Preliminary Study On Synthetic Microbial Community Of Biogenic Amines Metabolism In Chinese Rice Wine

Biogenic amines?BAs?are one type of the main risk substances in Chinese rice wine?CRW?,which are mainly produced by the decarboxylation of certain amino acids.Lactic acid bacteria?LAB?is considered to be the main bacteria capable of producing amino acid decarboxylase.Analysis of the mechanism of BAs formation in CRW and its relationship with LAB is essential for the safe brewing of CRW.In this paper,the dynamic changes of LAB in industrial-grade semi-dry CRW brewing process were studied in depth,and the relationship between LAB and BAs was further clarified.BAs-producing strains were isolated from CRW combined with high-throughput screening strategy,and then effects of different environment factors on the accumulation of BAs from isolated BAs-producing LAB strains were investigated,which provided a theoretical basis for the reduction of BAs during the brewing process of CRW.Finally,a synthetic microbial community containing BAs-producing LAB strains and non BAs-producing LAB strains was constructed,which further revealed the interactions between LAB strains and their effects on BAs formation.The main results are as follows:?1?Established an approach to detect viable LAB by quantitative PCR?qPCR?combined with propidium monoazide?PMA?treatment.And this method exhibited good specificity,high sensitivity(Limit of detection=4.0×10¹-1.7×10³ CFU?mL^-1),and the variation coefficients of Cq values were less than 1.5%.Additionally,it had no statistically significant?P>0.05?difference compared with plate count.PMA-qPCR method provided a feasible approach on tracking the real-time composition of viable LAB and detecting viable but nonculturable LAB in samples.Furthermore,the PMA-qPCR method was used to detect the dynamic changes of LAB during CRW brewing process.The results showed that the viable LAB in the CRW brewing process decreased with time.Lactobacillus and Lactococcus were the dominant LAB during CRW brewing process,accounting for 69.88%-96.45%and 2.75%-28.44%,respectively.Further we detected 8 kinds of viable Lactobacillus at species level,indicating that Lactobacillus fermentum had a high content in the whole CRW brewing process,which was about 3.08×10⁴-4.95×10⁵ CFU·mL^-1,accounting for 25.22%-44.60%of the Lactobacillus.?2?A total of 31 BAs-producing LAB strains were isolated using decarboxylase medium combined with high-throughput screening strategy and identified through 16S rRNA gene analysis,including 8 strains of Lactobacillus and 23 strains of Enterococcus.High-performance liquid chromatography was used to assess the BAs accumulation capability of 31 LAB strains,and it was found that the ability of BAs accumulation differed from strain to strain.Putrescine,cadaverine,histamine,tyramine,and phenethylamine were the main biogenic amines produced by LAB,and their highest yields were 781.49,990.03,79.85,692.16,and 284.39 mg·L^-1,respectively.Tyrosine decarboxylase genes?tdc?,ornithine decarboxylase gene?odc?and histidine decarboxylase gene?hdc?were found in 23,8 and 8 strains,respectively.The effects of different brewing environmental factors on the metabolic characteristics of BAs-producing LAB strains were investigated.It was found that pH 4.0-5.0,alcohol content 2%-6%,glucose5 g?L^-1 and temperature 35?were the optimal conditions for BAs-producing strains to accumulate BAs.Amino acids had a positive but limit effect on the formation of BAs,and the critical point was 0.1%.?3?Established a synthetic microbial community containing BAs-producing LAB strains and non BAs-producing LAB strains,and detected the number of viable LAB strains and BAs contents in different mixed-culture fermentations.The results showed that Lactobacillus sp.L1-2 inhibited the growth of L.fermentum LF-1 and Enterococcus sp.E1-9 in the Lf-Ls-Es mixed-culture.L.fermentum LF-1 and Enterococcus sp.E1-9 stimulated Lactobacillus sp.L1-2produced more putrescine(increased from 3.95 to 4.36-20.06 mg?L^-1).When L.plantarum was added to the Lf-Ls-Es mixed-culture,the maximum population of Lactobacillus sp.L1-2 and Enterococcus sp.E1-9 decreased from 1.39 and 1.49×10⁸ CFU?mL^-1 to 8.19 and 5.22×10⁷CFU?mL^-1,respectively,and the BAs content decreased 22.15%,which was 43.54 mg?L^-1.