Efeito do processamento de alta pressão isostática em enzimas coagulantes do leite

Author(s)

Bruno Ricardo de Castro Leite Júnior

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia de Alimentos
• Defense date: 2017-03-27

Examining board members

Marcelo Cristianini; Helia Harumi Sato; Walkíria Hanada Viotto; Ariene Gimenes Fernandes Van Dender; Daniel Augusto Cavalcante

• Advisor: Marcelo Cristianini

Abstract

The High Isostatic Pressure (HIP) processing promotes changes in enzyme activity. In this context, this study aimed to: optimize the process conditions by HIP to maximize milk clotting activity (MCA) of five coagulants (protease obtained from Rhizomucor miehei, calf rennet, recombinant chymosin, bovine rennet and porcine pepsin); evaluate MCA at different pH and temperature; evaluate the stability during storage; analyze the degree of ?-casein (?-CN) hydrolysis by proteases action; describe the structural changes in the enzymes induced by HIP; and monitor the coagulation process by different methods. Pressures up to 300 MPa for times and temperatures below 10 min and 25 °C, respectively, resulted in enzyme activation. On the other hand, under more drastic conditions a reduction of enzymatic activity was observed, with complete inactivation in processes performed above 550 MPa with times >10 min and temperatures >45 °C. Specifically, HIP processed coagulants (mainly enzymes with high chymosin concentration) showed a higher degree ?-CN hydrolysis compared to the non-processed enzymes. In addition, pressurization improved the stability of the enzymes at different pH values after processing and at optimum pH during storage. The protease obtained from R. miehei presented greater resistance to the process, being the only one without activation. Structural evaluation of the enzymes indicated that the HIP process increased the exposure of the aminoacid tryptophan (assessed by intrinsic fluorescence) under the activation conditions and promoted a drastic reduction of this exposure under inactivation conditions indicating a molecular unfolding after HIP. These results were confirmed by the increased surface hydrophobicity observed for samples processed by HIP. Similarly, the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy results confirmed that the HIP process produced significant changes in the secondary structures of the enzymes with the highest changes in higher pressures. To evaluate the practical effects of HIP-induced changes on enzymes, the coagulation of milk by these enzymes was accompanied by methods of near-infrared spectroscopy (which evaluates the degree of protein aggregation), rheological assay (which determines the gel consistency) and confocal microscopy (which characterizes the gel porosity). These results demonstrated that enzymes processed by HIP under optimized conditions promoted faster coagulation and the gels were more consistent and with a higher degree of protein network aggregation compared to those produced using the non-processed enzymes. These effects are attributed to the higher hydrolysis degree of k-CN fraction hydrolysis during the first coagulation phase. In terms of yield, gels obtained with HIP-processed enzymes were found to have a higher fresh yield compared to those obtained from the respective non-processed enzymes (up to 4.3% increase). Therefore, the HIP process application can improve the performance and competitiveness of theses coagulants with consequent reduction of costs (AU)

• FAPESP's process: 14/17782-4 - Effect of high isostatic pressure processing on milk-clotting enzymes
• Grantee: Bruno Ricardo de Castro Leite Júnior
• Support Opportunities: Scholarships in Brazil - Doctorate