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Abstract(s)
A beterraba “Beta vulgaris L.” pertence à família Chenopodiaceae, é um legume com elevada atividade antioxidante, capacidade de prevenção de doenças cardiovasculares, nomeadamente no controlo da dislipidemia. Estas características parecem encontrar-se diretamente relacionadas com a elevada quantidade de compostos bioativos (CBs) presentes neste legume. No entanto, apenas é normalmente consumida a raiz da beterraba, embora as suas folhas e talos sejam ricos em CBs, estes são muitas vezes considerados resíduos agrícolas. O objetivo deste estudo foi avaliar o potencial de valorização das folhas e talos da beterraba. Neste sentido foram quantificadas as atividades biológicas dos extratos aquosos de folhas de beterraba e talos, identificados e quantificados alguns CBs presentes, nomeadamente compostos fenólicos (CFs), e adicionalmente foi avaliada a capacidade da tecnologia de membrana no isolamento de CBs presentes no extrato.
Neste trabalho foram preparados vários extratos aquosos de folhas de beterraba e talos: Decocção (Bdec), extração assistida pelo enzima pepsina (BDG), extração assistida pelo enzima pancreatina (BDP) e extração assistida pelos enzimas pepsina e pancreatina (BDC), pretendeu-se evitar o uso de solventes orgânicos e simular a ingestão. Os extratos foram caracterizados fazendo as quantificações de fenóis totais (FT) (método Folin-Ciocalteu), proteínas (método Bradford), hidratos de carbono (método Antrona), e analisadas as atividades biológicas, atividade antioxidante (ensaio 2,2-difenil-1-picrilhidrazil (DPPH)) e a atividade inibidora da enzima acetilcolinesterase (AChE). O extrato BDP apresentou os melhores resultados com uma concentração de fenóis totais de 0.559 μg GAE/μL, em proteínas de 23.838 mg BSA/mL, em hidratos de carbono 5.427 mg Glucose/mL de extrato, e com as concentrações em FT de 3.8_μg_GAE/mL e 20.8 μg GAE/mL revelaram 50 % da atividade antioxidante e atividade inibitória da AChE, respetivamente.
A metodologia aplicada para o estudo do isolamento dos CBs do extrato aquoso com extração assistida pelo enzima pancreatina (BDP) foi a tecnologia de membranas por Ultrafiltração (UF), em modo de recirculação total. Nesta etapa, cinco membranas assimétricas de acetato de celulose foram preparadas através do método de inversão de fase, com um intervalo de limite de exclusão molecular (MWCO) entre 3.8 kDa a 62 kDa. Os ensaios de permeação com o extrato de BDP foram realizados a três pressões transmembranares (PTM) (1, 2 e 3 bar).
As amostras da alimentação e permeados do extrato de BDP das cinco membranas foram caracterizados em termos de FT, proteínas e hidratos de carbono e foram avaliadas as atividades biológicas, atividade antioxidante pelo método DPPH e atividade inibitória da AChE. Verificou-se que os permeados das cinco membranas estudadas à PTM de 3 bar apresentaram maiores quantidades de FT recuperados. O permeado obtido da membrana com MWCO de 4.6 kDa à pressão transmembranar de 3 bar foi o que apresentou atividades biológicas mais elevadas, obtendo-se 50% da atividade antioxidante e atividade inibitória do enzima AChE para uma concentração de 1.8 μg GAE/mL e 25.9 μg GAE/mL de extrato, respetivamente. Na generalidade, verificou-se que todos os permeados do extrato BDP apresentam melhores atividades antioxidantes que o extrato original.
Os coeficientes de rejeição aparentes (f) para os FT, proteínas e hidratos de carbono foram determinados para todas as membranas e às PTM de 1, 2 e 3 bar, verificou-se que estes parâmetros diminuem com pressão transmembranar. Os coeficientes de rejeição intrínseco (f’) diminuem com o aumento do MWCO das membranas preparadas, enquanto o coeficiente de transferência de massa (k) aumenta com a porosidade das membranas. Verificaram-se fenómenos de polarização de concentração/colmatação para as membranas com MWCO de 32_kDa e 62 kDa.
Os extratos preparados e os permeados do extrato BDP foram analisados por cromatografia líquida de elevada performance com detetor de fotodíodos (HPLC-DAD) para se efetuar a quantificação de alguns CFs presentes: derivado do ácido ferúlico, rutina, derivado da vitexina e derivado da isovitexina. Denotou-se que após o processo de UF, a concentração do derivado do ácido ferúlico foi maior nos permeados resultantes das membranas com MWCO de 3.8 kDa e 4.6 kDa e, o derivado da isovitexina apresentou maiores concentrações em todos os permeados, com exceção da membrana com MWCO de 3.8 kDa.
Recorreu-se à cromatografia líquida de fase reversa acoplada a espectrometria de massa (LC-QTOF-MS) para identificação dos compostos presentes no extrato BDP (alimentação) e dos permeados resultantes das cinco membranas estudas à PTM de 3 bar. Os compostos identificados foram na maioria compostos fenólicos (CFs), tais como: o ácido p-cumárico, o ácido ferúlico, rutina e derivados da vitexina (vitexina -2”-O-ramnoside, apigenina 6,8-glucosideo, a schaftoside ou corymboside e a vitexina-6”-O-malonyl-2”-O-xyloside), também foram identificados ácidos orgânicos, como o ácido málico e ácido cítrico, aminoácidos (fenilalanina e triptofano) e o pigmento betanina.
Ficou ainda demonstrada a segurança na ingestão do extrato BDP e dos seus permeados resultantes das membranas com MWCO com 4.6 kDa e 62 kDa à PTM de 3 bar, uma vez que foi verificado que estes não apresentam citotoxidade em células Hep-G2 do hepatocarcinoma do fígado humano.
Neste trabalho, verificou-se que o extrato aquoso e os permeados de folhas e talos da beterraba, um resíduo agrícola de alto valor, apresentam potencial para a sua valorização dada a sua composição em compostos bioativos exibindo propriedades antioxidantes relevantes para a saúde. Com a aplicação da tecnologia de membranas foi possível isolar os CBs presentes no extrato BDP, permitindo obter um permeado que demonstrou elevada atividade antioxidante e que, dada a sua segurança, demonstra potencial de aplicação para suplementar dietas ou incorporação em produtos alimentares, tais como massas, sumos ou molhos.
The beet “Beta vulgaris L.”, of the Chenopodiaceae family, is a vegetable with high antioxidant activity, proven to prevent cardiovascular diseases and control dyslipidaemia. These features were seen to be directly related to the bioactive compounds (CBs) present in this vegetable. However, only the root of beet is typically consumed, and although its leaves and stalks are rich in bioactive compounds, they are often considered agricultural residues. The aim of this study is to evaluate the levels of phenolic compounds (CFs) and antioxidant properties of the aqueous extracts of beet leaves and stalks, and the ability of the membrane technology in isolating this extract. In this work, to avoid the use of organic solvent and to simulate the intake, several aqueous extracts from beet leaves and stalks were obtained: Decoction (Bdec), aqueous extract with enzymatic extraction with pepsin (BDG), aqueous extract with enzymatic extraction with pancreatin (BDP) and aqueous extract with enzymatic extraction with pepsin and pancreatin (BDC). The extracts were characterized by quantifying its total phenols (Folin-Ciocalteu method), proteins (Bradford method), carbohydrates (Anthrone method), and the biological activities were assayed, namely antioxidant activity (2,2-difenil-1-picrilhidrazil assay(DPPH)) and the inhibitory activity of enzyme acetylcholinesterase (AChE). In the end the BDP extract has presented the best results showing a 0.559 μg GAE/μL of total phenols, in proteins of 23.838 mg BSA/mL, in carbohydrates of 5.427 mg Glucose/mL, and total phenols concentration to obtain 50 % of antioxidant activity and inhibitory activity of enzyme AChE of 3.8 μg GAE/mL and 20.8 μg GAE/mL of total phenols, respectively. Additionally, a separation methodology was developed for the purification of bioactive compounds from BDP extract using membrane technology by ultrafiltration in recirculation mode. In this stage, five asymmetric cellulose acetate membranes were prepared through the phase inversion method covering a wide range of Molecular Weight Cut-Offs (MWCO) between 3.8 kDa and 62 kDa. The permeation experiments with the BDP extract were carried out at three transmembrane pressures (PTM) (1, 2 and 3 bar). The feed and permeate streams of the BDP extract from the five membranes were characterized and biological activities assayed. It was also found that the permeate of the five membranes obtained at the transmembrane pressure (PTM) of 3 bar presented a higher quantification of total phenols. But the permeate obtained from the membrane with MWCO of 4,8 kDa and the PTM of 3 bar presents higher ability to recover bioactive compounds that the total phenols concentration to obtain 50 % of antioxidant activity and inhibitory activity of enzyme AChE of 1.8 μg GAE/mL and 25.9 μg GAE/mL of total phenols, respectively. The permeates of the BDP extract present higher antioxidant activities than the original extract. The apparent rejection coefficients (f) for FT, proteins and carbohydrates decrease with the increase of the PTM, but the intrinsic rejection coefficients (f') decrease with the increase in MWCO of the membranes, while the coefficient of mass transfer (k) increase with the porosity of membranes. There phenomena of fouling and concentration of polarization of membranes were detected in the membrane with MWCO of 32 kDa and 62 kDa. With high performance liquid chromatography with photodyte detector (HPLC-DAD) the following CFs were quantified: derived from ferulic acid, rutin, vitexin derivative and isovitexin derivative. It was observed that the concentration of ferulic acid derivative is higher in the permeates resulting from membranes with MWCO of 3.8 kDa and 4.6 kDa and the derivative of isovitexin has higher concentrations in all permeates, except for the membrane with MWCO of 3.8 kDa. In addition, the liquid reverse phase chromatography coupled to mass spectrometry (LC-QTOF-MS) was used to identify the compounds present in the BDP extract (feed) and the permeates resulting from the five membranes at PTM of 3 bar. The compounds identified were mostly phenolic compounds (CFs), such as: p-cumaric acid, ferulic acid, rutin and vitexin derivatives (vitexin -2"-O-ramnoside, apigenin 6,8-glucoside, schaftoside or corymboside and vitexin-6"-O-malonyl-2"-O-xyloside), it was also identified organic acids (malic acid and citric acid), amino acids (phenylalanine and tryptophan) and pigment betaine. The safety of ingestion of BDP extract and permeates resulting of the membranes with MWCO of 4.8 kDa and 62 kDa at PTM of 3 bar was also been demonstrated, as they do not present cytotoxicity in Hep-G2 cells. In this work, a high value agriculture residue, such as beet leaves and stalks, was seen to have advantages to be incorporated in diet, displaying valuable health properties. When the aqueous extract is processed using membrane technology, many active bioactive compounds can be obtained. which ultimately can be used to supplement diets or incorporate food products, such as pasta, juices or sauces.
The beet “Beta vulgaris L.”, of the Chenopodiaceae family, is a vegetable with high antioxidant activity, proven to prevent cardiovascular diseases and control dyslipidaemia. These features were seen to be directly related to the bioactive compounds (CBs) present in this vegetable. However, only the root of beet is typically consumed, and although its leaves and stalks are rich in bioactive compounds, they are often considered agricultural residues. The aim of this study is to evaluate the levels of phenolic compounds (CFs) and antioxidant properties of the aqueous extracts of beet leaves and stalks, and the ability of the membrane technology in isolating this extract. In this work, to avoid the use of organic solvent and to simulate the intake, several aqueous extracts from beet leaves and stalks were obtained: Decoction (Bdec), aqueous extract with enzymatic extraction with pepsin (BDG), aqueous extract with enzymatic extraction with pancreatin (BDP) and aqueous extract with enzymatic extraction with pepsin and pancreatin (BDC). The extracts were characterized by quantifying its total phenols (Folin-Ciocalteu method), proteins (Bradford method), carbohydrates (Anthrone method), and the biological activities were assayed, namely antioxidant activity (2,2-difenil-1-picrilhidrazil assay(DPPH)) and the inhibitory activity of enzyme acetylcholinesterase (AChE). In the end the BDP extract has presented the best results showing a 0.559 μg GAE/μL of total phenols, in proteins of 23.838 mg BSA/mL, in carbohydrates of 5.427 mg Glucose/mL, and total phenols concentration to obtain 50 % of antioxidant activity and inhibitory activity of enzyme AChE of 3.8 μg GAE/mL and 20.8 μg GAE/mL of total phenols, respectively. Additionally, a separation methodology was developed for the purification of bioactive compounds from BDP extract using membrane technology by ultrafiltration in recirculation mode. In this stage, five asymmetric cellulose acetate membranes were prepared through the phase inversion method covering a wide range of Molecular Weight Cut-Offs (MWCO) between 3.8 kDa and 62 kDa. The permeation experiments with the BDP extract were carried out at three transmembrane pressures (PTM) (1, 2 and 3 bar). The feed and permeate streams of the BDP extract from the five membranes were characterized and biological activities assayed. It was also found that the permeate of the five membranes obtained at the transmembrane pressure (PTM) of 3 bar presented a higher quantification of total phenols. But the permeate obtained from the membrane with MWCO of 4,8 kDa and the PTM of 3 bar presents higher ability to recover bioactive compounds that the total phenols concentration to obtain 50 % of antioxidant activity and inhibitory activity of enzyme AChE of 1.8 μg GAE/mL and 25.9 μg GAE/mL of total phenols, respectively. The permeates of the BDP extract present higher antioxidant activities than the original extract. The apparent rejection coefficients (f) for FT, proteins and carbohydrates decrease with the increase of the PTM, but the intrinsic rejection coefficients (f') decrease with the increase in MWCO of the membranes, while the coefficient of mass transfer (k) increase with the porosity of membranes. There phenomena of fouling and concentration of polarization of membranes were detected in the membrane with MWCO of 32 kDa and 62 kDa. With high performance liquid chromatography with photodyte detector (HPLC-DAD) the following CFs were quantified: derived from ferulic acid, rutin, vitexin derivative and isovitexin derivative. It was observed that the concentration of ferulic acid derivative is higher in the permeates resulting from membranes with MWCO of 3.8 kDa and 4.6 kDa and the derivative of isovitexin has higher concentrations in all permeates, except for the membrane with MWCO of 3.8 kDa. In addition, the liquid reverse phase chromatography coupled to mass spectrometry (LC-QTOF-MS) was used to identify the compounds present in the BDP extract (feed) and the permeates resulting from the five membranes at PTM of 3 bar. The compounds identified were mostly phenolic compounds (CFs), such as: p-cumaric acid, ferulic acid, rutin and vitexin derivatives (vitexin -2"-O-ramnoside, apigenin 6,8-glucoside, schaftoside or corymboside and vitexin-6"-O-malonyl-2"-O-xyloside), it was also identified organic acids (malic acid and citric acid), amino acids (phenylalanine and tryptophan) and pigment betaine. The safety of ingestion of BDP extract and permeates resulting of the membranes with MWCO of 4.8 kDa and 62 kDa at PTM of 3 bar was also been demonstrated, as they do not present cytotoxicity in Hep-G2 cells. In this work, a high value agriculture residue, such as beet leaves and stalks, was seen to have advantages to be incorporated in diet, displaying valuable health properties. When the aqueous extract is processed using membrane technology, many active bioactive compounds can be obtained. which ultimately can be used to supplement diets or incorporate food products, such as pasta, juices or sauces.
Description
Trabalho final de mestrado para obtenção do grau de mestre em Engenharia Química e Biológica
Keywords
Beta vulgaris L. BDP Extrato aquoso Aqueous extract Extração assistida Assisted extraction Enzima pancreatina pancreatin enzyme Tecnologia de membranas Membrane technology Ultrafiltração Ultrafiltration
Citation
RODRIGUES, Márcia Patricia de Melo - Extratos aquosos de folhas e talos de “Beta vulgaris L.” : isolamento de compostos bioativos utilizando a tecnologia de membranas. Lisboa: Instituto Superior de Engenharia de Lisboa, 2019. Dissertação de mestrado.
Publisher
Instituto Superior de Engenharia de Lisboa