肉制品生产工艺对沙门氏菌活的非可培养状态的诱导及检测方法研究

Induction and detection of viable but non-culturable Salmonella in meat production processing

目的探索肉制品生产工艺对活的非可培养(viable but non-cultruable,VBNC)状态沙门氏菌的诱导因素,并建立一种快速检测肉制品中VBNC状态沙门氏菌的检测方法加以应对。方法通过高温、低温、高渗透压、防腐剂、杀菌剂等生产工艺处理获取VBNC状态沙门氏菌,使用脱氧胆酸钠(sodium deoxycholate,SD)和PMAxx处理菌液,依据ttr基因进行荧光定量聚合酶链式反应(quantitative polymerase chain reaction,qPCR)建立SD-PMAxx-qPCR进行检测。采用人工污染的方式对VBNC状态沙门氏菌进行检测和验证。结果65℃处理40 min可使沙门氏菌快速进入VBNC状态,添加0.1 g/L山梨酸冷藏状态可使少量沙门氏菌缓慢进入VBNC状态。SD-PMAxx-qPCR能显著区分沙门氏菌的存活状态,最佳条件为0.08%SD,20μmol/L PMAxx,黑暗孵育10 min、光解10 min。纯培养时活菌的检出灵敏度为100 CFU/mL,添加高浓度死菌时,检出灵敏度不变。通过人工污染添加至样品时,需要对样品进行涂布确认其沙门氏菌不可培养,通过100℃加热样品10 min作为阴性对照,从而通过对比循环阈值实现样品中VBNC状态沙门氏菌的检测。结论沙门氏菌在肉制品生产过程中容易受到多种因素诱导进入VBNC状态,本研究建立了SD-PMAxx-qPCR快速区分沙门氏菌的存活状态,可用于肉制品中VBNC状态沙门氏的检测,为肉制品安全生产提供技术保障。

Objective To investigate the inducible factors of viable but non-cultruable(VBNC)Salmonella in meat production processing,and develop a method for rapid detection of VBNC Salmonella for meat product.Methods VBNC Salmonella was obtained through various meat production processes including high temperature,low temperature,high osmotic pressure,preservatives,antibiotic and so on.The bacteria were treated with sodium deoxycholate(SD)and PMAxx.The SD-PMAxx-qPCR was established by fluorescence quantitative polymerase chain reaction(qPCR)based on ttr gene for detection.Artificial contamination was used to detect and verify VBNC Salmonella.Results Treating at 65℃for 40 min could make Salmonella enter VBNC state quickly,and adding 0.1 g/L sorbic acid in cold storage state could make a small amount of Salmonella enter VBNC state slowly.SD-PMAxx-qPCR could significantly distinguish the survival state of Salmonella.The optimum conditions were 0.08%SD,20μmol/L PMAxx,incubation in the dark for 10 min and photolysis for 10 min.The detection sensitivity of live bacteria was 100 CFU/mL in pure culture,but it remained unchanged when high concentration of dead bacteria was added.When it was added to the sample through artificial pollution,it was necessary to coat the sample to confirm that the Salmonella was uncultured,and heat the sample at 100℃for 10 min as a negative control,so as to realize the detection of Salmonella in VBNC state in the sample by comparing the cycle threshold value.Conclusion Salmonella can easily be induced into VBNC state by many factors during meat production.In this study,SD-PMAxx-qPCR is established to quickly distinguish the survival state of Salmonella,which can be used for the detection of VBNC state of Salmonella in meat froduction and provide guarantee for the safe of meat production.