Assembly Of Core Microbial Communities And Biogenic Amine Regulation Mechanism In Chinese Rice Wine Fermentation

Chinese rice wine is a semi-solid,open,complex species fermentation alcoholic beverage with grain and koji as raw materials.Microbial ecosystem experienced frequent changes during fermentation,and maintain the stable coexistence of different species.Resource competition and metabolic interactions among microbial ecological members mediate succession and functional balance to ensure product safety and quality.Lactic acid bacteria(LAB)in Chinese rice wine and even all fermented food possess the core advantage,and participate in nitrogen metabolites accumulation of biogenic amines.LAB were divided into the homofermentative/facultative heterofermentative /obligate heterofermentative type based on carbohydrates utilization,individual metabolic characteristics of LAB and their internal interaction for the effect of microbial ecological succession and function are still not clear.Model for Chinese rice wine fermentation system,this paper apply the "top-down" and "bottom-up" microbial ecology research methods,analyses the core microbial flora of Chinese rice wine in different regions,studies the individual metabolic characteristics of different types of LAB,to illustrate the succession and interaction mechanism of different types of LAB,and realizes the control of biogenic amines on the industrial Chinese rice wine.The main research contents and results of this paper are as follows:(1)By comparing the microbial communities and physicochemical metabolites of Chinese rice wine fermentation in different regions,it was revealed that the change trend of physical and chemical indexes of Chinese rice wine fermentation in different regions was similar,and the content of total acid,ethanol and amino nitrogen in C producing area was higher,and the fermentation intensity was higher.Saccharomyces,Rhizopus,Lichtheimia,Puccinia,and Saitoella were the dominant fungal genera in three production regions,and the species and succession trend of fungi were conserved.The distribution of dominant bacterial genera was significantly different.LAB dominated the three regions,but there were differences in species.Based on the relative abundance of pathways related to precursor amino acid metabolism of biogenic amines during Chinese rice wine fermentation,the active metabolism of amino acids in different regions was concentrated in the Chinese rice wine pre-fermentation stage.The most abundant metabolic contribution of precursor amino acids in A factory was the reason for the highest accumulation of biogenic amines.Lactobacillus and Leuconostoc genera have always been the main contributors to biogenic amine accumulation in three regions.The differences of microbial succession,metabolic phenotype,and functional gene distribution in the fermentation process of Chinese rice wine in three regions are correlated with distance.The closer the distance,the higher the similarity.(2)Based on the microbial composition kinetics,the ratio of LAB(6.52%-99.51%)was always dominant in the fermentation process,and there was a phasic succession law among different fermentation types of LAB.The obligate heterofermentative LAB(83.86%-65.22%)was dominant in the pre-fermentation stage,and the homofermentative LAB(39.95%-52.32%)was dominant in the main fermentation stage.A variety of LAB was obtained through highthroughput screening.Analysis of individual metabolic characteristics based on resource constraints and metabolite inhibition levels revealed that the highest fitness of facultative heterofermentative LAB.The interactions of the three fermentation types in 48 paired communities were 63.5% positive,34.4% negative,and only 2.1% neutral.Co-culture promotes substrate depletion,while lactate presents to be utilized.The stability of non-transmissive competitive combinations under the influence of abiotic factors was found by reconstructing ternary interaction combinations in vitro.(3)To investigate the bio-factorial response of ternary assembled communities of different fermentation types LAB to microbial disturbances and to provide a basis for determining combination starter culture.Five groups of microbial community combinations with different nitrogen sources were constructed,and 11 invasion experiments were conducted using typical amine-producing strains(Lactobacillus brevis 367),non-amine-producing strains Lactobacillus casei),and biogenic amine-degrading strains(Staphylococcus xylosus),and the nontransmissive competitive combinations were propagated for 30 days under unsupplemented versus supplemented amino acid conditions,showing that amino acid supplementation caused dramatic fluctuations in the members of the non-transmissive competitive combinations in the early stage and a balanced steady state in the later stage.Combined with the invasion failure of L.brevis 367,it showed the resistance to disturbance and resilience of the non-transmissive competitive e combinations;invading the non-transmissive competitive combinations at different ratios and frequencies as well as different invasion targets,higher ratios and multiple batches of invasion all delayed the extinction of L.brevis 367,the relative abundance was less than 8%,illustrated the resistance of non-transmissive competitive combinations to invasion intensity and frequency.The relative abundance of L.brevis 367,L.casei and S.xylosus at the end of invasion in the transmission competition combination was 15.23 ± 0.75%,27.86 ± 3.46%and 29.42 ± 0.23%,respectively,showed the success of invasion,and resulting in differential changes of sugar depletion,lactate metabolism and biogenic amine accumulation.(4)Based on 16 SrRNA representative sequences and database alignment,we applied the PICRUSt2 to predict the functional contributions of decarboxylases and deiminases,showed that the decarboxylase gene families during Chinese rice wine fermentation were significantly affected by seven bacterial genera including Lactobacillus,Acinetobacter,Bacillus,Exiguobacterium,Citrobacter,Pseudomona,and Enterobacter.Lactobacillus plantarum and Staphylococcus xylosus were inoculated with different interference intensities,and 46.49%regulation of total biogenic amines in Chinese rice wine was achieved in the 1:2 inoculation group from both the perspectives of biogenic amine metabolic pathway blockage and degradation.Spearman analysis showed that Lactobacillus strongly inhibited Exiguobacterium and Pseudomona(ρ =-0.867 and ρ =-0.782;P < 0.05),which resolving the mechanism of biogenic amine reduction based on microbial interactions.