| Paocai is one of the most representative traditional fermented food of China.Sichuan paocai is a kind of natural functional fermented vegetable with unique flavor,high nutritional value and rich biological function.Studies showed that the flavor and functionality in fermented vegetables were strongly influenced by complex microbial populations.However,the study on the fermentation process of Sichuan paocai lacks systematic and in-depth attention.In this study,high-throughput sequencing and metatranscriptomics techniques were used to study the composition and dynamic changes of microbial community during the Sichuan paocai fermentation process.At the same time,chromatograph technology was used to monitor the main flavor compounds,such as short-chain fatty acid,amino acids and volatile compounds,and the contribution of key flavor compounds was studied.Besides,the relationship between the flavor components and microbes in Sichuan paocai was analyzed by the methods of metatranscriptiomics and partial least squares regression analysis.Finally,we discussed the effects of microorganism on the fermentation of Sichuan paocai at different levels,and studied the mechanism of how the functional microorganisms regulate the key genes and enzymes during fermentation process.This study provided the theoretical basis for functional microorganisms screening and the technology of industrial Sichuan paocai fermentation.Also,it would be conducive to the promotion of Chinese traditional fermented foods and culture.The main findings are as follows:1.High-throughput sequening results showed that Firmicutes and Proteobacteria were two major phyla during Sichuan paocai fermentation.At the genus level,Lactobacillus,Enterobacter,Lactococcus,Leuconostoc and Weissella were the dominant Genera.And the Lactobacillus pentosus was the dominant species.The bacterial diversity was the highest at the beginning of fermentation and then decreased gradually along with the fermentation time.In the early stage of fermentation,Enterobacter and some other environmental species such as Acinetobacter and Pseudomonas were the major microorganisms,the Weissella showed growth at the same time.The abundance of Citrobacter,Lactococcus and Leuconostoc increased in the middle fermentation period.The Lactobacillus was the major microorganism in the late fermentation period.2.The results of flavor compounds detection shows that,a total of 239 volatile compounds,17 amino acids and 6 short-chain fatty acid were detected in Sichuan paocai.Malic acid was the main initial organic acid,and at later stage,lactic acid and acetic acid were two major acids with a concentration of 3.0 mg/m L and 1.41 mg/ m L,respectively.The total concentration of organic acids increased rapidly to 0.71 mg/m L at day 6th,and then decreased to 0.50 mg/m L at the late stage after a little fluctuation.Serine,L-Aspartic acid and glutamate were the three major amino acids in the fermentation of Sichuan paocai.As for the volatile compounds,sulfur compounds,alcohols,terpenoids and esters are the four most abundant volatile organic compounds.16 potential key volatile compounds were identified by using the OAV method,including 9 sulfides,2 alcohols,2 terpene compounds,1 alkylbenzene,1 ketone and 1ester.The results showed that sulfur compounds,esters and alcohols provided rich and unique flavors,glutamic acid and L-Aspartic acid acid provided umami and sour taste for Sichuan paocai,Lactic acid,acetic acid and malic acid provide sour taste for Sichuan Paocai.3.O2PLS analysis showed that microorganisms were closely related to flavor compounds.4 organic acids,17 amino acids and 22 volatile compounds were strongly correlated with 32 genera.8 Genera were most closely related to flavor substances.Among them,Enterobacter was negatively correlated with 23 flavor compounds,Lactobacillus was positively correlated with 21 flavor compounds.13 genera were correlated with citric acid,lactic acid,acetic acid and formic acid.21 genera influenced the synthesis and metabolism of amino acids and Lactobacillus was the most important genera;29 genera are associated with volatile compounds,Lactobacillus,Enterobacter,Citrobacter,and Pectinobacterium are all related to more than 10 volatile compounds.4.Metatranscriptomics analysis.The metatranscriptomics analysis of 9 Sichuan paocai samples showed that the sequence data ranged from 6.39 to 7.28 Gbp,and each sample obtained 42.6 million to 49.1 million high-quality sequences and then got 140795 unigenes after assembling the sequence at Trinity.The results of OTU annotation showed that Lactobacillus,Lactococcus,Enterobacter and Leuconostoc were the dominant microorganisms,and the results were similar to the 16 s r RNA annotation at the genus level.As for the functional annotation,PICRUST could predict microbial function to understand the functional profile of microorganisms,but it differs from macrotranscriptome in gene expression and annotation at different levels.5.KEGG function annotation results showed that carbohydrate metabolism,amino acid synthesis and metabolism were the most active pathways.The results of CAZy function annotation showed that the glycosidase and glycosyltransferase genes were mostly expressed.Go function annotation indicated that metabolic process,cellular process,single-organism process,binding and catalytic activity were the five most prominent functions.The Egg NOG function annotation results indicated that energy,signal transport,carbohydrate and amino acid metabolism were most active.6.Cluster analysis of differentially expressed genes showed that the number of differentially expressed genes in the Mid-Lat stage of fermentation was much less than that of Pre-Mid fermentation stages.The differentially expressed genes were mainly involved in carbon metabolism,amino acid biosynthesis,purine metabolism,two-component system,ABC transporter,pyrimidine metabolism,pyruvate metabolism and glycolysis/gluconeogenic transporter.The differentially expressed genes in Pre-Mid and Mid-La were enriched in 220 and 182 metabolic pathways,respectively.Thereinto,carbon metabolism and biosynthesis of amino acids were two major active pathways.Four major pathways,including glycolysis,pyruvic acid metabolism and the biosynthesis and metabolism of amino acid involved in Sichuan paocai fermentation were reconstructed according to the results of metatranscriptome.Lactobaciliales and Enterobacteriaceae play the key roles in the fermentation of Sichuan paocai.7.The microorganisms in Sichuan paocai play a key role in the regulation of the key flavor compounds.Lactobaciliales,Enterobacteriaceae and Pseudomonadaceae are functional microorganisms involved in the synthesis and metabolism of organic acid,pyruvate metabolism is the main pathway.90 genes and 5 enzymes were involved in regulation of lactate biosynthesis.153 genes and 8 enzymes were involved in regulation of acetic acid.353 genes and 4 enzymes were involved in regulation of malate biosynthesis.Lactobaciliales,Enterobacteriaceae,Pseudomonadaceae and Moraxellaceae are the functional microorganisms for the synthesis and metabolism of glutamic acid and L-aspartic acid.Glutamic acid biosynthesis involved 369 genes and 6 enzymes.The biosynthesis and metabolism of L-aspartic acid involved 317 genes and 12 enzymes;Fungi were also involved in the regulation of L-aspartic acid metabolism.Lactobaciliales and Enterobacteriaceae were the two most important order in the regulation of volatile sulfur compounds.The reaction of methionine with L-Cysteine to sulfides involves 4 enzymes and 187 genes.Microbial lipase can catalyze the esterification of linalool and formic acid to produce linalool methyl ester. |
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