Isolation And Identification Of Bacillus With Penicillium-Resistant And Flavor-Producing Properties From Fermented Soybean Paste

Microorganisms play an important role in the production of soybean paste by traditional natural fermentation,which is closely related to the formation of color,flavor and nutritional components of soybean paste.Previous studies have shown that Bacillus is the dominant bacteria in traditional fermentation soybean paste.It exists in all stages of soybean paste fermentation,and its content increases significantly in the middle and late stages of soybean paste fermentation,and Bacillus is responsible for the formation of soybean paste.The specific mechanism of action is not yet clear,and needs to be further explored.In this study,16 samples of traditional fermented soybean paste were collected from 11 regions in Northeast China.Bacillus strains with potential probiotic characteristics were isolated from the samples,and their anti-penicillium and flavor-producing properties were studied.The following conclusions were drawn:1.In this experiment,62 strains of Bacillus were isolated from 16 samples of traditionally fermented soybean paste collected from 11 regions in Northeast China.14 strains of Bacillus with with good intestinal tolerance and significant antibacterial effects against Staphylococcus aureus,Listeria monocytogenes and Escherichia coli were screened by colony counting method and Oxford cup agar diffusion method.2.The 14 strains obtained by screening were identified by 16 S r RNA gene sequence homology analysis.And the strains were all confirmed to be Bacillus,including 3 strains of Bacillus licheniformis,3strains of Bacillus amyloliquefaciens and 8 strains of Bacillus subtilis.3.Two strains of Bacillus subtilis QQHE-2 and DQ1-3 which had a good inhibitory effect on Penicillium were screened by plate pairing method and agar column method.The inhibition rate of Penicillium can reach 83.42% and 46.83%.The external morphology and internal structure of Penicillium were observed by SEM and TEM.It was found that the bacteriostatic substance produced by strain QQHE-2made the spore stalk of Penicillium spoiled and slender,the number of spores decreased significantly,and vacuoles appeared in the cells.The expansion of the mitochondria has undergone significant changes.4.By adjusting the p H and temperature of the fermentation supernatant of the strain and treating with protease,it was found that the antibacterial substance secreted by the strain had poor acid resistance and heat resistance,and the inhibition rate against Penicillium was significantly reduced after protease treatment.The effective component of the antibacterial substance was determined as a protein substance.The molecular weight of the antibacterial substance produced by QQHE-2 strain was about35 k Da by SDS-PAGE gel electrophoresis,and the conditions for the production of antibacterial substances of the two strains were optimized by the orthogonal test.5.Screening of strains for flavor characteristics and 14 strains producing protease and amylase were screened by the transparent circle method.The viability of the two enzymes was combined to select 9strains for fermenting soybeans.The detection of free amino acid content and volatile flavor substances in the sample finally gave Bacillus subtilis DQ1-3,DQ1-1,JMS2-6 strain as the dominant Bacillus.The above results provide technical support for preventing mould contamination and improving the flavor quality of soybean paste,and provide theoretical basis for the development and utilization of Bacillus.