三磷酸腺苷荧光法在乳制品商业无菌快速检测中的适用性评价

Applicability evaluation of adenosine triphosphate fluorescence method for rapid detection of commercial sterility in dairy products

中文关键词:  乳制品  商业无菌  ATP荧光法  国标方法

英文关键词:dairy products  commercial sterility  adenosine triphosphate fluorescence method

中文摘要:

      目的 系统评估三磷酸腺苷荧光法在乳制品商业无菌快速检测中的适用性。方法 采用三磷酸腺苷荧光法, 通过包容性实验测试其对35种代表性微生物的检出能力; 通过排他性实验评价三磷酸腺苷荧光法在7类共70份乳制品基质中的抗干扰性能; 通过人工污染实验, 以平板划线法和GB 4789.26—2023《食品安全国家标准 食品微生物学检验 商业无菌检验》为参比方法, 系统比较其在7类乳制品中对7种代表性污染菌的检测灵敏度; 并组织两家实验室进行比对, 评估方法的重现性。结果 三磷酸腺苷荧光法的包容性和排他性结果均为100%。在灵敏度测试中, 对植物乳杆菌检测灵敏度(50%检出限, limit of detection 50%, LOD50)=0.32 CFU/样品)略优于平板划线法(0.37 CFU/样品)和GB 4789.26—2023 (0.37 CFU/样品), 对其他6种菌的50%检出限(LOD50)与参比方法基本一致(相对检出限, relative limit of detection, RLOD约等于1)。实验室间比对结果显示该方法具有良好的重现性(多数RLOD约等于1)。该方法对黑曲霉、粪肠球菌等可在培养3 d时实现早期检出, 相较GB 4789.26—2023(检测周期7~10 d), 检测周期缩短4~7 d。结论 三磷酸腺苷荧光法检测速度快、操作简便、包容性和特异性良好, 灵敏度与传统方法相当, 适用于乳制品商业无菌的快速筛查与过程监控。本研究为缩短乳制品商业无菌检测周期、推动快速检测技术应用提供了实验依据, 同时构建了可复用的方法学验证框架, 为其他快速检测技术在食品基质中的验证提供参考, 并为实践中“初筛—复核”流程提供了实验证据。

英文摘要:

      Objective To systematically evaluate the applicability of adenosine triphosphate fluorescence method in the commercial aseptic rapid detection of dairy products. Methods The detection ability of 35 kinds of representative microorganisms was tested by the inclusion experiment using the adenosine triphosphate fluorescence method. The anti-interference performance of adenosine triphosphate fluorescence method in 70 dairy product matrices of 7 types was evaluated through exclusive experiments. Through artificial contamination experiments, using the plate streptography method and GB 4789.26—2023 National food safety standard-Microbiological testing of food-Commercial sterility testing as reference methods, the detection sensitivity of 7 kinds of representative contamination bacteria in 7 types of dairy products was systematically compared. The 2 laboratories were arranged to perform inter-laboratory comparison tests for evaluating the reproducibility of the established method. Results The inclusive experiment and exclusive experiment results of the adenosine triphosphate fluorescence method were both 100%. In the sensitivity test, the limit of detection at 50% positive rate (LOD50) of the developed method for Lactobacillus plantarum was 0.32 CFU/ sample, slightly superior to the streak plate method (0.37 CFU per sample) and the national standard GB 4789.26—2023 (0.37 CFU per sample). The LOD50 values for the other 6 kinds of target bacterial strains were basically consistent with those of the reference methods, with the relative limit of detection (RLOD) approaching 1. Interlaboratory comparison results verified favorable reproducibility of this method, as the RLOD for most strains were close to 1. This method could achieve early detection of Aspergillus niger, Enterococcus faecalis and other microorganisms following 3 days of incubation. In comparison with GB 4789.26—2023, which required a detection period of 7–10 days, the assay duration was reduced by 4–7 days. Conclusion The adenosine triphosphate fluorescence method features rapid detection speed, simple operation, as well as good inclusivity and specificity. Its sensitivity is comparable to that of traditional methods, making it suitable for the rapid screening and process monitoring of commercial sterility in dairy products. This study provides experimental evidence for shortening the detection cycle of commercial sterility in dairy products and promoting the application of rapid detection technologies. Meanwhile, it constructs a reusable methodological validation framework, providing a reference for the validation of other rapid detection technologies in food matrices, and offers experimental evidence for the “primary screening-rechecking” process in practice.