为突破部分微生物在榨菜高盐环境难培养的局限,建立一种准确定量榨菜腌制过程中细菌和真菌数量的方法,并探讨动态变化过程。植物乳杆菌ATCC 8014的16S rRNA基因和酿酒酵母ATCC 9763的ITS基因分别与质粒载体连接作为标准参照物构建标准曲线,建立实时荧光定量PCR(real-time quantitative PCR, qPCR)方法,检测3个腌制时期腌制汁液中的细菌和真菌数量,同时检测理化指标。结果表明,所得qPCR标准曲线相关系数R2>0.99,扩增效率E均在95%～105%。腌制过程中,细菌和真菌分别在108.10~1010.43和105.29~107.75 copies/μL内变化,细菌在3个腌制阶段都有明显增殖,真菌仅在第3腌制阶段有明显增殖;腌制汁液中,pH值不断下降,NaCl的质量浓度和酸度主要在第2、3阶段先升高后下降。该结果为榨菜微生物过程控制提供参考数据。
In order to fast determine the whole microbial community in a high salt environment during the curing process of Zhacai (cured Tuber Mustard), a method for accurate quantification of all bacteria and fungi was developed. The 16S rDNA of Lactobacillus plantarum and the ITS of Saccharomyces cerevisiae cloned in a plasmid were used as standard references and a real-time quantitative PCR (qPCR) assay was established to detect the quantity of bacteria and fungi in brines during three curing periods. Besides, the physical and chemical indices of brine were also determined. The linear coefficients of the qPCR standard curves were greater than 0.99 and the amplification efficiencies were within 95%-105%. The pH value of the brine continuously decreased during the curing process. Whereas, the concentration of NaCl and acidity initially increased and then subsequently decreased during the second and third periods. Meanwhile, bacteria and fungi changed in the ranges of 108.10-1010.43 and 105.29-107.75 copies/μL, respectively. In addition, bacteria showed significant quantitative multiplication during all three curing periods while the count of fungi only increased during the third period. This study provides a reference for microbial control during Zhacai curing process
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