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Authors
Abstract(s)
Atualmente, grande volume dos resíduos gerados ao longo da cadeia alimentar Europeia é
produzido durante o processamento alimentar (19-39%). A indústria de extração de azeite não é
exceção, gerando anualmente grandes quantidades de subprodutos e resíduos com elevado
impacto fitotóxico, mas que são também ricos em diversos compostos bioativos e nutrientes com
potencial aplicação como ingredientes alimentares. Assim sendo, é imprescindível a adoção de
estratégias de redução do desperdício e de valorização dos subprodutos, no sentido de melhorar a
sustentabilidade económica e ambiental da indústria alimentar. Atualmente, o bagaço de azeitona é
o subproduto de maior relevo da indústria de azeite e a sua valorização adequada é também o maior
desafio do sector para alcançar uma cadeia produtiva sustentável alinhada com os princípios da
Bioeconomia Circular. Esta tese visou alcançar uma valorização “zero desperdício” para o bagaço
de azeitona, priorizando os produtos de alto valor acrescentado sobre os de baixo valor através da
produção e caracterização de ingredientes alimentares, nomeadamente ingredientes em pó ricos
em vários compostos bioativos, seguida da validação in vitro das suas atividades biológicas ligadas
a benefícios para a saúde, como a prevenção e redução de doenças cardiovasculares, diabetes,
problemas intestinais e posterior aplicação no desenvolvimento de alimentos funcionais.
Uma estratégia de “desperdício zero” foi alcançada para o bagaço de azeitona. Esta estratégia
consiste num procedimento simples de fracionamento, em que se obteve uma fração líquida que
revelou um alto teor de hidroxitirosol (513,61-625,76 mg/100 g de peso seco) e uma fração de polpa
caracterizada por ser uma boa fonte de fibra alimentar antioxidante. Adicionalmente, obteve-se
ainda uma fração de caroços que exibiu valores caloríficos significativamente elevados (18,65-18,94
MJ/kg). Estes resultados validaram o potencial valor acrescentado das frações líquidas e de polpa
como ingredientes alimentares e como biocombustível no caso da fração de caroços.
Após validação do potencial da fração líquida e de polpa do bagaço de azeitona como ingredientes
alimentares, dois novos ingredientes em pó, estáveis e seguros, foram desenvolvidos sem qualquer
etapa de extração, nomeadamente um pó enriquecido na fração líquida (LOPP) e um pó enriquecido
na fração de polpa (POPP). O LOPP exibiu uma quantidade significativa de manitol (141 g/kg),
potássio (54 g/kg) e hidroxitirosol/derivados (5 mg/g). O POPP exibiu uma grande quantidade de
fibra alimentar (620 g/kg) associada a um teor significativo de fenólicos (7,41 mg equivalentes de
ácido gálico/g fibra) com atividade antioxidante substancial. O POPP também revelou uma
composição de ácidos gordos insaturados semelhante à do azeite (76% do total de ácidos gordos),
sendo ainda uma fonte razoável de proteína (12%). As suas propriedades funcionais (solubilidade,
capacidade de retenção de água e de óleo), capacidade antioxidante e atividade antimicrobiana
foram também avaliadas, assim como a sua segurança biológica. Em síntese, a multifuncionalidade
e aplicabilidade dos pós de bagaço de azeitona como fortificantes nutricionais, promotores de
benefícios para a saúde e conservantes foram atestadas.
A digestão simulada in vitro permitiu concluir que os fenólicos e minerais do LOPP foram afetados
negativamente pela digestão, no entanto, uma bioacessibilidade significativa de potássio e
hidroxitirosol foi verificada ( 57%) e as suas bioatividades foram apenas moderadamente afetadas.
Em relação às suas bioatividades, pelo menos metade da atividade antioxidante (capacidade de
Resumo
x
absorção do radical de oxigénio), de inibição da -glucosidase e de inibição da enzima conversora
de angiotensina (91,98%) foram retidas e bioacessíveis após a digestão. Por sua vez, a digestão do
POPP revelou uma perda significativa de fenólicos na digestão oral (62,48%), mas o papel da fibra
como veículo de fenólicos permitiu recuperar uma quantidade significativa de fenólicos no estômago
(77,11%) e um índice de bioacessibilidade de pelo menos 50% (tirosol e o seu glucosídeo). A fibra
do POPP também interagiu positivamente com os ácidos gordos, diminuindo e facilitando
bioacessibilidade dos saturados (5-6%) e dos insaturados (4-11%), respetivamente.
Após realização da digestão in vitro dos pós de bagaço de azeitona, as frações disponíveis no cólon
de ambos os pós exibiram uma composição abundante em compostos associados a potenciais
benefícios para a saúde intestinal, nomeadamente fibra e compostos fenólicos e fibra. Assim sendo,
procedeu-se a fermentação fecal in vitro das frações não digeridas retidas no cólon após a digestão
gastrointestinal simulada in vitro do LOPP e POPP. Os resultados da análise do gene 16S rRNA e
dos ácidos gordos de cadeia curta mostraram que estes pós não tiveram um impacto negativo na
diversidade da microbiota intestinal e que promoveram uma maior produção de ácidos de cadeia
curta (acetato>butirato>propionato) do que os frutooligossacarídeos. A identificação dos metabólitos
fenólicos dos pós de bagaço de azeitona por LC-ESI-UHR-QqTOF-MS seguida das análises de
clustering supervisionada e clustering hierárquico permitiram avaliar os padrões destes metabólitos
ao longo da fermentação fecal in vitro, bem como explicar as potenciais atividades biológicas destes
pós no intestino. Ambos os pós apresentaram um conteúdo fenólico e uma capacidade de absorção
de radical de oxigénio significativas e capacidade de inibição da adesão de patógenos como o
Bacillus cereus (22,03%) e Listeria monocytogenes (20,01%), sobretudo pelo POPP.
Por fim, para validar o desempenho dos pós de bagaço de azeitona como ingredientes funcionais,
LOPP e POPP foram incorporados em iogurte para aumentar o seu conteúdo em fibra alimentar,
hidroxitirosol e ácidos gordos insaturados. A adição de POPP (2%) ao iogurte permitiu cumprir a
condição de “fonte de fibra”, por sua vez LOPP (1%) forneceu 5 mg de hidroxitirosol/ derivados num
iogurte (120 g). A adição de POPP e azeite juntamente com LOPP ou POPP permitiu uma melhoria
do perfil de ácidos gordos insaturados dos iogurtes. Além disso, todos os iogurtes fortificados
apresentaram um maior teor de fenólicos e atividade antioxidante. A digestão in vitro reduziu a
atividade antioxidante de todas as formulações e mostrou que a incorporação de azeite juntamente
com o LOPP e POPP aumentou a bioacessibilidade de hidroxitirosol e do hidroxitirosol glucosídeo,
respetivamente. Contudo, todas as formulações com os pós de bagaço de azeitona podem ser
consideradas boas fontes de hidroxitirosol, lípidos insaturados e fibras para os consumidores.
Em suma, os resultados deste trabalho irão contribuir para a sustentabilidade da indústria do azeite
no contexto da bioeconomia circular, através do desenvolvimento de ingredientes de valor
acrescentado destinados à indústria alimentar com potencial impacto positivo na saúde,
proporcionando ainda um biocombustível sólido.
Currently, a large amount of the total European food supply chain waste (between 19 and 39%) is produced during food processing. The olive oil industry is no exception, generating large quantities of by-products and wastes annually with high phytotoxic impact, but also rich in several bioactive compounds and nutrients with potential application as food ingredients. Thus, it is imperative to implement strategies to reduce waste and valorise by-products to improve the food industry's economic and environmental sustainability. Olive pomace is the most prominent by-product of the modern olive oil industry, and its proper valorisation is one of the major obstacles to olive oil industry achieve a sustainable production chain aligned with the principles of the Circular Bioeconomy concept. This project aimed at achieving a “zero waste” valorising approach to olive pomace that prioritised the high over low added-value products, through the production and characterisation of food ingredients, namely high-value powdered ingredients rich in several bioactive compounds, followed by the validation of their biological activities related with health benefits as prevention and reduction of the prevalence of cardiovascular diseases, diabetes and gut disorders and, their application in the development of functional foods. A “zero waste” strategy was achieved for olive pomace. This strategy is based on a feasible fraction approach, and it was obtained a liquid fraction that revealed a high amount of hydroxytyrosol (513.61- 625.76 mg/100 g dry weight) and pulp fraction that was characterised to be a good source of antioxidant dietary fibre. Additionally, it was achieved a stones fraction that exhibited substantial higher heating values (18.65-18.94 MJ/kg). These results supported the potential value-added of the liquid and pulp fractions from olive pomace as functional food ingredients and as biofuel to stones. After validating the liquid and pulp fraction potential as food ingredients, new two stable and safe, powdered ingredients without any extraction step were developed, namely a liquid-enriched powder (LOPP) and a pulp-enriched powder (POPP). LOPP exhibited a significant amount of mannitol (141 g/kg), potassium (54 g/kg) and hydroxytyrosol/derivatives (5 mg/g). POPP exhibited a high amount of dietary fibre (620 g/kg) associated with a significant amount of bound phenolics (7.41 mg gallic acid equivalents/g fibre dry weight) with substantial antioxidant activity. POPP also revealed an unsaturated fatty acid composition similar to that of olive oil (76% of total fatty acids) and a reasonable source of protein (12%). Their functional properties (solubility, water‐holding and oil‐ holding capacity), antioxidant capacity and antimicrobial activity were also assessed, and their biological safety was validated. The multifunctionality and applicability as nutritional enhancers, health-benefits promoters and preservatives of the olive pomace powders were recognised. The in vitro simulated digestion allowed concluding that phenolics and minerals from LOPP were negatively affected by digestion. However, a significant bioaccessibility of potassium and hydroxytyrosol was verified ( 57%) and its bioactivities were only moderately affected. Regarding its bioactivities, at least half of the antioxidant activity (measured by Oxygen Radical Absorbance Capacity assay), -glucosidase inhibition activity and angiotensin-converting enzyme inhibitory activity (91.98%) were retained and bioaccessible after in vitro digestion. In its turn, POPP digestion Abstract xii revealed a significant loss of phenolics in the oral step (62.48%), but the dietary fibre role as phenolics’ carrier allowed recovering a significant amount of phenolics in the stomach (77.11%) and a bioaccessibility index of at least 50% (mainly for tyrosol and its glucoside). POPP dietary fibre also positively interacts with lipids decreasing the saturated fatty acids bioaccessibility (5-6%) and facilitating the unsaturated fatty acid bioaccessibility (4-11%). After the in vitro digestion system of both olive pomace powders, the colon-available fractions LOPP and POPP exhibited an abundant composition in phenolics, but also dietary fibre with potential gut health benefits as antioxidants, antimicrobial and prebiotic agents. So, the gut's potential beneficial effects were analysed by in vitro faecal fermentation of the undigested fractions retained in the colon after in vitro simulated gastrointestinal digestion. The 16S rRNA gene analysis results showed that LOPP and POPP did not negatively impact gut microbiota diversity. The short-chain fatty acids analysis showed a higher production of these fatty acids (acetate > butyrate > propionate) by LOPP and POPP than by fructooligosaccharides. The identification of phenolic metabolites by LC-ESIUHR-QqTOF-MS followed by supervised cluster and hierarchical clustering analysis allowed to evaluate the metabolite patterns of olive pomace powders phenolics throughout faecal fermentation, explaining the potential biological activities exhibited in the gut by these powders. Both powders showed significant total phenolic content and oxygen radical absorbance capacity during faecal fermentation. But also, mucin‐adhesion inhibition ability against pathogens as Bacillus cereus (22.03%) and Listeria monocytogenes (20.01%), specially POPP. Finally, to validate olive pomace powders' performance as functional ingredients, LOPP and POPP were incorporated into yoghurt to increase its content in dietary fibre, hydroxytyrosol, and unsaturated fatty acids. POPP (2%) and LOPP (1%) addition to yoghurt allowed fulfilling the condition on being a “source of fibre” and provides 5 mg of hydroxytyrosol and derivatives in a standard yoghurt (120 g), respectively. The addition of olive oil, together with olive pomace powders, was also investigated in order to understand the lipids-phenolics interaction, and not only POPP addition improved the yoghurts’ unsaturated fatty acids profile, but the olive oil addition together both olive pomace powders also improved too. Besides that, all yoghurts fortified with olive pomace powders exhibited higher total phenolic content and antioxidant activity. The in vitro digestion of yoghurts showed that olive oil incorporation together with olive pomace powders increased the bioaccessibility of hydroxytyrosol (LOPP + olive oil) and hydroxytyrosol glucoside (POPP + olive oil). Despite the reduction of antioxidant activity after digestion, the yoghurts formulations with olive pomace powders could be considered suitable carriers to deliver hydroxytyrosol, unsaturated fatty acids and fibre to the consumers. In short, the results from this work will contribute to the sustainability of the olive oil industry in circular bioeconomy context, through the development of value-added ingredients to the food industry with a positive impact in health, offering also a solid biofuel.
Currently, a large amount of the total European food supply chain waste (between 19 and 39%) is produced during food processing. The olive oil industry is no exception, generating large quantities of by-products and wastes annually with high phytotoxic impact, but also rich in several bioactive compounds and nutrients with potential application as food ingredients. Thus, it is imperative to implement strategies to reduce waste and valorise by-products to improve the food industry's economic and environmental sustainability. Olive pomace is the most prominent by-product of the modern olive oil industry, and its proper valorisation is one of the major obstacles to olive oil industry achieve a sustainable production chain aligned with the principles of the Circular Bioeconomy concept. This project aimed at achieving a “zero waste” valorising approach to olive pomace that prioritised the high over low added-value products, through the production and characterisation of food ingredients, namely high-value powdered ingredients rich in several bioactive compounds, followed by the validation of their biological activities related with health benefits as prevention and reduction of the prevalence of cardiovascular diseases, diabetes and gut disorders and, their application in the development of functional foods. A “zero waste” strategy was achieved for olive pomace. This strategy is based on a feasible fraction approach, and it was obtained a liquid fraction that revealed a high amount of hydroxytyrosol (513.61- 625.76 mg/100 g dry weight) and pulp fraction that was characterised to be a good source of antioxidant dietary fibre. Additionally, it was achieved a stones fraction that exhibited substantial higher heating values (18.65-18.94 MJ/kg). These results supported the potential value-added of the liquid and pulp fractions from olive pomace as functional food ingredients and as biofuel to stones. After validating the liquid and pulp fraction potential as food ingredients, new two stable and safe, powdered ingredients without any extraction step were developed, namely a liquid-enriched powder (LOPP) and a pulp-enriched powder (POPP). LOPP exhibited a significant amount of mannitol (141 g/kg), potassium (54 g/kg) and hydroxytyrosol/derivatives (5 mg/g). POPP exhibited a high amount of dietary fibre (620 g/kg) associated with a significant amount of bound phenolics (7.41 mg gallic acid equivalents/g fibre dry weight) with substantial antioxidant activity. POPP also revealed an unsaturated fatty acid composition similar to that of olive oil (76% of total fatty acids) and a reasonable source of protein (12%). Their functional properties (solubility, water‐holding and oil‐ holding capacity), antioxidant capacity and antimicrobial activity were also assessed, and their biological safety was validated. The multifunctionality and applicability as nutritional enhancers, health-benefits promoters and preservatives of the olive pomace powders were recognised. The in vitro simulated digestion allowed concluding that phenolics and minerals from LOPP were negatively affected by digestion. However, a significant bioaccessibility of potassium and hydroxytyrosol was verified ( 57%) and its bioactivities were only moderately affected. Regarding its bioactivities, at least half of the antioxidant activity (measured by Oxygen Radical Absorbance Capacity assay), -glucosidase inhibition activity and angiotensin-converting enzyme inhibitory activity (91.98%) were retained and bioaccessible after in vitro digestion. In its turn, POPP digestion Abstract xii revealed a significant loss of phenolics in the oral step (62.48%), but the dietary fibre role as phenolics’ carrier allowed recovering a significant amount of phenolics in the stomach (77.11%) and a bioaccessibility index of at least 50% (mainly for tyrosol and its glucoside). POPP dietary fibre also positively interacts with lipids decreasing the saturated fatty acids bioaccessibility (5-6%) and facilitating the unsaturated fatty acid bioaccessibility (4-11%). After the in vitro digestion system of both olive pomace powders, the colon-available fractions LOPP and POPP exhibited an abundant composition in phenolics, but also dietary fibre with potential gut health benefits as antioxidants, antimicrobial and prebiotic agents. So, the gut's potential beneficial effects were analysed by in vitro faecal fermentation of the undigested fractions retained in the colon after in vitro simulated gastrointestinal digestion. The 16S rRNA gene analysis results showed that LOPP and POPP did not negatively impact gut microbiota diversity. The short-chain fatty acids analysis showed a higher production of these fatty acids (acetate > butyrate > propionate) by LOPP and POPP than by fructooligosaccharides. The identification of phenolic metabolites by LC-ESIUHR-QqTOF-MS followed by supervised cluster and hierarchical clustering analysis allowed to evaluate the metabolite patterns of olive pomace powders phenolics throughout faecal fermentation, explaining the potential biological activities exhibited in the gut by these powders. Both powders showed significant total phenolic content and oxygen radical absorbance capacity during faecal fermentation. But also, mucin‐adhesion inhibition ability against pathogens as Bacillus cereus (22.03%) and Listeria monocytogenes (20.01%), specially POPP. Finally, to validate olive pomace powders' performance as functional ingredients, LOPP and POPP were incorporated into yoghurt to increase its content in dietary fibre, hydroxytyrosol, and unsaturated fatty acids. POPP (2%) and LOPP (1%) addition to yoghurt allowed fulfilling the condition on being a “source of fibre” and provides 5 mg of hydroxytyrosol and derivatives in a standard yoghurt (120 g), respectively. The addition of olive oil, together with olive pomace powders, was also investigated in order to understand the lipids-phenolics interaction, and not only POPP addition improved the yoghurts’ unsaturated fatty acids profile, but the olive oil addition together both olive pomace powders also improved too. Besides that, all yoghurts fortified with olive pomace powders exhibited higher total phenolic content and antioxidant activity. The in vitro digestion of yoghurts showed that olive oil incorporation together with olive pomace powders increased the bioaccessibility of hydroxytyrosol (LOPP + olive oil) and hydroxytyrosol glucoside (POPP + olive oil). Despite the reduction of antioxidant activity after digestion, the yoghurts formulations with olive pomace powders could be considered suitable carriers to deliver hydroxytyrosol, unsaturated fatty acids and fibre to the consumers. In short, the results from this work will contribute to the sustainability of the olive oil industry in circular bioeconomy context, through the development of value-added ingredients to the food industry with a positive impact in health, offering also a solid biofuel.
Description
Keywords
Bagaço de azeitona Ingredientes em pó Bioacessibilidade Benefícios para a saúde Olive pomace Powder ingredients Bioaccessibility Health benefits