Imobilização de alcalase em pó de sabugo de milho: hidrólise das proteínas do soro de queijo bovino e obtenção de peptídeos bioativos
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Data
2017-12-21
Autores
Cruz, Clariana Zanutto Paulino da [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O atual trabalho teve como objetivo realizar a hidrólise das proteínas do soro do queijo bovino (SQB) utilizando a alcalase® imobilizada em pó de sabugo de milho, para obtenção, purificação parcial e avaliação das propriedades biológicas de peptídeos bioativos. O sabugo de milho (SM) é o principal resíduo formado da indústria de processamento de milho. Alcalase® foi usada para a imobilização em SM e agarose. O desempenho dos dois derivados foi estudado em relação aos parâmetros cinéticos de imobilização. A hidrólise das proteínas do SQB em sistema descontínuo foi realizada em reator de batelada (pH 9,0; 50ºC/48h) usando a alcalase® livre (AL) e derivados alcalase®-glioxil-agarose (AGA); alcalase®-glioxil-SM (AGSM) para fins de comparação. O grau de hidrólise (GH) para AL, AGA e AGSM foi de 59,63, 43,88 e 26,59% respectivamente. O derivado estável AGSM apresentou capacidade de cinco ciclos de reuso de 24 h consecutivos mantendo atividade maior que 70% e exibiu estabilidade térmica (65ºC/pH 7,0) 62 vezes maior do que a AL, retendo 12% de sua atividade inicial após 48 horas, enquanto que a AL e AGA foram totalmente inativados. Os hidrolisados proteicos do soro (HPS) obtidos com AGSM foram fracionados por RP-HPLC em três frações (F1, F2 e F3) de acordo com a hidrofilicidade dos peptídeos (F1: hidrofílicos, F2: hidrofilicidade intermediária e F3: hidrofóbicos). As frações foram analisadas por MALDI-TOF que identificou peptídeos de massa molecular (<1500 m/z). HPS obtido por AGSM antes do fracionamento apresentou elevada capacidade de redução do radical ABTS (57,82%) e elevada atividade quelante de ferro II (76,2%). Houve diferença significativa (P<0.001) na atividade antioxidante entre as três frações (F1>F2C>F3) e a atividade quelante de ferro para as frações (F1, F2 e F3) foram nulas. As espécies E. coli (ATCC 43895) e Listeria monocytogenes (ATCC 7644) foram susceptíveis à ação dos peptídeos obtidos neste presente estudo. Os peptídeos de hidrofilicidade intermediária (F2) apresentaram maior atividade antimicrobiana, tanto para E. coli quanto para L. monocytogenes, sendo mais efetivos contra a segunda (p<0,05). Apenas F2 mostrou ser efetivo contra a cepa de Candida albicans ATCC 18804 testada (MIC= 10 mg/mL). Para a hidrólise das soroproteínas em sistema contínuo (50oC/pH 9,0), empregou-se um reator de leito empacotado (PBR), contendo 25 g do derivado AGSM (8,66 U/g). Foi feita a caracterização hidrodinâmica do reator utilizando o corante vermelho de fenol (0,6 g.L-1) como traçador. O PBR operou numa vazão de 6 mL.h-1, o que representou um tempo de residência de 12,9 h, aproximadamente 19% maior do que o tempo espacial (10,8 h). Portanto, a formação de caminhos preferenciais foi mínima e o sistema apresentou alto grau de hidrólise (58,98-70,70%) durante 180 horas de reação. O tempo de meia-vida do derivado AGSM foi bastante elevado (246 h) e foram obtidos peptídeos bioativos de massa molecular (<2600 m/z) com alta capacidade antioxidante (65,27% de redução de ABTS). Neste trabalho, o SM apresentou grande potencial como suporte de baixo custo para a imobilização de alcalase®. Os resultados deste estudo mostram que a hidrólise das proteínas do SQB utilizando o derivado AGSM foi possível para sistemas descontínuos e contínuos. O processo utilizado evidencia uma grande perspectiva para a indústria alimentícia e farmacêutica, pois, além de contribuir para a melhoria do meio ambiente pelo reuso do sabugo, possibilita reutilização do soro e obtenção de peptídeos bioativos com elevada capacidade antioxidante e antimicrobiana.
The objective of the present work was to hydrolyze bovine cheese whey proteins (BCW) using the alcalase® immobilized on corn cob powder (CCP), to obtain partial purification and evaluation of the biological properties of bioactive peptides. Corn cob (CC) is the main residue formed in the maize processing industry. Alcalase® was used for the immobilization on CCP and on agarose. The performance of the two derivatives enzymatic was studied in relation to the kinetic properties of immobilization. The free alcalase® (FA) and the derivatives: alcalase®-glyoxyl-agarose (AGA); alcalase®-glyoxyl-corn-cob-powder (AGCCP) were used for the hydrolysis of bovine cheese whey proteins in a batch reactor (pH 9.0, 50oC/48h). The degree of hydrolysis (DH) for FA, AGA and AGCCP was of 59.63, 43.88 and 26.59% respectively. However, AGCCP derivative was very stable and presented reuse capacity of five consecutive 24-hour cycles maintaining its activity greater than 70%. In addition, AGCCP exhibited thermal stability (65ºC, pH 7.0) 62 times greater than FA, retaining 12% of its initial activity after 48 hours, while FA and AGA were totally inactive. The whey protein hydrolysates (WPH) obtained with AGCPP were fractionated by reverse-phase HPLC according to the hydrophilicity of the peptides (F1: hydrophilic, F2: intermediate hydrophilicity and F3: hydrophobic). The fractions were analyzed by MALDI-TOF and molecular weight peptides (<1500 m/z) were identified. Total WPH obtained by AGCCP presented high capacity reduction of the ABTS radical (57.82%) and high chelating activity of iron II (76.2%). A significant difference (P<0.001) in the antioxidant activity between the three fractions (F1>F2C>F3) was observed while the chelating activity of iron for the all fractions (F1, F2, and F3) was null. The obtained peptides showed high antimicrobial activity (87.75-100%) against the species E. coli (ATCC 43895) and Listeria monocytogenes (ATCC 7644). Only F2 was effective against the Candida albicans strain ATCC 18804 tested (MIC=10 mg/mL). A packed bed reactor (PBR) containing 25 g of the AGCCP derivative (8.66 U/g) was used for the hydrolysis of whey proteins in a continuous system process (50°C/pH 9.0). The hydrodynamic characterization of the reactor was performed using red phenol dye (0.6 g.L-1) as a tracer. The PBR operated at a flow rate of 6 mL.h-1, which represented a residence time of 12.9 h, approximately 19% higher than the spatial time value (10.8 h). Therefore, the formation of preferential paths was minimal and the system presented a high degree of hydrolysis (58.98-70.70%) during 180 hours of reaction. The half-life of the AGSM derivative was very high (246 h) and bioactive peptides of molecular weight (<2600 m/z) with high antioxidant capacity (65.27% ABTS reduction) were obtained. In this work, CCP presented great potential as a low cost support for alcalase® immobilization. The results of this study show that the hydrolysis of SQB proteins using the AGSM derivative was possible for discontinuous and continuous systems. The process used shows a great prospect for the food and pharmaceutical industry, as well as contributing to the improvement of the environment by the reuse of the cob, allows the reuse of the whey and the obtaining of bioactive peptides with high antioxidant and antimicrobial capacity.
The objective of the present work was to hydrolyze bovine cheese whey proteins (BCW) using the alcalase® immobilized on corn cob powder (CCP), to obtain partial purification and evaluation of the biological properties of bioactive peptides. Corn cob (CC) is the main residue formed in the maize processing industry. Alcalase® was used for the immobilization on CCP and on agarose. The performance of the two derivatives enzymatic was studied in relation to the kinetic properties of immobilization. The free alcalase® (FA) and the derivatives: alcalase®-glyoxyl-agarose (AGA); alcalase®-glyoxyl-corn-cob-powder (AGCCP) were used for the hydrolysis of bovine cheese whey proteins in a batch reactor (pH 9.0, 50oC/48h). The degree of hydrolysis (DH) for FA, AGA and AGCCP was of 59.63, 43.88 and 26.59% respectively. However, AGCCP derivative was very stable and presented reuse capacity of five consecutive 24-hour cycles maintaining its activity greater than 70%. In addition, AGCCP exhibited thermal stability (65ºC, pH 7.0) 62 times greater than FA, retaining 12% of its initial activity after 48 hours, while FA and AGA were totally inactive. The whey protein hydrolysates (WPH) obtained with AGCPP were fractionated by reverse-phase HPLC according to the hydrophilicity of the peptides (F1: hydrophilic, F2: intermediate hydrophilicity and F3: hydrophobic). The fractions were analyzed by MALDI-TOF and molecular weight peptides (<1500 m/z) were identified. Total WPH obtained by AGCCP presented high capacity reduction of the ABTS radical (57.82%) and high chelating activity of iron II (76.2%). A significant difference (P<0.001) in the antioxidant activity between the three fractions (F1>F2C>F3) was observed while the chelating activity of iron for the all fractions (F1, F2, and F3) was null. The obtained peptides showed high antimicrobial activity (87.75-100%) against the species E. coli (ATCC 43895) and Listeria monocytogenes (ATCC 7644). Only F2 was effective against the Candida albicans strain ATCC 18804 tested (MIC=10 mg/mL). A packed bed reactor (PBR) containing 25 g of the AGCCP derivative (8.66 U/g) was used for the hydrolysis of whey proteins in a continuous system process (50°C/pH 9.0). The hydrodynamic characterization of the reactor was performed using red phenol dye (0.6 g.L-1) as a tracer. The PBR operated at a flow rate of 6 mL.h-1, which represented a residence time of 12.9 h, approximately 19% higher than the spatial time value (10.8 h). Therefore, the formation of preferential paths was minimal and the system presented a high degree of hydrolysis (58.98-70.70%) during 180 hours of reaction. The half-life of the AGSM derivative was very high (246 h) and bioactive peptides of molecular weight (<2600 m/z) with high antioxidant capacity (65.27% ABTS reduction) were obtained. In this work, CCP presented great potential as a low cost support for alcalase® immobilization. The results of this study show that the hydrolysis of SQB proteins using the AGSM derivative was possible for discontinuous and continuous systems. The process used shows a great prospect for the food and pharmaceutical industry, as well as contributing to the improvement of the environment by the reuse of the cob, allows the reuse of the whey and the obtaining of bioactive peptides with high antioxidant and antimicrobial capacity.
Descrição
Palavras-chave
Enzimas proteolíticas, Hidrolisados de proteína, Peptídeos, Resíduos agrícolas, Soro do leite