Isolation Of Salt-tolerant Lactic Acid Bacteria In Fish Sauce And The Research Of Its Metabolic Characteristics

In this paper,several strains of salt-tolerant lactic acid bacteria were isolated from fish sauce,and their fermentation characteristics were further compared.The growth and metabolism pattern of a strain of Tetragenococcus halophilus,which had the best fermentation effect,was studied.The transcriptomes of it at different oxygen concentrations were sequenced.Some of the genes and metabolic pathways related to the formation of flavor components were obtained by comprehensive analysis of metabolite changes and transcriptome results.The main results of this paper are as follows:(1)Five strains of salt-tolerant lactic acid bacteria were isolated from fish sauce.Four of them were identified as by 16 S rDNA sequencing.They were named as JL-B,JL-C,JL-D,JL-E.The other strain is Staphylococcus epidermidis named JL-F.(2)Then four strains of T.halophilus and one strain of Staphylococcus epidermidis were separately inoculated into the hydrolyzate of Decapterus maruadsi.Their fermentation characteristics were compared.The flavor of the samle inoculated with strain JL-B,which had prominent sauce and umami flavor,was the most acceptable.The sample also had the lowest pH value,which kept stable at around 4.30.The amino acid nitrogen content of the sample was around 0.49 g/100 mL.(3)The analysis of the growth metabolites of halophilic Tetragenococcus JL-B showed that the hydrolyzate of Decapterus maruadsi fermented at 30°C had the lowest pH and the highest number of bacteria at different temperatures.The amount of amino acid nitrogen(AAN)produced at 30°C was also significantly higher than that at 23°C and 37°C.As the fermentation temperature increases,the amount of TVBN produced also increaes.,and TVBN content at 37°C was significantly higher than 23°C.Under anaerobic fermentation,the pH,total volatile base nitrogen(TVBN),amino acid nitrogen(AAN),soluble trichloroacetic acid(TCA-N),soluble total nitrogen(TSN),free sweetness and Flavor amino acids were lower than aerobic groups.Then the number of bacteria and protease activity was significantly higher than that of aerobic fermentation group.Whatever the oxygen concentrations,the activity of proteases in the fermentation broth was increased at 30°C during the initial fermentation period.The zymographic electrophoresis showed that the transparent bands produced by proteases were the most obvious at the 15 th day,the activity of proteases reached the highest at the same time,namely 7.23 U/mL.Thereafter,it gradually decreased.The change trend of protease activity was consistent with the number of Tetragenococcus halophilus.In addition,most of the volatile substances detected in the above fermentation broth were alcohols,followed by aldehydes and ketones and esters again.The proportions of acids,nitrogen compounds,sulfur compounds,and hydrocarbons are low.And alcohol content is always in a dominant position,showing a trend of increasing first and then decreasing.At 60 days of fermentation,the aldehydes in the anaerobic and aerobic fermentation broth increased by 1.26-fold and 0.41-fold respectively;the ketones decreased by 0.68-fold and 0.47-fold;the esters decreased by 0.96-fold and 0.87-fold.Within 30 days before fermentation,the relative content of acid substances increased dramatically,in which the anaerobic fermentation group increased from 2.93% to 15.79%,and the aerobic fermentation group increased from 0.39% to 14.95%.At 60 days of fermentation,the relative content of acids in the aerobic fermentation group was significantly higher than that in the anaerobic fermentation group,with values of 26.69% and 13.84%,respectively.(4)The components isolated from the fermentation broth were separated and purified.After the sensory evaluation,the part 4 with the most umami taste was selected for mass spectrometry analysis.There were 6 ion peaks in the primary mass spectrum of the sample part-4.The ratios were 119.95,165.95,273.95,317.96,361.96,and 405.97,respectively.It is initially determined that the composition of the sample is mainly a small molecule compound.The incorporation of secondary mass spectrometry deduces that peak-4 may contain porphyrin and DL-phenylalanine.(5)The transcriptomes of T.halophilus at different oxygen concentrations were sequenced and a total of 291 differential genes were obtained.The anaerobic fermentation group was used as a control.There 94 were up-regulated differentially expressed genes and 197 down-regulated differentially expressed genes under aerobic conditions.Further analysis revealed that there were 51 KEGG metabolic pathways that were significantly enriched.The up-regulated differentially expressed genes are mainly concentrated in peptidoglycan biosynthesis,glyoxylate and dicarboxylic acid metabolism,arginine and proline metabolism,glycine,serine and threonine metabolism,and glutathione metabolism pathways.Then the down-regulated differentially expressed genes are mainly enriched in amide biosynthesis,microbial metabolism in different environments,pentose phosphate pathway,glycolysis/gluconeogenesis,lysine biosynthesis and other metabolic pathways.Eight target differentially expressed genes associated with flavor development were obtained through analysis of metabolic pathways and metabolites.They were gene gdh(glutamate dehydrogenase(NADP +),EC: 1.4.1.4)and gene proB(glutamate 5-kinase).,EC: 2.7.2.11),gene ldh(L-lactate dehydrogenase,EC: 1.1.1.27),gene rpiA(ribose-5-phosphate isomerase A.,EC: 5.3.1.6),gene fni(isoprene)Thiodiphosphoric acid ?-isomerase,EC: 5.3.3.2),gene hprA(glycerate dehydrogenase,EC: 1.1.1.29),gene gshAB(glutamic acid-cysteine ligase,EC: 6.3.2.2)?gene tkt(transketolase,EC: 2.2.1.1).The gene proB regulates glutamate 5-kinase(EC: 2.7.2.11)and promotes glutamate production.The gene hprA regulates glycerate dehydrogenase(EC: 1.1.1.29)and directly promotes hydroxypyruvate production.Then it was converted from hydroxypyruvate to serine.The expression levels of these two genes were all higher under aerobic conditions than under anaerobic conditions,while the contents of glutamic acid and serine under aerobic conditions were 9.58% and 20.38% higher than those under anaerobic conditions,respectively,suggesting that the above two genes There is a correlation between the expression level and the corresponding amino acid content.L-glutamic acid and L-cysteine can be converted to L-?-glutamylcysteine through the regulation of the gene gshAB,and L-?-glutamylcysteine can be further transformed into L-Cysteine.The other five target differentially expressed genes may be related to the formation of flavor,and the specific regulation mechanism needs to be further explored.(6)To determine the reliability of the transcriptome data,q-PCR experiments were performed on the eight targets differentially expressed genes gdh,proB,ldh,rpiA,fni,hprA,gshAB,and tkt.The q-PCR results were consistent with the transcriptome results,indicating that the transcriptome data was reliable.