Rational Construction And Application Of The Combined Starter Of Pixian Douban Broad Bean Meju

Pixian Douban is a special condiment in China,known as the "Soul of Sichuan Cuisine",with hundreds of millions of consumers.However,the traditional Pixian Douban fermentation process is exposed to the environment.Due to the open fermentation mode which is always affected by uncontrollable factors,significant differences exist between various batches.It has gradually become difficult to meet consumers’ increasingly escalating consumption requirements for food safety,greenness,and health.Therefore,this study aims to propose a bottom-up strategy for rational construction of synthetic microbiome with the above problem as a starting point,and to design a microbial community with similar function to the in situ samples based on this strategy as a fermentor for Pixian Douban broad bean meju for preliminary application and further research.The main research contents and results of this study are as follows:(1)In order to solve the problem of unclear aroma-producing functional microorganisms in the fermentation process of Pixian Douban,the non-pathogenic and dominant microorganisms that might have an important role in the microbial community.The genera in the fermentation process were determined by three indicators including the relative abundance(> 1%),the sample coverage(> 50%)and the eigenvector centrality of the associated network nodes.Then,within these genera,the species corresponding to the OTUs with the highest relative abundance and widely used in industry or GRAS generally safe strains were identified as potential core microorganisms.Finally,C.versatilis,Z.rouxii,S.carnosus,P.acidilactici,P.pentosaceus,W.confusa,T.halophilus,L.plantarum were selected as potential core microorganisms.It provided a theoretical basis for the next step of synthetic microbiome construction.(2)In order to solve the problem of unclear flavor metabolism of the presumed core microorganisms of Pixian Douban,this study designed a fully synthetic medium that simulated the in situ system,and based on this medium,the flavor compound metabolism profiles of aroma-producing microorganisms were analyzed.Simulating the content of carbohydrates and amino acids in Pixian Douban,a fully synthetic medium which composed of 4 monosaccharides,17 amino acids,8 vitamins and several inorganic salts was designed.The type and concentration of the main fermentation substrate of the fully synthetic medium were consistent with the in situ fermentation system,and could meet the growth requirements of all aroma-producing functional microorganisms.The fermentation results showed that the use of fully synthetic medium for research had the advantages of short fermentation time and good batch repeatability.Thus,this study analyzed the aroma-producing profile of aroma-producing functional microorganisms based on the fully synthetic medium,and found that the aroma-producing microorganisms could be divided into three categories according to their flavor-producing abilities(yeast,lactic acid bacteria and others).And the "flavor complementarity" was observed among different categories.(3)In order to construct a synthetic microbiome that can be used for the closed fermentation of Pixian Douban,this study proposed a bottom-up and rational strategy for constructing a synthetic microbiom.Based on the selection of core aroma-producing microorganisms and the fully synthetic medium,this strategy toke the aroma-producing function of individual strains,interactions of core aroma-producing microorganisms,and the differences of flavor compounds among different two-strain communities with different inoculation sequences into account.A three-strain community composed of Pediococcus acidilactici,Staphylococcus carnosus and Candida versatilis was designed according to the strategy.And the number of volatile compounds produced by the three-strain community reached 63.1% of the in situ samples,and flavor compound type and structure were relatively similar.It is worth noting that the concentration of amino acid nitrogen in the combined inoculum sample increased by 21.8%,compared with the in situ sample.The starter and its construction strategy have a high value in synthetic microbiome construction and fermentation process optimization.