimuland fluidul intestinal la pH 7.4, aceasta fiind formata din KH2PO4 1.074g in 30 ml de 0.2N NaOH, si pancreatina 275 mg (utilizand “Triferment”)

• toate testările s-au efectuat la 37ºC cu barbotare continuă de CO2

IV.2. REZULATTE ŞI DISCUŢII

Eficinţa încapsulării e uleiurilor în capsule

Eficienţa încapsulării este reprezentata în graficul din Fig.IV.1. pentru diferite tipuri de capsule. Valorile prezentate sunt ale uleiului de cătină, însa pentru toata uleiurile analizate aceasta eficienţă a prezentat aceleaşi valori.

După cum se poate observa şi în graficul prezentat, eficienţa încapsulării este direct proporţionala cu creşterea concentraţiei matricilor. Dintre toate matricile şi complexele de matrici utilizate, după cum se poate observa şi in graficul din Fig.IV.1., s-a obţinut cea mai bună eficienţa a încapsulării utilizând ca şi matrici: alginatul în concentraţie de 2%, chitosanul in aceeaşi concentraţie, urmate de concentraţiile de 1.5%, şi de alginatul în complex cu k-caragenan şi gume în raport de 0.75:0.75%.


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Fig.IV.1. Reprezentarea grafica comparata a eficientei incapsularii a uleiurilor in capsule din complexul alginat cu k-caragenan, gume xantan si guar, alginat si chitosan : AG2% = capsule alginat 2% ; CH2% = capsule

chitosan 2% ; CH1.5% = capsule chitosan 1.5% ; AG1.5% = capsule alginat 1.5% ; AG-CAR (0.75:0.75) = complex alginat-k-carrageenan (raport 0.75:0.75) ; AG-XG (0.75:0.75) = complex alginate-guma xantan (raport

0.75:0.75); CH1% = capsule chitosan 1%; AG -GG (0.75:0.75) = complex alginate-guma guar (raport 0.75:0.75) ; AG1% = capsule alginat 1% ; AG -CAR (0.5:0.5) = complex alginat-k-carrageenan (raport 0.5:0.5); AG -GG (0.5:0.5) = complex alginate-guma guar (raport 0.5:0.5); AG-XG (0.5:0.5) = complex

alginate-guma xantan (raport 0.5:0.5)

Măsurarea ratei de eliberare a uleiurilor din capsule

Ca şi sisteme de eliberare s-au luat in considerare 3 solvenţi: tetrahidrofuran (THF), metanol si hexan. În toate cazuri s-a evidentiat o rapida eliberare in THF ca si solvent din toate capsule obtinute, si o mai lenta eliberare in cazul metanolului si o foarte slaba in cazul hexanului (vezi Fig.IV.2., graficele fiind parte din lucrarea publicata in revista Chemické Listy Journal (IF=0.683)). THF este considerat şi în litaratura ca fiind solventul cel mai eficient în extracţia carotenoidelor, lucru dovedit şi în acest studiu. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea şi solubilitatea uleiului în matrice.

Eliberarea uleiurilor din capsule a demonstrat faptul ca alginatul, complexul dintre alginat cu k-caragenan şi gume, precum şi chitosanul, sunt matrici pretabile la încapsularea uleiurilor vegetale.


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A.

B.

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

Eliberarea in vitro a uleiurilor din capsule

În cazul mimării mediului digestiv s-a demosntrat stabilitatea capsulelor la pH 1.2 si pH 4.5, dizolvarea acestora si eliberarea continutului realizandu-se la pH 7.4, atat in cazul solutiilor continand enzime cat si a celor fara continut enzimatic în cazul capsulelor din alginat şi alginat în complex cu k-caragenan şi gume (Fig.IV.3.), ceea ce demonstreaza aplicabilitatea viitoare a acestor capsule continand ulei de catina ca si nutraceutice sau in industria farmaceutica. Capsulele de chitosan nu s-au dizolvat la nici unul dintre pH-urile testate.

0

0.1

0.2

0.3

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Wavelenght (nm)

Abso

rban

ce (a

.u.) Chitosan 1.5% in methanol

Chitosan 1% in methanolChitosan 1% in hexaneChitosan 1.5% in hexaneAlginate 2% in methanolAlginate 2% in hexane

0

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0.15

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0.4

0 100 200 300 400

Wavelenght (nm)

Abs

orba

nce

(u.a

.)

Alginate-carrageenancomplex in hexaneAlginate-carrageenancomplex in methanolAlginate 2% in methanol

Alginate 2% in hexane


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A. B.

C. D.

Fig.IV.3. Eliberarea ”in vitro” a uleiului de catina din capsulele de alginate 2% de la stanga la dreapta in fiecare poza fluidele stimulatoare fara enzyme si cu adios de enzyme (Sanzyme): A. capsulele obtinute; B. dupa 1 ora in stimulatul fluid gastric la pH 1.2; C. dupa 3 ore in mixul dintre fluidul gastric si fluidul intestinal la pH 4.5; D. in

stimulatul fluid intestinal pH 7.4 dupa 30 minute

IV.3. CONCLUZII

Studiile referitoare la eficienţa încapsulării şi stabilitatea capsulelor conţinând uleiuri au demonstrat:

1. Creşterea concentraţiei matricilor sau a complexului de matrici determină obţinerea unei mai bune eficienţe la încapsulare. s-a obţinut cea mai bună eficienţa a încapsulării utilizând ca şi matrici: alginatul în concentraţie de 2%, chitosanul in aceeaşi concentraţie, urmate de concentraţiile de 1.5%, şi de alginatul în complex cu k-caragenan şi gume în raport de 0.75:0.75%.

2. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea şi solubilitatea uleiului în matrice. Eliberarea a fost mai lentă in cazul hexanului, mai ridicată în cazul metanolului şi cea mai buna eliberare fiind in THF, indiferent de matricea sau concentraţia utilizată la încapsulare.

3. Stabilitatea capsulelor la pH 1.2 si pH 4.5, dizolvarea acestora şi eliberarea continutului realizandu-se la pH 7.4.


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CAPITOL V. CARACTERIZAREA FTIR A OXIDĂRII ULEIURILOR

V.1. MATERIALE ŞI METODE

Analiza spectrala in domeniul IR s-a utilizat spectrofotometru FT-IR Shimadzu Prestige-21, echipat cu Reflectanta Totala Atenuata Orizontala (HATR), cu accesoriu de ZnSe. Masuratorile au fost efectuate in domeniul infrarosu 650-4000 cm-1, 100 scanari fiecare proba la rezolutia 2 cm-1. Dupa fiecare proba accesoriul a fost spalat cu acetona.

Uleiurile libere si capsulele cu uleiuri au fost supuse in vederea oxidarii la temperatura de 105ºC, si la iradiere UV (254µm) timp de o ora, 4 ore si 6 ore.

S-au inregistrat spectrele FTIR-ATR dupa fiecare oxidare, atat la uleiul liber cat si extras din capsule. In cazul analizelor FTIR-ATR au fost posibile inregistrarea spectrelor capsulelor cu ulei, nefiind necesara extracţia uleiului din capsule.

V.2. REZULTATE ŞI DISCUŢII

În cazul analizelor FTIR-ATR a fost stabilit stadiul oxidării, calculandu-se raportul între intensităţile principalele peakuri considerate markeri ai oxidarii conform literaturi (Guillén and Cabo, 1999, 2000, 2002): A2853/A3005, A1746/A3006, A1474/A3006, A1377/A3006 and A1163/A3006, înainte şi după tratamentul UV.

S-a constat ca uleiul liber avea un stagiu mai avansat al oxidarii 2 sau 3, in timp ce uleiul incapsulat se afla in stagiu 1 al oxidarii. S-a demosntrat astfel ca uleiul de catina incapsulat in diferitele capsule obtinute din matricile utilizate a fost mult mai protejat impotriva oxidarii in urma diferitelor tratamente, comparativ cu uleiul liber (ex: la uleiul de cătină, Fig.V.1.).

Cea mai bună protecţie împotriva oxidării a fost asigurată de urmatoarele capsule formate din matrcile şi concentraţiile urmatoare: alginat 1%, chitosan 1.5%, complexele alginate-gumă gum şi alginat-gum xantan în raport 0.5:0.5, şi alginat-k-caragenan complex în raport 0.75:0.75.


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0

1

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After 1h UV/Dupa 1hUV



Types of ratios on time/Tipul rapoartelor in timp

Rat

io v

alue

s/Va

loar

ea r

apor

telo

rOil free/Ulei liber

Oil from AG 1%/Ulei din AG 1%

Oil from AG 1.5%/Ulei din AG 1.5%


Fig. V.1. Reprezentarea grafica a uleiului de canepa liber si incapsulate (in diferite capsule de alginate) in timpul oxidarii

(A= A2853/A3005-3008, B= A1744/ A3005-3008, C= A1464/ A3005-3008, D= A1377/ A3005-3008, E= A1160/ A3005-3008)

V.3. CONCLUZII

Uleiurile încapsulate prezintă o mai buna stabilitate împotriva oxidării provocate de diferite conditii comparativ cu uleiurile libere.

Cea mai bună protecţie împotriva oxidării a fost asigurată de urmatoarele capsule formate din matrcile şi concentraţiile urmatoare: alginat 1%, chitosan 1.5%, complexele alginate-gumă gum şi alginat-gum xantan în raport 0.5:0.5, şi alginat-k-caragenan complex în raport 0.75:0.75.

Spectroscopia FTIR este considerată o foarte buna tehnică de monitorizare a oxidării uleiurilor libere şi încapsulate, dovedindu-se totodată a fi o tehnică rapidă, acurată şi simplă.


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CONCLUZII GENERALE

În conformitate cu scopul si obiectivele acestei teze de doctorat, s-a realizat bioîncapsularea a patru uleiuri funcţionale din plante în diferite matrici naturale, precum şi evaluarea eficienţei încapsulării, stabilitatea şi eliberarea uleiurilor din capsulele obtinute.

S-a realizat o evaluare comparativă si sistematică a calităţii celor patru uleiuri funcţionale din plante înaintea încapsulării lor: uleiul de cânepă (HP), uleiul extra virgin de măsline (EVO), uleiul de dovleac (PK) şi uleiul de cătină (SB) (de provenienţă din România şi Italia).

Având în vedere obiectivele propuse s-a realizat:

I. S-au identificat caracteristicile uleiurilor înainte de încapsulare, stabilindu-se markeri de calitate si autenticitate:

1. Majoritatea uleiurilor analizate prezintă indicele de iod în conformiatte cu specificaţiile din CODEX 210, excepţie uleiul de cătină care prezintă a valoare mai scazută.

2. Spectrele UV-Vis ale uleiurilor au relevant peakurile specifice, ca şi markeri ai autenticităâii.

3. Studiile FTIR-ATR au demonstrat relaţia dintre benzile aparute în spectre şi compoziţia specifică fiecarui ulei, putându-se astfel stabili fingerprintul specific uleiurilor studiate.

4. Analizele GC-FID au demonstrate faptul rpofilul acizilor din compoziţia uleiurilor analizate este în conformitate cu datele din literatură.

II. Studiile experimentale utilizând ca şi metodă gelarea ionicăde numită:‘’ionotropically crosslinked gelation’’, în vederea bioîncapsulării uleiurilor funcţionale în matrici naturale, au demonstrat stabilitatea şi eliberarea controlată a uleiurilor bioîncapsulate

1. S-a reusit obţinerea diferitelor tipuri de capsule utilizănd matrici naturale şi complexe dintre acestea, fiind incorporate uleiuri

2. Caracteristicile capsulelor obţinute (aria, perimetrul, compactitatea, sfericitatea şi elongaţia), în special mărimea lor, au fost influenţate de conţinutul de uleiuri încapsulate.

3. Cele mai bune matrici pentru bioîncapsularea uleiurilor au fost: alginat 2%, chitosan 2%, şi alginate în complex cu k-caragenan, gumă guar şi gumă xantan în raport de 0.75:0.75.

III. Caracterizarea capsulelor a fost realizată prin diferite metode: SEM, FTIR, analize DSC şi TGA

1. Suprafata capsulelor analizată prin SEM, a fost non-regulara, aceasta datorita picaturilor de ulei prezente, exceptand chitosanul care prezinta o suprafata mult mai mata


XXX

2. Prin analizele FTIR-ATR, diferitele capsule conţinând uleiuri au prezentat peakuri care sunt atribuite atat uleiurilor cât şi capsulelor goale (regiunile dintre 2800-2900 cm-1 şi 1700-900 cm-1).

3. Termogramele DSC au arătat faptul că temperature peakurilor creşte direct proportional cu cresterea temperaturii, fiecare peak fiind characteristic fiecărui tip de capsula obţinută.

4. Analizele TGA au demonstrate ca pierderea în greutate se datoreaza continutului ridicat in apa a unor capsule. Încapsularea uleiurilor nu afectează pierderea în greutate a capsulelor.

IV. Evaluarea eficienţei încapsulării

1. Creşterea concentraţiei matricilor sau a complexului de matrici determină obţinerea unei mai bune eficienţe la încapsulare. s-a obţinut cea mai bună eficienţa a încapsulării utilizând ca şi matrici: alginatul în concentraţie de 2%, chitosanul in aceeaşi concentraţie, urmate de concentraţiile de 1.5%, şi de alginatul în complex cu k-caragenan şi gume în raport de 0.75:0.75%.

2. Rata de eliberare a uleiurilor din capsule depinde de difuzitatea şi solubilitatea uleiului în matrice. Eliberarea a fost mai lentă in cazul hexanului, mai ridicată în cazul metanolului şi cea mai buna eliberare fiind in THF, indiferent de matricea sau concentraţia utilizată la încapsulare.

3. Stabilitatea capsulelor la pH 1.2 si pH 4.5, dizolvarea acestora şi eliberarea continutului realizandu-se la pH 7.4.

V. Protecţia bioîncapsulării a uleiurilor împotriva

1. Uleiurile încapsulate prezintă o mai buna stabilitate împotriva oxidării provocate de diferite conditii comparativ cu uleiurile libere.

2. Cea mai bună protecţie împotriva oxidării a fost asigurată de urmatoarele capsule formate din matrcile şi concentraţiile urmatoare: alginat 1%, chitosan 1.5%, complexele alginate-gumă gum şi alginat-gum xantan în raport 0.5:0.5, şi alginat-k-caragenan complex în raport 0.75:0.75.


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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.

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PUBLICAŢII PE DURATA STAGIULUI DOCTORAL SI PARTICIPARI LA SIMPOZIOANE ŞI CONFERINŢE NATIONALE ŞI INTERNAŢIONALE

2009

1. Socaciu C., Trif. M, A. Baciu, T. Nicula, A. Nicula, ‘’ Encapsulation of plant oleosomes and oleoresins in mixed carbohydrate matrices’’, COST865, Spring Meeting "Microcapsule property assesment", Luxemburg 2009, Proceeding

2008

1. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. „Bioencapsulated seabuckthorn oil: controlled release rates in different solvents”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania

2. Pece Aurelia, D. Vodnar, Monica Trif, C. Coroian, Camelia Raducu, G. Muresan, “Study of the physico-chemical parameters from buffalo raw milk during different lactations”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania

3. Pece Aurelia, D. Vodnar, Monica Trif, “Corelation between microbiological and physico-chemical parameters from buffalo raw milk during different lactations”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania

4. Carmen Socaciu, Baciu A., Trif M., “Oleosome-rich pectin network as a new, natural bioencapsulation matrix”, XVI International Conference on Bioencapsulation Dublin, Ireland ; September 2008, Proceeding

5. Monica Trif, Carmen Socaciu, Andreea Stanila, “The evaluation of encapsulated Seabuckthorn oil properties usind FTIR”, CIGR - International Conference of Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797

6. Andreea Stanila and Monica Trif, “Antioxidant activity of carotenoide extracts from HIPPOPHAE RHAMNOIDES”, CIGR - International Conference of Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797

7. Monica Trif, Carmen Socaciu and Horst Diehl, “Evaluation of effiency, release and oxidation stability of seabuckthorn encapsulated oil using FTIR spectroscopy”, 7th Joint Meeting of AFERP, ASP, GA, PSE & SIF, August 2008, Athens, Greece, Book of Abstracts, pg.39

8. Monica Trif and Carmen Socaciu, “Evaluation of effiency, release and oxidation stability of Seabuckthorn microencapsulated oil using Fourier Transformed Infrared Spectroscopy”, 4th Meeting on Chemistry and Life, and accepted to be published in Chemické Listy Journal (current IF=0.683)


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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

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 ________________________________________________________________________________________

II

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


III

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).


IV

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 k-carrageenan and gums: xanthan and guar, chitosan) to encapsulate functional oils (pumpkin oil, extra virgin olive oil, hemp oil and seabuckthorn oil)

Improvement and optimization of bioencapsulation methods for vegetable oils with functional properties

Investigations of different obtained beads: morphology (scanning electron microscopy), characterization of beads (area, diameter, perimeter, elongation, compactness), Fourier transform infrared spectroscopy (FTIR) analysis

Investigations of bioencapsulated functional oils: encapsulation efficiency and stability, control release of oils encapsulated, material and functionality of the beads obtained , FTIR characterization of: free oils, obtained beads and oxidation of free and encapsulated oils

The work presented was carried out in the Department of Chemistry and Biochemistry at the University of Agricultural Sciences and Veterinary Medicine (USAMV), Cluj-Napoca, Romania, in collaboration with the Technical University Berlin (TU Berlin), Germany, Department of Enzyme Technology, under supervision of Prof. Dr. rer. nat. Marion Ansorge-Schumacher. I would like to thank the sponsors who made this work possible providing scholarships to pursue doctoral studies: Deutsche Bündestiftung Umwelt (DBU) Germany and EU COST 865.


V

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. Poly-

cored (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.


VI

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


VII

Active ingredients for drug delivery Bacteria and cells

Matrices – materials for encapsulation

Enormous range of different materials can be used for encapsulation, such as synthetic polyelectrolytes (Sukhorukov G.B. et al., 1998; Donath E. et al., 1998), natural polyelectrolytes (Shenoy D.B. et al., 2003) inorganic nanoparticles (Caruso F. et al., 2001), lipids (Moya S. et al., 2000), dye (Dai Z. et al., 2001), multivalent ion (Radtchenko I.L. et al., 2005), and biomacromolecules (Yang H. et al., 2006).

Biopolymers are polymers that are generated from renewable natural sources, are often biodegradable, and not toxic to produce. They can be produced by biological systems (i.e. micro-organisms, plants and animals), or chemically synthesized from biological starting materials (e.g. sugars, starch, natural fats or oils, etc.).

Natural polymers and their derivatives: anionic polymers: HA, alginic acid, pectin, carrageenan, chondroitin sulfate, dextran sulfat; cationic polymers: chitosan, polylysine; amphipathic polymers: collagen (and gelatin), carboxymethyl chitin, fibrin; neutral polymers: dextran, agarose, pullulan.

The ability of carbohydrates, such as starches, maltodextrins, corn syrup solids and gums, to bind flavours is complemented by their diversity, low cost, and widespread use in foods and makes them the preferred choice for encapsulation.

Guar gum (E412, also called guaran) is extracted from the seed of the leguminous shrub Cyamopsis tetragonoloba, where it acts as a food and water store. Guar gum shows high low-shear viscosity but is strongly shear-thinning. Being non-ionic, it is not affected by ionic strength or pH but will degrade at pH extremes at temperature (for example, pH 3 at 50°C).

Alginates (E400-E404) are produced by brown seaweeds (Phaeophyceae, mainly Laminaria). Gelling properties depends on the ion binding (Mg2+ << Ca2+ < Sr2+ < Ba2+) with the control of the di-cation addition being important for the production of homogeneous gels.

Carrageenan (E407) is a collective term for polysaccharides prepared by alkaline extraction (and modification) from red seaweed (Rhodophycae). The strongest gels of κ-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.


VIII

Many components naturally present in vegetable oils have been shown to have beneficial properties.

Hempseed oil is pressed from the seed of the hemp plant (i.e., non-drug varieties of Cannabis sativa L). The Oleic Acid (Omega 9) contained in Hemp Seed Oil helps keep arteries supple because of its fluidity. In excess Oleic acid can interfere with EFA's and prostaglandin's.

Olive oil contains triacylglycerols and small quantities of free fatty acits, glycerol, pigments, aroma compounds, sterols, tocopherols, phenols, unidentified resinous components and others (Kiritsakis A., 1998). Among these constituents the usaponifiable fraction , which covers a small percentage (0,5-15%) plays a significant role on human health (Waterman and Lockwood, 2007). Olive oil is considerably rich in monounsaturated fats, most notably oleic acid.

Pumpkin oil is a healthy, high quality, specialty oil, ranked in the top 3 most nutritious. Pumpkin seed oil has an intense nutty taste and is rich in polyunsaturated fatty acids. Brown oil has a bitter taste. The tocopherol content of the oils is ranging from 27.1 to 75.1 μg/g of oil for α-tocopherol, from 74.9 to 492.8 μg/g for γ-tocopherol, and from 35.3 to 1109.7 μg/g for δ-tocopherol (Stevenson D.G. et al., 2007).

Most often seabuckthorn oil is called “Nature's anti-oxidant cocktail”, because it has a unique composition, combining a cocktail of components usually only found separately. The seabuckthorn oil is stored in extra-chromoplastic organelle, named oil bodies, a natural form of encapsulation (Socaciu et al., 2007, 2008). Seabuckthorn seed oil contains a high content of the two essential fatty acids, linoleic acid and α-linolenic acid (Chen et al., 1990), which are precursors of other polyunsaturated fatty acids such as arachidonic and eicosapentaenoic acids. The oil from the pulp/peel of seabuckthorn berries is rich in palmitoleic acid and oleic acid (Chen et al. 1990).

Oils include also flavonoids (Chen et al., 1991), carotenoids, free and esterified sterols, triterphenols, and isoprenols (Goncharova and Glushenkova, 1996). Carotenoids also vary depending upon the source of the oil.

The physical and chemical properties of functional oils

The physical and chemical properties of oils, including iodine, saponification, acid and peroxide values, refractive index, density and unsaponifiable matter are determined according to standard procedures. Iodine value measures the unsaturation of oil. The fact that the iodine value is lower than 100 shows that the oil is of lower degree of saturation (Pa Quart, 1979; Pearson, 1981). The saponification value is an indication of the average molecular mass of fatty acids present in oil. The acid value has been shown to be a general indication of the edibility of oils (AOAC, 1980; Pearson, 1981). The peroxide value is frequently used to measure the progress of oxidation of oil. It indicates the oxidative rancidity of oil. (deMan, 1992).

The techniques to characterize and authentify of functional oils

Several techniques to characterize and authentify the food products have been proposed. The authentication methods applied to oils and fats can be classified as chemical (=


IX

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 spray-dried 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.


X

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.


XI

Table 1. Chemical and physical characteristics of analyzed oils compared with literature

Hemp

Oil

Ulei de Canepa

Extra Virgin Olive Oil

Ulei de Măsline Extra Virgin

Pumpkin

Oil

Ulei de Dovleac

Sea buckthorn

Oil

Ulei de Cătina

Chemical and physical characteristics

Caracteristicile chimice si fizice

Acid value+

(mg KOH/g Oil)

Aciditatea+

(mg KOH/g ulei)

4.0 6.6 4.0 4.0

Iodine value

Indicele de iod 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”).


XII

A. B

C. D.

Fig.II.1.UV-Vis spectra of investigated oil - fingerprint for regions 3X0-600 nm with specific of maximum absorbance peak: A. hemp oil (HP); B. extra virgin olive oil (EVO); C. pumpkin

oil (PK); D. seabuckthorn oil (SB)

The chlorophyll content decreases as the fruit matures so olives picked green produce a greener oil with a "grassy" flavor. The fingerprint of the extra virgin olive oil we attributed to the “color equation” mentioned previously (Fig.II.1.B.).

Pumpkin (Cucurbita pepo) oil

The representative fingerprint of this oil accepted have the peak at 418 nm lower and the peak at 435 nm higher (Fig.II.1.C.) compared to the not accepted oils which have a high peak at 418 nm and a low one at 435 nm (Lankmayr et al., 2004).

Seabuckthorn (Hippophae rhamnoides) oil

The absorption maxima from seabuckthorn oil spectrum shows that the fingerprint of this oil has a broad absorption with the three maxima or shoulders in the blue spectral range between 400 and 500 nm, corresponding to the carotenoids (Fig.II.1.D.). The main nutrient in seabuckthorn oil is beta-carotene. According with literature and compared to the three maxima in the spectra of seabuckthorn oil, is it obviously that the fingerprint of this oil is given by beta-carotene (Lichtenthaler and Buschmann, 2001).


XIII

Fourier Transform Infrared Spectroscopy (FTIR) analysis of oils

FTIR studies of edible oils have proved the existence of relationships between frequency and absorbance values of certain bands of the oil FTIR and the oil composition as well as between some of these spectroscopic parameters and the oil oxidation level (Guillen, M. D. and Cabo, N, 1997, 1998, 1999, 2000, 2002).

According to these spectra, we identified the relevant infrared frequencies (bands) useful to and assign the specificity of the oils investigated ( Table 2.).

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

No. Bands

Nr. banda

HP

HP

(cm-1)

EVO

EVO

(cm-1)

PK

PK

(cm-1)

SB

SB

(cm-1)

Functional group

Grupul functional

Mode of vibration

Modul de vibratie

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

2 2956 2956 2956 2956 -C-H (CH3) stretching (asymetric)

3 2923 2923 2923 2922 -C-H (CH2) stretching (asymetric)

4 2853 2853 2854 2853 -C-H (CH2) stretching (symetric)

5 1742 1742 1742 1742 -C=O (ester) stretching

6 1654 1653 1653 1653 -C=C- (cis-) stretching

7 1463 1464 1464 1464 -C-H (CH2, CH3)

8 1456 1456 1456

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

10 1396 1402 1398 1402 bending

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

12 1317 1319 bending

13 1236 1238 1238 1238 -C-O, -CH2- stretching, bending

14 1155 1159 1157 1161 -C-O, -CH2- stretching, bending

15 1120 1118 1120 1116 -C-O stretching

16 1097 1097 1099 1095 -C-O stretching

17 1028 1028 1029 1033 -C-O stretching

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

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

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

bending (rocking)


XIV

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 %

Acizi graşi %

Hemp Oil

Ulei de Canepă

Extra virgin Olive oil

Ulei Extra Virgin de Măsline

Pumpkin oil

Ulei de Dovleac

Seabuckthorn oil

Ulei de Cătină

Palmitic acid (16:0)

7.48 7.28 6.29 7.76


XV

Stearic acid (18:0) 1.66 2.67 3.64 0.3

Arachidic acid (20:0)

1.06 - - 0.11

Σ saturated % 10.02 9.95 9.93 8.17

Palmitoleic (C16:1)

Oleic acid

(C18:1) Linoleic acid (C18:2)

Linolenic

(18:3n3)

Eicosadienoic acid (C20:2)

-

14.94

72.6

-

0.55

-

36.81

43.14

0.93

-

-

42.44

46.71

0.92

-

5.4

6.3

-

0.8

-

Σ unsaturated % 87.54 80.88 90.07 12.5

C18:1/C18:2 0.21 0.85 0.91 6.3

omega 3 : omega 6 fatty acids - 0.022 0.02 -

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.


XVI

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.


XVII

The surface morphology of freeze- and air-dried hydrogels was determined using a scanning electron microscope (Hitachi S-2700, iMOXS, with BSE detector). Beads samples were sputtered with gold and scanned at an accelerating voltage of 15 kV.

III.2. RESULTS AND DISCUSSIONS

Microscopic evaluation of emulsions before encapsulation Stability of emulsions (including the composition and microstructure) is a key element

for evaluation of the lifetime and temperature conditions for the storage and use of emulsion based products. The oil droplets sizes and shapes dispersed in the structure of matrices dissolved were compared in order to evaluate the stability of the emulsions.

The drop size distributions of emulsions were determined by optical microscopy associated to an image analysis technique. It was observed that oils droplets in emulsion coalescence after a few minutes when the matrices concentrations increased (because no emulsificator was used to help the emulsion formation), being necessary to drop it immediately into the hardening bath. Different concentrations of matrices were used to encapsulate the oils. The first evaluation of the solution of matrices dissolved was done.

The oils droplets were homogenized uniformly, they are smaller with the increasing of matrix (Fig.III.1.). This demonstrated that the good oils encapsulation increased with the increasing of matrix concentration.

A. B.

C. D.

Fig. III.1. Microscopic images with different emulsions using as matrices: A. alginate 2%; B. alginate 1%; C. alginate-guar gum complex; D. alginate-xanthan gum complex. The scale bar

represents 5 μm.


XVIII

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.

0

1

2

3

4

5

6

7

8

9

AG-CAR (0

.5:0.5

)

AG-CAR (0

.75:0.

75)

AG-XG (0

.75:0.

75)

AG-XG (0

.5:0.5

)

AG-GG (0

.75:0.

75)

AG-GG (0

.5:0.5

)

AGoil2%

AGoil1.5

%

AGoil1%

CHoil2%

CHoil1.5

%

CHoil1%

Samples/Probele

Para

met

er v

alue

s/Va

loar

ea p

aram

etril

or

Area / Aria (cm2)

Perimeter / Perimetru

Elongation (axes ratio)/Elongatia (raportul axelor)Roundness (up to 1) /Sfericitatea val. max. 1 Diameter / Diametrul (cm)

Compactness (up to 1)/Compactitatea (val. max. 1)

Fig.III.2. Comparative graphic representation of characteristics of alginate complex with k-carrageenan, xanthan and guar gums, alginate and chitosan beads obtained containing oil: AG-CAR (0.5:0.5) = alginate-k-carrageenan (ratio 0.5:0.5) complex beads containing oil;

AG-CAR (0.75:0.75) = alginate-k-carrageenan (ratio 0.5:0.5) complex beads containing oil; AG-XG (0.75:0.75) = alginate-xanthan gum (ratio 0.75:0.75) complex beads containing oil; AG-XG (0.5:0.5) = alginate-xanthan gum (ratio 0.5:0.5) complex beads containing oil; AG-GG (0.75:0.75) = alginate-guar gum (ratio 0.75:0.75) complex beads containing oil; AG-GG

(0.5:0.5) = alginate-guar gum (ratio 0.5:0.5) complex beads containing oil; AGoil2% = alginate 2% beads containing oil; AGoil1.5% = alginate 1.5% beads containing oil; AGoil1% = alginate 1% beads containing oil; CHoil2% = chitosan 2% beads containing oil; CHoil1.5%

= chitosan 1.5% beads containing oil; CHoil1% = chitosan 1% beads containing oil


XIX

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

AG CAR GG XG CH

O–H stretching vibration 3244 3514

PolyOH groups

3299 3302 3289

O-H +

N-H strech

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

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


XX

Deformations of CH2 group ( bending)

1408 1474, 1400 1408 1400 1428

O-H bending - 1223 ( S=O strech sulphate ester

1350 1247 -

C-O and C-C ring stretching 1200-1000 - 1145 1150 1151

–CH2OH stretching mode 1054 1063 1054 1061

C–OH alcoholic

(C-O stretching saccharide)

1024 1024 - 1025 1024

–CH2 twisting vibration 948, 902,

Gululonic & mannuronic

924, 910

Polyhydroxy groups

1016 - -

Glycosidic links 809 842

Galactose sulphate, glycosidic link

866,777

(1,4; 1,6) link galactose

and mannose

785 C-H rocking, bending

C-C stretching

892,

776

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 1700-900 cm-1) which are present also in the free oils.


XXI

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.


XXII

0

20

40

60

80

100

120

140

160

180

200

AG 2% AG 1.5%

Alginate 1%

AG-CAR(0.75%)

CH 2% CH 1% AG-GG AG-KG SB

Tem

pera

ture

(°C)

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

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

0

20

40

60

80

100

120

AG 2% AG 1.5% AG-CAR(0.75%)

AG-GG AG-KG SB

Rest

mas

s %

Fig.III.6. Graphic representation of restmass% of some samples of TGA analysis


XXIII

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 k-carrageenan, xanthan and guar gums in ratio concentrations of 0.75:0.75.

The results show that the amount of oil encapsulated in different matrices affected the mean diameter of the beads. The size of the gel beads increased with the amount of oil encapsulated. Also the other characteristics of capsules (area, perimeter, roundness and elongation) chanced after oil encapsulation.

By FTIR-ATR analysis, mixture of oils and different blank beads showed the peaks attributable to both oils and empty beads. This confirms the oils entrapment into the beads at the molecular level, the oil specific double peaks (regions between 2800-2900 cm-1 and 1700-900 cm-1) which are present also in the free oils.

CHAPTER IV. ENCAPSULATION EFFICIENCY AND RELEASE STUDIES

IV.1. MATERIALS AND METHODS

Encapsulation efficiency of the beads

The oils encapsulation was determined calculating the amount of β-carotene or total carotenoids content of each oil analyzed before and after encapsulation. The samples were assayed for β-carotene or total carotenoids content of each oil according previous analysis when was identified the UV-Vis fingerprint, spectrophotometrically.

Encapsulation efficiency (EE%) was calculated by using formulae:

EE% = C1/C2 x XL0, C1= carotenoid concentration in the oil

C2= carotenoid concentration after release from beads

Also from the hardening baths, after encapsulation process, were extracted the carotenoids with THF for a better efficiency calculation.


XXIV

Release rate measurements of oil from beads

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

In vitro release oils from the beads

The scheme of using the artificial simulated fluids at different pH was as follows:

• 1st hour: simulated gastric fluid of pH 1.2 • 2nd to 3rd hour: mixture of simulated gastric and intestinal fluid of pH 4.5 • 4th to 7th hour: simulated intestinal fluid of pH 7.4 In vitro oil release studies were performed as per scheme in different simulated fluids.

Simulation of gastrointestinal (GI) transit conditions was achieved by using different dissolution media.

Simulated gastric fluid (SGF) pH 1.2 consisted of 0.1N HCl and X ml Sanzyme (enzyme syrup containing 80 mg papain, 40 mg pepsin and XL mg sanzyme 2000); pH adjusted to 1.2 ±0.1.

Simulated intestinal fluid (SIF) pH 4.5 was prepared by mixing SGF pH 1.2 and SIF pH XX.4 in a ratio 3XX:61; pH adjusted to 4.X ±0.1.

Simulated intestinal fluid (SIF) pH 7.4 consisted of KH2PO4 1.0XX4g in 30 ml of 0.2N NaOH, and pancreatin 2XXX mg (using “Triferment”); pH adjusted to XX.4 ±0.1.

The experiment was performed into an incubator with a continuous supply of carbon dioxide at 37ºC.

IV.2. RESULTS AND DISSCUSIONS

Encapsulation efficiency of the beads

UV-Vis analysis of the extracts from hardening baths, did not show significant values. In the cases of low encapsulation efficiency the absorbance values were ranging from 0.0001 to 0.0003, we can say that the efficiency encapsulation is enough to be calculated using formulae mentioned before.

According with formulae described on Material and Methods, the encapsulation efficiency is presented on the following table (Fig.IV.1.) for the different types of beads, and related to each oil. The dates presented represent the average of values for the same beads and different oils.


XXV

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 k-carrageenan 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


XXVI

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.

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200 250 300 350 400Time (minutes)

Abs

orba

nce

(a.u

.)

MethanolHexaneTHF

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.


XXVIII

CHAPTER V. FTIR CHARACTERIZATION OF OIL OXIDATION

V.1. MATERIALS AND METHODS

The FTIR spectra were obtained with a Fourier transform spectrometer Spectrum One (PerkinElmer), equipped with the universal ATR as an internal reflection accessory which have Composite Zinc Selenide (ZnSe) and Diamond crystals. Each spectrum was from 4000 to 6X0 cm-1. Between measurements the crystal was cleaned with acetone.

The oxidation process under UV light on time (after 1h, 4h and 6h) was done using an UV lamp (2X4 μm), each oil an all obtained beads containing oils were exposed under these conditions.

V.2. RESULTS AND DISCUSSIONS

The oxidation process under UV light (2X4 μm) on time (after 1h, 4h and 6h) was monitored calculating the ratios between absorbance of some bands of the spectra of free oil, according with literature (Guillén and Cabo, 1999, 2000, 2002) and encapsulated oil in different type of beads obtained: A2853/A3005, A1746/A3006, A1474/A3006, A1377/A3006 and A1163/A3006, before and after treatment under UV. The values are given for these ratios could be considered as indicative parameters of the oxidation level of different kinds of oils.

All oils free obtained values showed SS or TS stage oxidation, comparing with the values of oils encapsulated in FS stage of oxidation (see as an example Fig.V.1., the oxidation on time of HP oil free and encapsulated).

The best protection from all this concentrations used against UV treatment was found to be alginate 1%, chitosan 1.5%, alginate-guar gum and alginate-xanthan gum complexs in ratio 0.5:0.5, and alginate-k-carrageenan complex in ratio 0.75:0.75.


XXIX

0

1

2

3

4

5

6

7

8

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




Types of ratios on time/Tipul rapoartelor in timp

Rat

io v

alue

s/Va

loar

ea r

apor

telo

rOil free/Ulei liber


Oil from AG 1.5%/Ulei din AG 1.5%


Fig. V.1. Graphic representation of the hemp oil free and encapsulated (in different alginate concentrations beads) under oxidation changes

(A= A2853/A3005-3008, B= A1744/ A3005-3008, C= A1464/ A3005-3008, D= A1377/ A3005-3008, E= A1160/ A3005-3008)

V.3. CONCLUSIONS

The usefulness of absorbance ratios and frequency data to measure the oxidative stability and oxidation degree of encapsulated oils directly into the beads was studied.

All free oils show SS or TS stage oxidation, compared with the values of encapsulated oils in FS stage of oxidation.

The best protection against UV treatment was found to be alginate 1%, chitosan 1.5%, alginate-guar gum and alginate-xanthan gum complexs in ratio 0.5:0.5, and alginate-k-carrageenan 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.


XXX

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.


XXXI

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

1. 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 33-1981 (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., MÖHWALD, 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. Kühtreiber, R.P. Lanza and W.L. Chick, eds., Birkhäuser, 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), 1281–1286.

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), 1788–1793.

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), 503–510.

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, 9072–9079.

23. HEINZEN, C., 2002, Microencapsulation solve time dependent problems for foodmakers. European Food and Drink Review, 3, 27–30.

24. KRAJEWSKA, B., 2005, Membrane-based Processes Performed with use of Chitin/Chitosan Materials, Separation & Purification Technology, 41, 305–312.

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.

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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.

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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.

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PUBLICATIONS RELEASED DURING PhD

2009

1. Socaciu C., Trif. M, A. Baciu, T. Nicula, A. Nicula, ‘’ Encapsulation of plant oleosomes and oleoresins in mixed carbohydrate matrices’’, COST865, Spring Meeting "Microcapsule property assesment", Luxemburg 2009, Proceeding

2008

1. Monica Trif, Ansorge-Schumacher M., Socaciu C., Diehl H.A. „Bioencapsulated seabuckthorn oil: controlled release rates in different solvents”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania

2. Pece Aurelia, D. Vodnar, Monica Trif, C. Coroian, Camelia Raducu, G. Muresan, “Study of the physico-chemical parameters from buffalo raw milk during different lactations”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania

3. Pece Aurelia, D. Vodnar, Monica Trif, “Corelation between microbiological and physico-chemical parameters from buffalo raw milk during different lactations”, Bull. USAMV-CN, 65/2008, ISSN 1454-2382, Romania

4. Carmen Socaciu, Baciu A., Trif M., “Oleosome-rich pectin network as a new, natural bioencapsulation matrix”, XVI International Conference on Bioencapsulation Dublin, Ireland ; September 2008, Proceeding

5. Monica Trif, Carmen Socaciu, Andreea Stanila, “The evaluation of encapsulated Seabuckthorn oil properties usind FTIR”, CIGR - International Conference of Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797

6. Andreea Stanila and Monica Trif, “Antioxidant activity of carotenoide extracts from HIPPOPHAE RHAMNOIDES”, CIGR - International Conference of Agricultural Engineering XXXVII Congresso Brasileiro de Engenharia Agrícola, Processing Conference - 4th CIGR Section VI International Symposium On Food And Bioprocess Technology, September 2008, Iguaccu, Brazil, ISSN 1982-3797

7. Monica Trif, Carmen Socaciu and Horst Diehl, “Evaluation of effiency, release and oxidation stability of seabuckthorn encapsulated oil using FTIR spectroscopy”, 7th Joint Meeting of AFERP, ASP, GA, PSE & SIF, August 2008, Athens, Greece, Book of Abstracts, pg.39

8. Monica Trif and Carmen Socaciu, “Evaluation of effiency, release and oxidation stability of Seabuckthorn microencapsulated oil using Fourier Transformed Infrared Spectroscopy”, 4th Meeting on Chemistry and Life, and accepted to be published in Chemické Listy Journal (current IF=0.683)


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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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