Study On Antibacterial Activity And Antibacterial Mechanisms Of Walnut Green Husk Extract

In this paper,the antibacterial activity and mechanism of walnut green husk extract were studied in order to provide reference for it to replace feed antibiotics.Walnut green husk was extracted with deionized water,50%hydroalcoholic or anhydrous ethanol,and investigated the effects of its extract on the diameter of bacteriostatic zone,minimal inhibitory concentration（MIC）,growth curve,activity of intracellular adenosine triphosphate,adenosine triphosphatase and extracellular alkaline phosphatase,electrical conductivity,degree of cell lysis,metabolic activity,fluorescence intensity of DNA and RNA and cell morphology of Staphylococcus aureus,Bacillus subtilis and Escherichia coli.The molecular action mechanisms of walnut green husk extract were analyzed by E.coli transcriptome sequencing.The results show that:（1）The contents of flavonoids and juglone in anhydrous ethanol extract were significantly higher than those in other groups（P<0.01）,and the content of polysaccharides in 50%hydroalcoholic extract was significantly higher than that in deionized water extract group（P<0.05）.（2）Among the antibacterial activities,the anhydrous ethanol walnut green husk extract had the best inhibitory effect on the tested bacteria,and the diameter of bacteriostatic zone against S.aureus was significantly higher than that of other groups（P<0.01）.The MIC of anhydrous ethanol extract to S.aureus and B.subtilis was 6.25 mg/m L,and the MIC to E.coli was 25 mg/m L.Compared with the control group,the cell growth was significantly inhibited when the concentration of the extract was 0.25,0.5×MIC,and there was almost no sign of cell growth at the concentration of 1,2×MIC.（3）After treated with 1,2 and 3×MIC of the extract,the A₂₆₀ of the tested bacteria was significantly higher than that of the control group（P<0.01）.After the bacterial solution was treated with 0.6,0.8,1,1.2×MIC of the extract,A₆₃₀ was significantly higher than that of the control group（P<0.05）.The fluorescence intensity of DNA and RNA of cells treated with 1×MIC of the extract for 4 hours was significantly lower than that of the control group（P<0.01）.All of them were treated with 1×MIC of the extract.The AKP of S.aureus was significantly higher than that of the control group at 2 h,4 h and 6 h,and the ATP content was significantly lower than that of the control group at 6 h and 12 h（P<0.05）.The AKP of B.subtilis was significantly higher than that of the control group at 2 h（P<0.01）,the ATP content was significantly lower than that of the control group at 18 h（P<0.01）,and the ATPase activity was significantly lower than that of the control group at 12 h,18 h and 24 h（P<0.05）.The AKP of E.coli at 6 h and 8 h was significantly higher than that in the control group,the ATP content at 6 h（P<0.01）,12 h,18 h and 24 h was significantly lower than that in the control group（P<0.01）,and the ATPase activity at 24 h was significantly lower than that in the control group（P<0.01）.（4）There were 3667 differential genes in E.coli treated with 1×MIC compared with the control group,of which 1676 genes were up-regulated and 1991 genes were down-regulated.GO functional enrichment analysis showed that there were 165 up-regulated genes and 33 down-regulated genes,which were enriched in biological processes,cellular components and molecular functions,respectively.KEGG enrichment analysis showed that the extract had significant effects on E.coli RNA degradation,bacterial chemotaxis and ABC transporter pathway（P<0.05）.Under the experimental conditions,the walnut green husk extract extracted by anhydrous ethanol can affect the nucleic acid synthesis and cell integrity,interfere with the cell energy metabolism,change the cell membrane permeability and promote the leakage of intracellular substances by acting on the metabolism,movement and efflux system of the tested bacteria,thus affecting its normal growth.