Comparative Genome Analysis Of Acetilactobacillus Jinshanensis And Its Function In Vinegar Fermentation

Acetilactobacillus jinshanensis is a novel genus scattered in the family Lactobacillaceae,and the type strain HSLZ-75^T was isolated from the cupei of Zhenjiang aromatic vinegar.Previous work showed that A.jinshanensis dominated the bacterial cmmunities during the solid-state fermentation process of vinegar and baijiu.In particular,the relative abundance of A.jinshanensis reached above 90%at the late stage of fermentation under the condition of high acidity.However,there is still a lack of understanding of its physiological and biochemical characteristics,genome analysis,and fermentation performance.Therefore,this study aims to(i)elucidate the growth and metabolism characteristics of A.jinshanensis HSLZ-75^T,and optimize the culture conditions;(ii)analyze the functional potential of HSLZ-75^T at the genomic level and the underlying mechanisms of of stress resistance such as acidproof behavior through comparative genome analysis;(iii)investigate the roles of HSLZ-75^T in the flavor formation of Zhenjiang aromatic vinegar through in situ bioaugmentation at different fermentation stages of acetic acid fermentation(AAF).This research systematically analyzed the growth,metabolism characteristics and brewing functions of A.jinshanensis HSLZ-75^T,in order to provide a theoretical basis for its rational application in production.The main findings are as follows:(1)The effects of different kinds of acids(acetic acid,lactic acid,hydrochloric acid,oxalic acid,pyruvic acid and citric acid)and oxygen conditions on the growth of HSLZ-75^T were studied.The results showed that HSLZ-75^T growed well only when the pH of medium was adjusted to 3.65-4.15(optimum pH 3.85)with acetic acid,and the optimum oxygen condition was is 5%CO2,10%H2 and 85%N₂.The culture conditions for growth and lactic acid accumulation of A.jinshanensis were optimized through single-factor experiments,and the optimum conditions were:MRS medium supplemented with 200 g·L^-1 malt extract and 3%(v/v)acetic acid,incubation temperature 37?,and anaerobic.After culture for 3.5 days under the optimal conditions,HPLC analysis revealed that the main organic acid produced by HSLZ-75^T was lactic acid(11.98±0.26 g·L^-1),followed by acetic acid(2.60±0.35 g·L^-1)and citric acid(1.93±0.21 g·L^-1).Twenty-one volatile matabolites were identified,mainly including phenethyl alcohol,furfuryl alcohol,caprylic acid,acetic acid,furfuryl acetate,ethyl acetate,benzaldehyde,nonanal,2,3-dimethylpyrazine,and 2,6-dimethylpyrazine.(2)The genome of A.jinshanensis HSLZ-75^T is composed of a 1,649,987 bp circular chromosome,and the length of its genome coding region is 1,616,430 bp.The genomic DNA GC content is 39.70%.There are 23,1061,1043,821 genes annotated against the CAZyme,COG,GO and KEGG databases.Comparative genome analysis of 11 strains was conducted,including HSLZ-75^T and another 10 kinds of bacteria with high abundance(9 Lactobacillus and 1 Acetobacter strains)in Zhenjiang aromatic vinegar fermentation process.Results showed that the genome size of HSLZ-75^T was smaller than the other 9 strains.It has low collinearity with other vinegar-brewing bacteria.Unique gene families include sodium ion transport,thiamine diphosphate biosynthesis,et al.The genome of HSLZ-75^T contains the largest number of genes related to energy metabolism,amino acid transport and acid stress resistance(25.48‰).(3)Specific primers with product size of 199 bp,GC content of 55%and Tm value of 59?were designed using the pheS gene of HSLZ-75^T.The optimized RT-qPCR method displayed potent specificity,high sensitivity,good repeatability and versatility.The amplification efficiency was 99.31%and the detection range was 2.24 lg(copies·?L^-1)-10.24 lg(copies·?L^-1).This method was evaluated and successfully applied for the detection of HSLZ-75^T in cupei and jiupei from different regions.This method was further used to analyze the copies of HSLZ-75^T in cupei of Zhenjiang aromatic vinegar during AAF.The number of HSLZ-75^T increased rapidly from 5.85±0.04 to 8.72±0.09 lg(copies·g^-1 dry pei)in 4-9 days,and until the end of fermentation(day 18),its copies kept around 108 copies·g^-1 dry pei.(4)To investigate the function of HSLZ-75^T in AAF,lab-scale bioaugmentation of HSLZ-75^T(9×105 copies·kg^-1 cupei)in different fermentation stages(i.e.,phase ?,day 0 of AAF;phase ?,day 3 of AAF;phase ?,day 1 of fengpei)of was conducted.Track and monitor the microbial content and physical and chemical indicators of cupei and vinegar.The results showed that bioaugmentation of A.jinshanensis HSLZ-75^T at the beginning of AAF(phase ?)could significantly increase the proportion of HSLZ-75^T in total bacteria in cupei during fermentation,and shorten the time to reach the maximum temperature.Compared with the control group,the contents of total acid,lactic acid and acetic acid in vinegar of bioaugmentation group in phase ? increased by 10.47%(P<0.001),76.22%(P<0.001)and 10.85%(P<0.001),respectively,and the proportion of lactic acid to total organic acid increased from 15.12%to 22.33%.Moreover,the contents of volatile compounds such as phenethyl alcohol,pentanoic acid,ethyl tetradecanoate,phenyl ethyl acetate were higher than those in the control group.The taste and aroma of bioaugmented vinegar were better than control based on sensory evaluation.(5)Bioaugmentation of A.jinshanensis HSLZ-75^T at the beginning of AAF was selected for field-scale experiments.HSLZ-75^T could increase the maximum temperature of cupei,speed up the consumption of glucose,and shorten the fermentation cycle,which was consistent with the lab-scale results.At the end of AAF,the contents of total acid,acetic acid,lactic acid,total amino acid,volatile compounds and esters were increased by 22.87%(P<0.001),10.88%(P<0.01),106%(P<0.001),12.48%(P<0.001),15.96%(P<0.05)and 10.58%(P<0.05),respectively.In comparison with the control the total acidity and lactic acid content of bioaugmented vinegar were increased by 14.28%(P<0.001)and 44.77%(P<0.001),which were 62.70±0.66 g·L^-1and 13.28±0.37 g·L^-1,respectively.Compared with the control group,the proportion of lactic acid to total organic acid increased from 17.86%to 21.54%.According to the OPLS-DA analysis,there were 15 volatiles(VIP?1)that contribute significantly to the differences between the control and bioaugmented groups,mainly including tetramethyl Pyrazine,Ethyl acetate,3-methyl butanoic acid,2-methyl propanoic acid,Furfural,Acetoin,3-acetyl-2-butanone.