The Effect Of Lactobacillus Bulgaricus Cell Wall Proteinase On Properties Of GDL Milk Gel

Cell wall proteinase(CEP)is a hydrolase anchored on the cell wall of lactic acid bacteria.Its substrate is casein,and casein hydrolysis is a key step in the fermentation proces s of yogurt.Therefore,cell wall proteinase will have a certain impact on the fermentation of yogurt.The previous studies have suggested that the cell wall proteinase improve the texture of the milk gel.This study focuses on the effect of cell wall proteinase on the properties of the milk gel.In this study,we identified the lactic acid bacteria CH9-3 strain as Lactobacillus delbrueckii subsp.bulgaricus by the 16 S r RNA sequence and API system.The gene fragment of cell wall proteinase in Lactobacillus bulgaricus CH9-3 strain was amplified and cloned and subsequently analyzed by using the NCBI database,DNAstar and Bio Edit software to obtain the information of bioinformatics.Results showed that the gene fragment encoded a protein containing 1976 amino acids,in which,1494 are hydrophilic amino acids,implied that the protein is a hydrophilic protein.Moreover,the protein has an instability coefficient of 18.75,means that this protein is stability.In addition,the irregular bristle accounts for 56.88% and the extended backbone accounts for 22.93%.The ?-helix accounts for 20.19%.The cell wall proteinase was extracted by 5 mol/L Li Cl and purified by DEAE Sefinose TM FF agarose gel and ultrafiltration.A single electrophoresis band of 190 k Da was observed in SDS-PAGE,and identified to be the proteinase by QE Mass spectrometry.Thereafter,the ability to hydrolyze casein of this protein was verified in milk.The gluconate-?-lactone(GDL)acidified skim milk model was optimized,and results showed that 1.75% of GDL was most suitable to simulate the yogurt fermentation process in the acidification model.The purified proteinase was then added to the GDL acidification model,and the properties of the milk gel were characterized.Results showed that the free amino acids of the milk gel were significantly increased(P < 0.05),the texture was significantly enhanced by significantly increase(P < 0.05)of the hardness,consistency,viscosity,and adhesion index.The distribution of larger particle size was significantly increased(P < 0.05),the zeta potential was significantly decreased(P < 0.05),the syneresis coefficient was significantly decreased(P < 0.05).This study found that cell wall proteinase derived from Lactobacillus bulgaricus CH9-3 strain could significantly improve the quality of the milk gel by increasing the hardness,consistency,viscosity index and degree of proteolysis of the milk gel,and reducing the syneresis rate.Furthemore,the performance and the formation of the milk gel were promot ed by lowering the surface charge of the milk gel by the cell wall proteinase.In conclusion,the cell wall proteinase of CH9-3 strain has a positive effect on the properties of milk gel,and has potential benefit for the manufacturing of yogurt and other protein products.