Study On The Effect And Mechanism Of NaCl Concentration And PH Value On Putrescine Metabolism Of Enterobacter Cloacae

Biogenic amines(BAs),which contained nitrogen with biological activity,were organic compounds with low molecular weight.An excessive intake of biogenic amines will give rise to adverse physiological reactions that can seriously threaten life.Putrescine,an aliphatic diamine,was one of the most common BAs in food.Its large occurrence in food not only led to the spoilage odor of food products,but can enhance the toxic effects of other BAs.It was also a precursor of carcinogenic N-nitrosamine.The production of biogenic amines in food was mainly due to the metabolism of food spoilage bacteria,and it was affected by the environmental factors.So it is particularly important to study the effect of environmental conditions on microbial metabolism to reduce the production of biogenic amines in food.In our previous study,it was found that Enterobacter cloacae was one of the main spoilage bacteria in chicken meat and it can produce a large amount of putrescine.The major metabolic pathway which determined the production of putrescine in E.cloacae was first evaluated,and then the effect and mechanism of different pH and Na Cl concentration on the putrescine production was studied in this paper.The specific research contents and conclusions were listed as follows:(1)The major metabolic pathway which determined the production of putrescine in E.cloacae was first studied.Two different nutrient culture medium which contained 0.5% arginine or0.5% ornithine,respectively,were prepared.The E.cloacae C4 broth of 1% was inoculated in the above different media,respectively,and then grown in a shaker for several hours at 37 oC.The OD value,p H value and putrescine contents in the suspension were determined at different time intervals.It was found that the putrescine contents in E.cloacae C4 suspension containing ornithine were higher than that suspension containing arginine.In summary,the putrescine production in E.cloacae was mainly through the ornithine mechanism.(2)The effect of different p H value on putrescine accumulation of E.cloacae was studied.The nutrient broth medium contained 0.5% ornithine was prepared,and the p H values of the media were adjusted to 9,8,5,4 respectively.The E.cloacae C4 broth of 1% was inoculated in the above different media,respectively,and then grown in a shaker for several hours at 37 oC.The OD value,pH value,ornithine and putrescine contents in the bacterial suspension were determined at different time intervals.It was found that the growth curve of E.cloacae was almost the same under the different conditions,while the ornithine content was decreased and putrescine content was increased as the decreasing of p H values.The expression levels of agm,arg and orn underdifferent initial p H treatments were further studied using real-time PCR.It was shown that the expressions of arg and agm did not change significantly,whereas the orn expression levels were significantly decreased with the increasing of p H value.In summary,the putrescine production was manly happened in low pH conditions due to the high expression levels of ornithine decarboxylase gene.(3)The effect of different Na Cl concentrations on putrescine accumulation of E.cloacae was studied.The nutrient broth medium contained 0.5% ornithine was prepared,and the Na Cl concentrations of the media were adjusted to 0%、 2%、 4%、6% and 8%,respectively.The E.cloacae C4 broth of 1% was inoculated in the above different media,respectively,and then grown in a shaker for several hours at 37 oC.The OD value,p H value,ornithine and putrescine contents in the bacterial suspension were determined at different time intervals.It was found that E.cloacae grew slowly in early growth stage under 4% Na Cl condition,but it continued to grow in the late stage.E.cloacae did not grow under 6% and 8% conditions.The putrescine accumulation was decreased as the increasing of Na Cl concentration.Real-time PCR was used to analyze the relative expression of agmatinase gene(agm),arginine decarboxylase gene(arg),ornithine decarboxylase gene(orn)in the logarithmic phase of E.cloacae C4.It was found that the expression levels were inhibited as the increasing of Na Cl concentrations.In summary,Na Cl stress can decrease the putrescine production by inhibiting the growth of E.cloacae and the expression of ornithine decarboxylase gene.(4)The recombinant putative protein of E.cloacae ornithine decarboxylase gene was heterologously expressed in Escherichia coli.The effect of environment factors on the activity of ornithine decarboxylase was further studied.The results showed that the Km and Vmax of ornithine decarboxylase were 1.666 m M and 6.398 U/mg,respectively.The optimum temperature was 30 oC,and the optimum p H was 6.0.The activity of ornithine decarboxylase was highest when NaCl concentration was 1.5%.In summary,although putrescine accumulation was high when the E.cloacae was in a low p H condition,the ornithine decarboxylase was in low activity.It was indicated that the putrescine production was mainly due to the high gene expression levels of ornithine decarboxylase.The putrescine accumulation was inhibited as the increasing of Na Cl concentrations,which is because the Na Cl stress inhibited the gene expression levels of ornithine decarboxylase.In this study,the main metabolic pathway for putrescine production of E.cloacae was first analyzed,and the effect and mechanism of Na Cl concentration and pH value on putrescine metabolism was studied.It provided a feasible research support for further control of the accumulation of biogenic amines in food,and for improving the quality and safety of processed chicken meat products by changing the processing parameters(acid,salt concentrations,etc).