| The effect of high pressure carbon dioxide (HPCD) on sublethal injury of Escherichia coli O157:H7, a well characterized strain, was investigated in this study. The recovery of sublethally injured cells was also studied. The mechanism of the induction of sublethally injured cells by HPCD and the recovery process was analyzed by proteomics method. Available data in this study will benefit the controlling of foodborne pathogen in low acid foods through HPCD processing.(1) Lethal and sublethal injury of E. coli cells in physiological saline (PS, pH5.60) and phosphate-buffered saline (PBS, pH7.00) by HPCD were determined using nonselective and selective media. The treatment pressure was5MPa, temperatures were25-45℃and treatment time5-70min. The sublthally injured cells in PS after HPCD treatment were very limited, but the injured cells occurred a lot in PBS. The proportion of sublethally injured E. coli cells in the survivors increased steady up after the lag phase and gradually increased with increasing the treatment time. The membrane permeability of the cells stained with SYTO9/PI was studied by flow cytometer (FCM). The FCM cytogram showed that HPCD induced significant damage in the membrane of the cells. The percentage of permeabilized cells (from FCM) in the HPCD treated E. coli cells was similar with the inactivation rate obtained in nonselective medium, and much lower than that obtained in selective medium. HPCD treatment induced alternation in both morphology and interior structure off. coli cells.(2) Comparing to the untreated cells, the sensitivity of sublehtally injured cells was enhanced to45℃, seven acids and nisin. Therefore, HPCD had synergistic effect with nisin on inactivation of E. coli when these two treatments were combined. The effect of HPCD and nisin on inactivation of E. coli in PBS and carrot juice was studied. The time for complete inactivation by HPCD was shortened in the presence of nisin. The inactivation kinetics of E. coli in PBS or carrot juice by HPCD followed slow-to-fast two-stage kinetics and was fitted by the modified Gompertz equation.(3) The effect of media, temperature, media pH and inhibitors of specific metabolic processes, Mg and Ca cations on the recovery of the sublethally injured cells was also investigated. The recovery could occur at4,25and37℃in tryptic soy broth (TSB), but the recovery was inhibited when the pH of TSB was lower than4.0. When PBS, peptone water, TSB and carrot juice were used as recovery media, the maximum repair rate of the sublethally injured cells was found in TSB and the minimum was in PBS. The addition of inhibitors such as sodium azide, chloramphenicol, rifampicin, penicillin G and cerulenin showed that repair of most sublethally injured cells required energy, protein and RNA synthesis, but was not dependent on peptidoglican synthesis. Moreover, Mg and Ca cations were also needed in the repair.(4) Proteomic method was used to analyze the possible mechanism of the induction and recovery of sublethally injured cells caused by HPCD.766differentially expressed proteins were found in live cells, dead cells, sublethally injured cells and recoved cells. By analyzing the functions of differentially expressed proteins in live cells, dead cells and sublethally injured cells, we found that both dead cells and sublethally injured cells had a lower acitivity in physiological metabolism, DNA damage repair and cell defense, but a higher activity in cell wall metabolism, secretion system, and stress responses. However, there were significant differences between sublethally injured cells and dead cells in accumulation and synthesis of protection material and the behavior of nuclear areas. These differences might be related to the formation of sublethally injured cells. Comparing to the sublethally injured cells, the metabolism acitivity in recovered cells increased significantly. |
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