Quantitation Analysis And Metabolic Profiling Of Lactic Acid Bacteria In Sesame-flavor Liquor Fermentation

Chinese liquor fermentation system contains complex microbial community,including a kind of bacteria,yeasts and molds.Among them,lactic acid bacteria group(LAB)is main bacteria and plays an important role in fermentation process.However,current studys about the communtity succession characteristics and flavor compounds metabolic function of LAB in the fermentation process are unknown.Uncovering the communtity succession characteristics and flavor compounds metabolic function of LAB can provide a powerful theoretical basis for regulating mirobiota structure and improving brewing quality.In order to resolve the problem that high-throughput sequencing only provide relative abundance data,which cannot accurately determine the communtity succession characteristics of LAB.In this study,sesame-flavor liquor fermentation system is selected as our research object,building an absolute quantitation method combined with quantitative PCR and amplicon sequencing technology.Meanwhile,combining with multi-statistics and metatranscriptomics,we revealed the flavor compounds metabolism characteristics of LAB.There will be a great theoretical value to reveal the microbial brewing mechanism,and it also have a practical guiding significance for transforming microbial community to improve the quality of liquor.The main results of this study are as follows:(1)In order to establish natural internal standard bacterial communtity absolute quantitation method.Lactobacillus acetotolerans,Lactobacillus sp.was selected as the natural internal standard microorganism base on which appeared on all samples and mean relative abundance was greater than 10%;Species-specific primers PLace and Plsp were designed for Lactobacillus acetotolerans and Lactobacillus sp..The natural internal standard bacterial communtity absolute quantitation method was builded by conbining quantitation PCR and high-throughput sequencing.Quantitation PCR had a strong linear,R~2 was greater than 0.99.Its sensitivity was high,the limit of quantification was 17.9 copies/μL and 10.7 copies/μL,respectively.The repeatability was well,coefficient of variation of Ct value were less than 1%.The quantitative PCR method was consistented with plate count method(pearson correlation coefficient was 0.997,slope was 1.16).The absolute content of Weissella was calculated using Lactobacillus sp.and Lactobacillus acetotolerans as internal standards,and the results were consistent(pearson correlation coefficient was 0.98,slope was 1.082).Distribution characteristics of two natural internal standard microorganisms in ten production areas was analyzed,Lactobacillus acetotolerans was detected in ten regions,Lactobacillus sp.was detected in eight regions,the result suggested that the natural internal standard bacterial communtity absolute quantitation method has a broad scope of application.(2)This study reveals the communtity succession characteristics of LAB using natural internal standard bacterial communtity absolute quantitation method.The composition of LAB in the fermentation process was Lactobacillus,Weissella,Leuconostoc and Pediococcus.The absolute content of LAB was determined,the absolute content of Lactobacillus and Weissella was the most than other LAB,the highest value was 7.67 Lg(copes/g)and 6.62 Lg(copes/g),respectively,which account for 86.18%and 7.77%of total bacterial absolute content.The communtity succession characteristics of LAB could be divided into two stages:the first stage was named as growth stage(from 0 to 15 days),the total content gradually increases,and reaches the highest value(7.72 Lg(copes/g))at 15 days.The second stage was named as stationary phase(from 15 to 46 days),the total content was maintained above 7.0Lg(copes/g)with a slow decline trend.The absolute quantitative data and relative quantitative data were compared in describing succession characteristics and growth trend of LAB.The results showed that relative quantitation data was consistent with absolute quantitation data at descriting growth trend for dominant LAB-Lactobacillus(pearson correlation coefficient was0.97),for other non-dominant LAB was incorrectly(pearson correlation coefficient<0.25);Meanwhile,on the description of composition characteristics of microbial community,the absolute quantitation data was more sensitive to observe the difference of microbial community than relative quantitation data.The results showed that absolute quantitation data could more accurately reveal the the communtity succession characteristics of LAB.(3)To uncover the metabolism contribution of LAB for flavor compounds,a total of 16core flavor compounds were detected in the fermentation process samples.Based on microbial-flavor compounds correlation network,nine core flavor compounds were associated with LAB.Metatranscriptomics was used to reveal metabolic contribution mechanism of LAB,a total of 114.07G high quality short sequences were obtained.After assembly and de-redundancy,45567 unigenes were obtained,44381 unigenes were annotated with NR database,the active LAB contained Lactobacillus,Weissella,Leuconostoc,Pediococcus and Acetobacter.24,377 unigenes were annotated with KEGG database,the mejor pathway was classfied into Metabolism and Brite Hierarchies.The carbohydrate metabolism and amino acid metabolism related to flavor synthesis were analyzed.Lactobacillus and Weissella had the most unigenes than other LAB,and reached 33.47%and 14.45%in carbohydrate metabolism pathway,24.60%and 14.90%in amino acid metabolism pathway,respectively.Meanwhile,compared with other microorganisms,Lactobacillus and Weissella had higher gene transcriptional abundance about the synthesis of acid compounds such as acetic acid,octanoic acid,citric acid and phenylethyl alcohol than other LAB.