The Impact Of The Usage Times On The Differences Of Microbiota And Metabolic Activity In The Strong-flavor Baijiu Workshop

The brewing of strong-flavor Baijiu(SFB)is a complex multi-strain spontaneous fermentation process involving diverse microbiomes that each plays their part and work together to advance the fermentation.During the production of Baijiu,not only microbiota in the Daqu and cellar were involved,but also microbiota from the workshop environment had the potential to penetrate the fermentation system,affecting the quality of Baijiu.However,there was a lack of systematic and comprehensive researches on the structure of microbial populations and their succession patterns in the environment of SFB.Therefore,in this study,we collected samples of Zaopei,raw materials,and environment from three SFB workshops with different usage times(named 70 a,30a,and new,respectively).Using 16 S r RNA and internal transcribed spacer(ITS)gene amplicon full-length sequencing and non-targeted metabolomics,we analyzed and compared the microbial communities in the SFB workshops to investigated the influence of microbiota on Zaopei fermentation in different workshops and to provided insights for achieving a highly controllable brewing process.The results of the study were as follows:(1)SFB workshops contained abundant and diverse microbial populations,and the microbial community in the Zaopei was affected by various sources of microbiome.The microbial communities in the fermentation workshops were analyzed at the species level.It is found that under different environments,the main bacterial genera at the beginning of fermentation include Lactobacillus acetotolerans,Acetobacter pasteurianus,and Saccharopolyspora rectivirgula,which mainly came from the raw material Daqu and the surface of equipments and tools.With the progress of fermentation,L.acetotolerans became the main source of Zaopei after fermentation.The predominant bacterial population,while the workshop environment forms a unique distribution of bacterial communities.The composition of the fungal community in the brewing microbiome was similar,fungi such as Saccharomycopsis fibuligera,Debaryomyces hansenii,Lichtheimia ramosa,Lichtheimia corymbifera,and Pichia kudriavzevii were the most abundant.The results of Source Tracker analysis showed that in addition to the main sources of fermentation microbiota,such as the Zaopei,pit mud,and equipment surfaces,microbiota from the ground and air were the main sources of Zaopei microbiomes at the beginning of fermentation.(2)Microbiota had preferences for specific environments,and unique microbial communities were formed in different locations of the workshop to gather,and bacterial microbial populations tended to stabilize in workshops with long usage time,dominated by unique non-brewing functional populations.With the increase of usage time,the diversity of microbiota in the workshop environment,including ground and air,increased.The analysis of microbial population differences showed that environmental samples from different workshops were unique.For the workshop ground samples,the 70 a workshop and the new workshop showed significant differences.The difference markers were Rubrobacter and Debaryomyces hansenii in the 70 a workshop,and the new workshops were Brevibacterium and Brachybacterium.The content of Bacillus licheniformis in the wall and air samples of the new workshop was significantly higher than that of other workshops.The Source Tracker tool was used to investigate the links between fermentation samples,raw materials and the environment.With the increase of usage time,the bacteria in the workshop environment tended to be unique,the source cannot be traced.The fungi in the SFB workshop came from a wide range of sources,fermented Zaopei,raw materials and surfaces of tools and equipment all contributed to it.The Beta Nearest Taxon Index(βNTI)and the Bray-Curtis dissimilarity-based Raup-Crick index(RCbray)showed that as the usage time increased,the bacterial community in the indoor environment was determined by variable selection The proportion of the deterministic process increased,and the community succession dominated by variable selection(80.77%)became the most important construction process of the 70 a workshop.During the long-term brewing process,the bacterial community structure of the70 a workshop tended to be stable.Homogeneous selection(48.57%)and homogenization diffusion(31.43%)dominated in the new workshop.On the contrary,the fungal community in the environment was affected by the resident functional fungi of Baijiu fermentation.The fungal community in the 70 a workshop was in a similar process of community structure construction in the new workshop.It was in a variable-dominated community succession and was not affected by the usage difference.(3)Under different environments,the microbial population structure was the direct cause of the difference in the metabolic profiles of Zaopei in different workshops.The results based on non-targeted metabolomics showed that 38 metabolites were significantly up-regulated and28 metabolites were significantly down-regulated in the Zaopei from the 70 a workshop compared to the 30 a workshop,and metabolic pathway analysis yielded 18 differential metabolic pathways.Pearson correlation analysis and Redundancy Analysis(RDA)showed that Trichosporon,Trichosporon coremiiforme,Pichia kudriazwe and Candida apicola had significant differences for the various metabolites in 70 a workshop,Aspergillus chevalieri,Aspergillus penicillioides and Saccharomyces cerevisiae were the main contributors to the differential metabolites of Zaopei in the 30 a workshop,proving that the metabolism of different microbiome in Zaopei may be the factor that caused the difference in flavor and quality of Baijiu in the different usage times workshop.