Study On Microflora Composition And Its Metabolic Characteristics During Pellicle Occurred And Recovery Of Cowpea Paocai In Sichuan

Sichuan paocai is one of the most representative paocai product in China,and the pellicle on the surface of paocai brine is one of the most common adverse phenomena in the fermentation process of home-made paocai in Sichuan.This research adopts the traditional Sichuan paocai fermentation method,and Cowpea(Vigna unguiculata(L.)Walp.)was used as raw materials,a stable fermentation system was obtained through multiple rounds of fermentation,and continue to carry out multiple rounds of fermentation without salt supplementation,after the pellicle occurred,the fermentation system was treated by replacing fresh cowpea,adding salt and/or Baijiu to explore the recovery method and clarify the metabolic characteristics of microorganisms.The metagenomic sequence was used to obtain the microbial metagenomic sequence data,and the important roles of microorganisms in the formation of paocai quality were deeply revealed from the perspectives of metabolites-microbes-metabolic properties-metabolic pathways-metabolic network.The main results were as follows:(1)A total of 111 components,including 5 sugar,6 organic acids,77 volatile components,17 amino acids and 6 biogenic amines were analyzed by gas chromatography-mass spectrometry,high performance liquid chromatography and amino acid automatic analyzer.The determination results showed that the consumption of glucose by microorganisms was the largest,but the formation of pellicle did not make a significant(P>0.05)change in the consumption of glucose by microorganisms,and replacing fresh cowpea,adding salt and/or Baijiu,the consumption of sugars by microorganisms was lower than other fermentation rounds.During multiple rounds of fermentation,there was no obvious accumulation of organic acids in paocai brine,but the content of lactic acid in paocai increased as the most important organic acid.Esters were the most abundant volatile substances in paocai,and the number fluctuates greatly in each fermentation cycle,and added Baijiu would significantly affect the volatile substances in paocai.The total content of free amino acids in paocai decreased at first,then increased and then decreased.There were 9 kinds of amino acids with high content,such as serine,alanine and leucine.The total content of biogenic amines and each biogenic amine were lower,and the formation of pellicle did not have a significant effect on the accumulation of biogenic amines.(2)Through species annotation of metagenomic sequencing data,a total of 197 genera and687 microorganisms were obtained,both in genius and in species the diversity of fungi increased when pellicle was formed,but bacterial composition was relatively stable,the main fungi were Kazachstania saulgeensis,Pichia membranifaciens,Pichia kudriavzevii and Candida inconspicua and the main bacterial were Lactobacillus pentosus,Lactobacillus plantarum,unclassified＿g＿Lactobacillus,Lactobacillus kisonensis,Lactobacillus parafarraginis and Lactobacillus buchneri.Treatment with added salt and/or Baijiu had a significant(P<0.05)effect on the microbial composition in the fermentation system,Adding Baijiu with a total volume of 1.5% of paocai brine has the best inhibitory effect on the microbes,and on this basis,increasing the salt concentration(adding Baijiu with a total volume of 1% of paocai brine and adjusting the salt concentration to 7%)has no obvious promoting inhibitory effect.(3)Through functional annotation of metagenomic sequencing data,it was found that carbohydrate metabolism and amino acid metabolism were the main metabolic processes,there were many metabolic pathways in microorganisms,and the metabolic pathways with higher abundance in each fermentation cycle were relatively stable,However,the differential metabolic pathways between IB(Initial brine,)and OP(Pellicle occurred),OP and the fourth fermentation cycle with different treatments belong to multiple KEGG Level 2 metabolic classifications.Among genes annotated in each fermentation stage,genes belong to glycoside hydrolases were the most,followed by glycosyltransferases and auxiliary activities.(4)Through the differential analysis of the species annotated by metagenomic sequencing data,it was found that the microorganisms associated with the formation of pellicle belonged to 10 fungi genera,4 bacterial genera and 3 unclassified bacterial genera,especially,Kazachstania,Lactobacillus,Pichia,Candida,Lachancea and Saccharomyces,which contained at least three species of microorganisms belonging to these six genera,these microorganisms have some metabolisms related to human diseases,indicating that the production of pellicle would cause food safety problems.Through the correlation analysis of volatile substances and the microorganisms related pellicle formation,it was found that Propanoic acid,hexyl ester,1,3-Dimethyl-1-cyclohexene,Oxime-,methoxy-phenyl-and Phenylethyl Alcohol may be the unpleasant odor produced by paocai during multiple rounds of fermentation.(5)To establish the metabolic network of microorganisms in the process of paocai pellicle formation and recovery,main flavor components,by combining KEGG functional annotation and species annotation,enzymes that were related to enzymatic reaction or metabolic pathways of glucose metabolism,organic acid metabolism and amino acid metabolism were collected,and encoded genes of these enzymes in gene catalogue were predicted at the genera Level by taxonomy annotation.This paper studies the recovery method of pellicle formation of paocai,as well as the metabolic characteristics of microorganisms in the fermentation process,then established the metabolic network relationship between specific substance and the main function microorganism in process of paocai pellicle formation and recovery by metagenomic sequencing and annotation.