基于三步荧光定量PCR技术揭示不同产区白酒酿造系统中Lactobacillus sp.的分布特征

Distribution of Lactobacillus sp. in Chinese liquor fermentation system from different producing location by three-step fluorescent quantitative PCR

[背景]Lactobacillus sp.是一株存在于白酒酿造系统中的功能微生物,但是针对Lactobacillus sp.的定量方法及其在中国白酒酿造系统中的分布尚未被研究。[目的]建立一种基于特异性引物的荧光定量PCR定量方法,并应用于实际生产检测,揭示Lactobacillus sp.在中国白酒酿造系统中的分布特征。[方法]基于16S rRNA基因序列设计特异性引物,并通过PCR验证引物特异性;优化荧光定量PCR反应程序,提高引物扩增效率;定量分析所采集样本中Lactobacillus sp.的含量,揭示Lactobacillus sp.在中国白酒酿造系统中的分布特征。[结果]设计了一对扩增产物大小为445 bp的特异性引物。通过优化扩增条件,构建含有变性、退火、延伸过程的三步荧光定量PCR方法,该方法扩增线性较强,R^2>0.99;灵敏度高,定量限为17.9 copies/μL;重复性好,Ct值的变异系数小于1%。跟踪检测全国10个产区代表产地的白酒酿造系统,其中8个产区均检测到Lactobacillus sp.,在相同产地不同酿造工艺的酿造系统中均检测到Lactobacillus sp.,但是含量上有显著差异。山东潍坊产地的芝麻香型白酒发酵体系含量最高(7.27±0.04 lgcopies/g),跟踪发酵时间样本发现Lactobacillus sp.的生长分为两个阶段:生长期(0-15 d)和稳定期(15-45 d)。[结论]基于特异性引物所建立的三步荧光定量PCR技术可实现对白酒酿造系统中Lactobacillus sp.的鉴定和定量,通过跟踪检测发现Lactobacillus sp.广泛分布在中国白酒发酵体系中,其中产地决定Lactobacillus sp.的分布,酿造工艺影响含量,在发酵过程中Lactobacillus sp.的含量具有明显的动态变化规律,为进一步研究该菌在发酵过程中的功能提供参考。

[Background] Lactobacillus sp. is an important function microorganism in Chinese liquor fermentation. However, there is no quantitative method to uncover the distribution of Lactobacillus sp. among different production locations. [Objective] This study aimed to build a quantitative method to uncover the distribution Lactobacillus sp. among different Chinese liquor production locations. [Methods] Specific primers were designed based on 16 S r RNA gene sequence, and the specificity was verified by PCR. The qPCR reaction procedure was further optimized to increase amplification efficiency and reveal the distribution of Lactobacillus sp. [Results] A pair of specific primers were designed and validated, its amplification product size was 445 bp. By optimizing annealing and extension conditions, a three-step qPCR quantitative method was developed with independent denaturation, annealing and extension process. The amplification linearity of this method was strong(R^2>0.99). The sensitivity was high, the quantitative limit was 17.9 copies/μL. The repeatability was well, the coefficient of variation of Ct value was less than 1%. Following and detecting the representative fermentation system from 10 producing locations in China, Lactobacillus sp. presented in 8 producing areas. In the different making process samples from the same region, Lactobacillus sp. was detected but its content was significantly different. The content of Lactobacillus sp. from sesame aroma fermentation system in Weifang, Shandong province was the highest(7.27±0.04 lgcopies/g). The growth curve of Lactobacillus sp. in this system was divided into two stages: the growth stage(0 to 15 d) and the stable stage(15 to 45 d). [Conclusion] The established novel qPCR method can accurately quantify and identify Lactobacillus sp. Through follow-up detection, it was found that Lactobacillus sp. was widespread in Chinese liquor fermentation system, the distribution of Lactobacillus sp. was determined by the origin, the content of Lactobacillus sp. was affected by the making process. During fermentation process, the content of Lactobacillus sp. has an obvious dynamic characteristic. This study will improve the development of function study about Lactobacillus sp.