Study On The Dynamic Changes Of Microbial Community During Fermentation Process Of Soy Sauce

Soy sauce is a kind of traditional condiment and has a long history , it is very popular inChina.Soy sauce production was produced by high-salt diluted state fermentation process inmost of Guangdong condiment enterprises.The character of this process is opened all day andlong time fermentation cycle,the production is sweet-smelling and taste-well.During thefermentation period,microoganism is very important to control the quality of soy sauceproduction,but rarely relevant researching papers were pubilished.The fermentation process isrelated to the collabaration of many beneficial microoganisms,especially lactic acid bacteriaand yeast take very important role in saccharides fermentation to yield flavor materials,whichis the main route to generate the flavor of soy suace including alcohols,aldehydes,carboxylicacids,esters and phenols.In this study, the changes of amino nitrogen content, total acid content, pH value andNaCl content in fermentation process was analyzed 16S rDNA DGGE(denaturing gradient gelelectrophoresis) fingerprinting and phylogenetic analysis were used to reveal the dynamicchanges of flora during fermentation of soy sauce. The main results were as follows:1. During the fermentation period, within former 15 days, amino nitrogen contentincreased rapidly, approximate to 0.8g/100mL, 15 days later, its content increase slowly,finally approximate to 1.0g/100mL. The content of total acid increased rapidly, pH value andNaCl content decreased slowly within the former 15 days, after then, the changes of the threeproperties almost remain stable.2. The effect of DNA extraction of two kinds DNA extraction kits were compared,agarose electrophoresis map shows DNA extraction effect of Soy sauce DNA extraction kitswas better.3. The DNA concentration in PCR system and annealing temperature of Touch downPCR was optimized. The results demonstrate the optimum conditions of PCR amplificationwere as following: DNA concentration is 6μL, annealing temperature is 65℃.4. Effect of DNA concentration and electrophoresis time on DGGE fingerprint wasstudied. The optimum condition were as following: DNA concentration is 20μL,electrophoresis time is 3.5h.5. The flora diversity during fermentation of soy sauce was analysed. DNA of the totalbacteria was extracted from the samples, and the touch down PCR was applied to amplify theV3 region of 16S rDNA, judging from DGGE fingerprints,18 specific gel bands were excised,then sequenced and analysed by GenBank database. The results were following: 4 bands represent Weissella cibaria.5 bands represent uncultured microoganism, unculturedFirmicutes bacterium, uncultured bacterium, uncultured bacterium, Uncultured Enterobactersp. and Uncultured Klebsiella sp. Respectively. The others are Weissella paramesenteroides,Lactobacillus fermentum, Citrobacter freundii, Enterobacter sp. BF1-8, Chromohalobactersp., Pantoea sp., Tetragenococcus halophilus, Tetragenococcus sp..6. The flora diversity during fermentation of soy sauce was analysed. PCR-DGGE wasused to detect the dynamic changes of flora in the process of soy sauce fermentation.Weissella cibaria was the dominating microbial specie in the whole fermentation process,while Weissella paramesenteroides, Lactobacillus fermentum, Citrobacter freundii andEnterobacter sp. BF1-8 showed the trend that their content decreased gradually asfermentation processing. Tetragenococcus halophilus and uncultured bacterium occurred 15days latter in the fermentation process, and had the trend to become the dominate bacterias.The other 8 stains disappeared at the beginning of fermentation process. In conclusion, theregularity of the dynamic changes of flora in the process of soy sauce fermentation wasmultiplicity to simplicity. Meanwhile,to some extent, the changes of total acid, pH value,NaCl conten and amino nitrogen content was related to the regularity of the dynamic changesof flora in the process of soy sauce fermentation.