Diversity And Metabolic Characteristics Of Lactic Acid Bacteria And Lactate-degrading Bacteria In Fermented Grains Of Luzhou-flavor Liquor

Luzhou-flavor liquor, also named strong-aroma liquor, is a typical representative of Chinese liquor, which is produced from sorghum through multispecies and solid-state fermentation. As the main driving force to generate flavor compounds of liquor, it is essential to unravel the diversity and succession of microbial community during the fermentation process for dissecting the mechanism of solid-state fermentation. Lactate and its derivates are important flavor substances in Luzhou-flavor liquor, and they are indispensible for the fragrance and taste of liquor, whereas the information about the microbial community associated with lactate metabolism are still incomprehensive. In this study, the diversity and succession of lactic acid bacteria(LAB) during the fermentation process was unravelled, the origin source of abundant LAB species in fermented grains was identified, and the predicted functions of LAB community was conducted with PICRUSt and vertified by simulatively anaerobic fermentation. In order to eliminate the excessive accumulation of lactate during the fermentation process, diverse lactate-degrading bacteria were isolated from fermented grains, and then specific metabolic characteristics were determined. The main results showed as follows:(1) Monitoring the biomass and relative abudance of LAB during the fermentation process of Luzhou-flavor liquor. Real-time quantity PCR(RT-qPCR) results showed that the biomass of LAB keeps increasing from 4.05×108 copies?g-1 culture to 2.53×1010 copies?g-1 culture during the fermentation process, which increased by nearly a hundred times. At initial, the relative abudance of LAB accounted for more than 50% of the total bacteria in fermented grains, but dramatically decreased to 11.20%, and then increased rapidly during the first 7 d, afterthat increased in volatility up to 78.80% in the end, becoming the dominant microorganism in fermented grains.(2) Unravelling the diversity and succession of LAB community during the fermentation process of Luzhou-flavor liquor. At genus level: A total of 15 genera of LAB were detected via high-throughput sequencing(HTS), wherein Lactobacillus was the dominant population, and genera Weissella, Leuconostoc, Pediococcus, Vagococcus, Erysipelothrix, Listeria, Gemella, Aerococcus, Atopobium, Carnobacterium were firstly reported in fermented grains of Luzhou-flavor liquor. Weissella was predominant population in the early stage of the fermentation process, but which was replaced by Lactobacillus after 2 d fermentation.At species level: A total of 49 LAB species were found, thereinto Lactobacillus acetotolerans was the absolutely dominant species, followed by Weissella confusa. Similarly, the dominant position of Weissella confusa was replaced by Lactobacillus acetotolerans after 2 d fermentation.(3) Exploring the original source of abundant LAB species in fermented grains.The comparative results of LAB communities in Daqu, fermented grains and pit mud showed that, among the 14 abundant LAB species in fermented grains, 8 species were also frequently found in Daqu, and Lactobacillus acetotolerans was dominant both in fermented grains and pit mud, so we speculated that most of abundant LAB species in fermented grains originated from Daqu, while Lactobacillus acetotolerans derivated from pit mud.(4) Predicting the potentional functions of LAB community and vertified by simulatively anaerobic fermentation. PRICUSt results showed that LAB mainly participated in glycolysis, and possessed the functional potential to generate butanoate and propanoate during the fermentation process of Luzhou-flavor liquor, while barely involved in ascorbate and aldarate metabolism, C5-branched dibasic acid metabolism and inositol phosphate metabolism. Moreover, 19 LAB species were isolated from fermented grains, with the exception of generating lactic acid and acetic acid, which could also produce propionic acid, butanoic acid, caproic acid and ethyl caproate under anaerobic fermentation conditions, especially Lactobacillus senioris.(5) Unravelling the diversity of lactate-degrading bacteria in fermented grains. A total of 17 lactate-degrading bacteria species were iaolated from fermented grains, which belonged to 7 genera, including Clostridium, Terrisporobacter, Bacillus, Ilyobacter, Desulfotomaculum, Staphylococcus and Raoultella, wherein Clostridium was dominant population.(6) Determining the characteristics of flavor compounds metabolism and environmental factor tolerance of lactate-degrading bacteria. In total, 64 kinds of volatile flavor compounds were detected by using GC-MS, mainly including alcohols, acids, esters, and whose metabolic characteristics of flavor compounds showed significant differences among genera and species. The results of environmental tolerance evaluation indicated that ethanol content and pH show great effects on lactate-degrading efficiency, and the best inoculation amount was 1%. Ilyobacter delafieldii worked best when ethanol content is no more than 2% vol, pH is no less than 5.5 and sodium lactate concentration is no more than 40 g?L-1, while all tested strains showed low lactate-degrading efficiency under the conditions of ethanol content is no less than 4% vol and sodium lactate content is less than 5.0.