Source Tracking Of Microbial Community And Functional Microbiota Analysis For Polypeptide-degrading In Pixian Broad Bean Paste

Pixian broad bean paste is a fermented condiment with a spicy and mellow taste,which is known as the "soul of Sichuan cuisine".However,as a typical representative of traditional fermented foods,the production of Pixian broad bean paste still relies on experience.The source of substrates,the evolution of functional microorganisms,and the anabolic pathway of important flavor substances are not clear during the fermentation process.It has restricted the standardization,precision and controllability of traditional brewing.Therefore,clarifying functional microorganisms and their sources is an important prerequisite for the modernization of Pixian broad bean paste.In this study,amplicon sequencing,proteomics,source tracking and other methods were used to first explain the volatile and non-volatile compound of Pixian broad bean paste and the microbial and material contributions of broad bean meju and chili moromi to the moromi-meju mixture.Secondly,this study focused on the analysis of functional microbes in broad bean meju.Finally,three microorganisms with peptide degradation function during the brewing process of hyperosmotic moromi environment are finally obtained.The main research contents include:(1)Diversity and source tracking of microorganisms and material in moromi-meju mixture.This study analyzed the microbial structure by using high-throughput sequencing methods.And we quantified the volatile/nonvolatile flavor compounds by using the GC-MS and other methods.We traced the origin of microorganisms and materials by using multiple bioinformatic methods.In the end,we found that 44%-59% of the moromi-meju mixture's bacteria were derived from chili moromi,and 5%-22% of the moromi-meju mixture's bacteria were derived from broad bean meju.At the same time,42%-77% of the moromi-meju mixture's fungi originated from chili moromi,and 2%-18% of the moromi-meju mixture's fungi originated from broad bean meju.In addition,16 bacterial genera are specifically contributed by chili moromi;2 bacterial genera and 2 fungal genera are specifically contributed by broad bean meju.Chili contributed main microorganisms to moromi-meju mixture while broad bean meju contributes more fermentative substrates(amino acids and glucose).(2)Functional microbial analysis for polypeptide-degrading during broad bean meju brewing.Therefore,the efficiency of protein and peptide degradation to amino acids during broad bean meju fermentation is critical for product quality.In this study,we found that amino acids concentration increased and the peptide concentration decreased significantly at the late stage of the broad bean meju fermentation.It was speculated that there were functional microorganisms with the capability of peptide-degrading during the fermentation of broad bean meju.This study explored the candidate of polypeptide-degrading functional microorganisms by using correlation analysis and extracellular proteome analysis.Results of correlation analysis suggested that Candida,Saccharomyces,Aspergillus and Pseudomonas may have the capability of polypeptide-degrading during the broad bean meju fermentation.A total of 318 proteins were detected in the proteome analysis.Among them,five peptidases were found from Aspergillus,two peptidases were found from Bacillus,and two peptidases were found from Staphylococcus.Therefore,it is speculated that Aspergillus,Bacillus and Staphylococcus might have peptide degradation ability.(3)The microbial screening from broad bean meju and batch fermentation for amino acids production.In this study,117 salt-tolerant strains including 54 bacteria,45 yeasts and 18 molds were cultivated which were selected from the broad bean meju fermentation samples,and all candidate peptide-degrading microbes were verified by pure culture simulated fermentation.Batch fermentation and enzymatic analysis were conducted and evaluated the functional microbiota for polypeptide-degrading during hypertonic moromi fermentation,involving Aspergillus niger,Candida zeylanoides and Bacillus licheniformis.These 3 strains might be the functional microbiota for polypeptide-degrading during the borad bean fermentation.