Occurrence And Resuscitation Of A Viable But Non-Culturable State In Salmonella Typhimurium By Thermosonication

VBNC(viable but nonculturable)cells are alive bacterial cells,but they lose their culturability in conventional culture media,and thereby fail to be detected by traditional plate counting techniques.Common pathogenic bacteria,like Salmonella Typhimurium,can enter a VBNC state under unfavourable conditons,which usually escape detection by the plate counting method and pose a serious risk to public health.Based on the plate counting method,food-borne pathogens in fruit-vegatable juice beverage are removed efficiently by thermosonication(TS).However,considering the occurrence of VBNC state,it is necessary to investigate a quantitative detection method on VBNC state in S.Typhimurium,the occurrence of VBNC state in S.Typhimurium induced by TS treatments and the factors affecting resuscitation.The main research results were summarized as follows:(1)A method combing reverse transcription quantitative PCR(RT-qPCR)assay with the plate counting method was established for the quantification of S.Typhimurium in the VBNC state.And qPCR method was also established for total bacterial enumeration.The detection method provided a useful tool for calculating the population of viable S.Typhimurium.The population of VBNC cells was obtained by the difference between viable cell number and culturable cell number.The acetic acid and H2O2 validation assay incidated that the established RNA-based method can be used to evaluate VBNC bacteria.(2)The induction conditions of TS treatments for inducing S.Typhimurium into VBNC state were determined.The influence of the growth phase of S.Typhimurium,temperature of TS treatments,the time of TS treatments,treatment times of TS and free radical scavenging agent on the survival and culturability were investigated.The results indicated that logarithmic and decline phase cells were more sensitive to TS treatment than other growth phases,and were induced into the VBNC state more easily.S.Typhimurium was induced into the VBNC state after TS treatments at high temperatures and on prolonged exposure.The treatment times of TS had no influence on the induction of VBNC state in S.Typhimurium cells.Sodium pyruvate,a kind of free radical scavenging agent,decreased the occurrence of VBNC state in S.Typhimurium during TS treatments.(3)The factors affecting resuscitation were studied.The resuscitate ways included the direct warming resuscitation,adding chemical substance resuscitation,adding nutrient substance resuscitation and adding maltose resuscitation.The results showed that the optimal growth temperature(37oC),sodium pyruvate(20.00 mmol/L)and 4% peptone acculated the resuscitation of VBNC state cells induced by TS treatments,while Tween 20(24.44 mmol/L)reduced compared to BPY broth alone.S.Typhimurium in the VBNC state failed from resuscitation when incubating in BPY broth at 4oC and in maltose system.This research was a successful attempt for induction of VBNC state in S.Typhimurium and its quantitative detection.The RT-qPCR was proved efficient to detect the population of VBNC state cells quantitativly and overcome the leak detection on VBNC state cells by the plate counting method.Although the application of RT-qPCR method in food systems needs further research,the detection method has a vast potential in the future development.