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Development and characterization of functional ingredients through valorization of spent brewer yeast : peptide concentrates and ß-glucans with biological activities
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A crescente preocupação da sociedade com o bem-estar e a utilização de práticas verdes e sustentáveis tem levado à promoção de ações que visam explorar recursos de alto valor, e que podem ser convertidos em compostos de interesse biológico. Entre essas ações, encontra-se o aproveitamento da levedura de cerveja, representando o segundo maior subproduto da indústria cervejeira, e que possui elevado valor nutricional e biológico. Por este motivo, existe um grande interesse em encontrar aplicações sustentáveis para a valorização deste subproduto. Uma estratégia de valorização integrada que pode passar por métodos de autólise, hidrólise e fracionamento, pode ser uma metodologia eficiente para à recuperação de compostos diferenciados e de interesse biológico. Assim, o principal objetivo desta tese de doutoramento foi utilizar o subproduto de levedura de cerveja no desenvolvimento de ingredientes funcionais: concentrados peptídicos e glucanas; caracterizar e validar atividades biológicas em doenças crónicas que afetam grande parte da população atual, tais como, doenças cardiovasculares, obesidade e distúrbios imuno-inflamatórios. Inicialmente, foi avaliado o efeito da autólise e a subsequente hidrólise da levedura com um extrato enzimático vegetal de Cynara cardunculus, com o objetivo de definir as condições ótimas para maximizar a atividade inibitória da enzima conversora da angiotensina (I-ECA), indicado por valores de concentração necessária para inibir 50% da ECA (IC50). De acordo com os modelos de resposta, as condições que permitiram obter autólise máxima foram à temperatura de 70 ºC com um tempo de reação de 5 h, seguida de uma hidrólise com uma relação de enzima/substrato de 4% (v/v) durante 4.5 h com valor de IC50 de 146.1 ± 8.3 μg mL-1 de I - ECA. Finalmente, a realização de uma nanofiltração com membrana de 3 kDa permitiu a separação de péptidos de baixo peso molecular que exibiram uma maior atividade I- ECA (IC50 84.2 ± 11 μg mL-1), permanecendo estável ao longo do trato gastrointestinal simulado em condições in vitro. As condições de autólise e hidrólise, previamente otimizadas, foram selecionadas para aplicação em processos de ultrafiltração e nanofiltração à escala piloto para fracionamento de diferentes compostos. Foram obtidas quatro frações de diferentes pesos moleculares, e foram avaliadas quanto às suas atividades biológicas in vitro: I-ECA (com intervalos de IC50 84 - 260 μg mL-1) e atividade antioxidante (7 - 4 μM TE mg-1). A fração proteica retida (PRH >3 kDa e PRH <3 kDa) mostrou os melhores valores in vitro, sendo desta forma usada posteriormente nos testes in vivo. Foi determinado o efeito anti-hipertensivo oral num modelo in vivo de ratos espontaneamente hipertensos (SHR), e demonstrou-se que a fração de baixo peso molecular (PRH <3 kDa) causou uma diminuição mais percetível na pressão arterial sistólica, diastólica e média dos SHR. Desta forma, esta fração foi estudada (PRH <3 kDa) em um modelo animal de obesidade induzida por uma dieta hiperlipídica (HF), e observou-se que a ingestão diminuiu a ingestão de alimentos e de energia dos animais na dieta HF. Os níveis de peso corporal e leptina diminuíram a partir da décima semana de tratamento. Em relação à microbiota intestinal, não foi observado impacto negativo na microbiota dos animais. Assim, essas propriedades destacam o uso como ingrediente nutracêutico ou funcional para o tratamento da obesidade. Por outro lado, as frações obtidas foram avaliadas quanto a sua capacidade de proteção na mucosa gástrica em um modelo animal com lesões ulcerativas causadas pela administração oral de etanol absoluto. Verificou-se que a fração de baixo peso molecular (PRH <3 kDa), reduziu as lesões gástricas a níveis significativos e o efeito citoprotetor poderá estar relacionado com mecanismos mediados por prostaglandinas e não específicos. Foram testadas as atividades antiproliferativas das frações em nove linhas celulares tumorais humanas e os resultados exibiram uma promissora atividade antiproliferativa contra a linha celular K-562 (leucemia). No que diz respeito aos componentes da parede celular de levedura contidos na fração de alto peso molecular (PRH >3 kDa), estes foram separados, caracterizados quimicamente e o efeito pré-biótico e imunoestimulante foi avaliado. O extrato obtido é composto por um complexo polisacarídico semelhante ao glicogênio, com prevalência de ligações 1,4-glucanas e mostrou um estímulo do crescimento das de estirpes probióticas (lactobacilos e bifidobactérias) selecionadas. Os resultados mostraram a capacidade de assimilação destes substratos atuando como fontes de carbono e em consequência, a elevada produção de ácidos orgânicos e ácidos gordos de cadeia curta. Em relação aos ensaios imunológicos, foi observado uma ativação significativa de células B em baixas concentrações de polissacarídeo (10 μg mL-1). Finalmente, como prova de conceito, foi testada a inclusão da fração (PRH >3 kDa) numa matriz alimentar de forma a avaliar o impacto no sabor e que pode influenciar a escolha do consumidor. Assim, foi formulada uma barra de cereal à base de uma mistura de aveia, arroz e banana desidratada com 2% de extrato de levedura. Os testes de aceitação do consumidor mostraram uma resposta positiva para todos os atributos avaliados. Assim, as barras de cereais mostraram ser uma matriz alimentar adequada para incorporar ingredientes bioativos de levedura. Os resultados deste trabalho experimental contribuirão para a sustentabilidade da indústria cervejeira, no conceito de economia circular, através do desenvolvimento de compostos de valor acrescentado, num modelo de biorefinaria, com impacto biológico em doenças crônicas.
The establishment of a bio-based and green society enables the appearance of actions to explore high value resources that can be converted into valuable biological ingredients. On the list of these resources appears Spent brewer's yeast, the second biggest byproduct generated from brewing industry, with a high biological and nutritional value. Nowadays, there is an increasing demand towards sustainable practices for the valorization of such byproduct by social, economic and environmental reasons. The processing of spent brewer's yeast through autolysis and hydrolysis methods, fractioning and recovering molecules with interesting biological value, is one of the possible approaches to perform an integrated valorization of this byproduct. Hence, the main objective of this PhD research work was to use spent brewer's yeast in the development of functional ingredients such as protein/peptides concentrates and glucans, and to characterize and validate biological activities related with chronic diseases that affects a large part of the current population, namely cardiovascular diseases, obesity and immune-inflammatory disorders. First, the effect of autolysis and subsequent hydrolysis of spent brewer yeast was studied using a plant enzymatic extract of Cynara cardunculus, to define the optimal conditions with maximized angiotensin converting enzyme inhibitory activity (ACE-I), measured by the IC50 values (defined as the concentration of yeast extract needed to inhibit 50% of the ACE activity). The response models determined that maximum autolysis may be obtained using temperatures of 70 ºC and reaction times of 5 h, followed by hydrolysis with a ratio of 4% (v/v) Enzyme/Substrate during 4.5 h with IC50 146.1 ± 8.3 μg mL-1 for ACE-I. Finally, nanofiltration produced an extract with molecular weight (MW) <3 kDa that exhibited the highest ACE-I (IC50 84.2 ± 11 μg mL-1), which remained stable throughout gastrointestinal passage tested using an in vitro system. The optimal hydrolysis conditions were tested at pilot scale and resulting hydrolysate was fractionated combining ultrafiltration and nanofiltration processes. Four fractions with different MW and composition were obtained. These fractions were evaluated for their biological activities, such as in vitro ACE-I (IC50 range values 84 – 260 μg mL-1) and antioxidant activity (7 – 4 μM TE mg-1). Protein retentate hydrolyzed fractions (PRH > 3kDa and PRH < 3kDa) showed the best effects in vitro, being thus used for in vivo assays. Short-term oral antihypertensive effect was tested on the spontaneously hypertensive ats (SHR) model. The fraction with low MW (PRH <3 kDa) caused the most noticeable decrease in systolic, diastolic and mean blood pressure of SHR. In sequence, this fraction – PRH <3 kDa, was studied in an animal model of high-fat (HF) diet-induced obesity and decreased food and energy ingestion of the animals were observed. The body weight and leptin levels decreased since the 10th week of yeast ingestion. Concerning gut microbiota analysis, no negative impact on the microbiota of animals was observed. These properties highlight the use as nutraceutical or functional ingredient for the management and control of obesity. The protection of gastric mucosa by these fractions were also tested in an animal model with ulcerative lesions caused by oral administration of absolute ethanol. It was found PRH <3 kDa fraction could reduce gastric injuries to significant levels and the cytoprotective effect appears to depend on prostaglandin-mediated and nonspecific mechanisms. The antiproliferative activities of the extracts in nine different human tumoral cell lines were tested and results exhibited a promising antiproliferative activity against cell line K-562 (leukemia). High MW fraction (PRH >3 kDa) were submitted to sequential purification steps to produce a yeast glucans complex, chemically characterized as well as its prebiotic and immunobiological potential. The extract obtained was composed by 75% of a complex of glycogen-like polysaccharide, mostly 1,4-glucans linkages. The stimulation of growth of the probiotic strains (lactobacilli and bifidobacteria) was evaluated and results showed the ability of these substrates to enhance probiotic bacteria growth acting as carbon sources. When in colon conditions, a high production of short chain fatty acids (with relevance for propionic acid) envisaging relating benefits for intestinal health. The immunological assays showed a significant activation of B cells at low concentrations (10 μg mL-1). Finally, the inclusion of PRH > 3kDa fraction in a food matrix was tested to evaluate their particular taste and flavor. Thus, a cereal bar was formulated based on a mixture of oat, rice and banana raisin with 2% of extract. Positive consumer acceptance of tested cereal bar showed to be an adequate matrix to incorporate this bioactive ingredient to produce a functional food product. The results from this experimental work will contribute for the sustainability of brewing industry in circular economy context, through the development of value-added ingredients with biological impact on chronical diseases.
The establishment of a bio-based and green society enables the appearance of actions to explore high value resources that can be converted into valuable biological ingredients. On the list of these resources appears Spent brewer's yeast, the second biggest byproduct generated from brewing industry, with a high biological and nutritional value. Nowadays, there is an increasing demand towards sustainable practices for the valorization of such byproduct by social, economic and environmental reasons. The processing of spent brewer's yeast through autolysis and hydrolysis methods, fractioning and recovering molecules with interesting biological value, is one of the possible approaches to perform an integrated valorization of this byproduct. Hence, the main objective of this PhD research work was to use spent brewer's yeast in the development of functional ingredients such as protein/peptides concentrates and glucans, and to characterize and validate biological activities related with chronic diseases that affects a large part of the current population, namely cardiovascular diseases, obesity and immune-inflammatory disorders. First, the effect of autolysis and subsequent hydrolysis of spent brewer yeast was studied using a plant enzymatic extract of Cynara cardunculus, to define the optimal conditions with maximized angiotensin converting enzyme inhibitory activity (ACE-I), measured by the IC50 values (defined as the concentration of yeast extract needed to inhibit 50% of the ACE activity). The response models determined that maximum autolysis may be obtained using temperatures of 70 ºC and reaction times of 5 h, followed by hydrolysis with a ratio of 4% (v/v) Enzyme/Substrate during 4.5 h with IC50 146.1 ± 8.3 μg mL-1 for ACE-I. Finally, nanofiltration produced an extract with molecular weight (MW) <3 kDa that exhibited the highest ACE-I (IC50 84.2 ± 11 μg mL-1), which remained stable throughout gastrointestinal passage tested using an in vitro system. The optimal hydrolysis conditions were tested at pilot scale and resulting hydrolysate was fractionated combining ultrafiltration and nanofiltration processes. Four fractions with different MW and composition were obtained. These fractions were evaluated for their biological activities, such as in vitro ACE-I (IC50 range values 84 – 260 μg mL-1) and antioxidant activity (7 – 4 μM TE mg-1). Protein retentate hydrolyzed fractions (PRH > 3kDa and PRH < 3kDa) showed the best effects in vitro, being thus used for in vivo assays. Short-term oral antihypertensive effect was tested on the spontaneously hypertensive ats (SHR) model. The fraction with low MW (PRH <3 kDa) caused the most noticeable decrease in systolic, diastolic and mean blood pressure of SHR. In sequence, this fraction – PRH <3 kDa, was studied in an animal model of high-fat (HF) diet-induced obesity and decreased food and energy ingestion of the animals were observed. The body weight and leptin levels decreased since the 10th week of yeast ingestion. Concerning gut microbiota analysis, no negative impact on the microbiota of animals was observed. These properties highlight the use as nutraceutical or functional ingredient for the management and control of obesity. The protection of gastric mucosa by these fractions were also tested in an animal model with ulcerative lesions caused by oral administration of absolute ethanol. It was found PRH <3 kDa fraction could reduce gastric injuries to significant levels and the cytoprotective effect appears to depend on prostaglandin-mediated and nonspecific mechanisms. The antiproliferative activities of the extracts in nine different human tumoral cell lines were tested and results exhibited a promising antiproliferative activity against cell line K-562 (leukemia). High MW fraction (PRH >3 kDa) were submitted to sequential purification steps to produce a yeast glucans complex, chemically characterized as well as its prebiotic and immunobiological potential. The extract obtained was composed by 75% of a complex of glycogen-like polysaccharide, mostly 1,4-glucans linkages. The stimulation of growth of the probiotic strains (lactobacilli and bifidobacteria) was evaluated and results showed the ability of these substrates to enhance probiotic bacteria growth acting as carbon sources. When in colon conditions, a high production of short chain fatty acids (with relevance for propionic acid) envisaging relating benefits for intestinal health. The immunological assays showed a significant activation of B cells at low concentrations (10 μg mL-1). Finally, the inclusion of PRH > 3kDa fraction in a food matrix was tested to evaluate their particular taste and flavor. Thus, a cereal bar was formulated based on a mixture of oat, rice and banana raisin with 2% of extract. Positive consumer acceptance of tested cereal bar showed to be an adequate matrix to incorporate this bioactive ingredient to produce a functional food product. The results from this experimental work will contribute for the sustainability of brewing industry in circular economy context, through the development of value-added ingredients with biological impact on chronical diseases.