Isolation,Identification And Fermentation Process Of Main Fermented Microorganism In Weining Bean Saucee

With its unique flavor,Weining bean paste is very popular in Weining area.Weining area is mainly natural fermentation production,long-term production cycle,uneven product quality,low production efficiency and so on.Therefore,it is quite necessary to study the fermentation strain and technology of Weining soybean paste.In this study,the sauce koji produced in the traditional way of Weining area in Guizhou Province was used as a sample,and the microorganisms in the sauce were separated by conventional microbiological separation method.The isolated and purified strains were separately prepared into single bacteria suspension and inoculated into soybean powder.Pure fermentation is conducted.After repeated trials,the strains similar in taste to the traditional Weining bean paste were screened by sensory evaluation.According to the colony morphology and microscopic morphology,the two strains were initially identified,and the universal primers were used to amplify the 16 S r DNA or r DNA internal transcribed spacer gene was ligated into the p MD18-T vector and transformed into DH5? competent cells.The positive strain was selected for plasmid validation and sequence.Combined with the micro-automatic identification system for identification and its growth characteristics were studied.Combining the growth characteristics of the strains,the fermentation process was initially explored,and the physical and chemical indicators of the finished bean paste were analyzed.Finally,volatility substances of pure fermented soybean paste and natural fermented soybean paste were determined by gas chromatography-mass spectrometry(GC-MS).The volatility substances were compared and analyzed.The research results are as follows.1.22 strains of bacteria and 10 strains of fungi were isolated and purified from Weining sauce koji.The sensory evaluation results showed that the flavor of soybean paste fermented by bacterial DX-9 and fungus DZ-3 was similar to that of traditional Wining bean paste.DX-9 was identified as Bacillus subtilis,DZ-3 as Wickerhamomyces anomalus.Identification by the methods of molecular biology and the automatic identification system of microorganism.The results showed that the optimum growth temperature and p H of DX-9 were 38? and 7.0,respectively.The optimum growth temperature and p H of DZ-3 were 30? and 5.5,respectively.2.Through the orthogonal experiment,the optimum addition amount of the auxiliary material in soybean paste was determined as follows: salt 11%,chili powder 6%,five flavor powder 1%.The optimum koji-making time of DX-9 was 12 days,the temperature was 38?,the after-fermentation time was 90 days,the temperature was 40? and the amount of inoculu was 2%.The optimum koji-making time of DZ-3 was 18 days,the temperature was 34?,the fermentation time was 90 days,the temperature was 36? and the amount of inoculu was 3%.3.The results showed that the total acid,amino acid nitrogen,water content,salt content and p H of DX-9 pure fermented soybean paste were 1.66 g / 100 g,0.96 g / 100 g,55%,11.5 %,and 4.8,respectively.The total acid,amino acid nitrogen,water content,salt content and p H of DZ-3 pure fermented soybean paste were 1.53 g / 100 g,0.75 g / 100 g,52%,11.8 % and 5.6,respectively.The detection of coliform group and 2 pathogenic bacteria(Salmonella,Staphylococcus aureus)in 3 kinds of soybean sauce showed that their contents were lower than the corresponding requirements in the National Food Safety Standard GB 2718—2014 Brewing Sauce.Compared with the three kinds of soybean paste,natural fermentation and DX-9 fermented bean paste were brown,while DZ-3 fermented bean paste was reddish brown,and the flavor of natural fermented soybean paste was rich in odour.The pure seed fermented soybean paste has the flavor of sauce and ester.In taste,three kinds of finished soybean paste taste fresh and mellow,salty and sweet,but pure fermented bean paste is more delicate.From the body state,natural fermentation is sparser than pure strain fermentation.4.GC-MS determination of volatile substances showed that 66 kinds of volatile substances were detected in natural fermented soybean paste,including 6 kinds of alcohols,4 kinds of ketones,17 kinds of esters,2 kinds of acids,9 kinds of aldehydes.There are 1 kind of furan,22 kinds of hydrocarbons,1 pyrazine,1 pyrrole,1 sulfur compound,and 1 other compound.73 kinds of DZ-3 fermented soybean paste were detected,including 10 kinds of alcohols,7 kinds of ketones,12 kinds of esters,6 kinds of aldehydes,3 kinds of acids,3 kinds of nitriles,2 kinds of furans,21 kinds of hydrocarbons.There are kinds 5 kinds of pyrazines,1 kind of pyrrole,1 kind of sulfur-containing compound,and 2 kinds of other compounds.52 species were detected in DX-9,including 6 alcohols,2 ketones,11 esters,3 acids,4 aldehydes,1 furan,16 hydrocarbons,and 1 phenol.There are 3 kinds of pyrazines,1 kind of pyrrole,1 kind of sulfur-containing compound,and 1 kind of other compounds.The principal component analysis of volatile substances showed that the main non-volatile substances in Weining bean paste were hydrocarbons and esters.To sum up,the microbial separation,rejoining test and identification of the main fermentation in Weining bean paste koji were carried out.The fermentation flavor of one strain of Wickerhamomyces anomalus and one strain of Bacillus subtilis was similar to that of natural fermentation.Combined with the growth characteristics of two strains,the production technology was explored to determine the amount of supplementary materials in the fermentation process,and the time and temperature of making koji,the time and temperature of fermentation,and the inoculum quantity of two strains of fermentation bacteria.Main volatile substances in Weining bean paste were identified by GC-MS.In order to improve the quality and stability of the product,stabilize the flavor of soybean paste,reduce the production cost and improve the production efficiency,two main strains of fermentation were applied to the pure seed fermentation of Weining soybean paste,and the reference strains were also provided for the pure seed fermentation of Weining soybean paste.