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Abstract(s)
Atualmente existe uma tendência para a reutilização de subprodutos da indústria alimentar, com foco na extração de compostos bioativos e aplicação no desenvolvimento de novos ingredientes para nutrição humana e animal, cosméticos e nutracêuticos. A indústria vitivinícola não é exceção, pois gera anualmente até 7 milhões de toneladas de biomassa, desperdiçada na forma de bagaço de uva, descrito como sendo rico em compostos de alto valor, tais como polissacáridos neutros, proteínas estruturais e compostos fenólicos. O objetivo deste trabalho foi a valorização de um subproduto representativo da indústria vitivinícola, o bagaço de uva, através da produção de um extrato com elevado valor agregado para a indústria alimentar, a sua caracterização composicional e bioactiva, melhoria da biodisponibilidade através da libertação controlada no intestino e prova de conceito da sua aplicação numa bebida funcional.
A otimização da produção do extrato de bagaço de uva (EBU) foi realizada com base na extração de xilo-oligossacáridos (XOS), utilizando métodos convencionais (extrações ácida e alcalina) ou enzimáticos. Os métodos convencionais recuperaram entre 21.8 a 74.6% e entre 5.2 a 96.3% do total de XOS, para o tratamento ácido e alcalino, respetivamente. Os métodos enzimáticos permitiram extrair até 88.7 ± 0.12% do total de XOS. Tendo em conta o rendimento de extração, a qualidade do extrato e o fato de tratar-se de um processo verde, selecionou-se o extrato produzido enzimaticamente com xilanases de A. niger (EBU) para a continuação deste estudo.
O EBU foi caracterizado em termos da sua composição química e bioatividades, e submetido à simulação da digestão gastrointestinal para compreender como essas propriedades são afetadas pelo trato gastrointestinal. O EBU apresentou elevado teor de fibra (26.1 ± 1.59 g.100g-1) e outros hidratos de carbono, incluindo XOS, minerais e polifenóis. O uso de 2% (m/v) de EBU provou ser uma potencial fonte de carbono, podendo ser fermentada por diferentes espécies de probióticos, mesmo após a digestão, confirmando o seu potencial prébiótico. O EBU também apresentou elevada atividade antioxidante e antimicrobiana contra diferentes microrganismos patogénicos. A simulação in vitro da digestão permitiu concluir que os XOS foram resistentes às condições gástricas, ao contrário dos compostos fenólicos, que foram determinantes para a sua atividade antioxidante e antimicrobiana.
Dessa forma, a fim de melhorar a sua biodisponibilidade, o EBU foi encapsulado em micropartículas (MPs) de quitosano ou alginato. O impacto da digestão gastrointestinal simulada sobre as propriedades biológicas e acessibilidade dos compostos core foi avaliado, bem como a permeabilidade de fenólicos e XOS através da co-cultura de células Caco-2/ HT29-MTX. Além disso, o quitosano foi modificado com uma sonda fluorescente para estudos de uptake celular. As MPs de alginato apresentaram uma dimensão de 523 nm, polidispersão de 0.112, potencial zeta de 15.0 mV e eficiência de associação de polifenóis de 68%. As MPs de quitosano apresentaram dimensão de 853 nm, polidispersão de 0.358, potencial zeta de 14.9 mV e eficiência de associação de polifenóis de 65%. Ambos os sistemas permitiram a libertação do EBU no intestino, aumentando a biodisponibilidade dos compostos fenólicos, bem como as atividades antioxidante e antimicrobiana. A permeabilidade das células intestinais dos XOS diminuiu de 45% para 7.9 e 15.7%, após a encapsulação em MPs de alginato ou quitosano, respetivamente. A modificação de MPs de quitosano com Cyanine 5.5 para ensaios de uptake celular não afetou a biocompatibilidade das MPs com EBU em relação às células intestinais e a análise de microscopia confocal confirmou a integridade das tight junctions após a internalização das MPs.
Como prova de conceito, foi desenvolvida uma bebida funcional à base de água de côco com o EBU encapsulado. A estabilidade química foi avaliada em duas condições de armazenamento, liofilizado à temperatura ambiente ou em estado líquido a 4 ºC, ao longo de 60 dias. O armazenamento à temperatura ambiente acelerou a taxa de degradação de fenólicos e antocianinas, comparando com o armazenamento a 4 ºC. Os potenciais antimicrobiano e prebiótico, após digestão da bebida, também foram testados, verificando-se uma redução no crescimento dos diferentes patogénicos e promoção no desenvolvimento das bifidobacterias e lactobacilos. A análise sensorial permitiu concluir que a incorporação das MPs não promoveu diferenças significativas na maioria dos atributos avaliados, relativamente à água de côco.
Os resultados deste trabalho poderão contribuir para a sustentabilidade da indústria vitivinícola num contexto de economia circular, através do desenvolvimento de ingredientes de alto valor agregado com impacto positivo na saúde humana.
There is a current trend for reutilization of food industry by-products, focused on the extraction of bioactive compounds and application in the development of new feed, food, cosmetic or nutraceutical ingredients. The wine industry is no exception and generates up to 7 million tons of wasted biomass in the form of grape bagasse, which is reported to be rich in high added value compounds such as neutral polysaccharides, structural proteins and phenolic compounds. This project aimed to valorize a major wine production by-product, grape pomace, through the production and characterization of a high valued extract for food industry, improvement of intestinal bioavailability through a gastrointestinal delivery system, and application in the development of a functional beverage as a proof-of-concept. Optimization of grape pomace extract (GPE) was performed through the optimization of xylooligosaccharides (XOS) production, using conventional (acid and alkaline extraction) or enzymatic processes. Conventional methods recovered 21.8 to 74.6% and 5.2 to 96.3% of total XOS, for acid and alkaline processes, respectively. Enzymatic process extracted up to 88.7 ± 0.12% of total. Taking into account the extraction yield, the quality of the extract and the fact of being considered a green process, the enzymatically produced extract with A. niger xylanases (GPE) was selected to continue this study. GPE was characterized for its chemical composition and biological activities, and submitted to the simulation of gastrointestinal digestion to understand how these properties are affected by the gastrointestinal environment. The extract presented high content of dietary soluble fiber (26.1 ± 1.59 g.100g-1) and other carbohydrates, including XOS, minerals and phenolics. In vitro simulated digestion allowed to conclude that XOS were resistant to gastric conditions but phenolics were not. The use of 2% (w/v) of GPE proved to be a potential carbon source that could be fermented by different probiotics, even after digestion. GPE also exhibited strong antioxidant and antimicrobial activity against different pathogens, however, after digestion, these bioactivities were strongly reduced. These results allow to conclude that GPE had low intestinal bioavailability due to action of digestion enzymes and acidic pH. Aiming at improving its bioavailabiliy, GPE was then encapsulated into chitosan or alginate microparticles (MPs) , in order to improve its bioavailability. MPs were characterized for their size, polydispersity, zeta potential and total phenolics (TPC) association efficiency, and the simulation of gastrointestinal digestion was performed to evaluate the release profile of polyphenols, the antioxidant and antimicrobial activities, and permeability of phenolics and XOS across Caco-2/ HT29-MTX cell layer. Also, chitosan was modified with a fluorescent probe for cellular uptake studies. GPE-loaded alginate MPs presented size of 523 nm, polydispersity of 0.112, zeta potential of -15.0 mV and 68% of association efficiency of polyhenols. GPE-loaded chitosan MPs presented size of 853 nm, polydispersity of 0.358, zeta potential of 14.9 mV and 65% of association efficiency of polyphenols. Both systems allowed the delivery of GPE in the intestine, increasing the bioavailability of different polyphenols, the antioxidant and antimicrobial activities. Permeability of XOS across the intestinal cell layer decreased from 45% to 7.9 and 15.7%, after encapsulation in alginate or chitosan MPs, respectively. Modification of chitosan MPs with Cyanine5.5 for cellular uptake studies did not affect the biocompatibility with intestinal cells, and confocal microscopy analysis confirmed the integrity of these cells tight junctions. As a proof of concept, it was developed a coconut beverage through the incorporation 2.5% (w/v) of the encapsulated GPE. Physicochemical stability under two storage conditions (freeze-dried at room temperature or liquid at 4 ˚C) was evaluated along 60 days. The antimicrobial and prebiotic potential after the digestion of the beverage were assessed using different microbial strains. Room-temperature storage accelerated the rate of degradation of phenolics, comparing to storage at 4 ˚C. Alginate and chitosan functional beverages decreased the growth of different pathogens and promoted the growth of different probiotics. Sensory analysis allowed to conclude that the incorporation of particles did not promoted significant differences in most of evaluated attributes. The results from this work will contribute for the sustainability of wine industry in circular economy context, through the development of value-added ingredients with positive biological impact.
There is a current trend for reutilization of food industry by-products, focused on the extraction of bioactive compounds and application in the development of new feed, food, cosmetic or nutraceutical ingredients. The wine industry is no exception and generates up to 7 million tons of wasted biomass in the form of grape bagasse, which is reported to be rich in high added value compounds such as neutral polysaccharides, structural proteins and phenolic compounds. This project aimed to valorize a major wine production by-product, grape pomace, through the production and characterization of a high valued extract for food industry, improvement of intestinal bioavailability through a gastrointestinal delivery system, and application in the development of a functional beverage as a proof-of-concept. Optimization of grape pomace extract (GPE) was performed through the optimization of xylooligosaccharides (XOS) production, using conventional (acid and alkaline extraction) or enzymatic processes. Conventional methods recovered 21.8 to 74.6% and 5.2 to 96.3% of total XOS, for acid and alkaline processes, respectively. Enzymatic process extracted up to 88.7 ± 0.12% of total. Taking into account the extraction yield, the quality of the extract and the fact of being considered a green process, the enzymatically produced extract with A. niger xylanases (GPE) was selected to continue this study. GPE was characterized for its chemical composition and biological activities, and submitted to the simulation of gastrointestinal digestion to understand how these properties are affected by the gastrointestinal environment. The extract presented high content of dietary soluble fiber (26.1 ± 1.59 g.100g-1) and other carbohydrates, including XOS, minerals and phenolics. In vitro simulated digestion allowed to conclude that XOS were resistant to gastric conditions but phenolics were not. The use of 2% (w/v) of GPE proved to be a potential carbon source that could be fermented by different probiotics, even after digestion. GPE also exhibited strong antioxidant and antimicrobial activity against different pathogens, however, after digestion, these bioactivities were strongly reduced. These results allow to conclude that GPE had low intestinal bioavailability due to action of digestion enzymes and acidic pH. Aiming at improving its bioavailabiliy, GPE was then encapsulated into chitosan or alginate microparticles (MPs) , in order to improve its bioavailability. MPs were characterized for their size, polydispersity, zeta potential and total phenolics (TPC) association efficiency, and the simulation of gastrointestinal digestion was performed to evaluate the release profile of polyphenols, the antioxidant and antimicrobial activities, and permeability of phenolics and XOS across Caco-2/ HT29-MTX cell layer. Also, chitosan was modified with a fluorescent probe for cellular uptake studies. GPE-loaded alginate MPs presented size of 523 nm, polydispersity of 0.112, zeta potential of -15.0 mV and 68% of association efficiency of polyhenols. GPE-loaded chitosan MPs presented size of 853 nm, polydispersity of 0.358, zeta potential of 14.9 mV and 65% of association efficiency of polyphenols. Both systems allowed the delivery of GPE in the intestine, increasing the bioavailability of different polyphenols, the antioxidant and antimicrobial activities. Permeability of XOS across the intestinal cell layer decreased from 45% to 7.9 and 15.7%, after encapsulation in alginate or chitosan MPs, respectively. Modification of chitosan MPs with Cyanine5.5 for cellular uptake studies did not affect the biocompatibility with intestinal cells, and confocal microscopy analysis confirmed the integrity of these cells tight junctions. As a proof of concept, it was developed a coconut beverage through the incorporation 2.5% (w/v) of the encapsulated GPE. Physicochemical stability under two storage conditions (freeze-dried at room temperature or liquid at 4 ˚C) was evaluated along 60 days. The antimicrobial and prebiotic potential after the digestion of the beverage were assessed using different microbial strains. Room-temperature storage accelerated the rate of degradation of phenolics, comparing to storage at 4 ˚C. Alginate and chitosan functional beverages decreased the growth of different pathogens and promoted the growth of different probiotics. Sensory analysis allowed to conclude that the incorporation of particles did not promoted significant differences in most of evaluated attributes. The results from this work will contribute for the sustainability of wine industry in circular economy context, through the development of value-added ingredients with positive biological impact.
Description
Keywords
Bagaço de uva Enzimas Biodisponibilidade Encapsulação Grape pomace Enzymes Bioavailability Encapsulation