发酵乳制品中乳酸菌的流式检测方案探索与研究

Research on the flow cytometric testing scheme of lactic acid bacteria in fermented dairy products

中文关键词:  乳酸菌  流式细胞术  细菌计数

英文关键词:lactic acid bacteria  flow cytometric  bacteria counting

中文摘要:

      目的 建立基于流式细胞术的发酵乳制品中乳酸菌快速计数方法, 实现发酵乳制品中乳酸菌的快速定量检测。方法 使用5(6)-羧基荧光素二乙酸酯[5-(and 6)-carboxyfluorescein diacetate, cFDA]和碘化丙啶(propidium iodid, PI) 2种荧光染料对乳酸菌进行染色, 经流式细胞仪检测乳酸菌的荧光信号, 从而实现细菌定量计数。在分析过程中对染色剂浓度、荧光通道阈值进行优化, 并将流式计数结果与现行国标方法进行对比研究。结果 在样品浓度为104 CFU/mL (g)时, 通过0.05 μg/mL的PI和10 μmol/L的cFDA染色, 在绿色荧光通道增益为100, 红色荧光通道增益达到1000时, 能够达到最佳的流式检测条件, 且样品检测结果与现行国标检测方法存在显著的正相关关系(P<0.01), 2种方法测试结果相关系数值为0.949。结论 流式细胞术具有灵敏度高、检测时间短、稳定性高、重现性好等优点, 对于货架期普遍较短, 放行压力大的乳制品生产企业来说, 能够缩短检测时间, 实现产品快速放行上架。

英文摘要:

      Objective To establish a rapid counting method for lactic acid bacteria in fermented dairy products based on flow cytometry and realize rapid quantitative detection of lactic acid bacteria in fermented dairy products. Methods Lactic acid bacteria were stained with 2 kinds of fluorescent dyes including 5-(and 6)-carboxyfluorescein diacetate (cFDA) and propidium iodid (PI) fluorescein diacetate, fluorescence signal of lactic acid bacteria was detected by flow cytometry, so as to realize the quantitative count of bacteria. In the analysis process, the concentration of dye and the threshold of fluorescence channel were optimized, and the flow counting results were compared with the current national standard method. Results When the concentration of the sample was 104 CFU/mL (g), 0.05 μg/mL PI and 10 μmol/L cFDA staining were performed, and the green channel gain was 100, and the red channel gain was 1000, the optimal flow detection conditions could be achieved. There was a significant positive correlation between the test results of the samples and the current national standard test method (P<0.01), and the correlation value of the test results of the 2 kinds of methods was 0.949. Conclusion Flow cytometric has the advantages of high sensitivity, short testing time, high stability, and good reproducibility. For dairy product manufacturers with generally short shelf life and high release pressure, the inspection time can be shortened and the product can be released quickly on the shelf.