Study On The Mechanism Of Putrescine Biosynthesis And Growth And Decline Based On The Amino Acid Metabolism Of Shewanella Baltica 73 In Large Yellow Croaker

Biogenic amines(BA)are basic nitrogenous compounds of low molecular weight with biological activity and widely distributed in aquatic products which are abundant in proteins and amino acids.As one of the valuable commercial fishes,large yellow croaker has surging demand in domestic market.However,microorganisms are easily multiplied and produce large amount of BAs during storage,process and transportation,resulting in the aquatic corruption,terrible economic losses,resource waste and environmental pollution.However,the biosynthesis profile and formation mechanism of BAs in large yellow croaker are still poorly understood.Thus,the research on the mechanism of main BA production based on the regulation of specific spoilage bacteria in ice-stored large yellow croaker was conducted,which may provide a good theoretical foundation and guarantee for better detection of the aquatic quality and inhibition targets for aquatic spoilage.The study contains the following four parts.(1)The BAs contents,aerobic bacterial count(TVC),several characteristic physicochemical indexes(pH,TVB-N,TMA,P.I.%)and free amino acids(FAAs)contents were monitored in large yellow croaker during storage at 4 ℃.It’s forcefully uncovered that the putrescine and cadaverine were the main BAs and the sum value of them could be used as a novel quality indicator instead of BAI(BAI = Put + Cad + Him + Tym)to evaluate the freshness of large yellow croaker rapidly and directly.The regression model(Rput = 0.88,P<0.05;RCad = 0.87,P<0.05)between the main BAs and physicochemical indexes offered a new interpretation for the physiochemical properties of BAs in aquatic products.And the dynamic correlation between BAs and their precursors indicated the relative complexity of putrescine production.(2)According to our previous results that S.baltica 73 was the specific spoilage organism in cold-storaged large yellow croaker,the putrescine biosynthesis of S.baltica 73 was performed.Based on the reports of putrescine biosynthesis,11 genes(ARG,ADC,AgDI,OCT,odc,speF,potE,TF,TG,TH,Potl)involved in putrescine formation were identified in S.baltica 73 through DNA alignment and PCR.Furthermore,the effects of ornithine and putrescine on the ODC system were explored.The speF and potE gene-deleted mutants were established to determine the function mechanism of the ODC system.Our results indicated that speF and potE genes had a critical effect on the putrescine biosynthesis,and a model of "PUT positive regulation" was proposed in S.baltica 73.(3)The regulation of environmental pH on the putrescine accumulation based on ODC system was explored.Five different pH stimulus(5.5,6.0,6.5,7.0,7.5)were set to explore the the effect of environmental pH on the gene expression in ODC system,enzyme activity of speF,and the putrescine production were evaluated.Results indicated that acidic environment had a significant induction on the transcription of speF and potE genes,especially at 6.0.And the enzyme activity of speF at five cultivation conditions was ranked as 6.0>6.5>7.0>5.5>7.5.Finally,the putrescine production in strain respectively were 536.74 mg/L(pH =5.5),679.16 mg/L(pH = 6.0),604.25 mg/L(pH = 6.5),538.54 mg/L(pH=7.0),340.91 mg/L(pH = 7.5),respectively.To conclude,acidic environment had a significant induction on the putrescine production.(4)As for protein activity,the global acetylproteome analysis of S.baltica 73 was performed through high affinity purification of acetylated peptides and mass spectrometry(MS)detection.2929 lysine acetylation sites were identified in 1103 proteins.It’s also found that the acetylated proteins participated in a wide variety of biological processes.Notably,three pivotal enzymes(ADC,OCT,speF)involved in the biosynthesis of putrescine were all acetylated,which indicated the potential regulation on the putrescine biosynthesis.Thus,the △KAT and △KDAC were constructed to explore the relation between the level of acetylation and putrescine formation by ELISA and putrescine detection.The results showed a negative correlation between them.In conclusion,the research showed the main BAs(putrescine and cadaverine)in large yellow croaker and proposed a novel quality indicator of the sum of putrescine and cadaverine for the fish.Besides,the mechanism of putrescine biosynthesis and its special accumulation pattern"PUT positive regulation" in&baltica 73 were revealed and interpreted.The acid-induction mechanism of putrescine production was also revealed.Finally,we proposed a potential negative relevance between acetylation and putrescine accumulation.The study may provide a theoretical basis for controling the putrescine production and the aquatic preservation.