A oleuropeína é o mais abundante biofenol presente nas folhas de oliveira (Olea Europaea), com importantes funções antimicrobiana e antioxidante. Estudos visando à obtenção deste composto têm sido conduzidos, porém, muitos deles utilizam solventes tóxicos e métodos caros. A presente dissertação teve por objetivo estudar diferentes metodologias para a obtenção de extratos de folhas de oliva contendo quantidades significativas de oleuropeína. Os extratos foram obtidos a partir de folhas de oliva micronizadas, com ou sem pré-tratamento para redução do teor de clorofila, submetidas a contatos simples ou múltiplos com diferentes solventes, como dietil éter, clorofórmio, acetona, etanol, 1-propanol, 2-propanol, água e soluções hidroalcoólicas com diferentes concentrações. O contato das folhas micronizadas com os solventes foi promovido pelos seguintes métodos: agitação manual em temperatura ambiente, agitação mecânica a 50 ºC, ultrassom ou uma combinação desses dois últimos, totalizando 38 experimentos, sendo que em 17 destes os extratos foram produzidos na forma líquida e 21 na forma sólida. Os resultados mostraram que, de maneira geral, a etapa prévia de redução do teor da clorofila (realizada através de sucessivos contatos com hexano, diclorometano ou ainda, com CO2 supercrítico) não foi vantajosa, devido à elevada quantidade de solventes utilizados em relação às quantidades de extratos obtidos. Nestes experimentos, a maior concentração de oleuropeína, 1,88%, foi detectada no procedimento em que as folhas micronizadas foram previamente umedecidas com etanol e limpas com CO2 supercrítico, e posteriormente colocadas em contato com a mistura etanol e água, na proporção 1:1, utilizando o ultrassom combinado com a agitação mecânica como método de extração. Nos experimentos finais do trabalho, foi estudada ainda a adição de ácidos orgânicos (cítrico ou acético), juntamente com os solventes hidroalcoólicos (diferentes teores de água) no momento da extração da oleuropeína em banho de ultrassom, utilizando amostras que não foram previamente tratadas para remoção da clorofila. A combinação do ácido acético com a solução etanólica contendo 30 % de água resultou em um extrato com 2,17 % de oleuropeína, em apenas 1 contato com o solvente. Quando três contatos foram utilizados, nestas mesmas condições, o teor de oleuropeína aumentou para 4,8 %, maior do que alguns valores encontrados na literatura, utilizando o mesmo método de extração, indicando que processo ainda pode ser otimizado, utilizando técnicas simples e solventes que não agridam o meio ambiente. / Oleuropein is the most abundant biofenol present in olive leaves (Olea europaea), presenting important antioxidant and antimicrobial functions. Studies focusing on obtaining this compound have been conducted; however, many of them use toxic solvents and expensive methods. The present work aimed to study different methodologies for obtaining extracts from olive leaves containing significant amounts of oleuropein. The extracts were obtained from micronized olive leaves, with or without pretreatment for reducing the chlorophyll content, submitted to single and multiple contacts with different solvents such as diethyl ether, chloroform, acetone, ethanol, 1- propanol, 2 -propanol, water and hydroalcoholic solutions at different concentrations. The contact between the micronized leaves and the solvents was promoted by the following methods: manual shaking at room temperature, mechanical agitation at 50 °C, ultrasound or a combination of these last two methods, totaling 38 experiments, from which in 17 of them the extracts were produced in the liquid form, and in 21 in the solid form (as a powder). The results show that, in general, previous step of reducing the content of chlorophyll (performed by successive contacts with hexane, dichloromethane or with supercritical CO2) was not advantageous due to the high amount of solvent used in relation to amounts of extracts. In these experiments, the highest concentration of oleuropein, 1.88 % was detected in the procedure in which the micronized leaves were previously soaked with ethanol and cleaned with supercritical CO2 and then placed in contact with the mixture of ethanol and water in the proportion 1:1, using ultrasound combined with mechanical agitation as extraction method. In the last experiments of the work, the addition of organic acids (citric or acetic acid) together with hydroalcoholic solvents (different water contents) in the extraction of oleuropein in ultrasound was studied, using samples that have not previously been treated for removal of chlorophyll. The combination of acetic acid to the ethanolic solution containing 30 % of water resulted in an extract with 2.17 % of oleuropein, with only one contact to the solvent. When three contacts were used, under the same conditions, the oleuropein content increased to 4.8 %, larger than some values found in the literature, using the same extraction method, indicating that the process can be further optimized using simple techniques and solvents that do not harm the environment.

Programa de Apoio aos Planos de Reestruturação e Expansão das Universidades Federais / Olive leaves are an agricultural by-product generated by the pruning of trees. Recently, it was reported that these leaves have high levels of phenolic compounds with biological activity, which increase the interest of both academic and economic exploitation in relation to the their use for feed and food. However, few information is available regarding to the presence of other nutritional compounds, such as fatty acids and mineral elements. Thus, this study aimed to determine the fatty acids profile and also different elements present in different varieties of olive leaves grown in southern Brazil, as well as develop a procedure for extraction of phenolic compounds with the aid of ultrasound, using a low-toxicity solvent (ethanol 60 % v/v, with 1 g L-1citric acid), and compare it with the traditional extraction method of extraction (maceration) to its future application in food products. For varieties studied, Ascolano, Arbosana, Negrinha do Freixó, Koroneiki and Grappolo concentrations of ash, protein, lipid and total carbohydrates levels ranged from 4.37% to 6.00%, 10.50% to 13.10%, 9 13% to 9.80% and 8.74% to 32.63%, respectively. The variety Arbosana showed the highest concentration of phenolic compounds when was carried out one extraction followed by re-extraction by maceration (35.71 mg GAE g-1), and the highest concentration of saturated fatty acids (total of 37.26%, composed by 1.54 ± 0.04% myristic acid, 26.90 ± 0.50% palmitic acid, 5.55 ± 0.14% stearic acid and 3.26 ± 0.13% arachidic acid). The varieties Ascolano, Koroneiki and Grappolo had the highest amounts of fatty acids considered beneficial to health (68.03%, 68.63% and 68.18% respectively, data for the sum of fatty acids oleic, linoleic and linolenic). The Ascolano variety showed generally higher concentrations of most minerals determined. The elements present in highest concentration in the five varieties studied were Al, Ca, Fe, K, Mg, P and S, but the levels found for Fe, Cu, Zn, Mn, and Ca were more significant in relation to the recommended daily intake. All these results show the importance of the formation of these varieties which can be used as supplements in the feed. In the extraction of phenolics from Arbequina variety assisted by ultrasound were optimized the position of the probe (1 and 3 cm), the extraction temperature (20 °C, 40 °C and 60 °C) and the extraction time (0.5 - 20 min), using 40% amplitude and 20 kHz frequency. The results showed that the use of 20 °C during 20 min for extraction lead to 75.33% of recovery of phenolic compound (20.50 ± 0.26 mg GAE g-1) when compared with the conventional method of extraction (maceration, 22 °C, 5 h, 27.32 ± 0.90 mg GAE g-1). The position of the probe did not significantly affect the results and the main effect caused by the ultrasound was stirring. Thus, we developed a fast and effective method of extraction, confirming the benefits of using ultrasound to obtain extracts from natural sources. / As folhas de oliveira são um subproduto agrícola gerado pela poda de oliveiras. Recentemente, foi relatado que essas folhas apresentam elevados teores de compostos fenólicos com atividade biológica, o que despertou o interesse tanto acadêmico quanto econômico com relação ao aproveitamento das mesmas para a alimentação animal e humana. Contudo, há pouca informação sobre a presença de outros compostos de interesse nutricional, tais como ácidos graxos e elementos minerais. Assim, este trabalho teve como objetivo determinar o perfil de ácidos graxos e elementos presentes em diferentes variedades de folhas de oliveira cultivadas no sul do Brasil, assim como desenvolver um procedimento de extração de compostos fenólicos com auxílio do ultrassom, utilizando um solvente de baixa toxicidade (etanol 60% v/v, adicionado de ácido cítrico 1 g L-1), e compará-lo com a extração por metodologia tradicional de extração (maceração), visando sua futura aplicação em produtos alimentícios. Para as variedades estudadas, Ascolano, Arbosana, Negrinha do Freixó, Koroneiki e Grappolo as concentrações de cinzas, proteínas, lipídios e carboidratos totais variaram de 4,37% a 6,00%; 10,50% a 13,10%, 9,13% a 9,80% e 8,74% a 32,63%, respectivamente. A variedade Arbosana apresentou a maior concentração de compostos fenólicos totais quando se realizou uma extração seguida de re-extração por maceração (35,71 mg GAE g-1), e a maior concentração de ácidos graxos saturados (total de 37,26%, sendo 1,54 ± 0,04% ácido mirístico; 26,90 ± 0,50% ácido palmítico; 5,55 ± 0,14% ácido esteárico e 3,26 ± 0,13% ácido araquídico). As variedades Ascolano, Koroneiki e Grappolo apresentaram as maiores quantidades dos ácidos graxos considerados benéficos à saúde (68,03%; 68,63% e 68,18% respectivamente, dados relativos ao somatório dos ácidos graxos oleico, linoleico e linolênico). A variedade Ascolano apresentou de modo geral as maiores concentrações da maioria dos minerais determinados. Os elementos presentes em maior concentração nas cinco variedades estudadas foram Al, Ca, Fe, K, Mg, P e S, mas os teores encontrados para Fe, Cu, Zn Mn e Ca foram mais significativos com relação à ingestão diária recomendada. Estes resultados demonstram a importância da constituição destas variedades que podem ser utilizadas como suplementos na alimentação animal ou humana. Na extração dos compostos fenólicos assistida por ultrassom utilizando a variedade Arbequina foram otimizados a posição da sonda (1 e 3 cm), a temperatura de extração (20 °C, 40 °C e 60 °C) e o tempo de extração (0,5 - 20 min) utilizando 40% de amplitude e 20 kHz. Os resultados indicaram que a utilização de 20 °C, durante 20 min de extração levaram a uma recuperação de 75,33% dos compostos fenólicos (20,50 ± 0,26 mg GAE g-1), quando comparado com o método convencional de extração (maceração, 22 °C, 5 h , 27,32 ± 0,90 mg GAE g-1). A posição da sonda não interferiu significativamente nos resultados e o principal efeito provocado pelo ultrassom foi agitação. Assim, desenvolveu-se um método rápido e eficaz de extração, confirmando os benefícios da utilização de ultrassom na obtenção de extratos a partir de fontes naturais.

A oleuropeína é o mais abundante biofenol presente nas folhas de oliveira (Olea Europaea), com importantes funções antimicrobiana e antioxidante. Estudos visando à obtenção deste composto têm sido conduzidos, porém, muitos deles utilizam solventes tóxicos e métodos caros. A presente dissertação teve por objetivo estudar diferentes metodologias para a obtenção de extratos de folhas de oliva contendo quantidades significativas de oleuropeína. Os extratos foram obtidos a partir de folhas de oliva micronizadas, com ou sem pré-tratamento para redução do teor de clorofila, submetidas a contatos simples ou múltiplos com diferentes solventes, como dietil éter, clorofórmio, acetona, etanol, 1-propanol, 2-propanol, água e soluções hidroalcoólicas com diferentes concentrações. O contato das folhas micronizadas com os solventes foi promovido pelos seguintes métodos: agitação manual em temperatura ambiente, agitação mecânica a 50 ºC, ultrassom ou uma combinação desses dois últimos, totalizando 38 experimentos, sendo que em 17 destes os extratos foram produzidos na forma líquida e 21 na forma sólida. Os resultados mostraram que, de maneira geral, a etapa prévia de redução do teor da clorofila (realizada através de sucessivos contatos com hexano, diclorometano ou ainda, com CO2 supercrítico) não foi vantajosa, devido à elevada quantidade de solventes utilizados em relação às quantidades de extratos obtidos. Nestes experimentos, a maior concentração de oleuropeína, 1,88%, foi detectada no procedimento em que as folhas micronizadas foram previamente umedecidas com etanol e limpas com CO2 supercrítico, e posteriormente colocadas em contato com a mistura etanol e água, na proporção 1:1, utilizando o ultrassom combinado com a agitação mecânica como método de extração. Nos experimentos finais do trabalho, foi estudada ainda a adição de ácidos orgânicos (cítrico ou acético), juntamente com os solventes hidroalcoólicos (diferentes teores de água) no momento da extração da oleuropeína em banho de ultrassom, utilizando amostras que não foram previamente tratadas para remoção da clorofila. A combinação do ácido acético com a solução etanólica contendo 30 % de água resultou em um extrato com 2,17 % de oleuropeína, em apenas 1 contato com o solvente. Quando três contatos foram utilizados, nestas mesmas condições, o teor de oleuropeína aumentou para 4,8 %, maior do que alguns valores encontrados na literatura, utilizando o mesmo método de extração, indicando que processo ainda pode ser otimizado, utilizando técnicas simples e solventes que não agridam o meio ambiente. / Oleuropein is the most abundant biofenol present in olive leaves (Olea europaea), presenting important antioxidant and antimicrobial functions. Studies focusing on obtaining this compound have been conducted; however, many of them use toxic solvents and expensive methods. The present work aimed to study different methodologies for obtaining extracts from olive leaves containing significant amounts of oleuropein. The extracts were obtained from micronized olive leaves, with or without pretreatment for reducing the chlorophyll content, submitted to single and multiple contacts with different solvents such as diethyl ether, chloroform, acetone, ethanol, 1- propanol, 2 -propanol, water and hydroalcoholic solutions at different concentrations. The contact between the micronized leaves and the solvents was promoted by the following methods: manual shaking at room temperature, mechanical agitation at 50 °C, ultrasound or a combination of these last two methods, totaling 38 experiments, from which in 17 of them the extracts were produced in the liquid form, and in 21 in the solid form (as a powder). The results show that, in general, previous step of reducing the content of chlorophyll (performed by successive contacts with hexane, dichloromethane or with supercritical CO2) was not advantageous due to the high amount of solvent used in relation to amounts of extracts. In these experiments, the highest concentration of oleuropein, 1.88 % was detected in the procedure in which the micronized leaves were previously soaked with ethanol and cleaned with supercritical CO2 and then placed in contact with the mixture of ethanol and water in the proportion 1:1, using ultrasound combined with mechanical agitation as extraction method. In the last experiments of the work, the addition of organic acids (citric or acetic acid) together with hydroalcoholic solvents (different water contents) in the extraction of oleuropein in ultrasound was studied, using samples that have not previously been treated for removal of chlorophyll. The combination of acetic acid to the ethanolic solution containing 30 % of water resulted in an extract with 2.17 % of oleuropein, with only one contact to the solvent. When three contacts were used, under the same conditions, the oleuropein content increased to 4.8 %, larger than some values found in the literature, using the same extraction method, indicating that the process can be further optimized using simple techniques and solvents that do not harm the environment.

Tesis por compendio / [EN] The main goal of this Thesis was to determine the influence of the main processing stages involved in obtaining natural extracts with high antioxidant potential from byproducts originating in the olive oil industry. Firstly, the effect of freezing and/or the drying methods applied to olive oil byproducts on the polyphenol content and antioxidant capacity of the extracts subsequently obtained was addressed. For this purpose, two byproducts were considered: olive leaves and olive pomace. Secondly, the feasibility of intensifying the extraction of olive leaf polyphenols by means of a new technology, such as power ultrasound, was approached taking both compositional and kinetic issues into account. Thirdly, how the processing conditions (drying and extraction) influence the extract's stability was evaluated. Thus, on the one hand, extracts obtained from olive leaves were subjected to in vitro digestion or dehydrated and stored at different conditions. Finally, the possibility of obtaining a dried vegetable matrix (apple) rich in olive leaf phenolic compounds was explored by addressing the influence of apple pretreatments (blanching and freezing) and drying on the final retention of infused phenolics. The antioxidant potential of extracts and the retention of infused polyphenols in apple were evaluated by means of the total phenolic content and antioxidant capacity analysis, as well as the identification and quantification of the main olive leaf polyphenols by HPLC-DAD/MS-MS. Moreover, in apple samples, the polyphenol oxidase and peroxidase activity and microstructure were also analyzed. The experimental results highlighted that both drying and freezing methods significantly (p<0.05) influenced the concentration of the main polyphenols identified in the olive leaf extracts. Thus, drying at the highest temperature tested was the best processing condition in which to obtain extracts with high antioxidant capacity and phenolic content. Ultrasound application was found to be a relevant, non-thermal way of speeding-up the antioxidant extraction from olive leaves. Thus, by appropriately tuning-up the process variables, the ultrasonic assisted extraction shortened the extraction time from the 24 h needed in conventional extraction to 15 min, without modifying either the extract composition or the antioxidant potential. As far as extract stability is concerned, the processing conditions used for obtaining the olive leaf extracts did not have a meaningful influence on bioaccessibility. Regardless of the method used, stabilizing the extracts by means of dehydration only reduced both the antioxidant capacity and the total phenolic content by around 10 %. Moreover, storage conditions did not show a significant (p<0.05) effect on the antioxidant potential of the extracts for 28 days of storage. A stable dried product (apple), rich in natural phenolic compounds (from olive leaves or tea extracts), was obtained by combining drying-impregnation-drying steps. However, it should be considered that the role of fresh apple drying on the retention of infused olive leaf polyphenols was more important than the further drying of the impregnated apple. In overall terms, olive leaves can be considered a potential source of natural phenolic compounds. Notwithstanding this, the previous drying and freezing steps applied in the raw material processing are decisive factors in the obtaining of natural extracts with high antioxidant potential. Moreover, enhancing the extraction by applying power ultrasound was stated as a non-thermal way of shortening processing times. The stability of olive polyphenols during storage and in vitro digestion was closely related to the individual component considered. Finally, the exploitation of olive leaf extracts as a means of enriching solid foodstuffs requires the use of porous solid matrices free of oxidative enzymes. / [ES] El objetivo principal de esta Tesis fue determinar la influencia de las principales etapas de procesado implicadas en la obtención de extractos naturales con alto potencial antioxidante a partir de los subproductos originados en la industria del aceite de oliva. En primer lugar, se evaluó el efecto de los métodos de congelación y/o secado de la materia prima (hojas y orujo), sobre el contenido polifénolico y la capacidad antioxidante de los extractos. En segundo lugar, se abordó la intensificación de la extracción de polifenoles de hoja de olivo con ultrasonidos de potencia, teniendo en cuenta: composición y la cinética del proceso. A continuación, se estudió cómo las condiciones de procesado (secado y extracción) podían influir en la estabilidad de los extractos. Así, extractos de hojas de olivo fueron sometidos a digestión in vitro o deshidratados y almacenados a distintas condiciones. Por último, se exploró la posibilidad de obtener una matriz vegetal deshidratada (manzana) y rica en compuestos fenólicos de hoja de olivo. Para ello, se evaluó la influencia de los pretratamientos de la manzana (escaldado y congelación) y del secado en la retención final de los polifenoles impregnados. El potencial antioxidante se determinó a través del contenido total en compuestos fenólicos y la capacidad antioxidante y la identificación y cuantificación (HPLC-DAD/MS-MS) de los principales polifenoles. Además, en manzana, se midió la actividad enzimática de la polifenol oxidasa y peroxidasa y se analizó la microestructura. Los resultados manifestaron que el método de secado y el de congelación influyeron significativamente (p<0.05) en la concentración de los principales polifenoles en los extractos. Así, el secado a mayor temperatura resultó ser el mejor tratamiento para obtener extractos con alta capacidad antioxidante y alto contenido fenólico. La aplicación de ultrasonidos resultó ser una alternativa no térmica muy interesante para acelerar la extracción de antioxidantes de hojas de olivo. Con la combinación adecuada de las variables del proceso, la aplicación de ultrasonidos redujo el tiempo de extracción de 24 h necesarias en extracción convencional a 15 min, sin modificar la composición de los extractos y su potencial antioxidante. En cuanto a la estabilidad del extracto, las condiciones de procesado no tuvieron una influencia significativa en la bioaccesibilidad de los extractos. Independientemente del método utilizado, la estabilización de extractos por deshidratación sólo redujo la capacidad antioxidante y el contenido total en compuestos fenólicos en torno a un 10 %. Además, las condiciones de almacenamiento no mostraron ningún efecto significativo (p<0.05) sobre el potencial antioxidante durante los 28 días de almacenamiento. Combinando secado-impregnación-secado, fue posible desarrollar un producto deshidratado (manzana), estable y rico en compuestos fenólicos naturales (de hojas de olivo o extractos de té). No obstante, cabe destacar que el secado de la manzana fresca jugó un papel más importante en la retención de los polifenoles de hoja de olivo infundidos que el secado final de la manzana impregnada. En términos generales, las hojas de olivo pueden considerarse como una fuente potencial de compuestos fenólicos naturales. No obstante, el secado y la congelación durante el procesado de la materia prima son factores decisivos para la obtención de extractos naturales con alto potencial antioxidante. Además, la aplicación de ultrasonidos de potencia durante la extracción puede resultar una alternativa no térmica muy interesante de cara a acortar el tiempo de procesado. La estabilidad de los polifenoles de la hoja de olivo, durante el almacenamiento y la digestión in vitro, dependió claramente del compuesto individual considerado. Finalmente, el empleo del extracto de hoja de olivo como medio para enriquecer alimentos sólidos requiere del uso de matrices s / [CA] L'objectiu principal d'aquesta tesi va ser determinar la influència de les principals etapes de processament implicades en l'obtenció d'extractes naturals amb alt potencial antioxidant procedents de subproductes de la indústria de l'oli d'oliva. En primer lloc, es va estudiar l'efecte de la congelació i/o els mètodes d'assecatge aplicats a fulles d'olivera i pinyolada sobre el contingut fenòlic i la capacitat antioxidant dels extractes. En segon lloc, es va avaluar, tenint en compte la composició i la cinètica del procés, la intensificació de l'extracció de polifenols de fulla d'olivera amb ultrasons de potència. En tercer lloc, es va avaluar com les condicions de processament (assecatge i extracció) poden influir en l'estabilitat dels extractes. Així, extractes de fulles d'olivera van ser sotmesos a una digestió in vitro o deshidratats i emmagatzemats a distintes condicions. Finalment, es va explorar la obtenció d'una matriu vegetal deshidratada (poma) i rica en compostos fenòlics de fulla d'olivera considerant la influència del pretractament de la poma (escaldament i congelació) i de l'assecatge sobre la retenció final dels fenòlics introduïts en la poma. El potencial antioxidant es va avaluar determinant el contingut fenòlic total i la capacitat antioxidant, així com identificant i quantificant els principals polifenols (HPLC-DAD/MS-MS). A més, en poma l'activitat enzimàtica de la polifenoloxidasa i la peroxidasa i la microestructura. Els resultats experimentals van destacar que el mètode d'assecatge i el de congelació van influir significativament (p<0,05) en la concentració dels principals polifenols identificats en els extractes. L'assecatge a la temperatura més alta que es va provar va resultar la millor condició de processament per a obtenir extractes amb una alta capacitat antioxidant i un alt contingut fenòlic. L'aplicació d'ultrasons va ser una manera rellevant i no tèrmica d'accelerar l'extracció d'antioxidants de les fulles d'olivera. Així, amb la combinació adequada de les variables del procés, l'extracció assistida per ultrasons va escurçar el temps d'extracció, de les 24 h requerides en l'extracció convencional a 15 min, sense modificar la composició de l'extracte ni el potencial antioxidant. Quant a l'estabilitat de l'extracte, les condicions de processament utilitzades per a l'obtenció dels extractes de fulla d'olivera no van tenir una influència significativa en la bioaccessibilitat. Independentment del mètode utilitzat, l'estabilització dels extractes per mitjà de la deshidratació només va reduir la capacitat antioxidant i el contingut fenòlic total al voltant d'un 10 %. A més, les condicions d'emmagatzematge (temperatura i forma de l'extracte: líquid o pols) no van mostrar cap efecte significatiu (p<0,05) en el potencial antioxidant dels extractes durant els 28 dies d'emmagatzematge. Combinant etapes d'assecatge-impregnació-assecatge fou possible obtenir un producte assecat estable (poma) i ric en compostos fenòlics naturals (de fulles d'olivera o te). No obstant això, cal destacar que l'assecatge de la poma fresca va ser més important i determinant en la retenció dels polifenols de fulla d'olivera que no l'assecatge de la poma impregnada. En termes generals, les fulles d'olivera es poden considerar com una font potencial de compostos fenòlics naturals. No obstant això, l'aplicació d'assecatge i congelació durant el processament de la matèria primera són factors decisius per a l'obtenció d'extractes naturals amb un alt potencial antioxidant. A més, l'aplicació d'ultrasons de potència durant l'extracció resultà ser una forma no tèrmica de millorar el procés, tot reduint-ne el temps d'extracció. L'estabilitat dels polifenols d'olivera durant l'emmagatzematge i la digestió in vitro va dependre del compost individual considerat. Finalment, la utilització d'extractes de fulla d'olivera per a desenvolupar aliments sòlids enriquits requ / Ahmad-Qasem Mateo, MH. (2015). ASSESSMENT OF THE INFLUENCE OF PROCESSING CONDITIONS ON THE ANTIOXIDANT POTENTIAL OF EXTRACTS OBTAINED FROM OLIVE OIL INDUSTRY BYPRODUCTS [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/53452 / Premios Extraordinarios de tesis doctorales / Compendio

The olive oil extraction industry represents a substantial share of the economies of Mediterranean countries but leads to serious environmental problems by producing huge amounts of wastes (by-products) within a short production period. The production rate of olive oil is about 1.4-1.8 million tonnes per year in the Mediterranean, resulting in 30 million m3 of by-products and 20 million tonnes of olive pomace. A small portion of these wastes can be used as raw materials in different industries as they contain valuable natural resources. Greece has about 2300 small-scale, rural, agro-industrial units that extract olive oil. These are generally three-phase systems and their by-products include olive mill residual solids (olive pomace and leaves) and olive mill waste water. Olive mills produce significant quantities of solid wastes with outputs of 0.35 tonnes of olive pomace and 0.05 tonnes of leaves per tonne of olives. The huge quantities of olive pomace and olive leaves produced within the short oil extraction season cause serious management problems in terms of volume and space. The solid wastes (olive pomace and olive leaves) that are produced contain almost 95% organic matter and although they could be highly beneficial to agricultural soils, it has been shown that they also contain toxic compounds and lipid which increase soil hydrophobicity and decrease water retention and infiltration rate. The soils of most Mediterranean countries have low organic matter contents (<1%) which has negative impacts on agriculture. Frequent application of composted organic residues increases soil fertility, mainly by improving aggregate stability and decreasing soil bulk density. Organic amendments play a positive role in climate change abatement by soil carbon sequestration. Recurrent use of composted materials enhances soil organic nitrogen content by up to 90%. To replenish soil organic matter content and promote eco-friendly crop production, the application of olive pomace compost could be a good solution. To examine olive mill solid waste composting, four pilot-scale experiments were carried out to produce good quality compost using three phase olive mill solid waste (olive pomace, OP) and different bulking agents such as rice husk (RH), olive leaves (OL) sawdust (SD), wood shavings (WS), and chromium treated reed plants (RP). A series of parallel experiments was carried out to examine the effect final compost quality of: (a) initial moisture content, (b) water addition during the composting process, and (c) material ratios, and to also determine the toxicity level in plants and human blood lymphocytes (genotoxicity and cytotoxicity). For each experiment, six trapezoidal bins were used with dimensions 1.26 m long, 0.68 m wide and 0.73 m deep, and a total volume of 0.62 m3. The study was carried out in the facilities of the Department of Environmental and Natural Resources Management, University of Patras, Agrinio, in a closed area to maintain controlled temperature conditions. To monitor the composting process and evaluate compost quality, physicochemical parameters (temperature, moisture content, pH, electrical conductivity, organic matter, volatile solids, total organic carbon, total nitrogen, total phosphorus, potassium, sodium, and water soluble phenols) were measured at different phases. The respirometric test (O2 uptake) was performed to determine compost stability. Experimental results showed that even after short composting periods, the quality of the final product remained high. The final product had excellent physicochemical characteristics (C/N: 12.1–17.5, germination index (GI): 88.32–164.43%, Cr: 8–10 mg/kg dry mass, that fulfill1 EU requirements and can be used as a fertilizer in organic farming. To achieve higher quality of the final product, Olive pomace should be used in higher ratios than the other materials (OL, RH, WS, SD and RP). The amount (volume of humidifying agents) and time (frequency) of moisture addition also played an important role during composting. Based on the experimental results, olive mill wastes can produce a high quality soil amendment which has no phytotoxic, genotoxic or cytotoxic effects. Nevertheless, composting duration and bulking agents and their ratios are crucial factors that determine the quality of the final product. Finally, the revision of EU regulations is proposed to include genotoxic and cytotoxic evaluation of composts that enter the human food chain. A full-scale compost unit was designed based on the experimental results. For a typical small-sized olive mill, processing 30 tonnes of olives per day for a 100-day operation period, a total area of about 850 m2 is needed to compost the mill’s entire annual waste production. / Η βιομηχανία παραγωγής ελαιόλαδου αποτελεί ένα σημαντικό κομμάτι της οικονομίας στις χώρες της Μεσογείου, προκαλώντας ταυτόχρονα σημαντικά περιβαλλοντικά προβλήματα, λόγω της παραγωγής μεγάλων ποσοτήτων αποβλήτων κατά τη σύντομη περίοδο λειτουργίας των ελαιοτριβείων. Η μέση ετήσια παραγωγή ελαιολάδου στην Μεσόγειο κυμαίνεται στους 1.4-1.8 χιλιάδες τόνους, ενώ παράγονται επίσης περίπου 30 χιλιάδες m3 παραπροϊόντων και 20 χιλιάδες τόνους ελαιοπυρήνα. Μόνο ένα μικρό μέρος αυτών των παραπροϊόντων μπορεί να χρησιμοποιηθεί ως πρώτη ύλη σε διάφορες βιομηχανίες. Η Ελλάδα έχει περίπου 2300 ελαιοτριβεία μικρής κλίμακας διασπαρμένα στην ύπαιθρο. Τα ελαιοτριβεία αυτά είναι κυρίως τριφασικά και τα παραπροϊόντα τους συμπεριλαμβάνουν στερεά υπολείμματα (ελαιουρήνας και φύλλα) και υγρά απόβλητα ελαιοτριβείου. Τα ελαιοτριβεία παράγουν σημαντικές ποσότητες στερεών υπολειμμάτων παρέχοντας περίπου 0.35 τόνους ελαιοπυρήνα και 0.05 τόνους φύλλων ανά τόνο ελαιοκάρπου, παρακαλώντας σημαντικά προβλήματα στη διαχείρισης τους. Τα στερεά υπολείμματα (ελαιοπυρήνας και φύλλα) περιέχουν 95% οργανική ύλη, καθιστώντας τα δυνητικά κατάλληλα ως εδαφοβελτιωτικά, καθώς τα εδάφη των περισσότερων Μεσογειακών χωρών έχουν χαμηλή περιεκτικότητα σε οργανική ύλη (<1%) επηρεάζοντας αρνητικά την γεωργία. Τα υπολλείματα αυτά περιέχουν ωστόσο τοξικές ουσίες και έλαια, τα οποία αυξάνουν την υδροφοβικότητα του εδάφους και μειώνουν την κατακράτηση του νερού και την ρυθμό διήθησης. Έχει αποδειχθεί ότι συχνές εφαρμογές κομποστοποιημένων οργανικών υπολειμμάτων αυξάνουν την γονιμότητα του εδάφους, αυξάνοντας κυρίως τη συνολική σταθερότητα και την πυκνότητα του εδάφους. Η συχνή χρήση κομποστοποιημένων υλικών βελτιώνει την περιεκτικότητα των εδαφών σε οργανικό άζωτο του εδάφους έως και 90%. Η κομποστοποίηση ελαιοπυρήνα θα μπορούσε να αποτελέσει μια πιθανή λύση για την αναπλήρωση του περιεχομένου σε οργανική υλη των εδαφών και για την προώθηση μιας οίκοφιλικής αγροτικής παραγωγής. Για να εξεταστεί η κομποστοποιήση στερεών υπολειμμάτων ελαιοτριβείων, διεξήχθησαν 4 πειράματα πιλοτικής κλίμακας για την παραγωγή κομποστ, χρησιμοποιώντας στερεά υπολείμματα τριφασικών ελαιοτριβείων (ελαιοπυρήνας) και διαφόρους διογκωτικούς παράγοντες, όπως φλοιό ρυζιού, φύλλα ελιάς, πριονίδια, ροκανίδια, και καλάμια με υψηλή περιεκτικότητα σε χρώμιο. Σκοπός των παράλληλων πειραμάτων ήταν η εξέταση της επίδρασης στην ποιότητα του τελικού κομπόστ των: (α) αρχικού περιεχόμενου υγρασίας, (β) της προσθήκης νερού κατά την διάρκεια της κομποστοποιήσης, (γ) των ποσοστών ανάμιξης των υλικών, καθώς επίσης και ο προσδιορισμός της φυτοτοξικότητας και της γενοτοξικότητας των τελικών κομπόστ. Σε κάθε πείραμα χρησιμοποιήθηκαν 6 τραπεζοειδή πλαστικά δοχεία διαστάσεων 1.26 m σε μήκος, 0.68 m σε πλάτος και 0.73 m σε ύψος, με ολικό όγκο 0.62 m3. Οι πιλοτικές μονάδες ήταν τοποθετημένες σε κλειστό χώρο του Τμήματος Διαχείρισης Περιβάλλοντος και Φυσικών Πόρων του Πανεπιστημίου Πατρών στο Αγρίνιο, ώστε να επικρατούν σταθερές συνθήκες θερμοκρασίας. Η παρακολούθηση της κομποστοποίησης και η εκτίμηση της ποιότητας του κομπόστ, έγινε μέσω του προσδιορισμού διαφόρων φυσικοχημικών παραμέτρων (θερμοκρασία, περιεχόμενο υγρασίας, pH, ηλεκτρική αγωγιμότητα, περιεχόμενη οργανική ύλη, πτητικά στέρεα, ολικός οργανικός άνθρακας, ολικό άζωτο, ολικό φώσφορος, κάλιο, νάτριο, και ολικές φαινόλες). Για την εκτίμηση της ποιότητας του κομποστ πραγματοποιήθηκαν επίσης ρεσπιρομετρικά τεστ (κατανάλωση O2). Τα πειραματικά αποτελέσματα απέδειξαν ότι ακόμα και μετά από σύντομες περιόδους κομποστοποιήσης η ποιότητα του τελικού κομπόστ παρέμενε υψηλή. Το τελικό προϊόν είχε εξαιρετικά φυσικοχημικά χαρακτηριστικά (C/N: 12.1–17.5, δείκτης βλαστικότητας (GI): 88.32–164.43%, Cr: 8–10 mg/kg ξηρής μάζας), τα οποία είναι εντός των νομοθετικών ορίων της ΕΕ για την χρήση λιπασμάτων σε βιολογικές καλλιέργειες. Για την παραγωγή υψηλής ποιότητας κομπόστ ο ελαιοπυρήνας πρέπει να χρησιμοποιείτε σε μεγαλύτερη αναλόγια σε σχέση με τα υπόλοιπα υλικά. Η ποσότητα και η συχνότητα προσθήκης νερού παίζει επίσης σημαντικό ρόλο κατά τη κομοστοποιήση. Με βάση τα πειραματικά αποτελέσματα αποδείχθηκε ότι τα στερεά υπολείμματα ελαιοτριβείων μπορούν να παράξουν ένα υψηλής ποιότητας εδαφοβελτιωτικό, το οποίο δεν εμφανίζει φυτοτοξικότητα, γενοτοξικότητα και κυτταροτοξικότητα. Παρόλο αυτά η διάρκεια της κομποστοποίησης, οι διογκωτικοί παράγοντες και τα ποσοστά ανάμιξης των υλικών είναι κρίσιμοι παράγοντες, που επηρεάζουν την ποιότητα του τελικού προϊόντος. Επίσης αναφέρουμε ότι η νομοθεσία της ΕΕ θα πρέπει να αναθεωρηθεί συμπεριλαμβάνοντας τόσο τη γενοτοξική και την κυτταρτοξική εκτίμηση του κομπόστ πριν χρησιμοποιηθεί για βρώσιμες καλλιέργειες. Τέλος με βάση τα πειραματικά αποτελέσματα διαστασιολοήθηκε μια μονάδα πλήρους κλίμακας για την κομποστοποίηση στερεών υπολειμμάτων ελαιοτριβείου. Έτσι για ένα τυπικό μικρής κλίμακας ελαιοτριβείο, που επεξεργάζεται ημερησίως 30 τόνους ελιών και για περίοδο κομποστοποίησης 100 ημερών, χρειάζεται μια συνολική έκταση περίπου 850 m2 για τη κομπστοποίηση όλης της ετησίας ποσότητας του ελαιοπυρήνα.