Study On The Synergistic Antibacterial Effect And Mechanism Of Nisin And Sodium Lactate On Spoilage Bacteria Of Sauced Duck Neck

Stewed meat products with soy sauce are kinds of traditional Chinese meat products,which are deeply loved by consumers.However,because of its rich water and nutrients and open circulation environment,it is easy to be contaminated by spoilage microorganisms,resulting in a short shelf life.Nisin and sodium lactate,as natural preservatives,meet the food demand of green label,which aim to prolong the shelf life of stewed meat products and reduce the harm to human health.This paper mainly studied the synergistic bacteriostatic effect of nisin and sodium lactate on the dominant spoilage bacteria in the sauced duck neck.Six pure strains were isolated and identified from the sauced duck neck.Three strains of different genera were selected to study their characteristics and the bacteriostatic effect of nisin and sodium lactate on the three spoilage bacteria.Through combined bacteriostatic effect test and dynamic germicidal curve study,two strains Bacillus cereus strain and Lactobacillus plantarum strain with synergistic bacteriostatic effect were selected to study.The synergistic bacteriostatic mechanism of nisin and sodium lactate on these two spoilage bacteria was explored from the aspects of cell membrane permeability,intracellular alkaline phosphatase(AKP)enzyme activity and adenosine triphosphate(ATP)enzyme activity,cell morphology and cell integrity.And I combined with transcriptome to study the pathway influence and regulation mechanism of the differentially expressed genes(DEGS)of the two strains under the synergistic inhibitory effect of nisin and sodium lactate system.This study combined the bacteriostatic effect with transcriptome to provide a theoretical basis for the synergistic bacteriostatic mechanism of nisin & sodium lactate system on spoilage bacteria of sauced duck neck.And it laid a foundation for finding potential antibacterial targets to control spoilage bacteria more effectively,so as to promote the application of nisin & sodium lactate system in meat products.The main research contents were as follows:1.Six strains of bacteria were isolated from the sauced duck neck.They were Bacillus subtilis strain KA9、Bacillus cereus strain KUBOTAB5、Bacillus cereus strain MD152 、Citrobacter sp.strain E1-G-103 、 Enterobacter sp.LB9 、Lactobacillus plantarum strain LLY 606.Three strains of different genera were selected to study the characteristics of spoilage bacteria.The results showed that B.cereus had a strong ability to decompose protein,mainly through the decomposition of protein to produce amines,which led to meat spoilage.The acid-producing ability of L.plantarum was strong,which led to the rancidity of sauced duck neck.On the other hand,Enterobacter sp.had a strong ability of microbial reproduction,which could accelerate the growth of microorganisms in the sauced duck neck and lead to its decay.2.Minimum inhibitory concentration(MIC)and fractio bacteriostatic concentration index(FICI)showed that nisin and sodium lactate had synergistic effect on B.cereus and L.plantarums,but had additive effect on Enterobacter sp.Two dominant spoilage bacteria with synergistic effect were selected as the objects of subsequent bacteriostatic experiments.This study explored the synergistic bacteriostatic mechanism of nisin and sodium lactate on spoilage bacteria from the aspects of cell membrane permeability,intracellular AKP enzyme activity and ATP enzyme activity,cell morphology and cell integrity.The synergistic bacteriostatic effect of nisin and sodium lactate system inhibited the growth of B.cereus mainly by aggravating the destruction of cell wall and cell membrane.The destruction of cell membrane increased the permeability of cell membrane and the leakage of contents.Besides,the destruction of cell wall increased the activity of AKP,which made B.cereus cells shrink and die.The synergistic bacteriostatic effect of nisin and sodium lactate system was mainly through the destruction of cell membrane and the reduction of energy metabolism to inhibit the growth of L.plantarums.The destruction of cell membrane reduced the activity of ATP enzyme,thus reduced the cell energy metabolism and increased the permeability of cell membrane,which made L.plantarums cells shrink,cell membrane disintegrate and die.3.Transcriptome analysis further studied the mechanism of synergistic bacteriostasis at the molecular level.The results showed that there were 2071 DEGS in B.cereus after co-treatment with Nisin & sodium lactate system,in which the affected pathways were mainly concentrated in the biosynthesis of valine,leucine and isoleucine,photosynthesis,pantothenate and Co A.The 1085 down-regulated DEGS were mainly related to the cell membrane integrity,cell wall composition,amino acid metabolism,energy metabolism and oxidative phosphorylation related genes,while the up-regulated DEGS were mainly related to DNA repair and stress-induced quorum sensing related genes.After co-processing with Nisin & sodium lactate system,there were 331 DEGS in L.plantarum,in which the affected pathways were mainly concentrated in citric acid cycle(TCA cycle),β-lactam resistance,nicotinate and nicotinamide metabolism.The down-regulated 211 DEGS were mainly related to protein transport ABC transporter related genes,amino acid synthesis metabolism and energy metabolism related genes,while the up-regulated 211 DEGS were mainly related to DNA repair related genes and quorum sensing related genes.Real-time fluorescence quantitative PCR(RT-q PCR)was used to further verify the regulation of B.cereus and L.plantarums related genes in the transcriptome,and the results were consistent with the results of RNA-seq analysis.