RAPORT STIINTIFIC SI TEHNIC
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1 RAPORT STIINTIFIC SI TEHNIC Exploatarea potentialului antioxidant al strugurilor negri pentru producerea de vinuri de calitate inalta autentificata (ANTIOXWIN) Cod : PN-II-PT-PCCA Contract 101/2012 ETAPA 2, 1 ianuarie-31 decembrie 2013 : Evaluarea initiala a a procesului de vinificatie pentru extractia controlata a nivelelor maxime de antioxidanti si dezvoltarea unor metode de clasificare a vinurilor REZUMATUL ETAPEI Pentru etapa 2 (ianuarie-decembrie 2013 au fost prevazute conform actului aditional nr 1/2013 la contractul 101/2012 si au fost realizate 4 activitati, respectiv : Activitatea 2.1 (responsabil: P2) Metoda MALDI pentru determinarea antocianilor Partenerul P2 a dezvoltat o metoda MALDI pentru determinarea antocianilor. Au fost testate doua proceduri de preparare a probelor (metoda dried droplet si metoda sandwich ) si diferite matrici pentru a determina care dintre acestea conduc la o mai buna ionizare a antocianilor de interes. Rezultatele asteptate la activitatea 2.1 (Raport metoda MALDI) au fost obtinute. Activitatea 2.2 (responsabil : P1, P2) a vizat evaluarea influentei etapelor critice ale procesului de fabricatie al vinurilor asupra potentialului antioxidant al acestora. Partenerul P1 a identificat etapele tehnologice critice care afecteaza potentialul antioxidant al vinurilor : evaluarea maturitatii strugurilor, macerarea-fermentarea si invechirea vinurilor. Potentialul antioxidant al vinurilor a fost urmarit de P1 si P2 folosind determinari spectrofotometrice pe grupe de compuri fenolici, teste de activitate antioxidanta totala (TEAC) si determinari ale 1
2 compozitiei in compusi fenolici prin HPLC-DAD-MS folosind metode dezvoltate de cei 2 parteneri in etapa anterioara. Dinamica parametrilor determinati a fost specifica soiului si variantei tehnologice. Rezultatele asteptate la activitatea 2.2 (Fisa tehnologica preliminara) au fost obtinute. Activitatea 2.3 (responsabil : Co, P2) a vizat dezvoltarea si validarea unui (bio)senzor pentru evaluarea rapida a activitatii antioxidante totale, in conditii de laborator. Coordonatorul, cu sprijinul P2 a dezvoltat o metoda electrochimica simpla bazata pe un sistem de analiza in flux, pentru evaluarea rapida a activitatii antioxidante pe baza radicalului 2,2-diphenyl-1- picrylhydrazyl DPPH (Figura 1). Sistemul se bazeaza pe capacitatea antioxidantilor de a neutraliza radicalul DPPH, conform reactiei: DPPH + RH DPPH-H +R unde RH reprezinta un antioxidant. Figura 1. Sistem experimental de tip FIA pentru determinarea electrochimica a capacitatii antioxidante totale: 1.) calculator pentru achizita datelor, controlul potentiostatului si a pompei 2.) pompa cu seringa, 3.) cuva electrochimica de tip wall jet care poate acomoda electrozii serigrafiati si este alimentata in mod continuu cu solutia care vine de la pompa seringa prin injector), 4.) injector manual cu 6 port -uri, 2 pozitii si bucla de injectie externa de 500 µl si 5.) potentiostat care permite polarizarea electrodului serigrafiat aflat in cuva electrochimica. Cu cat concentratia antioxidantului este mai mare, cu atat semnalul electrochimic inregistrat pentru un amestec de DPPH si antioxidant este mai mic (Figura 2). 2
3 Figura 2. Analiza electrochimica a capacitatii antioxidante totale a vinurilor pe baza inactivarii radicalului DPPH, folosind un sistem de analiza in flux. Pentru realizarea curbei de calibrare s-a utilizat Trolox (6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid), iar rezultatele au fost exprimate in mm echivalent Trolox. Sistemul a fost studiat in conditii de laborator, in solutii tampon. Au fost studiate influenta asupra intensitatii curentului inregistrat a mai multor factori precum potentialul aplicat, concentratia de DPPH, dilutia vinului si materialul din care este confectionat electrodul. De asemenea, senzorul TAC a fost comparat cu metoda clasica spectrofotometrica TEAC de determinare a capacitatii antioxidante, bazata pe radicalul cation 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid ABTS +. Rezultatele asteptate la activitatea 2.3. ((Bio)senzor TAC validat in conditii de laborator ) au fost obtinute. La activitatea 2.4. (Co, P1) s-a realizat dezvoltarea preliminara a unui set de senzori care pot fi inclusi intr-un dispozitiv de tip e-tongue pentru evaluarea profilului antioxidant al vinurilor si clasificarea vinurilor. A fost vizata imbunatatirea si diversificarea senzorilor din cadrul dispozitivului e-tongue, pornind de la studiile din etapa precedenta a proiectului. Pentru 3
4 realizarea electrozilor modificati s-au luat in considerare diferite materiale de electrod bazate pe carbune (electrozi serigrafiati de grafit si electrod de carbune vitros), doi compusi cu Co (o ftalocianina comerciala si un compus nou-sintetizat), nanoparticule, un polimer (polipirol) precum si o enzima (lacaza). Prin imbunatatirea si diversificarea senzorilor din cadrul dispozitivului e-tongue s-a ajuns la un sistem care cuprinde 5 senzori pe baza de grafit: un electrod nemodificat si 4 electrozi modificati cu diverse materiale. Vinurile furnizate de Partenerul 1, din recolta 2012, au fost analizate cu electrozi de grafit nemodificati. Probele de vin diluate corespunzator au fost supuse unui baleiaj de potential prin voltametrie cu unda patrata, inregistrandu-se intensitatea curentului electric rezultat (Figura 3a). Datele extrase din voltamograme au fost utilizate mai departe pentru analiza de componenti principali (Figura 3b). NV PN NM CS M BM NA FN (a) (b) Figura 3. Analiza a 8 vinuri rosii prin voltametrie cu unda patrata (SWV) pe electrozi de grafit (a) si interpretarea datelor prin analiza Componentilor Principali (b). PN: Pinot Noir, NV: Negru Vartos, NM: Negru Moale, NA: Negru Aromat, M: Merlot, CS: Cabernet Sauvignon, FN: Feteasca Neagra, BM: Burgund Mare. Pentru fiecare senzor aditional din cadrul dispozitivului e-tongue se adauga un set nou de voltamograme. Datele extrase din voltamogramele inregistrate cu toti senzorii alcatuiesc, pentru fiecare proba de vin, un `profil`unic al acelui vin. Rezultatele prezentate in figura 3 a si b ilustreaza principiul pe baza va functiona dispozitivul e-tongue pentru diferentierea vinurilor. Pe 4
5 baza datelor obtinute pentru toti senzorii a fost alcatuit un raport preliminar de dezvoltare a unui set de senzori de tip ``e-tongue``. Rezultatele asteptate la activitatea 2.4 (Raport preliminar de dezvoltare a unui set de senzori tip e-tongue ) au fost obtinute. Obiectivele propuse in etapa 2 a proiectului ANTIOXWIN au fost atinse. DISEMINARE Rezultatele obtinute in aceasta etapa de toti partenerii au fost diseminate prin 4 articole (in reviste ISI) in curs de evaluare, 6 participari la conferinte nationale si internationale prin 4 postere si 2 comunicari orale precum si 2 lucrari stiintifice in reviste nationale. Lucrari stiintifice publicate Porumb, R. La typicité phenolique des raisins noir par rapport aux conditions climatiques de la millėsime, in Lucrari stiintifice, Seria Horticultura USAMV Ion Ionescu de la Brad vol. 56, nr. 2, 2013, p.297, ISSN: print Tudorache A., Pircalabu, L., Porumb, R., The specificity of the area doc dealu marevalea calugareasca for thermal necessary of varieties for red wines, Lucrari stiintifice, Seria Horticultura, USAMV Ion Ionescu de la Brad vol. 56, nr. 2, 2013, p. 309, ISSN: print Articole in evaluare Andrei, V., Sharpe, E., Vasilescu, A., Andreescu., S., Disposable nanoceria based electrochemical sensor for the detection of phenolic wine antioxidants, in evaluare la Analytica Chimica Acta Albu Camelia, Litescu, S.C, Eremia, S.A.V, Radu, L.G., A novel HPLC-PDA-MS method for polyphenolic compounds analysis from red wines A HPLC method for polyphenols analysis, in evaluare la Food Analytical Methods Diaconu., M., Chira, A., Radu, L.G., Modulating ITO electrode properties for laccase electron transfer enhancement, in evaluare la Thin Solid Films 5
6 Alecu, A., Eremia, S.A.V, Radu, L.G, Litescu, S.C, Profile screening of anthocyanins in red wines by matrix assisted laser desorption/ionization time of flight mass spectrometry, in evaluare la Talanta Participari la conferinte Andrei, V., Nedelcu, G., Porumb, R., Tudorache, A., Vasilescu, A., Electrochemical sensor based on ceria nanoparticles for the detection of phenolic compounds in red wines from Valea Calugareasca vineyard, poster, Scoala de Vara de Electrochimie, Cluj- Napoca, iunie 2013 Nedelcu, G., Andrei, V., Alecu, A., Albu., C., Vasilescu, A., Characterization of some Romanian red wines by electrochemical and total antioxidant capacity assays, poster, Scoala de Vara de Electrochimie, Cluj-Napoca, iunie 2013 Andrei, V., Nedelcu, G., Vasilescu, A., Brinduse, E., Tudorache, A., Porumb, R., Albu, C., Litescu, S.C, Eremia, S.A.V, Profile of three red Romanian wines by electrochemical, chromatographic and total antioxidant capacity data, poster, In Vino Analytica Scientia, Reims, Franta, 3-5 iulie 2013 Mihai, I., Vezeanu, A., Vasilescu., A., A new thiol-coated interface for the development of an aptasensor for lysozyme, prezentare orala, Phototech for Biosensors and Energy, Atena, Grecia, octombrie 2013 Vasilescu, A., Mihai, I., Vezeanu., A., Andrei, V., Nedelcu, G., Szunerits, S., Un nou nanomaterial pentru studiul interactiunilor proteinelor, prezentare orala, Seminarul National de Nanostiinta si Nanotehnologie, Bucuresti, 16 mai Hosu, I., Peteu, S.F, Vasilescu, A., Detection of peroxynitrite with chemically modified electrodes based on reduced graphene oxide, poster, Priorichem, Bucuresti, octombrie
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