The Microbial Diversity In Fermented Grains Of Xi-feng Liquor

Based on the temperature variation of the fermented grains,we collected 8 grain samples from upper and middle grain layers in different spatial and temporal fermentation stages of Xi-feng liquor.To investigate the genetic diversity of microorganism during the fermentation,firstly,the traditional dilution plate method was used to get individual clones of bacteria and mold,and then statistical method of repeated data was used to describe the variation trend of number of cultivable clones.Secondly,the PCR-RFLP of 16S rDNA(for bacteria)and 5.8S rDNA ITS(for mold)to were conducted to the obtained strains,then the phylogenetic trees were constructed based on the sequences of representative strains.Furthermore,a culture-independent approach referred to as PCR-DGGE to reveal the composition and dynamical changes of microorganism in grain during fermentation.Taken together,we concluded that the follows:In this study,both the amount of bacteria and fungi showed a rise followed by a decline in the early and mid-stage of fermentation,while in the late fermentation the bacteria increased but fungi decreased.The difference of bacteria and fungi population was showed in different position of the cellar pool,the descending order of the amount of bacteria is from the upper to the blow,from the edge to the center,the similar distribution was shared by the fungi.However,the number of bacteria is 2 to 3 orders of magnitude higher than that of fungi in the whole process of the fermentation.16S rDNA PCR-RFLP of 286 bacterial isolates resulted in 128 genotypes,while 86 genotypes were obtained from 340 mold strains using 5.8S rDNA-ITS PCR-RFLP.The phylogenetic analysis indicated that all bacteria belonged to 48 species in 28 genera,of which Bacillus,Rothia,Kocuria and Pseudomonas were dominant,Bacillus spp.accounting for 56%of all the isolates.For the mold,32 species in 18 genus were isolated,the majority of the strains belonged to the Galactomyces,Aspergillus and Penicillium,of which Galactomyces spp.were widely distributed,accounting for 64%of total fugi.The bacterial composition changed significantly when the PCR-DGGE was used.The analysis of DGGE showed five bands with the same brightness,indicating they dominated bacteria populations,only two bands were presented in prophase of fermentation.In the mid-stage,a total of nine bands were visualized,showing an increase of preponderant populations of bacteria.Followed by it,a sharply declines in the bands number to five were observed in the later stage of fermentation.Sequence analysis of each band showed they belonged to 15 species in 6 genera,of which 10 were assigned into Lactobacillus and the remaining 5 into Paenibacillus,Acinetobacter,Pediococcus,Cellulosimicrobium and Klebsiella,respectively.Interestingly,Bacillus spp.were not detected by DGGE even if they were dominant using plate count method.Conclusively,the combined methods of culture-based and uncultured-based revealed the high genetic diversity of functional bacteria in the entire fermentation process.The Bacillus and Lactobacillus share a major contribution to fermentation of Xi-feng liquor.