| A large amount of pig manure is discharged at will,which is easy to cause environmental pollution and unhealthy growth of pigs.In addition,the irrational use of antibiotics and other drugs makes some strains show pathogenicity and drug resistance,which is very easy to cause growth retardation,thus bringing great economic losses to the pig industry.Therefore,the study of new bacteriostatic agents is becoming more and more important.This study aims to synthesize Nepetalol and perillaldehyde into a new plant-derived bacteriostatic agent against Escherichia coli(E.coli)and salmonella in pig feces,and study its potential bacteriostatic mechanism.It provides reference for further improving the ecological environment of piggery and pig intestinal function.The feces of pigs raised in local pig houses in Yan’an were selected.The target strains were isolated and identified,and the inhibitory effects of Nepetalol and perillaldehyde on two kinds of pig fecal bacteria were determined by Oxford cup method,double dilution method and plate coating method,respectively.The preliminary inhibitory mechanism was explored by measuring growth curve,cell membrane integrity,cell membrane permeability,cell wall integrity,biofilm formation ability and intracellular protein were determined to explore the preliminary bacteriostatic mechanism.The results results are as follows:(1)The target strains were isolated and identified.The preliminary screening found that the colony growth was in line with the morphological characteristics of E.coli and Salmonella.Further,the required strains were identified by molecular biology.(2)Effects of Nepetalol and perillaldehyde on Escherichia coli and Salmonella in pig feces,the two drugs showed good bacteriostasis effect against E.coli and salmonella in pig feces.The MIC and MBC values of Nepetalol against Escherichia coli and Salmonella were 1.45%,the MIC and MBC values of perillaldehyde against E.coli were0.73% and 1.45%,and the MIC and MBC values against Salmonella were 1.45%.(3)The optimal ratio of the combination of Nepetalol and perillaldehyde was 4:1,and the MIC and MBC values were 0.73% and 1.45% for Escherichia coli in pig faecal stool,and 0.73% for Salmonella.The combination of the two improved the antibacterial effect of perillaldehyde alone.(4)By measuring the growth curve,it was found that Escherichia coli and Salmonella were consistent with the normal growth characteristics of bacteria without adding complex essential oil.After the complex essential oil treatment,the growth curve changed obviously,and the growth of bacteria was inhibited,or even did not grow at high concentration.(5)The effect of the combined use of Nepetalol and perillaldehyde on the cell membrane of E.coli and Salmonella,It was found that the electrical conductivity of E.coli and salmonella was significantly higher than that of the group without essential oil at2 h after treatment with high concentration of complex essential oil,it was higher than that of the group without essential oil,and the damage to the cell membrane of Salmonella was higher.(6)Under the combined action of Nepetalol and perillaldehyde on E.coli and Salmonella,it was found that the absorbance values at 260 nm and 280 nm under 2×MIC treatment were significantly higher than those in the control group(P<0.05),and showed at 2 h the electrical conductivity of E.coli and Salmonella bacterial fluid increased significantly,and the damage degree of Salmonella cell membrane was higher.(7)By measuring the effects of the combination of Nepetalol and perillaldehyde on the cell wall of bacteria,it was found that the AKP activity of E.coli and salmonella was increased with the increase of the concentration of essential oil,indicating that the compound essential oil could increase the permeability of cell wall and destroy cell growth by destroying the cell wall of bacteria.(8)The bacterial biofilm formation was measured,and it was found that when the concentration of complex essential oil was 1/4×MIC,it showed a significant difference in the ability of E.coli biofilm formation.With the increase of the concentration of complex essential oil,it was found that the inhibition of E.coli biofilm formation ability was stronger,and for Salmonella,the biofilm formation amount was significant under the condition of 1/8×MIC treatment(P<0.05).The inhibitory effect of the compound oil showed better inhibitory ability.(9)In the treatment of different concentrations of complex essential oils,the effect of complex essential oils on the expression of Escherichia coli protein was not obvious after 12 h.It could inhibit the normal protein expression of Salmonella,and may affect the growth of inhibitory bacteria.Conclusion: The combined action of Nepetalol and perillaldehyde has a good inhibitory effect on Escherichia coli and Salmonella in pig feces.It can destroy the permeability and integrity of the cell membrane and the integrity of the cell wall,and inhibit the biofilm formation and protein synthesis,and affect the normal growth and development of bacteria.. |
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