鼓盖果粒酸奶相关酵母的分离、鉴定及实时荧光定量聚合酶链式反应检测方法建立

Isolation and identification of yeast in bulging fruit flavored yogurt and development of real-time fluorescence quantitative polymerase chain reaction detection method

中文关键词:  果粒酸奶  污染酵母  鼓盖  快速检测  实时荧光定量聚合酶链式反应

英文关键词:bulging fruit flavored yogurt  contaminated yeast  rapid detection  real-time fluorescence quantitative polymerase chain reaction

中文摘要:

      目的 分离鉴定导致果粒酸奶鼓盖的污染酵母, 并针对主要污染酵母建立一种快速检测的方法。方法 从不同的鼓盖酸奶样品中分离酵母菌, 分析其产气特性, 利用分子生物学方法对分离株进行鉴定, 基于26S rDNA序列设计特异性引物, 应用SYBR Green I染料的实时荧光定量聚合酶链式反应法快速检测酵母。结果 从鼓盖的蓝莓果粒酸奶、芒果果粒酸奶和黄桃果粒酸奶的3个样品中分离出9株酵母, 经分子生物学方法鉴定为异常威克汉姆酵母(Wickerhamomyces anomalus)、平常假丝酵母(Pichia inconspicua)、葡萄牙棒孢酵母(Clavispora lusitaniae)和喜仙人掌毕赤酵母(Pichia cactophila), 其中葡萄牙棒孢酵母为主要优势酵母, 且产气能力最强。根据26S rDNA基因设计的引物特异性良好; 所构建的标准曲线相关系数均大于0.99; 该方法检测酵母菌的灵敏度为102 CFU/mL; 重复性检验中组内与组间的变异系数均小于2%, 表明方法具有良好的可靠性与稳定性。结论 导致果粒奶产气的主要污染酵母为葡萄牙棒孢酵母, 构建的检测方法可用于果粒发酵乳酵母菌的快速检测, 为果粒发酵乳的食品安全提供了技术保障。

英文摘要:

      Objective To separate and identify the contaminated yeast that causes the fruit yogurt to bulge and establish a rapid detection method for the main contaminated yeast. Methods Yeasts were isolated from different samples of fermented yogurt and theirs gas production characteristics were also analyzed. Molecular biology methods were used to identify isolated strains. Specific primers were designed based on the 26S rDNA sequence, and real-time fluorescence quantitative polymerase chain reaction using SYBR Green I dye was applied for the rapid detection yeasts. Results The 9 yeast strains were isolated from 3 samples of Vaccinium uliginosum L. yogurt, Mangifera indica L. fruit yogurt and Prunus persica ‘Yellow Peaches’ fruit yogurt. The 9 yeasts were identified as Wickerhammomyces anomalus, Pichia inconspicua, Clavispora lusitaniae and Pichia cactus through molecular biology methods. Among them, Clavispora lusitaniae was the main dominant yeast and had the strongest gas production ability. The primer designed based on the 26S rDNA gene had good specificity. The correlation coefficients of the constructed standard curves were all greater than 0.99. The sensitivity of this method for detecting yeast was 102 CFU/mL. The coefficient of variation within and between groups in the repeatability test was less than 2%, indicating that the method had good reliability and stability. Conclusion In this study, the main contaminating yeast causing gas production in fruit milk is Clavispora lusitaniae. The developed detection method can be used for rapid detection of yeast in fruit fermented milk. It provides a technical support for the food safety of fermented fruit milk.