Philosophiae Doctor - PhD / The aim of this thesis is first, to evaluate various culture conditions to improve embryo development, and secondly, to analyze spent culture media for any biomarkers that may be predictive of embryo health. Single-step and sequential culture media were compared in both Planer and EmbryoScope™ incubators. Single-step media resulted in better blastocyst development compared to sequential media and the EmbryoScope™ incubation system showed slight improvements in embryo development than the Planer system. The benefits of supplementing the culture medium with either insulin or insulin-like growth factor 1 (IGF-1) or culturing in a 2% O2 environment, using two different strains of mice (hybrid and C57), as well as the suitability of these strains for quality control were compared. In insulin, hybrid embryos were slower to blastulate and had a lower blastocyst rate, whereas C57 embryos were slower to the morula and faster to blastocyst stages, and lower blastocyst rate than the controls. IGF-1 showed no difference in time-lapse morphokinetics (TLM) or blastocyst rates compared to controls in both hybrid and C57 embryos. Under 2% O2, hybrid embryos showed no significant difference in TLM up to the 8-cell stage, but slowed down afterwards, resulting in blastocysts with significantly lower cell counts than the 6% O2 group. The C57 embryos were slower to reach morula and expanded blastocyst, and had lower blastocyst rates in 2%O2 vs 6%O2. The C57 strain had significant slower overall embryo development for all time points than hybrid embryos in insulin, IGF-1 and ultra-low O2, as well as lower blastocyst rates. Measurement of growth differentiation factor 9 (GDF-9) and oxidation-reduction potential (ORP) in spent media as markers for embryo health were evaluated. Day 5 human blastocysts yielded higher pregnancy rates and GDF-9 levels in spent media compared to Day 6 blastocysts, but TLM parameters showed no impact on pregnancy outcome. In Day 6 blastocysts, the non-pregnant group showed significantly faster embryo development compared to the clinically pregnant group up to the 8-cell stage and start of blastulation. GDF-9 did not show any significant differences between non-pregnant and pregnant groups of Day 5 or Day 6 embryo transfers. ORP in spent media from good quality Day 3 embryos that developed into blastocysts were significantly higher than from those that did not, with no difference in control medium ORP. Spent media from arrested embryos showed lower ORP than their corresponding controls. Arrested embryos had slower development at syngamy, morula, blastulation and blastocyst stages. The single step medium in the EmbryoScope™ is the preferred choice for embryo culture. Insulin or IGF-1 media supplementation or 2% O2 culture did not provide any benefit to embryo development. The C57 mouse strain is more sensitive and may be better to detect changes in culture conditions, and therefore better model for quality control assays. GDF-9 values decrease from Day 5 to Day 6 which gives new insight to understanding the role of GDF-9 during embryogenesis. ORP in spent media indicate that embryos that developed into blastocysts did not contribute to ROS, but maintained ORP balance.

Объектами исследования являются полифенольные антиоксиданты. Целью выпускнoй квалификационной работы является исследование антиоксидантных свойств полифенольных соединений с позиции механизмов их действия. В работе рассмотрены причины возникновения окислительного стресса, система антиоксидантной защиты и существующие методы исследования антиоксидантных свойств соединений. В процессе работы предложенными методами оценена хелатирующая способность полифенолов по отношению к ионам железа (II) и (III), исследована зависимость антиоксидантных свойств от рН, определены значения условных констант устойчивости и их зависимость от рН. / The objects of research are polyphenolic antioxidants. The purpose of the final qualifying work is to study the antioxidant properties of polyphenolic compounds from the perspective of their mechanisms of action. The paper considers the causes of oxidative stress, the system of antioxidant protection and existing methods for studying the antioxidant properties of compounds. In the process of work, the proposed methods evaluated the chelating ability of polyphenols with respect to iron (II) and (III) ions, investigated the dependence of antioxidant properties on pH, determined the values of conditional stability constants and their dependence on pH.

Tesis por compendio / [ES] El uso masivo de plásticos y su impacto medioambiental, así como la necesidad de mejorar la conservación de los alimentos, hace necesario el desarrollo de alternativas biodegradables para el envasado activo de alimentos. La presente Tesis Doctoral, se ha centrado en el desarrollo de películas activas, basados en polímeros biodegradables con propiedades complementarias, con el fin de obtener materiales con propiedades mecánicas y de barrera adecuadas para cumplir con los requisitos del envasado alimentario. Para ello, se consideraron el poli (vinil alcohol) (PVA) y el poli (ácido láctico) PLA. Asimismo, el carvacrol y los ácidos ferúlico y cinámico fueron elegidos como compuestos activos naturales. Se estudió la encapsulación del carvacrol en liposomas de diferentes tipos de lecitina para mejorar su retención durante la etapa de formación del film. La lecitina de soja enriquecida en fosfatidilcolina fue la más eficaz para mantener la estabilidad de la emulsión de carvacrol, resultando en films con mayor retención. La incorporación de carvacrol (encapsulado o no), modificó ligeramente la microestructura y las propiedades físicas de los films, así como la cristalinidad y el comportamiento térmico del polímero. El PVA totalmente hidrolizado proporcionó films con un mejor desempeño mecánico y capacidad de barrera al oxígeno, pero con menor retención de carvacrol. Por el contrario, el PVA parcialmente hidrolizado dio lugar a films más homogéneos, con mayor contenido de carvacrol. Mientras que los grupos acetilos protegieron al polímero de la termodegradación, permitiendo la obtención de films mediante las técnicas habituales del procesado industrial. Por lo tanto, el PVA parcialmente hidrolizado tiene un gran potencial para la producción de films activos, con más posibilidades de procesamiento que el PVA totalmente hidrolizado. Se incorporaron también ácido cinámico y ferúlico, con menor impacto sensorial que el del carvacrol, al PVA parcial y totalmente hidrolizado, mediante casting de las soluciones acuosas con glicerol para mejorar la solubilidad de los ácidos. Los films de PVA plastificados con glicerol mostraron una menor capacidad de barrera que los no plastificados. La incorporación del ácido ferúlico promovió mayores cambios en las propiedades de los films que el ácido cinámico, debido a un efecto de reticulación, que promovió la cristalinidad, la rigidez y la capacidad de barrera del material. También se obtuvieron películas plastificadas a base de PVA parcialmente hidrolizado con ácidos fenólicos, mediante mezclado en fundido y moldeo por compresión. Los films termoprocesados fueron menos extensibles y resistentes a la rotura y más permeables al oxígeno y al vapor de agua. Estudios in vitro demostraron que los films con ácidos fenólicos obtenidos por casting o termoprocesado presentaron actividad antioxidante y antimicrobiana, especialmente con ácido ferúlico. Con el objetivo de cumplir los requisitos de envasado de alimentos, se obtuvieron films tricapa por termocompresión, compuestos por una capa central de PVA, cargada con compuestos activos (carvacrol o ácido ferúlico), y dos capas externas de PLA. La termocompresión fue eficaz para la adhesión interlaminar. Las multicapas mostraron un rendimiento mecánico próximo al de los films de PLA y una elevada capacidad de barrera al oxígeno y al vapor de agua. Todos los laminados con compuestos activos incorporados a la capa de PVA fueron eficaces para controlar el crecimiento microbiano en filetes de carne de vacuno envasada. Por tanto, el laminado de films de PVA y PLA parcialmente hidrolizado representa una estrategia adecuada para obtener materiales de envasado con propiedades funcionales más próximas a las de algunos plásticos convencionales utilizados en el envasado alimentario. Asimismo, estos materiales con carvacrol o ácido ferúlico incorporados podrían utilizarse para el envasado de alimentos altamente susceptibles a procesos oxidativos o de degradación microbiológica para alargar su vida útil. / [CA] L'ús massiu de plàstics i el seu impacte mediambiental, així com la necessitat de millorar la conservació dels aliments, fa necessari el desenvolupament d'alternatives biodegradables per a l'envasament actiu d'aliments. La present Tesi Doctoral, s'ha centrat en el desenvolupament de pel·lícules actives, basats en polímers biodegradables amb propietats complementàries, amb la finalitat d'obtindre materials amb propietats mecàniques i de barrera adequades per a complir amb els requisits de l'envasament alimentari. Per a això, es van considerar el poli (vinil alcohol) (PVA) i el poli (àcid làctic) PLA. Així mateix, es van triar el carvacrol i els àcids ferúlic i cinàmic, com a compostos actius. Es va estudiar l'encapsulació del carvacrol en liposomes de diferents tipus de lecitina per a millorar la seua retenció durant l'etapa de formació del film. La lecitina de soja enriquida en fosfatidilcolina va ser la més eficaç per a mantindre l'estabilitat de l'emulsió de carvacrol, resultant en films amb major retenció. La incorporació de carvacrol (encapsulat o no), va modificar lleugerament la microestructura i les propietats físiques dels films, així com la cristalinitat i el comportament tèrmic del polímer. El PVA totalment hidrolitzat va proporcionar films amb un millor exercisc mecànic i capacitat de barrera a l'oxigen, però amb menor retenció de carvacrol. Per contra, el PVA parcialment hidrolitzat va donar lloc a films més homogenis, amb major contingut de carvacrol. Mentre que els grups acetils van protegir el polímer de la termodegradació, permetent l'obtenció de films mitjançant les tècniques habituals del processament industrial. Per tant, el PVA parcialment hidrolitzat té un gran potencial per a la producció de films actius, amb una més àmplia gamma de possibilitats de processament que el PVA totalment hidrolitzat. Es van incorporar també àcid cinàmic i ferúlic, amb menor impacte sensorial que el del carvacrol, al PVA parcial i totalment hidrolitzat, mitjançant càsting de les solucions aquoses amb glicerol per a millorar la solubilitat dels àcids. Els films de PVA plastificats amb glicerol van mostrar una menor capacitat de barrera que els no plastificats. La incorporació de l'àcid ferúlic va promoure majors canvis en les propietats dels films que l'àcid cinàmic, a causa d'un efecte de reticulació, que va promoure la cristalinitad, la rigidesa i la capacitat de barrera del material. També es van obtindre pel·lícules plastificades a base de PVA parcialment hidrolitzat amb àcids fenòlics, mitjançant barrejat en fos i emotlament per compressió. Els films termoprocessats van ser menys extensibles i resistents al trencament i més permeables a l'oxigen i al vapor d'aigua. Estudis in vitro van demostrar que els films amb àcids fenòlics obtinguts per càsting o termoprocessat van presentar activitat antioxidant i antimicrobiana, especialment amb àcid ferúlic. Amb l'objectiu de complir els requisits d'envasament d'aliments, es van obtindre films tricapa per termocompressió, compostos per una capa central de *PVA, carregada amb compostos actius (carvacrol o àcid ferúlic), i dues capes externes de PLA. La termocompressió va ser eficaç per a l'adhesió interlaminar. Les multicapes van mostrar un rendiment mecànic pròxim al dels films de PLA i una elevada capacitat de barrera a l'oxigen i al vapor d'aigua. Tots els laminatges amb compostos actius incorporats a la capa de PVA van ser eficaces per a controlar el creixement microbià en filets de carn de boví envasada. Per tant, el laminatge de films de PVA i PLA parcialment hidrolitzat representa una estratègia adequada per a obtindre materials d'envasament amb propietats funcionals més pròximes a les d'alguns plàstics sintètics utilitzats en l'envasament alimentari. Així mateix, aquests materials amb carvacrol o àcid ferúlic incorporats podrien utilitzar-se per a l'envasament d'aliments altament susceptibles a processos oxidatius o de degradació microbiològica per a allargar la seua vida útil. / [EN] The massive use of synthetic plastics and their environmental impact, as well as the need of improving food preservation, makes necessary the development of biodegradable alternatives for food active packaging. The use of active compounds from natural origin to obtain this kind of materials is a new trend based of the consumer demand for safer and healthier products. The present Doctoral Thesis, has been focused on the development of active packaging films, based on biodegradable polymers with complementary properties, in order to obtain materials with adequate mechanical and barrier properties to meet food packaging requirements. To this end, poly (vinyl alcohol) (PVA) and poly (lactic acid) (PLA) were considered. Likewise, carvacrol and ferulic and cinnamic acid, were chosen as active natural compounds. The liposomal encapsulation of carvacrol, using different kinds of lecithin was evaluated in order to improve the carvacrol retention in the polymer matrix during the film formation step. Soy lecithin enriched in phosphatidylcholine was the most effective at maintaining the stability of the carvacrol emulsion, leading to its highest retention in the PVA films. Incorporation of carvacrol (lecithin encapsulated or not) slightly modified the film microstructure and physical properties and polymer crystallinity and thermal behaviour. The fully hydrolysed PVA provided films with a better mechanical performance and oxygen barrier capacity, but with less carvacrol retention. In contrast, partially hydrolysed PVA gave rise to more homogenous films with a higher carvacrol content. Acetyl groups protect the polymer for thermodegradation, allowing the film production using the common industrial thermoplastic processing techniques. Therefore, partially hydrolysed PVA has a great potential for the production of active films, with a wider range of processing possibilities than fully hydrolysed PVA. Cinnamic and ferulic acids, with lower potential sensory impact than of carvacrol, were also incorporated into partially and fully hydrolysed PVA by casting of aqueous solutions containing glycerol to improve the acid solubility. Glycerol plasticized PVA films exhibited poorer barrier capacity than those non-plasticized. The incorporation of ferulic acid promoted greater changes in the film properties than cinnamic acid, due to a crosslinking effect, which promoted crystallinity, stiffness and barrier capacity of the material. Glycerol plasticized films based on partially hydrolysed PVA with phenolic acids were also obtained by melt blending and compression moulding. Termoprocessed films were less stretchable and resistant to break and more permeable to oxygen and water vapor. In vitro studies demonstrated that cast and termoprocessed films containing phenolic acids exhibited antioxidant and antimicrobial activity, especially with ferulic acid. Three-layer films composed of a central layer of PVA loaded with active compounds (carvacrol lecithin encapsulated or not, or ferulic acid) and two external PLA layers were obtained by thermo-compression, aimed to meet food packaging requirements. Thermocompression was effective for the interlaminar adhesion. Multilayers exhibited mechanical performance close to the PLA sheets and high oxygen and water vapour barrier capacity. All laminates with active compounds incorporated into the PVA sheet were effective at controlling microbial growth in packaged beef meet. Therefore, lamination of partially hydrolysed PVA and PLA films represents a successful strategy to obtain packaging materials with functional properties closer to those of some conventional plastics commonly used in food packaging. Likewise, these materials incorporating carvacrol or ferulic acid could be used for packaging of foods highly susceptible to oxidative or microbiological degradative process to extend their shelf life. / The authors acknowledge the financial support of the Agencia Estatal de Investigacion, Ministerio de Economía y Competitividad (MINECO) of Spain through the projects AGL2016-76699-R and PID2019-105207RB-100. Author Johana Andrade thanks the Departamento de Nariño-Colombia y la Fundación CEIBA for the doctoral grant. The authors also thank the services rendered by the Electron Microscopy Service of the UPV. / Andrade Chapal, JC. (2021). Biodegradable materials based on poly(vinyl alcohol) (PVA) and poly (lactic acid) (PLA) with antioxidant and antimicrobial activity for food packaging applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/175566 / Compendio

<p>Podizanje novih zasada i povećanje kapaciteta prerade<br />grožđa povećava količinu otpada sa kojim se suočava<br />industrija proizvodnje vina i rakije. Najbolji i najodgovorniji<br />način upravljanja otpadom je njegovo dalje iskori&scaron;ćenje<br />kao nusproizvoda. U ovoj disertaciji sagledavana je mogućnost iskori&scaron;ćenja<br />nusproizvoda iz različitih faza proizvodnje u vinarijama i<br />destilerijama za dobijanje semenki grožđa koje su<br />kori&scaron;ćene za proizvodnju hladno presovanog ulja. Hipoteza<br />se zasniva na činjenici da semenke grožđa raznih sorti iz<br />različitih faza proizvodnje vina (bela, roze ili crvena vina),<br />odnosno, rakije mogu imati sasvim različit hemijski sastav i<br />kvalitet koji svakako može da se reflektuje na kvalitet,<br />bioaktivne komponente i održivost hladno presovanog ulja.<br />Ovakav proizvod, obzirom da je dobijen hladnim<br />presovanjem, može biti veoma atraktivan za potro&scaron;ače,<br />zbog svojih specifičnih senzorskih i nutritivnih<br />karakteristika.<br />Za potrebe izrade disertacije prikupljeni su nusproizvodi<br />i pripremljeni su uzorci iz vinarija i destilerija fru&scaron;kogorskog<br />vinogorja. Dobijene su semenke i proizvedeno hladno<br />presovano ulje od semenki crnog grožđa sorte Merlot,<br />belog grožđa sorte Italijanski rizling i belog grožđa sorte<br />Sila, kao autohtone sorte vinove loze. Hladno presovana<br />ulja su proizvedena od semenki grožđa koje nisu pro&scaron;le<br />nikakav tretman, odnosno dobijene su nakon presovanja,<br />prilikom proizvodnje belih (Italijanski rizling i Sila) i roze<br />(Merlot) vina, zatim od semenki koje su pro&scaron;le proces<br />fermentacije prilikom proizvodnje crvenih vina (Merlot) i od<br />semenki koje su pro&scaron;le proces fermentacije i destilacije<br />prilikom proizvodnje rakije (Merlot, Italijanski rizling i Sila).<br />Pored navedenih semenki i ulja, pripremljen je i prosečan<br />proizvodni uzorak koji predstavlja uzorak dobijen od svih<br />prikupljenih semenki. Kao uporedni uzorci kori&scaron;ćeno je<br />nerafinisano i rafinisano ulje od semenki grožđa nabavljeno<br />na trži&scaron;tu. Za realizaciju postavljenog cilja, rad na izvođenju ove<br />disertacije obuhvatio je različite faze. U prvoj fazi<br />prikupljene su semenke grožđa i ispitivane su njihove<br />tehničko-tehnolo&scaron;ke karakteristika i kvalitet. Zatim su od<br />navedenih semenki proizvedena hladno presovana ulja.<br />U sledećim fazama, ovako dobijena uja, zajedno sa<br />uzorkom nerafinisanog i rafinisanog ulja od semenki<br />grožđa sa trži&scaron;ta ispitivana su sa aspekta senzorskog i<br />nutritivnog kvaliteta i praćene su razlike u održivosti ulja.<br />Izvr&scaron;ena je senzorska analiza, određivane su frakcije<br />pigmenata, karotenoidi i hlorofili, merena je transparencija,<br />a parametri boje ulja određivani su i instrumentalno.<br />Nutritivni kvalitet ulja sagledavan je na osnovu sadržaja i<br />sastava bioaktivnih komponenti, pre svega tokoferola i<br />tokotrienola, fenola i sterola. Budući da ova jedinjenja<br />ispoljavaju značajne antioksidativne aktivnosti izvr&scaron;eno je i<br />ispitivanje antiradikalske aktivnosti uzoraka. Odživost ulja<br />od semenki grožđa sagledana je na osnovu početnog<br />kvaliteta i oksidativnog stanja, kao i rezultata ubrzanih<br />testova, kao &scaron;to je Rancimat test, Schaal-oven test i<br />fluorescentni test.<br />Na osnovu dobijenih rezultata, može se konstatovati da<br />su svi dobijeni uzorci semenki imali dobru skladi&scaron;nu vlagu,<br />koja, obzirom na mali sadržaj ulja u semenkama, može da<br />osigura čuvanje semenki u dužem periodu. Sadržaj ulja u<br />semenkama, pored uticaja sorte vinove loze, zavisi i od<br />uticaja procesa kome su podvrgnute semenke pre<br />presovanja (fermentacija, destilacija).<br />Senzorska analiza ulja od semenki grožđa pokazala je<br />značajne razlike u karakteristikama koje su posledica ne<br />samo načina dobijanja ulja (hladno presovano ili rafinisano), sorte vinove loze, već i postupka dobijanja,<br />porekla i kvaliteta semenki grožđa. Uslovi kojima su<br />semenke grožđa izložene u toku alkoholne fermentacije i<br />posebno destilacije utiču na formiranje specifične arome<br />hladno presovanog ulja. I pored toga &scaron;to se pojavljuje<br />izuzetno &scaron;irok spektar različitih aroma, mirisa i ukusa u ulju<br />i &scaron;to postoje značajne razlike u aromi ulja u zavisnosti od<br />sorte grožđa, moguće je prepoznati da li je hladno<br />presovano ulje dobijeno od semenki grožđa bez<br />fermentacije, posle fermentacije ili posle destilacije.<br />Takođe, sa aspekta boje ulja može se kazati da rafinisano<br />ulje od semenki grožđa ima svetlo žutu boju sa<br />zelenkastom nijansom, dok su hladno presovana ulja<br />intenzivnijih boja i kreću se od žuto-zelenkaste, preko<br />zelenkasto žute i svetlo zelenkaste do tamno zelene. Veći<br />udeo zelene boje imaju hladno presovana ulja od semenki<br />grožđa dobijenih posle destilacije.<br />U radu je pokazano da sadržaj pigmenata (karotenoida i<br />hlorofila) u velikoj meri zavisi od porekla semenki.<br />Postupak fermentacije doprinosi povećanju sadržaja<br />pigmenata, dok postupak destilacije, zbog visokih<br />temperatura ima negativan efekat.<br />Transparencija uzoraka hladno presovanih ulja od<br />semenki grožđa proizvedenih za potrebe disertacije kretala<br />se od 32,8% do 53,8%.<br />Sadržaj nezasićenih masnih kiselina u svim uzorcima<br />ulja od semenki grožđa veći je od 90%, pri čemu<br />dominantnu masnu kiselinu čini linolna, omega-6, masna<br />kiselina.<br />Sadržaj tokotrienola je veći od sadržaja tokoferola u uljima od semenki grožđa, a dominantni tokoferol je alfatokoferol.<br />Njegov sadržaj je veći u uzorcima ulja dobijenim<br />iz semenki nakon fermentacije i destilacije.<br />Sadržaj fenolnih jedinjenja, u zavisnosti od sorte, u<br />proseku je ne&scaron;to vi&scaron;i u hladno presovanim uljima dobijenim<br />od crvene sorte grožđa, ali uočava se i značajan porast<br />(akumulacija) fenola u hladno presovanim uljima dobijenim<br />iz semenki grožđa nakon procesa fermentacije i destilacije.<br />Najveći pojedinačni sadržaju u ulju od semenki grožđa ima<br />ursolna kiselina, a pored nje značajniji sadržaj, mada<br />mnogo manji, pokazuju rezveratrol, kemferol i vanilinska<br />kiselina.<br />Najzastupljeniji steroli ulja semenki grožđa su &beta;-<br />sitosterol sa udelom od 62,59-69,74%, stigmasterol sa<br />udelom od 12,13-15,00% i kampesterol sa udelom od 6,59-<br />11,94% u ukupnim sterolima. Na sadržaj fitosterola u<br />uljima od semenki grožđa nemaju uticaja procesi<br />fermentacije i destilacije kojima su podvrgnute semenke<br />pre preosvanja.<br />U radu je dokazan negativan uticaj procesa fermentacije<br />i destilacije kojima su povrgnute semenke grožđa na<br />antiradikalski potencijal dobijenih ulja. Antiradikalski<br />potencijal ulja od semenki grožđa zavisi i od sorte vinove<br />loze, kao i od načina dobijanja ulja (hladno presovano ili<br />rafinisano).<br />Procesi fermentacije i destilacije utiču i na osnovni<br />hemijski kvalitet dobijenih hladno presovanih ulja na način<br />da dovode do povećanja kiselinskog i peroksidnog broja.<br />Hladno presovana ulja od semenki grožđa pokazuju<br />dobru oksidativnu stabilnost. Rancimat test je pokazao razlike u indukcionom periodu hladno presovanih ulja od<br />semenki grožđa kao posledicu razlike u sorti, kao i u<br />načinu dobijanja semenki. Proces fermentacije utiče na<br />povećanje, a proces destilacije na smanjenje indukcionog<br />perioda.<br />Na osnovu svih dobijenih rezultata istraživanja može se<br />konstatovati da su hladno presovana ulja od semenki<br />grožđa pokazala različit nutritivni kvalitet i oksidativnu<br />stabilnost, zbog specifičnih razlika koje su posledica razlika<br />u sorti i poreklu semenki, tj. zbog specifičnog efekta<br />fermentacije i destilacije na semenke od kojih je ulje<br />proizvedeno, čime je i potvrđena hipoteza.</p> / <p>Raising new plantations and increasing the grape<br />processing capacity increases the amount of waste faced<br />by the wine and brandy industry. The best and most<br />responsible way of waste management is its further<br />exploitation as by-products.<br />In this dissertation, the possibility of using by-products from different stages of production in wineries and distilleries for<br />obtaining grape seeds, which were used for the production<br />of cold pressed oil, was examined. The hypothesis is<br />based on the fact that grape seeds of various varieties<br />from different stages of wine production (white, rose or red<br />wine), that is, brandy can have a completely different<br />chemical composition and quality that can certainly be<br />reflected on the quality, bioactive components and stability<br />of cold pressed oil.<br />Such a product, as it is obtained by cold pressing, can<br />be very attractive to consumers, due to its specific sensory<br />and nutritional characteristics.<br />For the needs of the dissertation, the by-products were<br />collected and samples were prepared from the wineries<br />and distilleries of the Fru&scaron;ka Gora vineyard. Seeds were<br />obtained and cold pressed oil produced from Merlot grape<br />seed, as representatives of red grape varieties, Italian<br />Riesling, as representatives of white grape varieties and<br />Sila, as new white grape varieties. Cold pressed oils were<br />produced from grape seeds that did not undergo any<br />treatment, that is, they were obtained after pressing, during<br />the production of white (Italian Riesling and Sila) and rose<br />(Merlot) wines, then from the seeds that were fermented<br />during the production of red wines (Merlot ) and seeds that<br />have undergone fermentation and distillation process<br />during the production of brandy (Merlot, Italian Riesling and<br />Sila). In addition to the mentioned seeds and oils, an<br />average production sample was prepared, which is a<br />sample obtained from all collected seeds. Unrefined and<br />refined grape seed oil purchased on the market was used<br />as comparative samples. For realization of the set goal, the work on the<br />performance of this dissertation encompassed different<br />phases. In the first phase, grape seed were collected and<br />their technical and technological characteristics and quality<br />were examined. Then cold-pressed oils were produced<br />from these seeds.<br />In the following phases, the resulting ear, together with a<br />sample of unrefined and refined grape seed oil from the<br />market, were examined from the aspect of sensory and<br />nutritional quality and differences in oil stability were<br />observed. Sensory analysis was performed, pigmentate<br />fractions, carotenoids and chlorophylls were determined,<br />transparency was measured, and oil color parameters<br />were also determined instrumentally. The nutritive oil<br />quality was examined based on the content and<br />composition of bioactive components, primarily tocopherols<br />and tocotrienols, phenols and sterols. Since these<br />compounds exhibit significant antioxidant activities, the<br />antiradical activity of the samples was also tested. The<br />stability of grape seed oils was examined based on the<br />initial quality and the oxidative state, as well as the results<br />of accelerated tests, such as Rancimat test, Schaal-oven<br />and fluorescence test.<br />On the basis of the obtained results, it can be concluded<br />that all the obtained seed samples had good storage<br />moisture, which, given the small content of oil in the seeds,<br />can ensure the storage of seeds for a longer period. The<br />content of oil in the seed, in addition to the influence of the<br />grape variety, depends on the influence of the process to<br />which the seeds are subjected to pressing (fermentation,<br />distillation). Sensory analysis grape seed oils showed significant<br />differences in characteristics that are due not only to the<br />method of obtaining oil (cold pressed or refined), grape<br />varieties, but also the method of obtaining, the origin and<br />quality of grape seed. The conditions for grape seeds<br />exposed during fermentation and especially distillation<br />affect the formation of a specific cold pressed oil. Although<br />an extremely wide range of different flavors, odors and<br />flavors in oil appear, and there are significant differences in<br />the aroma of the oil, depending on the grape variety, it is<br />possible to recognize whether the cold-pressed oil is<br />derived from the grape seed without fermentation, after<br />fermentation or after distillation. Also, from the aspect of oil<br />color it can be said that refined grape seed oil has a light<br />yellow color with a greenish shade, while cold pressed<br />grape seed oils have more intense colors range from<br />yellowish-greenish, over greenish yellow and light greenish<br />to dark green. A higher proportion of green color has cold<br />pressed grape seed oils obtained after distillation.<br />The thesis has shown that the content of pigments<br />(carotenoids and chlorophyll) depends to a great extent on<br />the origin of the seed. The fermentation process<br />contributes to increasing the content of pigments, while the<br />distillation process has a negative effect due to high<br />temperatures.<br />Transparency of cold pressed grape seed oils produced<br />for the dissertation ranged from 32.8% to 53.8%.<br />The content of unsaturated fatty acids in all samples of<br />grape seed oil is greater than 90%, with the dominant fatty<br />acid being linoleic, omega-6, fatty acid. The content of tocotrienols is higher than the content of<br />tocopherols in grape seed oils, and the dominant<br />tocopherol is alpha-tocopherol. Its content is higher in oil<br />samples obtained from the seed after fermentation and<br />distillation.<br />The content of phenol compounds, depending on the<br />variety, is somewhat higher in cold pressed oils obtained<br />from red grape varieties, but there is also a significant<br />increase in accumulation of phenol in cold pressed oils<br />obtained from the grape seed after the fermentation and<br />distillation process. The largest individual oil content of<br />grape seeds has ursolic acid, and besides it, significant<br />content, although much smaller, shows rezveratrol,<br />kemferol and vanillinic acid.<br />The most prevalent sterols of grape seed oils are &beta;-<br />sitosterol with a share of 62.59-69.74%, stigmasterol with a<br />share of 12.13-15.00% and campesterol with a share of<br />6.59-11.94% in total sterols. Fermentation and distilation<br />processes, to which the seeds have been exposed, have<br />no effect on the content of phytosterols in grape seed oils.<br />The paper has shown the negative influence of the<br />fermentation and distillation process, to which the seeds<br />have been exposed, onto the antiradical potential of the<br />obtained oils. The antiradical potential of grape seed oils<br />depends on grape varietes, as well as on the way oil is<br />obtained (cold pressed or refined).<br />Processes of fermentation and distillation also affect the<br />basic chemical quality of obtained cold pressed oils in such<br />a way as to increase the acid and peroxide values.<br />Cold pressed grape seed oils show good oxidative stability. The Rancimat test showed differences in the<br />induction period of cold pressed grape seed oils as a result<br />of the variation in the variety, as well as in the method of<br />obtaining the seed. The fermentation process affects the<br />increase, and the process of distillation decreases the<br />induction period.<br />Based on all the obtained results, it can be concluded<br />that cold pressed grape seed oils showed different<br />nutritional quality and oxidative stability due to specific<br />differences resulting from differences in variety and seed<br />origin, i.e. due to the specific effect of fermentation and<br />distillation processes on the seeds from which the oil was<br />produced, thus confirming the hypothesis.</p>

Les régions de Midi-Pyrénées (France) et de Chongqing (Chine) sont riches en plantes aromatiques et médicinales dites oubliées (ou médiévales). Afin de valoriser pleinement les différentes bio-molécules extractibles de ces plantes, le concept de MAP-raffinerie a été créé et appliqué à une sélection de plantes issues de ces deux régions. Plusieurs technologies d’extraction utilisant l’eau comme solvant vert (hydrodistillation, distillation à la vapeur et extraction par eau sub-critique) ont ainsi été employées et leur impact tant sur la composition des huiles essentielles que sur la récupération des molécules anti-oxydantes a été évalué. Dans un premier temps, une liste de plantes aromatiques et médicinales oubliées, voire sous-utilisées dans les deux régions a été établie selon des règles de sélection prédéfinies. Six plantes modèles de la région de Midi-Pyrénées (Tussilago farfara L., Calendula arvensis L., Robinia pseudoacacia L., Geranium robertianum L., Cytisus scoparius L. et Spartium junceum L.) et trois plantes de la région de Chongqing (Tussilago farfara L., Citrus aurantium L. et Saussurea costus) ont finalement été retenues. Puis, le concept de MAP-raffinerie a été appliqué à ces plantes afin d’étudier leur possible valorisation globale. L’étude des compositions chimiques des extraits volatils des racines de Tussilago farfara L. et de Calendula arvensis L., ainsi que des boutons de fleurs de Spartium junceum L. a été réalisée par GC et GC-MS pour la première fois. Les principaux composés chimiques dans l’extrait volatil de racines de Tussilago farfara L. étaient des hydrocarbures sesquiterpéniques et des composés aliphatiques tandis que les principaux composés chimiques dans l’extrait volatil de racines de Calendula arvensis L. étaient des sesquiterpènes oxygénés, des monoterpènes oxygénés et des diterpènes oxygénés. L’extrait volatil de boutons de fleurs de Spartium junceum L. était principalement composé de composés aliphatiques. Par ailleurs, les résultats de l’évaluation des capacités anti-oxydantes des extraits (par les tests DPPH, ABTS, FRAP, ORAC et Folin-Ciocalteu) ont montrés que plusieurs plantes comme Cytisus scoparius L., Tussilago farfara L., Citrus aurantium L. ou Robinia pseudoacacia L. pourraient être des sources potentielles d’anti-oxydants naturels. D’un point de vue technologique, les comparaisons de l’utilisation de l’hydrodistillation (HD), de la distillation à la vapeur (SD) et de l’extraction par eau sub-critique (SWE) ont montrées que si la HD et la SD ont des effets limités sur la composition des huiles essentielles, la HD semble être une méthode plus efficace pour la récupération des composés anti-oxydants à partir des résidus de distillation que la SD tandis que la SWE s’avère être une technologie prometteuse pour l’extraction directe de ces molécules à partir des plantes. Si la composition minérale de l’eau lors de l’hydrodistillation n’a que des effets très limités sur les rendements d’extraction, les teneurs en ions calcium et bicarbonate des eaux ont par contre des effets décroissants significatifs sur la capacité anti-oxydante et sur la teneur phénolique totale des extraits aqueux et méthanoliques. Au vue de ces résultats, un concept amélioré de MAP-raffinerie a été développé en intégrant une extraction à l’eau sub-critique pour l’extraction des composés anti-oxydants des résidus d’extraction primaire. Selon ce nouveau concept, cinq extraits peuvent être obtenus à partir des matières végétales: un extrait volatil, un extrait aqueux, un extrait méthanolique, un extrait à l’eau sub-critique et in fine un résidu solide. Les premiers résultats ont montrés que la "MAP-raffinerie améliorée" augmente de manière significative la récupération des antioxydants par rapport à la MAP-raffinerie originale et permet d’envisager une valorisation plus facile du résidu solide en agro-matériaux du fait de sa faible teneur en eau résiduelle. / In both Midi-Pyrénées region (France) and Chongqing region (China), there are rich and underutilized medicinal and aromatic plants (MAP). Aiming at fully exploiting different molecules in these plants, the concept of MAP-refinery was developed and applied to several underutilized medicinal and aromatic plants in these two regions. Several water-based green extraction technologies of natural products (e.g. hydrodistillation, steam distillation and subcritical water extraction) were also investigated to look at their effects on essential oil composition and antioxidants recovery from selected plants. Firstly, lists of forgotten or underutilized medicinal and aromatic plants in both regions were established according to the rules of selection. From the lists, six plants in the Midi-Pyrénées region (Tussilago farfara L., Calendula arvensis L., Robinia pseudoacacia L., Geranium robertianum L., Cytisus scoparius L. and Spartium junceum L.) and three plants in the Chongqing region (Tussilago farfara L., Citrum aurantium L. and Saussurea costus) were finally selected for investigations. Then the MAP-refinery was applied to the selected plants in two regions in order to realise their global valorisation. Volatile extracts composition in the roots of Tussilago farfara L. and Calendula arvensis L., as well as flower buds of Spartium junceum L. were firstly investigated. The main chemical compounds in volatile extract from Tussilago farfara L. roots were sesquiterpene hydrocarbons and aliphatic compounds while main chemical compounds in volatile extract from Calendula arvensis L. roots were oxygenated sesquiterpenes, oxygenated monoterpenes and oxygenated diterpenes. The volatile extract from flower buds of Spartium junceum L. was mainly composed of aliphatic compounds. Antioxidant capacity evaluation results (by DPPH, ABTS, FRAC, ORAC and Folin-Ciocalteu tests) showed that several plant samples like Cytisus scoparius L., Tussilago farfara L., Citrum aurantium L. and Robinia pseudoacacia L. could be potential sources of natural antioxidants. Comparisons of hydrodistillation (HD), steam distillation (SD) and subcritical water extraction (SWE) showed that HD and SD had limited effects on essential oil composition but HD, SD and SWE had significant impacts on the recovery of antioxidants. Hydrodistillation seemed to be a better method for recovery of antioxidant compounds from residues of distillation than steam distillation. However, SWE appeared to be a more efficient method for direct extraction of antioxidant molecules (or phenolic compounds) from plants. In the hydrodistillation process, mineral contents in water were found to have very limited effects on yields of extracts but calcium and bicarbonate ions, had significant decreasing effects on antioxidant capacity and total phenolic content of both aqueous and methanolic extracts. Finally, an improved MAP-refinery was developed. Subcritical water was used for further extraction of antioxidant compounds from residues in original MAP-refinery. In this way, five parts could be obtained from plant materials: volatile extract, aqueous extract, methanolic extract, subcritical water extract and the final residue. The results showed that the improved MAP-refinery significantly increased the recovery of antioxidants compared with original MAP-refinery. This promising process will also allow a better valorisation of the final solid residue due to the lower content of residual water.