采用纯培养和免培养(culture-independent)分析方法,对宜宾多家规模以上白酒企业成熟窖泥细菌多样性进行了研究。采用改良牛肉膏蛋白胨培养基(NA)和高氏I号分离培养基分离细菌(包括放线菌),并评价菌株的乙醇耐受能力和低pH耐受能力。构建了窖泥样品总DNA的16S rRNA基因克隆文库。分别挑选不同培养特征的89株细菌纯培养物和427个基因克隆的16S rRNA基因序列,进行系统发育分析。89株纯培养物分属于13个属,以Streptomyces(39株)和Bacillus(27株)为优势属。纯培养菌株中,具备7%浓度乙醇耐受能力和低pH(4.5)耐受能力的分别有16株和10株。挑取的克隆子经测序、去除嵌合体后,得到了406个有效序列,并在97%的序列相似性水平上可将其分为75个OTU(Operational Taxonomic Unit),分属于Clostridia、Bacilli、Acti-nobacteria、γ-Proteobacteria、α-Proteobacteria、Deinococci、Planctomycea、Bacteroidia 8个纲。其中Clostridia为绝对优势菌群,包含50个OTU、227个克隆子;Bacilli包含11个OTU、128个克隆子,为次优势菌群。证明宜宾浓香型白酒窖泥中细菌群落存在丰富的系统发育多样性。

The aim of this study was to investigate bacterial diversity of mud samples from fermentation pits of multi-grain strong-flavor liquor factories in Yibin Sichuan province.We employed modified nutrient agar medium and Gogan-I medium for isolation and cultivation,and constructed 16S rRNA gene clone library with universal bacteria-specific primers.Then the 16S rRNA gene sequences of the isolates and clones were analyzed,and the isolates were tested for capacity of ethanol and low-pH value tolerance.Eighty-nine strains with different culture characteristics and 427 clones from sample were selected for 16S rRNA gene sequences analysis.Phylogenetic analysis based on 16S rRNA gene sequences showed that the 89 strains belonged to 13 genera,and Streptomyces,Bacillus are dominant genera with 39 strains and 27 strains,respectively.Four hundred and six sequences of clones(21 chimeras wiped off) were defined into 75 operational taxonomic units(OTUs) according to the 97% similarity threshold for OTU assignment by the software program DOTUR.Phylogenetic analysis based on 16S rRNA gene sequences showed that these OTUs belonged to Clostridia,Bacilli,Actinobacteria,γ-Proteobacteria,α-Proteobacteria,Deinococci,Planctomycea,Bacteroidia.Clostridia and Bacilli are dominant classes with 50 OTUs(227 clones) and 11 OTUs(128 clones),respectively.Sixteen and 10 strains of 89 strains showed the capacity of ethanol and low-pH value tolerance.Bacteria of fermentation pits mud present plentiful diversity.