Research On The Antimicrobial Mechanism Of Black Pepper Chloroform Extracts On Escherichia Coli And StapPhylococcus Aureus

The inhibitory activities of black pepper extracts have been investigated against food spoilage and food pathogenic bacteria.This article explored the antimicrobial activity of black pepper chloroform extract(BPCE)against Escherichia coli and staphylococcus aureus.The effects on cell wall and membrane,the respiratory metabolism,and ATP levels of bacteria were examined to elucidate their antibacterial mechanism.The scanning electron microscopy(SEM)was used to explore the cell morphology;the effect of BPCE on end enzyme and Na+/K+-ATPase were assayed.And use the respiratory rate to investigate the effect of BPCE on bacterial respiratory metabolism;meanwhile,the assay kits were used to measure the key enzymes activities and ATP levels;what's more,organic acids were assayed by HPLC.The results were as below:(1)The results of effect of BPCE on cell morphology by scanning electron microscope showed that BPCE had severely effect on E.coli.After 24h,the cells began to rupture and concentrate,cell walls have been severely impaired,the cell boundaries became unclearly.S.aureus cells became deformation in comparison to normal control and ethanol treatment.After 24h,the cells were adhesion and the cell boundaries became unclearly,the cell wall were fractured.(2)Transaminase activities were measure by using the classical Reitman-Frankel colorimetric endpoint reaction.The results showed that ALT activity in the treatment group was higher than that in control within the initial 12 h.Moreover,AST activity in the treatment group was lower than that in the control at 12 h for E.coli.Transaminases in the S.aureus,bacterial cells were lower in the control culture without BPCE treatment.The current results indicated that transaminases were leakage when treated with BPCE in both bacteria,extracellular transaminases activities were increased.(3)Na+/K+-ATPase activity was assayed by Na+/K+-ATPase assay kit.It was showed that Na+/K+-ATPase activity was hindered after adding BPCE.(4)The results of effect of BPCE on cell respiratory rate showed that the BPCE could inhibit cell respiration.The superposition rate of BPCE and model inhibitor indicated that BPCE had strongest inhibition on cell respiration by TCA cycle,EMP in the second place and HMP in the last.(5)Bacterial pyruvate kinase(PK)and hexokinase(HK)were assayed by pyruvate kinase and hexokinase assay kit,respectively.The results showed that PK and HK in both E.coli and S.aureus were inhibited;the activities of both enzymes in both cells were decreased.(6)The pyruvic acid content was measured according to the method of 2,4-dinitrophenylhydrazine.The results showed that pyruvate acid was discharged and concentrated in extracellular,which inhibited the normal metabolism of bacteria.(7)Succinate dehydrogenase(SDH)and malate dehydrogenase(MDH)assay kits were used to measure the activity of SDH and MDH.The results explained that BPCE could inhibit the activity of SDH in both bacteria,but it had no effect on MDH of E.coli and S.aureus.(8)Organic acids,such as lactic acid(LA),critic acid(CA),?-ketoglutaric acid(KGA),succinic acid(SA)and L-mallic acid(MA)were measured via the HPLC system.The results showed that LA was concentrated in both bacteria,normal metabolism was disturbed;CA in both only had slightly changes in the entire incubation period for both bacteria.In the control groups,CA level decreased within initial several hours and then significantly increased in both bacteria.CA was not detected in control groups of E.coli and S.aureus after incubation for 10 and 8 h,respectively.CA content was lower in the treatment group than those in the control group at the end of the incubation period;KGA of both bacteria in treatment groups were higher than that of control;SA concentration of both bacteria in control groups were lower than that of the treatment groups.However,MA was normally metabolized and BPCE did not influence the MA metabolism in the both bacterial cells.(9)The ATP level was assayed by ATP assay kit.The results showed that for the treated groups,the ATP levels were decreased in the first 8h in E.coli while in S.aureus was decreased after 4 h.Finally,the ATP levels of control groups are much more than that of the treated group.In summary,BPCE could damage the cell wall and cell membrane at first,thus intracellular material was leakage.Secondarily,BPCE has mainly effect on bacteria aerobic respiration.In other words,BPCE could inhibit CA and KGA metabolism,decrease the activity of SDH,and affect the metabolism of SA.Absolutely,glucose metabolism in EMP was hindered and the synthesis of pyruvate was inhibited.Thus,all the metabolic activity was decreased;the energy production efficiency in cell was fell down,which leaded to the death of bacteria.