Molecular Biological Identification Of Lactic Acid Bacteria From Yanggaomeijiu Daqu

As the fermentation starters of Yanggaomeijiu,Yanggaomeijiu Daqu was the starter culture and saccharifying agent,which was the impetus of brewing and contained a large diversity of microorganisms and enzymes.Yanggaomeijiu Daqu was the object of study.For understanding the lactic acid bacteria(LAB)composition of Daqu,microorganism separation technology,the classical microbiological method,Rep-PCRanalysis,16SrDNA PCR-RFLP analysis and PCR-denaturing gradient gel electrophoresis were used in the study.From one side,this study could help us to reveal special flavour formation mechanism of Yanggaomeijiu,for further study,to lay the theoretical foundation improving the traditional ingredients and brewing process.In this work,a culture-dependent and a culture-independent approachs has been used to study the LAB communities in Yanggaomeijiu Daqu.The microorganism separation technology and classical microbiological method were used in studying LAB of Yanggaomeijiu Daqu.Combined with conventional culture-dependent approach,new molecular biological identification including Rep-PCR analysis and 16SrDNA PCR-RFLP analysis was used in this work.The culture-independent approach involve the extraction of total genomic DNA directly and PCR-denaturing gradient gel electrophoresis.A total of 65 LAB isolates were obtained from Yanggaomeijiu Daqu,including 18 strains bacillus and 47 strains cocci.65 LAB isolates could be divided into 9 different groups by conventional microbiological analyses.There were 3 different groups bacillus in Daqu,and type 2 was the predominant bacillus.On the other hand,there were 6 different groups cocci and type 4 and 9 were the predominant cocci.The study maked use of Rep-PCR as molecular biological identification method to analyze 9 different morphologica groups.(GTG)5-PCR was more suitable than BOXAIR-PCR in molecular biological identification for LAB of Yanggaomeijiu Daqu.LAB in Daqu could be divided into 5 different groups by Rep-PCR at a level of 75%similarity and group II was the big one including 4 types of cocci.9 different groups of LAB classified by classical microbiological method could be divided into 6 genetical types by RFLP analysis of the 16SrDNA.By gene sequencing,6 different LAB genetical types belonging to Lactobacillus plantarum subsp.,Lactobacillus fermentum,Pediococcus sp.,Pediococcus pentosaceus,Enterococcus faecalis and Leuconostoc fallax,involving Lactococcus,Pediococcus,Enterococcus and Leuconostoc.By a culture-independent approach combining with PCR-DGGE,the extraction of total genomic DNA from sample directly,the amplification of 16SrDNA V3 variable region and DGGE analysis,Weissella hanii,Weissella paramesenteroides,Pediococcus pentosaceus,Lactobacillus sp.and Lactobacillus namurensis,involving Weissella,Lactobacillus,Pediococcus were detected in Yanggaomeijiu Daqu.In addition to this,there were 4 uncultured bacterium in the fermentation starter.In this study,the microbial compositions of LAB in Yanggaomeijiu Daqu were investigated.This work shows that a culture-dependent approach combined with the culture-independent approachs is a rapidly and all-sided method to investigate LAB communities diversity.In conclusion,this study could help us to reveal special flavour formation mechanism of Yanggaomeijiu,for further study,to lay the theoretical foundation improving the traditional ingredients and brewing process.