Studies On The Changes Of Microbiome Structure And The Formation Mechanisms Of Characteristic Flavor In The Plant-derived Brine During Fermentation

"Eatting odor" has existed since ancient times.Although its odor was not friendly,they still display their charms because of their unique flavor,strong local and culture characteristics.Changsha Stinky Tofu is one of the representatives of local flavor snacks,and its characteristic flavor originates from fermented brine which is mainly composed of plant materials.At present,the brine was usually made using traditional techniques.However,due to unclear fermentation microorganisms and unclear flavor formation mechanism,the process cannot be qualitative and quantitative,resulting in unstable product quality.Therefore,this study,on the basis of studying the characteristic flavor substances of plant-derived brine in Changsha Stinky tofu production enterprise,aimed at solving a "bottleneck" technical problem in the production of brines using plant-based raw materials such as winter bamboo shoots,mushrooms,dried prunes,beancurd jelly,and Douchi.In this work,the HS-SPME-GC-MS combined with internal standard was used to quantify the volatile components,characteristic flavor components and their dynamic changes in the process of synthetic brine fermentation.The changes of microbial community structure in stinky tofu brine during different fermentation periods was investigated based on metagenomics.The associated bacteria,associated enzymes,major differential metabolites,and their changes in the aromatic amino acid metabolic pathway were investigated by combining the metagenomics,metabonomics,and bioinformatics.In addition,the network map of the main characteristic flavor substances in brines was constructed and the formation mechanism of the main characteristic flavor compounds of plant-derived brine was revealed at the genetic level.These results will provide a scientific basis for the standardization and normalization of industrial brines production and,strengthen or improve the flavor quality of traditional fermented foods.The main results are given below:(1)The results of the study on the taste of stinky tofu soaked by plant-derived brine in Changsha showed that the four brines had the greatest influence on the odor of stinky tofu,and the stinky tofu soaked by Z1 had the highest odor and total sensory score.The electronic nose(EN)can distinguish the overall flavor of the four kinds of brine samples.The most common odors in the brine that can be recognized by EN are mainly aromatic compounds,amines,and aliphatic hydrocarbons containing oxygen derivatives.Phenols and indoles were,the main aromatic compound and amine substance in four kinds of brine,determined by HS-SPME-GC-MS.OAV analysis showed that phenols and indoles such as phenol,4-methylphenol,indole,and3-methylindole are ubiquitous and are the main characteristic flavor substances found in the four brines.(2)In reference to the brine Z1,using plant materials such as winter bamboo shoots,mushrooms,dried prunes,beancurd jelly,and douchi as the substrate to prepare brine.And the volatile components,characteristic flavor substances and their changes during brine fermentation were investigated.Seventy-five volatile flavor compounds were identified during the mixed fermentation of stinky tofu brine,including 22 kinds of alcohols,8 kinds of acids,2 kinds of aldehydes,9 kinds of ketones,18 kinds of esters,3 kinds of ethers,4 kinds of phenols,2 kinds of indole,and 7 kinds of others.The total types of volatile flavor substances in fermented brines were 16,30,31,42,27,33,25,and 14 with a total of 3.01,305.42,562.05,365.8,629.56,120.62,21.87,and 2.49 μg/m L in content after 0,6,15,30,45,60,90,and120 days of fermentation,respectively.Phenols and indoles were the main characteristic flavor substances in the whole fermentation process,phenols mainly include phenol and p-methylphenol,both of which showed a trend of increasing first and then decreasing.The maximum content of p-methylphenol and phenol were389.62 and 89.96 μg/m L after 45 and 15 days of fermentation.Indoles mainly include Indole and Skatole,both of which show a tendency of rising and then falling and again in the whole fermentation process.The maximum content of indole and3-methylindole were found to be 242.92 and 3.00 μg/m L after 15 and 45 days of fermentation,respectively.(3)Based on Metagenomics,the predominant microorganisms in the stinky tofu brine samples before fermentation(0d)were mainly Exiguobacterium unclassified、Exiguobacterium alkaliphilum 、 Exiguobacterium sp.AB2 、 Exiguobacterium chiriqhucha and Enterococcus Faecalis.Eleven dominant bacteria were screened out in the samples after 15～60 days of fermentation,there were four Genera and five species in the family Clostridiales(Clostridium bacterium SYSU GA17129,Clostridiales unclassified,Clostridium ultunense,sporanaeromonas Acetenes,Keratinibaculum paraultunense),three Genera and three species in the family blastomycegalense(massiilibacterium Senegalense,Bacillus thiobacillus and gracilianus auburnensis),one Genu and one specie in the family of alkaliaceae(Paencaligenes Hominis),and two species in the genus Ignatzschineria(Ignatzschineria sp.F8392、Ignatzschineria indica).Among them,clostridium(5species)and Bacillus(3 species)were the dominant bacteria in the brine during the fermentation stage,especially during the early and middle fermentation stages of brine,and gradually reduced in the later stages.The results showed that there were significant correlations between phenol,p-methyl phenol,indole and Skatole with Clostridiales bacterium SYSU GA17129 、 Aneurinibacillus aneurinilyticus when calculating the Pearson Correlation Coefficient between the bacteria and the main characteristic flavor components(phenols and indoles)in the brines.(4)The associated bacteria,associated enzymes,differential metabolites and their changes of aromatic amino acid metabolism in brine were analyzed based on metagenomics,metabonomics and bioinformatics.Meanwhile,a network map of the formation pathway of characteristic flavor substances in brine was constructed.The results indicate that the main microorganisms involved in the metabolism of aromatic amino acid in the stinky tofu brine before fermentation(0 d)were Exiguobacterium unspecified,Exiguobacterium Aliphilum,Exiguobacterium SP.AB2 and Enterococcus Faecalis,the early and middle stage(15,30 d)were Massilibacterium Senegalense,sporanobacter Aceticenes,aneurinobacillus inilyticus,Clostridiales syga17129 and Clostridiales unclassified,the late stage(45,60 d)were Paenaligenes Hominis.The associated enzymes were transaminase,aldehyde dehydrogenase,decarboxylase,monoamine oxidase and lyase.These enzymes account for a high proportion of each aromatic amino acid metabolism pathway before and during fermentation.The main different metabolites were 7 kinds of organic acids and their derivatives,3 kinds of indole and its derivatives,and 11 kinds of benzene and its derivatives.The relative contents of the three kinds of aromatic amino acid were higher in brine(0 d)and were degraded gradually with the advancing of fermentation time.The relative contents of phenols and indoles flavor compounds were higher after 30 d and 45 d of fermentation.The highest contents of Phenylpyruvateb,4-Hydroxyphenylpyruvic acida were found in brines without fermentation(0 d).The highest relative content of tyramine,phenylethylamine and phenylacetaldehyde were found after 15 d of fermentation.Meanwhile,the other metabolites varied continuously during the whole fermentation process.The phenols and indoles in brine were mainly formed by a series of biochemical reactions such as transamination,degradation,decarboxylation,oxidation and hydroxylation of the precursors of L-phenylalanine,tyrosine and tryptophan,under the action of transaminase,aldehyde dehydrogenase,decarboxylase,monoamine oxidase and lyase.Phenol compounds were mainly formed by a series of reactions such as transamination,decarboxylation,oxidation and hydroxylation of tyrosine and L-phenylalanine,respectively.The formation of indole compounds was mainly the decomposition of tryptophan to indole by tryptophan lyase,a portion of indole was converted to indole-3-acetate by a carboxyl transferase and then to3-methyl indole by a lyase.In the whole brine fermentation process,decarboxylation and ammonia conversion were the main reactions in the early stage(15 days before the fermentation),and oxidation,decarboxylation and ammonia conversion were the main reactions in the middle stage(15～45 d),at the later stage(60 days after the fermentation),the metabolic pathways of aromatic amino acids producing flavor substances is gradually closed.