Dynamic Changes Of Microbial Communities And Screening Of Dominant Mold During The Preparation Of Daqu Of Shanxi Aged Vinegar

The traditional Shanxi aged vinegar Daqu was made by barley and pea as raw materials,enriching the microorganisms in the environment naturally.In this study,Illumina Miseq high-throughput sequencing technology was used to study the diversity and dynamic changes of bacteria and fungi community during the whole process of Daqu preparation,and the microbial changes were studied by species isolation;CCA analysis method was used to study the relationship between microbial community composition and basic physical and chemical analysis,and PLS analysis methods were used to study the relationship between microbial community composition and flavor changes.A strain of excellent enzyme-producing was isolated from Shanxi aged vinegar Daqu,then its growth and enzyme production were studied.The results were as follows:(1)Bacterial and fungal communities of 11 samples in the preparation of Daqu were studied by Illumina Mi Seq high-throughput sequencing platform combined with bioinformatics analysis.The results of bacterial and fungal diversity analysis showed that the abundance of bacteria and fungi increased in the whole process of Daqu production at 25000 bp sequencing depth,and the abundance of bacteria and fungi in the mature stage decreased overall.Moreover,the results of ?-diversity analysis of bacteria and fungi showed that the clustering effect of OTU abundance was good in the whole process of Daqu preparation.The dynamic monitoring results of bacterial and fungal communities in the preparation of Shanxi aged vinegar Daqu by high-throughput sequencing technology showed that 5 bacterial phylum were detected,mainly Firmicutes(59.77%)and Proteobacteria(38.73%);12 bacterial genus were detected.Bacillus(23.26%),Lactobacillus(21.79%),Pantoea(35.31%)and Pediococcus(10.62%)were major bacteria.The main bacteria in the early stage of fermentation(dq1-dq3)are Lactobacillus;Pantoea and Lactobacillus,Pediococcus became dominant bacteria in the mid-term(dq4-dq5);In the later stage,that is,the mature stage(dq7.1w-dq7.9w)Bacillus and Pantoea became dominant gradually.A total of 3 fungal phylum were detected in the preparation of Shanxi aged vinegar Daqu,mainly Ascomycota(96.96%);16 fungal genuswere detected.Candida(43.31%),Aspergillus(30.80%),Alternaria(11.9%)and Wickerhamomyces(7.82%)played an important role.Candida was the dominant in the early stage of fermentation(dq1-dq3);Yeast and mold were about 50% respectively in the medium-term(dq4-dq5);In the later stage,that is,mature stage(dq7.1w-dq7.9w)Aspergillus became dominant gradually.The yeast was reduced gradually throughout the fermentation process,and the mold played a significant role gradually of the finished product by growing and propagation.(2)In the process of Daqu production,with the time going,the number of yeasts and molds in Daqu increased as a whole,and entering the mature stage,the number showed a downward trend.Using strain isolation methods combined with fungal ITS identification,14 kinds of molds were detected during the preparation of Shanxi aged vinegar Daqu.The main molds were Alternaria tenuissima(7.33%),Aspergillus niger(23.67%),Aspergillus flavus(20.61%),Rhizopus oryzae(8.46%),Rhizopus microsporus(13.09%),Lichtheimia ramosa(10.27%),Lichtheimia corymbifera(8.07%).Aspergillus niger and Aspergillus flavus were at a high level in the whole preparation process of Daqu,and increased gradually from about 10% in the pre-preparation period to about 30% in the maturity stage.Rhizopus oryzae and Rhizopus microsporus were at a high level relatively in the early stage of preparation(both around 15%),and both decreased to about 4% in the later period.Alternaria tenuissima,Lichtheimia ramosa,and Lichtheimia corymbifera were in a fluctuating change basically.(3)With the number of times of buckling and the prolongation of time increasing,the total acid content and amino nitrogen content of Daqu increased first and then decreased;The moisture,starch content and reducing sugar content of Daqu showed a downward trend;The changes of various enzyme activities(protease,glucoamylase,cellulase)in Daqu increased first and then decreased and then stabilized basically.The canonical correspondence analysis between the physical and chemical environment of Daqu and the level of microbial genus showed that Enterococcus,Lactobacillus,Pediococcus,Candida,Wickerhamomyces were correlated positively with moisture,total acid and reducing sugar.Bacillus,Pantoea,Alternaria,Aspergillus were correlated positively with room temperature and core temperature.(4)The corresponding analysis of Daqu flavor substances and microbial genus levels showed that the organic acid spectrum and volatile aroma components of Daqu preparation(dq1~dq6)were similar.And theorganic acid spectrum and volatile aroma components were similar at the mature stage of Daqu preparation(dq7.1w~dq7.9w).During the preparation of Daqu,the contents of oxalic acid,tartaric acid,malic acid,acetic acid and citric acid increased first and then decreased,and the mature period rose slowly to a stable trend,while the contents of pyruvic acid,succinic acid and lactic acid were opposite basically.Daqu(dq7.5w-dq7.9w)at the maturity stage were correlated highly with tartaric acid,acetic acid,and citric acid.Bacillus and Streptomyces were associated with citric acid,acetic acid,etc,and Leuconostoc,Pediococcus,Pantoea,and Clostridium played an important role in the formation of lactic acid,pyruvic acid and succinic acid during the preparation of Daqu.Aspergillus and Rhizopus were associated with citric acid,acetic acid,tartaric acid,malic acid,oxalic acid,etc,and Penicillium,Alternaria,Wickerhamomyces and Candida played an important role in the formation of lactic acid,pyruvic acid and succinic acid during the preparation of Daqu.The volatile aroma components raised first and then decreased gradually and tended to be stable.PLS analysis of correlation with volatile aroma components and bacterial community showed that Bacillus,Streptomyces and Pantoea with 2-ethyl-1-Hexanol,3,7-dimethyl-1,6-Octadien-3-ol,acetoin,2,3-butanediol,5-methyl-3-Heptanone,and ethyl acetate had high correlation.Leuconostoc,Pediococcus and Clostridium played an important role in the formation of Dibutyl phthalate,6-methyl-3-Heptanone,3-octanone,3-octanol,1-Hepten-3-ol,1-octene-3-ol,2-octanol,2-methyl-Butanoic acid,2-heptanone,etc in the preparation of Daqu.PLS analysis of correlation with volatile aroma components and fungal community showed that Penicillium,Aspergillus and Rhizopus were associated with3,7-dimethyl-1,6-Octadien-3-ol,ethyl acetate,1-Hexanol,2-ethyl-1-Hexanol,acetoin,5-methyl-3-Heptanone,and Candida and Wickerhamomyces played an important role in the formation of Dibutyl phthalate,3-methyl-2-Pentanol,2-methyl-1-Butanol,(S)-2-methyl-1-Butanol,3-methyl-1-Butanol,1-octene-3-ol,3-octanol,Eucalyptol,2-heptanone,6-methyl-3-Heptanone,3-octanone,etc in the preparation of Daqu.(5)A strain with high glucoamylase production was screened out from the mold isolated from Shanxi aged vinegar Daqu,and the results showed that the ability to produce protease and cellulase were also strong.The activities of protease,glucoamylase and cellulase activities(filter paper activity)of the high-yield and excellent strain can reach 368.80 U/g,4271.11 U/g and 4746.60 U/g,respectively.The strainwas identified as Aspergillus niger.The glucoamylase gene was amplified by PCR,and studied by sequence alignment and mutation examination,the length of DNA of glycosylase gene was 1449 bp,which was consistent with the coding sequence of the glucoamylase gene of Aspergillus niger TCCC41661(accession number: KM488271.1).The DNA sequence encoded a protein of 482 amino acids.The calculated molecular weight and isoelectric point of the enzyme were 51.75 k D and p H 3.99,respectively.The effects of different conditions on the growth and characteristics of Aspergillus niger SAV186 showed that under the condition of moisture content of 40% and fermentation temperature of 30 °C,Aspergillus niger SAV186 grew best and had the highest acid-producing and the enzyme of protease,glucoamylase and cellulose-producing.