Analysis Of Microbial Community Structure And Function During Broad Bean Paste Fermentation

Broad bean paste(BBP),a traditional Chinese fermented food with broad beans,wheat flour and salt as raw material,is mainly prepared by koji making and pei fermentation.In general,the production of traditional BBP is characterized by an open process of multi-species solid-state fermentation,which involves complex communities of microorganisms.BBP microbiota performs essential ecosystem functions and plays an important role in the fermentation of BBP.However,due to the open fermentation environment and non-sterile raw materials,the microbial community would vary over time,especially in the case of seasonality and succession,which further led to inconsistencies in flavor and quality among different batches,and the open environment also increases the risk of food safety.In addition,humans have used high-salinity for the production of bean-based fermented foods over thousands of years.Although high-salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment,it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people's health.However,spontaneous fermentation with low salinity often leads to growth of harmful microorganisms,resulting in low quality and off-flavor products.Therefore,it is imperative to elucidate the microbial community structure and function during BBP fermentation and develop novel defined starter cultures for improving food safety,stability and guiding the transformation of traditional industries.In response to the above issues,this study provided a bottom-up approach for the development of simplified microbial community model with desired functions,to better understand the underlying mechanisms of microbial assembly and function by deconstructing and reconstructing the microbial structure and function,guide the industrialization transformation of traditional industries and achieve the standardized and low-salt production of bean-based fermention foods.The main research content and results of this paper were as follows:(1)To better understand the impact of different seasons on microbiota and its effects on quality,the physicochemical metabolism and microbial communities in different seasons were compared by inter-group discriminant analysis to distinguish the discriminant metabolites and unique taxa.Comparative analysis revealed significant differences in the physicochemical metabolism of pei in different seasons,with higher concentration of volatile flavor compounds in summer and autumn,and the highest concentration of amino acid nitrogen and amino acids in cold winter.In addition,amplicon sequencing analysis indicated that microbial composition and diversity varied with seasons.LEf Se analysis revealed the main biomarkers of spring,summer and autumn koji were Staphylococcus,Bacillus and Aspergillus,respectively.Among fermented pei,Staphylococcus,Tetragenococcus and Aspergillus were the main biomarkers of spring pei while Bacillus,Agaricostilbum,Sterigmatomyces,Fusicolla,Trichoderma and Meyerozyma were the main biomarkers of summer pei.In addition,autumn pei was characterized by Zygosaccharomyces while winter pei was characterized by Lactobacillales(Weissella,Lactococcus,Streptococcus,Pediococcus,Enterococcus),Kurthia,Proteus and Cronobacter.Then,correlation analysis revealed that temperature,salinity,and acidity together led to the seasonal distribution of microbiota.And differential microorganisms further contributed to the inconsistency of flavor quality,in which Lactobacillales was responsible for the higher titratable acid and amino acid nitrogen concentration in winter pei,while Saccharomycetales benefited the formation of higher content volatile flavor substances in summer and autumn pei.(2)To explore the succession mechanisms and functional roles of microbial communities throughout the fermentation of traditional Chinese BBP,high-throughput sequencing and quantitative real-time PCR were used to resolve the microbial community succession.It was found that microbial diversity and biomass decreased sharply in the week before fermentation and rapidly reached a steady state.And the dominant microbial genera were determined to be Staphylococcus,Bacillus,Weissella,Aspergillus,and Zygosaccharomyces.Among them,the biomass of Staphylococcus declined in the first two weeks of fermentation,and then slowly recovered,while Aspergillus,Bacillus and Weissella decreased rapidly during fermentation.Zygosaccharomyces increased slowly during the fermentation,and reached the peak in the middle stage of fermentation,then declined.Correlation analysis revealed that the salinity and microbial interactions together drove the dynamic of community.And pointed out that five dominant genera may play different key roles in different fermentation stages,i.e.,Aspergillus,Bacillus and Weissella mainly degraded macromolecule substances in the early stage of fermentation,while salt-tolerant Staphylococcus and Zygosaccharomyces played pivotal roles in the formation of flavor substances in the mid-late stages of fermentation.(3)Metagenomic sequencing was performed on representative samples from different stages of BBP fermentation to gain a deeper insight into the metabolic mechanism and the functional roles of microbial taxa in different fermentation stages.First,we reconstructed metabolic pathways for the substrate degradation and flavor formation during BBP fermentation based on functional annotation.Then based on the analysis of microbial contribution to functional genes,Staphylococcus,Bacillus,Weissella,Aspergillus and Zygosaccharomyces were found to be the potential predominant populations responsible for substrate breakdown and flavor biosynthesis,and their contribution to metabolic functional genes was proportional to their abundance.In addition,stress response genes responsible for adaption to osmotic stress were also found in Staphylococcus and Zygosaccharomyces,belonging to different families of genes involved in the pathway of salt stress,suggesting that they may have the ability to maintain the balance of intracellular and external osmotic stress.(4)To further validate the microbial functional properties and develop a microbial community model with specific functions,five core species(Aspergillus oryzae,Bacillus subtilis,Staphylococcus gallinarum,Weissella confusa and Zygosaccharomyces rouxii)with fermentative functions were isolated from BBP on the basis of microbial community structure and functional analysis.And their salinity tolerance,interaction,bacteriostatic effects and metabolic characteristics were evaluated.The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function.The results showed that A.oryzae and B.subtilis had high protease and amylase activities,which were responsible for the degradation of macromolecular substances in the early stage of fermentation.A.oryzae,S.gallinarum and W.confusa had higher organic acid and amino acid producing ability.Salttolerant Z.rouxii played a key role in the formation of volatile flavor substances in the midlater stages of fermentation.Then,simplified microbial community model was reconstructed using these five strains to simulate the fermentation process of BBP,and it was found that the synthetic community had a similar microbial succession with the traditional fermentation conditions.In addition,the differences in microbial community succession and fermentation performance between different salinity and traditional fermentation conditions were compared,which further confirmed that salinity was an important factor driving microbial community succession and preliminarily achieved low-salt fermentation of BBP.(5)The potential pathogenicity of S.gallinarum hinders its application as a food starter.To find food safe strains that could replace the conditional pathogen S.gallinarum,we compared the distribution mechanism and functional characteristics of different Staphylococcus species,revealed that the competitive relationship between different Staphylococcus species resulted in the dominance status of S.gallinarum,and determined that S.carnosus could replace the S.gallinarum for the fermentation production of BBP.Finally,the optimized synthetic community was further applied in BBP fermentations with different salinity,and found that low salinity could effectively improve fermentation efficiency and flavor quality of BBP.