| Under the optimization of extract technology, the antibacterial mechanism of black pepper petroleum ether extract (BPPE) against Listeria monocytogenes (L, monocytogenes)ATCC19115, Salmonella typhimurium (S, typhimurium) CMCC 50115 and Pseudomonas aeruginosa (P, aeruginosa) ATCC9027 and its application in meat products were studied.The antibacterial activity of BPPE and the effects on the cell morphology, cell respiration,energy metabolism and respiratory metabolism of bacteria were examined to elucidate their antibacterial mechanism. At the same time with the comparison of the antiseptic which been used in meat products to explore the value in the practical application, which have provide the reliable theoretical basis for it as safe, natural, non-toxic preservatives.The gradient dilution and plate count method were used to determine the antibacterial activity of BPPE. The effects on cell morphology was investigate by Propidium Iodide (PI),ALP-ase, Na+/K+-ATPase activity and the scanning electron microscopy(SEM). And the assay kits were used to measure the key endoenzyme activities of glycolysis (EMP) and tricarboxylic acid cycle (TCA) and use high performance liquid chromatography (HPLC)to measure the content of organic acids which to reveal the impact of BPPE on bacterial respiratory metabolism; meanwhile the assay kits were used to determine the ATP levels to reveal the impact of BPPE on bacterial energy metabolism. Through determine the pH value, the total number of bacterial and volatile in sterile meat pulp to compare with preservative and its practical value was obtained. The results were as follows:(1) Use gradient dilution and plate count method for the determination of antibacterial activity of BPPE could found that the different organic phase pepper extract has significant inhibitory effect to different strains. And the antibacterial activity of BPPE was the best which compared with other three kinds of organic extracts of pepper, on the contrary the black pepper n-butyl alcohol extract. What's more, the results showed that the BPPE have a influence on L, monocytogenes, S, typhimurium and P, aeruginosa.(2) The results of effect of BPPE on cell morphology by scanning electron microscope,on cellular structure was determined by ALP-ase, PI, Na+/K+-ATP-ase activity and the leakage of nucleic acid and large protein molecules The results showed that: The cell of L,monocytogenes, S, typhimurium and P, aeruginosa were adhesion and the cell boundaries became unclearly and the cell wall were fractured. The integrity and permeability of cell wall and membrane were been affected which resulted the nucleic acid and large protein molecules were leaked out of cell with the BPPE been added into the culture medium.(3) The activity of pyruvate kinase (PK) was determined by assay kit. Using the method of 2, 4-dinitrophenylhydrazine to measure the content of pyruvic acid and level of lactic acid was determined by HPLC. The results showed that the the PK in L,monocytogenes, S, typhimurium and P, aeruginosa were inhibited, the activity of PK were decreased in all bacterial. Pyruvate acid was discharged and concentrated in extracellular that effection the normol metabolism of bacteria. And Lactic acid was concentrated in bacteria which showed that the normal metabolism was disturbed.(4) The dehydrogenase of Succinate dehydrogenase (SDH) and malate dehydrogenase(MDH) activity were measured by SDH and MDH assay kits, respectively. And the change of the content of organic acids in the metabolic pathway of the tested bacteria was determined by HPLC. The results explained that BPPE could inhibit the activity of MDH in all bacteria, but the activity of SDH showed in was increased with the influence of BPPE. The a-ketoglutaric acid and L-mallic acid were concentrated in the incubation period; At the same time, the critic acid and succinic acid concentration of bacteria in treatment group were lower than that of control group which showed that the normal metabolism was been destroyed.(5) The ATP level in the tested bacteria was assayed by ATP assay kit. The results showed that the content of ATP in the treated group was significantly different from the control group and the ATP level was decreased to the lowest value at 24 h.(6) Through determine the pH and the total bacteria in meat pulp culture environment to explore application value. The results showed that the pH value rise was retard, the total number of bacteria decreased and the content of volatile basic nitrogen increased which compared with the commonly used preservative.In summary, the antibacterial mechanism of BPPE was that the intracellular material leakage and extracellular substances into the cell when the cell wall and cell membrane been destroyed at the first time. And then the BPPE inhibited the respiratory metabolism of bacteria which the activity of PK was reduced results the pyruvate metabolism been effected. Absolutely, the normal metabolism of a-ketoglutaric acid was disturbed, the content of succinic acid was decreased, and the activity of SDH was inhibited which resulted the L-mallic acid was concentrate in the environment, the normal metabolism of TCA was effected. Thus, the energy production efficiency in cell was fell down which leaded to the death of bacteria. The BPPE could significantly effect the growth of spoilage bacteria in meat products when compared with good antibacterial property. So the BPPE have a great potential to be a new natural preservative. |
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