食品检验用解脂耶氏酵母菌标准物质制备研究

Study on the preparation of Yarrowia lipolytica reference materials for food inspection

目的制备解脂耶氏酵母菌检验用标准物质,以满足实验室酵母菌日常检验质量控制及实验室间比对需求。方法通过生化、基质辅助激光解吸电离飞行时间质谱法(matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF MS)及转录间隔区(internal transcribed spacer,ITS)位点测序,对研究用菌株进行菌种鉴定确认后冻干,制备10^(4)CFU/样品浓度的标准物质。参照CNAS—GL003:2008《能力验证样品均匀性和稳定性评价指南》等的要求进行均匀性检验后,采用单因素方差分析对结果进行评价。样品放于-20、4、25和37℃条件下,分别进行运输稳定性检验及储存稳定性检验。依据GB 4789.15—2016《食品安全国家标准食品微生物学检验霉菌和酵母计数》,验证标准物质在真实食品样品中适用性。结果CMCC98025经生化鉴定为解脂耶氏酵母菌,准确度为93%;MALDI-TOF MS鉴定结果为解脂耶氏酵母,分数为2.012;ITS位点测序与美国国家生物信息中心基因库中序列比对,匹配最优结果为解脂耶氏酵母(Yarrowia lipolytica)。均匀性测试结果符合正态分布,F0.05(19,20)=2.137,F=1.697<F0.05,符合均匀性要求。25及37℃中放置7 d稳定,标准物质中菌含量仍保持在10^(4)CFU/样品;4℃储存28 d及-20℃储存90 d,菌含量为10^(4)CFU/样品,复苏率均在91%以上。本标准物质加入7类食品样品基质均可检出,回收率为81.3%。经3家实验室协作标定,本标准物质平均浓度为2.0×10^(4)~3.0×10^(4)CFU/样品。结论本研究制备的解脂耶氏酵母菌标准物质均匀性、稳定性、真实食品样品中应用验证及协作标定结果均符合要求,可应用于食品中解脂耶氏酵母菌检验。

Objective To prepare reference material for Yarrowia lipolytica,so as to meet the requirements of quality control and comparison between laboratories.Methods The strains were identified by biochemical,matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS)and internal transcribed spacer(ITS)site sequencing and then lyophilized to prepare reference materials with 10^(4)CFU/sample concentration.According to the requirements including CNAS—GL003:2008 Guide for evaluation of homogeneity and stability of proficiency testing samples,the results were evaluated by one-way analysis of variance(ANOVA).The samples were placed at-20,4,25 and 37℃for transport stability test and storage stability test respectively.According to GB 4789.15—2016 National food safety standard-Microbiological examination-Food molds and yeasts count,the applicability of the reference materials in real food samples was verified.Results CMCC98025 was biochemically identified as Yarrowia lipolytica with an accuracy of 93%;The result of MALDI-TOF-MS identification was Yarrowial ipolytica with a fraction of 2.012;The site sequencing was compared with National Center for Biotechnology Information gene bank,and the best result was Yarrowia lipolytica.The uniformity test results accorded with the normal distribution,F0.05(19,20)=2.137,F=1.697<F0.05,which met the uniformity requirements.The bacteria content in the reference material remained at 10^(4)CFU/sample after 7 d at 25 and 37℃;When stored at 4℃for 28 d and-20℃for 90 d,the bacterial content was 10^(4)CFU/sample,and the recoveries rates were above 91%.The reference substances could be detected in 7 kinds of food samples,and the recoveries were 81.3%.The average concentration of the reference materials were 2.0×10^(4)-3.0×10^(4)CFU/sample.Conclusion The homogeneity,stability,application verification and collaborative calibration results of the reference materials of Yarrowia lipolytica prepared in this study meet the requirements,and can be applied to the detection of Yarrowia lipolytica in food.