Study On The Regulation Mechanism Of Aromatizing And Reducing Amine In Fishweed Fermentation Process Based On LuxS/AI-2 Quorum Sensing System

Fish sauce as a typical traditional condiment in China,which possesses a long history,is a precious tangible cultural heritages in China.However,the production of fish sauce is greatly restricted due to many reasons,such as the long production period in traditional way,the difficulty in overcoming the flavor variation,and the difficulty in controlling the accumulation of biological amines in the fermentation process.AI-2/LuxS quorum sensing system,as the main inter species group sensing system of bacteria,can enhance the probiotic of bacteria and promote the fermentation of bacteria.Based on the main biogenic amines and the main volatile compounds in fish sauce,the strains with the ability of producing volatile compounds and degrading biogenic amines in the fermentation broth of fish sauce were screened.The ability of AI-2/LuxS quorum sensing system to effect volatile compounds producing,the ability of reducing amines and the salt tolerance of the strains were explored by adding AI-2.The purpose of this study provide theoretical basis to shorten the fermentation period,reduce the accumulation of biogenic amines during fish sauce fermentation.The main research contents are as follows:1.Seven strains with high protease production,four strains with high lipase production and one strain with both protease and lipase activity were isolated.The main biogenic amines in Xing Ye fish sauce were cadaverine(67.31 mg/L),histamine(102 mg/L),putrescine(59.94mg/L),tyramine(101.15 mg/L).Alcohol(1-octanol,phenylethanol,furfuryl alcohol,isoamyl alcohol),aldehydes(nonanal,benzaldehyde,Phenylacetaldehyde,3-methylthiopropionaldehyde,3-Methylbutyraldehyde,2-methylbutyraldehyde),acids(2-Methylbutyric acid,3-methylbutyric acid,acetic acid,caprylic acid),pyrazines and furans etc,are the main volatile compounds in Xing Ye fish sauce.According to the main biogenic amines and volatile substances in Xing Ye fish sauce,five strains with the ability of biogenic amines degradation and volatile substances production were determined.After 16 S rRNA and physiological and biochemical identification,they were preliminarily identified as Tetragenococcus halophilus MJ4,Bacillus amyloliquefaciens,Bacillus vietnamensis,Oceanobacillus iheyensis,Virgibacillus halodenitrificans.The degradation capacity ofTetragenococcus halophilus MJ4 to cadaverine,histamine,putrescine and tyramine was24.39%,15.8%,24.65% and 25.30%,respectively.Nonanal,methyl butyric acid,benzaldehyde,furfural and phenylethanol are the main volatile compounds in the simulated fish sauce which inoculum Tetragenococcus halophilus MJ4 and also the main volatile compounds in Xing Ye fish sauce.Thus,Tetragenococcus halophilus MJ4 was determined as specific flavor forming and amine reducing bacteria.2.Based on the results of the previous chapter,this experiment investigated the regulation of fermentation related capacity of Tetragenococcus halophilus MJ4 by exogenous addition of AI-2.Firstly,LuxS / AI-2 quorum sensing system was proved by the reaction between V.harveyi BB170 and AI-2.And AI-2 activity is the strongest at the end of logarithmic growth.The experimental results showed that the appropriate dose of AI-2 could significantly strengthen the lipase,protease,aminopeptidase,amylase activity,salt tolerance and amine reducing ability of Tetragenococcus halophilus MJ4.The tryptophan aminopeptidase,methionine aminopeptidase,glutamate aminopeptidase and amylase activities of Tetragenococcus halophilus MJ4 were increased by 16.5%,22.3%,19.3% and37.38% respectively at the concentration of 550 nmol/L of AI-2.Lipase activity increased by51.3% at AI-2 concentration of 850 nmol/L.Moreover,the quantity of Tetragenococcus halophilus MJ4 at 850 nmol/L AI-2 in the content of 15%,20% and 25% NaCl was significantly higher than the other without the addition of AI-2,the ability of reducing amines is also being greatly enhanced.The expression of lipase,aminopeptidase,?-amylase,salt tolerance and biogenic amine adjust related genes of Tetragenococcus halophilus MJ4 was determined by qPCR,and the regulation mechanism of AI-2 was preliminarily predicted.Finally,AI-2 was added to simulated fish sauce and inoculated with Tetragenococcus halophilus MJ4 for fermentation.The content of volatile flavor compounds such as nonanal,phenylethanol,methyl butyric acid and furfural was increased.3.Five amines producing bacteria were screened from the fermented fish sauce.They were identified as Paraliobacillus ryukyuensis? Staphylococcus cohnii,Bacillus flexus,Paenibacillus alvei and Ornithinibacillus halophilus by 16 S rRNA and physiological and biochemical identification.Among them,Paraliobacillus ryukyuensis has strong tyramine production ability,which can produce 481 mg/L tyramine at 5% salt content,142 mg/Ltyramine at 15% salt content.Staphylococcus cohnii has strong cadaverine and histamine production ability,can produce 156mg/L cadaverine and 72 mg/L histamine at 5% salt content,can produce 61.54 mg/L cadaverine and 68.43 mg/L histamine at 15% salt content.Paraliobacillus ryukyuensis,Staphylococus cohnii and Tetragenococcus halophilus MJ4 were cultured,and 850 nmol/L was selected as the amount of AI-2.According to the previous experimental results,so as to explore the growth and accumulation of amine producing bacteria and biogenic amines under the co culture system.The results showed that Tetragenococcus halophilus MJ4 had little effect on the growth of the two strains of amines producing bacteria growth in the early stage of culture.After 36 hours,Tetragenococcus halophilus MJ4 showed inhibition on the growth of the two strains.The content of biogenic amines in the co culture system decreased compare to control after 36 hours incubation.The addition of AI-2 not only enhanced the inhibition of Tetragenococcus halophilus MJ4 on the growth of amine producing bacteria,but also reduce the accumulation of biogenic amines in co-culture system with the addition of AI-2.Tetragenococcus halophilus MJ4 was identified as a specific flavor forming and amine reducing bacteria through the test of amine reducing and flavor forming ability.Based on the influence of volatile compounds forming ability and amine reducing ability by AI-2,the mechanism of AI-2 to regulate the fish sauce fermentation was revealed.The growth of amine producing strains and the accumulation of biogenic amine in the co-culture system reveal the optimization of micro community of AI-2.The results provide a reference for shortening the fermentation period and reducing the accumulation of biogenic amines in fish sauce.