Abstract:In this study, the induction and resuscitation of viable but non-culturable state (VBNC) of a typical hard-to-culture beer-spoilage Lactobacillus acetotolerans were investigated. Lb. acetotolerans strain 2011-11 in the exponential growth phase was repeatedly subcultured in degassed beer to reappear the survival situation in the actual beer brewing process, and the culturability on MRS agar media was examined. The viable cells were monitored based on the DNA-binding fluorescent dye (SYTO-9 and PI) plus the fluorescent microscopy assay and flow cytometry technology. Moreover, the beer-spoilage ability of VBNC cells was determined. Secondly, the effects of up shifting the temperature and adding the cytokine on resuscitation of VBNC state Lb. acetotolerans were observed. After 19 generations of culture in degassed beer, Lb. acetotolerans strain was found to be no longer detected on MRS agar despite the presence of major viable cells appearing green fluorescence, indicating that a large population of Lb. acetotolerans 2011-11 cells had entered into the VBNC state. Meanwhile, VBNC cells still exhibited the beer-spoilage ability. In addition, addition of catalase enabled cells to become culturable again before plating the MRS agar under anaerobic incubation at 26 ℃. Taken together, VBNC strain was successfully obtained from beer-spoilage Lb. acetotolerans by the prolonged adaptation to beer. It was also shown that addition of catalase in the culture media was an effective resuscitation method for the VBNC cells of Lb. acetotolerans strain.
