Sub-lethal Injury And Recovery Of Escherichia Coli O157:H7 Induced By High Pressure Processing

High pressure processing (HPP) has become an effective technique for the control of Escherichia coli O157:H7 (E. coli O157:H7) on beef. However, the sub-lethal injury will be induced by HPP treatoent which will be underestimate or ignored in the detection because of their lack of normal characteristics. It will bring potential risk to food safety and shelf-life. Researchers mainly focused on the inactivation effects of HPP to E. coli, and the literatures of sublethal injury and recovery were limited. The composition of contaminated bacterial community in meat is complex, whose growth will be influenced due to the interactions among them under certain conditions. Until now, the researches on recovery of sub-lethal injured E. coli O157:H7 induced by HPP when the background bacteria existing have not been reported. Therefore, one of the objectives of this study was to screen appropriate selective media for the accurate determination of injured E. coli O157:H7. Then, the variations of pressure resistance among four E. coli O157:H7 strains and their inactivation and injury were analyzed. Based on the above results, one pressure-resistant strain was selected and inoculated into ground beef to research the recovery of sub-lethal injured E. coli O157:H7 indeuced by HPP treatment; meanwhile, E. coli O157:H7 together with Lactobacillus sakei, one kind of predominant spoilage bacteria in vacuum-packed meat, were inoculated into ground beef to research the injury and recovery after HPP treatment. The study would direct the scientific application of HPP in the manufacture and process of food and provide the risk assessment technique for effective storage of food after HPP treatment. The contents and results are as follows:1、Screening the selective media of E. coli O157:H7The virulence genes of four E. coli O157:H7 strains were detected by multiplex PCR. E. coli CICC 21530 was stxl (+), stx2 (+), eae (+), NCTC 12900 and one of strains isolated from beef was eae (+), and the other beef isolate was negative for all virulence genes, which indicated that the pathogenecity of four strains was different. For accurate enumeration of injured cells of E. coli O157:H7 in subsequent experiments, counts of fresh bacterial broth were determined with three kinds of selective media. The results showed that the counts of four strains on selective media were all lower than those on TSA but there were no significant difference between them except for CT-SMAC (P< 0.05) The results indicated that CT-SMAC was not suitable for counting injured E. coli O157:H7 due to a greater inhibition of normal bacteria, while SMAC or mEMB could be used. When the bacterial broth was stored in 4℃, the coumts on four selective media were decreased, and counts of three strains on TSA at 10th day were significantly decreased (P< 0.05), which indicated that some bacteria were dead. So if the bacterial broth need to be stored in refrigerate, the time should be as short as possible, otherwise injured or even lethal cells were occurred.2^ Variation in pressure resistance among E. coli O157:H7 strainsFour E. coli O157:H7 strains were treated by HPP with different pressure and time. The higher the pressure was and the longer the holding time was, the stronger the inbibitory effects on bacteria were. The pressure resistance was different among strains. E. coli CICC 21530 was more resistant to HPP while one beef isolate was sensitive. Each bacterium was appeared different levels of lethality and injury by different high pressure processing conditions, but The lethal effects and the injury effects of different HPP conditions on strains were not consistent. All strains could not be detected with selective media after HPP treatment (400 MPa,5 min) except for E.coli CICC 21530, which indicted that there were not any normal cells of other three strains. Then E. coli CICC 21530 was selected for next experiment.3、Recovery of injured E. coli O157:H7 in beef induced by HPPE. coli O157:H7 was inoculated into ground beef, vacuum-packed and treated by HPP (400 MPa,5 min). After HPP treatment, the samples were stored at 20℃ and 10℃ to investigate the growth of the strain. The results showed that E. coli O157:H7 was inactivated (1.7 log CFU/mL loss) and injured after HPP treatment. Then the recovery of injured bacteria and the proliferation of normal bacteria were showed from the bacterial growth curves. The injured bacteria were recovered mostly at 2.5 day (20℃) and 9 day (10℃), and then stationary phase was reached at 4.5 days (20℃) and 12days (10℃). Meanwhile, E. coli O157:H7 together with L. sakei, one kind of predominant spoilage bacteria in vacuum-packed meat, were inoculated into ground beef to research the injury and recovery after HPP treatment. First, we confirmed that mEMB and MRSA could be used to count E. coli O157:H7 and L. sakei respectively in the combined inoculation experiment. The counts of E. coli O157:H7 were not changed significantly during the whole storage, which indicated that the growth of E. coli O157:H7 was obviously inhibited when L. sakei exiting while E. coli O157.H7 had no significant effect on the growth of L. sakei.