Effect of Thermosonication Treatment on the Viable but Non-culturable State of Shewanella putrefaciens
[Objective]The study focuses on the physiological characteristics and resuscitation status of the common spoilage bacterium Shewanella putrefaciens in aquatic products as it enters the viable but non-culturable (VBNC) state under thermosonication (TS) treatment.[Methods]S.putrefaciens was used as the research subject.Initially,the bacterial counts were determined using plate counting under various TS treatments at different temperatures (35,40,45,50,55,60,65,and 70 ℃) and power levels (100,150,200,250,and 300 W).Based on these results,further investigations focused on the VBNC state and morphological characteristics of S.putrefaciens under TS treatment at 70 ℃ with varying power levels (100,150,200,250,and 300 W).Specifically,flow cytometry was employed to enumerate bacterial populations in different states,and trypan blue staining was used to validate cell viability.Transmission electron microscopy was then applied to observe morphological changes,and DNA amplification assays were performed to detect VBNC characteristics.Finally,bacterial cultures treated at 70 ℃ and 300 W were selected for resuscitation experiments.[Results]After TS treatment under different conditions,the mortality rate of S.putrefaciens was found to be positively correlated with both temperature and ultrasonic power.Results from flow cytometry and trypan blue staining indicated that even after high TS treatment (50 kHz,300 W,ultrasonic treatment for 30 min,and heating at 70 ℃),a significant number of bacteria died,but 5.87％ of the cells remained viable.Combined with plate counting results,this portion of bacteria had entered the VBNC state.Transmission electron microscopy observations revealed that the cell wall and cell membrane of VBNC bacteria were thickened,the cytoplasmic density increased,and the nucleoid region appeared blurry.In vitro amplification results showed that TS treatment degraded bacterial DNA,and resuscitation experiments demonstrated that S.putrefaciens in the VBNC state still retained the ability to recover after TS treatment.[Conclusion]TS treatment at 300 W and heating at 70 ℃ induce S.putrefaciens to enter the VBNC state.In this state,the bacteria undergo a series of physiological changes to resist external environmental conditions.When placed in a suitable environment,S.putrefaciens in the VBNC state can resuscitate and continue to grow and proliferate.Comprehensive monitoring of VBNC bacteria in food safety is necessary,even after rigorous treatment processes.
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