TEMPERATURE AND PH OPTIMIZATION FOR ANTIOXIDANT ACTIVITY AND DEGREE OF HYDROLYSIS OF WHEY PROTEIN HYDROLYZED BY PROTEASE OBTAINED FROM BACILLUS STRAIN ISOLATED FROM KINEMA

Abstract

This study aimed to assess identification of strain using morphological study and biochemical tests and protease enzyme derived from that strain, isolated from Kinema. The refinement process for strain involved media optimization, involving optimization of pH, temperature, glucose concentration and nitrogen source along with time of incubation. Extraction of protease was carried out using same strain, followed by purification using ammonium sulfate. Subsequently, the pH and temperature conditions were optimized through response surface methodology (RSM), employing DPPH and DH as indicators. The antioxidant activity of the optimized protease was assessed through fractionation into <3kDa and >3kDa, with a comparative analysis against unhydrolyzed whey protein using both DPPH and ABTS scavenging. Finally, electrophoresis was employed using SDS PAGE to determine the extent of hydrolysis on different whey proteins, providing insights into the enzymatic specificity of the Bacillus-derived protease. Biochemical tests revealed the strain is similar to Bacillus subtilis. The optimum condition for protease production from strain was evaluated as pH 8, temperature 40°C and glucose concentration was 0.5%(w/v) with nitrogen source as defatted soyabean meal at incubation time of 48 hours. The extracted protease was partially purified by a factor of 5.62 with specific activity 5.88U/mg and 76.4% yield with using ammonium sulphate precipitation (0-80%) followed by dialysis. The optimum conditions for enzymatic hydrolysis of WPC to achieve maximum degree of hydrolysis (10.24%) and DPPH radical scavenging activity (18.02%) were observed at 54.963°C and pH 8. Comparative antioxidant activity among fractioned whey protein hydrolysate and unhydrolyzed hydrolysate revealed <3kDa fraction possessed maximum antioxidant activity in both DPPH and ABTS scavenging activity with lowest IC50 value of 29.09 mg/ml and 6.70 mg/ml respectively. Finally electrophoretic analysis resulted in higher degree of hydrolysis β-lactoglobulin compared to α-lactalbumin in interval of 1hour hydrolysis. On comparison of higher molecular weight peptides i.e. lactoferrin(80kDa) and BSA (60kDa) shows that lactoferrin has gone higher degree of hydrolysis compared to BSA.