Screening,Compounding Of Lactic Acid Bacteria And Study On Their Degradation Of Nitrite In Salted Grass Carp

Nitrite is one of the food additives allowed by the country for limited use and is widely used in the food industry.However,the toxic,carcinogenic and teratogenic effects caused by excessive intake of nitrite cannot be ignored.Therefore,it is of great significance for human health and food industry to study the effective degradation of nitrite in food.Lactic acid bacteria have important physiological functions.The bacterial antagonistic substances produced during the metabolic process can effectively inhibit the growth of contaminated bacteria.Moreover,the acids and enzymes produced by lactic acid bacteria have good degradation effect on nitrite.Accordingly,the following researches have been carried out.Firstly,the spontaneously ferment radish and flavored fermented milk were used to screen lactic acid bacteria.Three strains of lactic acid bacteria were selected,including Lactobacillus acidophilus XLA1731,Lactobacillus paracasei XLP1732 and Lactobacillus plantarum XLP1733.The 16S rDNA sequences of the three bacteria were submitted to NCBI,whose GenBank accession numbers were MG983979,MG983983 and MG983982,respectively.Then,the growth curves and acid production curves of the three strains were studied,and the results showed that there was a positive correlation between acid production rate and strain growth rate.The study of acid resistance and sodium chloride resistance showed that the biological characteristics of the three strains were good and met the requirements of starters.The antagonistic experiment showed that there was no antagonistic effect among the three strains,so they could be used for compounding studies.Finally,the degradation effect of three strains of lactic acid bacteria on different concentrations of nitrite was studied.It was found that the degradation rates of nitrite by the three lactic acid bacteria were all above 74%.Secondly,the compound study on the three strains of lactic acid bacteria were conducted,and the results showed that when the total inoculation amount of lactic acid bacteria was1.0×10~7cfu/mL,andtheproportionofstrainstobecompoundedwas XLA1731:XLP1732:XLP1733=1:2:2,the nitrite degradation effect in the MRS medium was the best,and the nitrite residue in the medium was the least.The compounding method was then applied to the salting and fermentation of grass carp.The effects of fermentation temperature,time and inoculum amount on the nitrite content,histamine content and volatile basic nitrogen content in fish were studied.Under the following salting conditions:10%of salt,100 mg/L of nitrite,3 h for salting and the following fermentation conditions:fermentation temperature of 30?,fermentation time of 24 h and the total inoculation amount of 1.0×10~9cfu/mL,the degradation rate of nitrite in fish was 87.11%,and the content of histamine and volatile basic nitrogen(TVB-N)was low,within the permitted range of national standard.Meanwhile,the quality of fish products was better.Under the above conditions,the changes of volatile flavor compounds in the fish of the control group(without inoculant)and the experimental group(with inoculant)were tested.The results showed that87 and 91 flavor substances were detected in the control group and the experimental group,respectively.The types and relative contents of the flavor substances in the two groups were different.The relative percentage of various flavor compounds in the experimental group was generally greater than the control group.Thirdly,the genome DNA of the strain L.plantarum XLP1733 was used as a template to clone the nitrite reductase(NiR)gene.Subsequently,the recombinant expression plasmid pET30a(+)-NiR was successfully constructed and realized the expression of Ni R in E.coli.The enzymatic activity assay demonstrated that the enzymatic activity was low.After sequencing and comparison of amino acid sequences,the important sites affecting the activity of the enzyme were explored in order to find an effective method to improve the enzyme activity,which provided a theoretical basis for the study of the enzymatic activity increase.