A CRISPR-Cas12a-Based Assay for Efficient Quantification of Lactobacillus panis in Chinese Baijiu Brewing Microbiome

Yanfeng Liu,Jianghua Li,Guocheng Du,Jian Chen,Li Wang,Mengchuang Wang,Xiaolong Zhang,Fan Yang

doi:10.3390/fermentation8020088

Abstract

Although the quantification of key microorganisms in fermentation microbiomes is important for monitoring and regulating fermented food production, conventionally used methods are generally time-consuming, involve complicated operations, and have a high level of equipment dependence. We developed a CRISPR-Cas12a-based quantitative Chinese Baijiu brewing microorganism assay (CQAOB) for Lactobacillus panis, the most important lactic acid-producing fermentative microorganism. We initially verified the feasibility of CQAOB for detecting L. panis, and then optimized the reaction parameters to enhance Cas12a detection sensitivity. The specificity of the developed method was verified via the accurate distinction of L. panis nucleic acids from those of other lactic acid bacteria. The quantitative range and detection time for L. panis were 106–109 copies/μL and 40 min, respectively. Finally, we successfully applied CQAOB for quantifying L. panis count in fermented grains. Given its rapid detection and low level of equipment dependence, CQAOB may make an important contribution to quantifying key microorganisms in brewing processes.

Highlights

Maotai-flavored Baijiu is a typical traditional fermented product produced using solid-state fermentation based on a brewing microbiome containing a diverse range of microorganisms [1]
Among the lactic acid-producing bacteria, Lactobacillus panis has been identified as the most important in the fermentation of Maotai-flavored Baijiu, relative abundances typically vary in the brewing microbiome during the fermentation process [6,7]
We developed a CRISPR-Cas12a-based quantitative Chinese Baijiu brewing microorganism assay (CQAOB) for L. panis, for which we optimized key parameters of the detection system to enhance detection sensitivity

Summary

Introduction

Maotai-flavored Baijiu is a typical traditional fermented product produced using solid-state fermentation based on a brewing microbiome containing a diverse range of microorganisms [1]. Among the lactic acid-producing bacteria, Lactobacillus panis has been identified as the most important in the fermentation of Maotai-flavored Baijiu, relative abundances typically vary in the brewing microbiome during the fermentation process [6,7]. In this latter regard, the quantification of important brewing microorganisms can characterize metabolic activity during fermentation and provide guidance for the regulation of the fermentation process.

Methods

Results

Conclusion