VBNC状态啤酒易感乳杆菌的诱导及复苏

Induction and Resuscitation of VBNC State Beer-spoilage Lactobacilli

本文研究了一种典型的难培养啤酒易感乳杆菌-耐酸乳杆菌(Lactobacillus acetotolerans)形成"活的但不可培养"(VBNC)的诱导方式,以及从此状态复苏至可培养状态的方法。将培养至对数期的耐酸乳杆菌2011-11菌株采用啤酒内连续传代驯化法还原其在啤酒酿造过程中的实际生存状况,并通过荧光染料染色对诱导后的耐酸乳杆菌细胞进行活性检测;利用逐步升温和添加促进物法对VBNC菌进行复苏试验。当连续传代至第19代,MRS检测平板上可培养菌数降为零,此时菌体多数仍存在呈现绿色荧光的活细胞,表明耐酸乳杆菌已形成VBNC状态,且仍具有污染啤酒能力;将刚进入VBNC状态的耐酸乳杆菌菌悬液经过氧化氢酶处理后涂布于MRS平板,于26℃厌氧培养,可使耐酸乳杆菌复苏至可培养状态。本研究证实,可通过啤酒内连续传代诱导获得VBNC状态耐酸乳杆菌,而添加过氧化氢酶改良常规MRS平板是一种有效的复苏方法。

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 sWain 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.