Characteristic Of Protease From Bacillus Cereus And Its Effects On Milk Quality

As one of the food-borne pathogens with a high detection rate in milk,Bacillus cereus is heat-resistant and toxicogenic.Besides,the metabolic enzymes from Bacillus cereus can also seriously affect the quality and safety of milk,especially the thermostable protease,which can limit shelf life of the milk and induce large economic losses by causing milk age gelation and bitter.It is significant to investigate the characteristics of protease from Bacillus cereus and its effect on the quality of milk.The object in this research was the most thermotolerant protease secreted by B.cereus C58,which was screened from many strains.The molecular weight and characteristics of the protease purified from B.cereus C58 were identified;In addition,the proteolytic activity of B.cereus C58were analyzed;Moreover,we also investigated the hydrolysis of milk proteins,the changes of physical stability and the trends in flavor of the milk added protease and stored at different conditions?28°C 24 h?10°C 6 d?.The conclusions obtained through above trials were as follows:?1?Bacillus cereus showed a strong ability to produce protease and their proteases were heat-resistant to some extent.23 strains were able to produce proteases and lipases of the 55 strains,the protease activity and lipase activity were among 10?29.20 U/m L and 1?4.40 U/m L respectively?37°C 48 h?.Overall,the proteases showed high heat resistance than lipases from Bacillus cereus strains,of which the protease from B.cereus C58 was the most thermotolerant.After the heat treatment at 70°C for 30 min and 100°C for 10 min,the relative proteolytic activity was 81.97%and 40.98%respectively,which had a serious threat to milk quality.?2?The characteristics of the protease from B.cereus C58 were clarified.The protease was purified by ammonium sulphate precipitation,DEAE-Sepharose F.F ion-exchange chromatography and Sephadex G-100 gel-filtration chromatography.The total protein was 2.74 mg,the purification fold was 78.81,and the yield was 11.63%.The molecular mass was identified as 43.907 k Da and described as Protease Hho A,which had serine endopeptidase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?and mass spectrometry.The optimum p H and temperature were 9 and 60°C.The protease was alkaline thermostable enzyme.Ca²⁺and Fe²⁺increased protease activity,and Cu²⁺decreased the activity,Mg²⁺had no effect.The protease was stable in the temperature from 110 to 130°C,The D-values were among 195.13?677.23 s,the half-lives were among 58.74?203.87 s.?3?The production of protease from B.cereus C58 was increasing with the proliferation of bacteria.B.cereus C58 could synthesize large variety of extracellular protease in the exponential phase.When the incubation temperature was 28°C and 10°C,The proteolytic activity increased slowly in lag phase,was 0?2 U/m L and 0?0.40 U/m L respectively,and were 2?29.70 U/m L and0.40?14.40 U/m L in exponential phase,while were 29.70?40.09U/m L in the early stage of decline phase at 28°C,which indicated the proteolytic activity increased quickly in the exponential phase?4?The effect of protease from B.cereus C58 on milk quality was investigated.The protease hydrolyzed?-casein preferably,followed with?-casein and?-casein,?-lactalbumin and?-lactoglobulin of whey proteins are resistant to degradation.The hydrolysis milk led the particle size,viscosity and degree of proteolysis to increase,the stability of the dispersion system to disorder,and the formation of gels.Besides,the differences of the content of protein and fat in the top and bottom layers became larger.At 28°C,the milk fat globules showed a tendency to settle first and then float up,and at 10°C,the milk fat globules showed a tendency to settle.The main flavor characteristics of milk were bitter,volatile odors of oxynitride,sulfur-containing compounds,methyl compounds,aldehydes and ketones during the hydrolysis of milk.