Lactic Acid Metabolic Mechanism Investigation And Control Strategy Development During Chinese Jiang-flavor Liquor Making

Lactic acid is produced by microorganism,which is a major organic acid in Chinese Jiangflavor liquor.Moreover,lactic acid helps modulate the microbiota pH,which coordinates the microbial community.Therefore,lactic acid plays a crucial role in the flavor of Jiang-flavor liquor and the balance of the brewing microbial community,and whether lactic acid can be controlled in the appropriate range during the brewing process directly affects the quality of Jiang-flavor liquor.The Jiang-flavor liquor adopts the process of twice feeding and no more sorghum is added between the seven rounds of distillation.The lactic acid in Zaopei is not easily volatile and continues to accumulate.Unraveling the mechanism of lactic acid metabolism for Chinese liquor making is important to develop effective measures to modulate lactic acid content.However,the knowledge of lactic acid metabolic mechanism and development strategies for control lactic acid metabolism during Chinese Jiang-flavor liquor brewing were constrained by the following three aspects: 1)unidentified predominant lactic acid-producing microorganism during liquor making,2)uncharacterized lactic acid biosynthesis pathway,3)lack of methods for modulating microbial community for lactic acid metabolism control.This dissertation focuses on the identification of predominant lactic acid-producing microorganisms,the regulation mechanism of lactic acid metabolism pathway,and the development and application of lactic acid regulation strategies in Jiang-flavor liquor production.By analyzing the diversity of the microorganisms for Jiang-flavor liquor brewing,the major lactic acid-producing microorganisms in the production process were isolated and identified as Lactobacillus panis.The effects of environmental factors on lactic acid synthesis and its mechanism of L.panis were investigated.Based on the results,L.panis was mutated by Atmospheric Room Temperature Plasma(ARTP)mutagenesis,followed by high-throughput screening of mutagenic strains with different lactic acid production capabilities.And then,molecular mechanism between these mutants were analyzed through comparative genomics.And regulation of lactic acid synthesis through perturbation of the microbial community in fermentation by L.panis was verified by lab-scale solide state fermentation.With the guidance of lactic acid metabolism regulation,the strategy to regulate lactic acid synthesis in liquor production was proposed,and eventually verified through liquor production process.The main research results were as follows:(1)The 16 S rDNA high-throughput amplicon sequencing was adopted to analyze the prokaryotic microbial community diversity during the pit fermentation in the Zaosha round,which had significant accumulation of lactic acid.The results showed that during 0-7 days of fermentation,the dominant bacteria in Zaopei were Bacillus,Lactobacillus,Pediococcus,etc.,and in the middle and late stages,Lactobacillus were the absolute dominant bacteria.151 strains of lactic acid bacteria and 84 strains of Bacillus were isolated by cultivable technology,belonging to 10 species and to 5 species separately.Among them,the content of Lactobacillus panis was the highest,accounting for 67.5% of the total lactic acid bacteria,followed by Lactobacillus plantarum,accounting for 7.3% of the total lactic acid bacteria.At the same time,Bacillus amyloliquefaciens accounted for more than half of the 5 Bacillus species.Comprehensive analysis of the acid-producing ability of different lactic acid bacteria and Bacillus and their proportion in the microbial community,L.panis was identified as the main lactic acid-producing microorganism during the fermentation in the pit,and was named L.panis L7.(2)Whole genome sequencing of L.panis L7 was conducted through the Illumina HiSeq 4000 sequencing platform.And genome assembly,gene prediction,and function annotation were performed.127 scaffolds were obtained with a total length of 2,026,099 bp and an average GC content of 47.0%.A total of 1,932 genes,51 t RNAs and 2 rRNAs were annotated in the genome.Analysis of lactic acid metabolism pathways revealed that L.panis L7 is obligate heterolactic fermentation bacteria,and its major fermentation products are D-lactic acid,Llactic acid,and other by-products such as acetic acid.The analysis of L.panis L7 under different environmental conditions indicated that L.panis L7 had a relative higher heat-resistant ability,resulting no significant effect of temperature on its growth and lactic acid synthesis capability.Moreover,the addition of lactic acid promoted the lactic acid production.However,ethanol showed a significant inhibitory effect on the growth and lactic acid production of L.panis L7,and the inhibitory effect increased with the increase of ethanol concentration.Glucose showed a promoting effect on lactic acid formation by L.panis L7 at a lower concentration.With further increase of glucose concentration,glucose had an inhibitory effect on the growth and lactic acid production.The lactic acid metabolism response of L.panis L7 to environmental factors was consistent with the characteristics of Jiang-flavor liquor technology,indicating that it had achieved directional domestication during the liquor production.(3)Mutant libraries of L.panis L7 strains was constructed by ARTP mutagenesis,and a high-yield lactic acid mutant strain R6 and a low-yield mutant strain R13 were obtained through high-throughput screening.Their lactic acid yield increased by 21% and decreased by 11%,respectively.Comparative genomics analysis identified the glutamic acid racemase gene murI and methylated DNA protein cysteine methyltransferase gene ybaZ were potential key genes to improve lactic acid biosynthesis.Mutations of fructokinase gene rbsK,the ribokinase gene scrK,arginine succinate lyase gene argH,and the large subunit ribosomal protein L30 gene rpm D were potential key factors that reduce lactic acid biosynthesis.(4)Perturbation of the microbial community by adding different L.panis mutants during brewing process showed that the addition of L.panis mutants with different lactic acidproducing ability during fermentation could significantly increase the lactic acid content,but had no obvious influence on the structure of microbial community,the proportion of Lactobacillus,content of acetic acid,ethanol and the composition of flavor substances.When mutant R6 was added to perturb the microbial community during pit fermentation,when fermented for 14 days,the lactic acid concentration increased by 23.6%.Therefore,adding L.panis could promote lactic acid production and achieved the target of increasing lactic acid content.(5)Augmentation of the low-yield lactic acid mutant strain R13 to disturb the pit fermentation process could not achieve the goal of lactic acid reduction.Therefore,an effective lactic acid reducing strategy was established by controlling the lactic acid bacteria amount in Daqu during the fermentation process.Compared with the control,controlling the amount of lactic acid bacteria in Daqu could effectively reduce the growth and proliferation of lactic acid bacteria,and gradually reduce the content of Lactobacillus from 80% to 60%.And at the end of fermentation,the content of lactic acid was reduced by 31.9%.Therefore,this strategy could significantly reduce the production of lactic acid,as well as reduce acidity accumulation during the fermentation process.Moreover,the control of lactic acid bacteria in Daqu had little effect on theliquor yield,while the quality of the base liquor was close to that of the control group.