Characterization Of The Milk Clotting Enzyme From Bacillus Screening In Jiuqu And The Application In Cheese-making

Milk clotting enzyme(MCE)is the key enzyme used in cheese production to solidify raw milk,which not only acts as a milk clotting agent but also affects the texture and flavor of the cheese.A strain producing MCE was screened in this study from the rice wine“wheat Qu”,and it was identified as Bacillus methanolicus.The enzymatic and structural properties of the MCE were studied.Finally,the MCE was applied to make Mozzarella cheese.The results of this study were as follows:(1)In this study,the microbes in the rice wine“wheat Qu”were screened and two excellent bacterial strains of LB-1 and LB-2 producing MCE were obtained,which were further identified as Bacillus methanolicus and Bacillus subtili.According to the MCA curve,the highest MCA of the enzymes from the two strains were 269.66±0.78 SU/m L and 187.50±1.4 SU/m L at 24 h fermentation,while the PA were 1.476±0.49 U/m L and1.29±1.41 U/m L,respectively.The C/P were 187.50 and 145.34,respectively.B.methanolicus LB-1,which had relatively high clotting activity,was selected for subsequent experimental studies.(2)The medium components were optimized using single-factor test and respose surface methodology(RSM)and determined to be 4.14g/L lactose,7.47g/L of casein,0.6g/L of K₂HPO4 and 8.31g/L of yeast extract.Under this condition,the MCA was329.90 SU/m L,which was close to the theoretical prediction of 379.53 SU/m L.On the basis of this,the optimum fermentation conditions were optimized which were as follows,fermentation time 57 h,fermentation temperature 32?,p H 6.7,loaded culture volume40%(v/v),inoculum size 4%(v/v)and agitation speed 120 r/min.Under this condition,the MCA was 578.40 SU/m L,which was close to the theoretical prediction of 530.68SU/m L,and the MCA was 2.14 times that before optimization.(3)The MCA of the purified MCE reached 597,310±0.13 SU/g,the optimal temperature was determined to be 50?,and the optimal p H was 6.5.The MCE was identified by LC-MS to be a putative protein(ID I3EB99),there was less?-helix than?-sheet in the secondary structure,and the prokaryotic expression vector of E.coli was constructed to obtain the recombinant MCE of B.methanolicus with MCA of 15870±1.17 SU/g,which met the requirements of cheese processing.(4)In order to study the effect of B.methanolicus MCE on the processing characteristics of mozzarella cheese,the single B.methanolicus MCE and the enzyme mixture containing 10%of B.methanolicus MCE and 90%of commercial rennet were used in mozzarella cheese preparation,respectively,while cheese made with commercial rennet was prepared as the control group,the texture and flavor during ripening of the cheeses were determined.The cheese made with single B.methanolicus MCE had relatively poor shape-preserving property with low sensory score.The cheese made with the enzyme mixture was comparable with that of the control in terms of texture and sensory scores,indicating that the MCE from B.methanolicus could be used as a partial substitute for commercial rennet in cheese production.