Design And Construction Of CRISPR-based Quantification For Lactobacillus Panis And Lactobacillus Plantarum In Jiang-flavor Liquor Fermentation

The brewing process of Jiang-flavor liquor adopts a solid-state multi-strain fermentation.Monitoring and analysis of the number and dynamic changes of key microorganisms in the brewing process play an important role in the regulation of the brewing process.Lactobacillus panis and Lactobacillus plantarum are the key microorganisms for lactic acid production,which have important effects on the composition of brewing microbial community and the flavor of base liquor.However,quantification of key microorganisms in brewing process mainly adopts methods such as plate counting,fluorescence quantitative PCR and high-throughput sequencing methods,which have the problems of time consuming,high equipment dependence and high detection cost.How to establish a rapid quantitative method for brewing microorganisms,obtain optimal detection conditions and verify the applicability of the method in the brewing system are three key issues to be solved.In this study,aiming at the problems of microbial quantification in Jiang-flavor liquor brewing,taking L.panis and L.plantarum as the research objects,the construction of a CRISPR-Cas12a-based quantitative method for brewing microorganisms,the optimization of the key detection conditions in the detection process,and the optimization of the detection of fermented grains in the fermentation process of Jiang-flavor liquor were studied,and a rapid quantitative method for L.panis and L.plantarum was established.The main research results are as follows:（1）The expression system of Francisella novicida Cas12a（Fn Cas12a）in Escherichia coli was constructed,and the expression of the protein was improved by replacing the promoter and optimizing the fermentation temperature.The optimal promoter type was P₂₂₄,and the optimal fermentation temperature was 25℃.Compared to the initial condition P_T7 promoter and 37℃ fermentation temperature,the expression of protein increased by 31.6%and 115.1%;the fermentation broth of Fn Cas12a was purified by AKTA protein purifier,and the final yield was0.94 mg·m L^-1;the trans-cleavage activity of Fn Cas12a was detected,reaching 9.72×10^-6a.u.·nmol^-1.A fluorescent reporter system based on CRISPR-Cas12a was constructed,and cr RNA designed based on 16S r DNA sequence of L.panis was used to verify the activation of the fluorescent reporter system.Eight designed cr RNAs can activate both synthetic and amplified sequence targets.The feasibility of the system to test L.panis nucleic acid was verified.（2）By optimizing the key components Fn Cas12a,cr RNA and ss DNA probes based on the CRISPR-Cas12a fluorescent reporter system,the system response signal value was improved.The optimal Fn Cas12a concentration after optimization is 100 n M,and the signal value increases to 2.1-fold before optimization;the optimal cr RNA concentration after optimization is 100 n M,and the signal value increases to 1.5-fold before optimization;the optimized probe type is HEX-BHQ1,the signal-to-noise ratio is 11.7-fold that before optimization.Based on the results of the gyr B gene sequence alignment of 13 Lactobacillus strains,8 cr RNAs were designed for L.panis and L.plantarum,and the target strain genome and the non-target strain genome were selected for specificity verification,and two pieces with the strongest activation ability were screened.The specific cr RNA sequences g7 and CR6-1 serve as the activation sequences for L.panis and L.plantarum,respectively.The quantitative standard curves of L.panis and L.plantarum were constructed.The detection ranges were 10⁶～10⁹ CFU·μL^-1 and10⁵～10⁹ CFU·μL^-1,respectively,and the correlation coefficients of the standard curves reached0.99.（3）Consistency detection of single bacteria samples of L.panis and L.plantarum was carried out,and the correlation coefficient reached 0.99,showing the good accuracy of the system in detecting single bacteria;the q PCR standard curve of L.panis and L.plantarum was established.The mixed bacteria samples were tested,and the detection results are in the same order of magnitude,which showed the good accuracy of the system in detecting mixed bacteria samples.The fermented grain samples during the fermentation process of Jiang-flavor liquor were tested,and the results showed that with the increase of fermentation time,the number of L.panis increased gradually during 28 days fermentation,and the number of L.plantarum initially increased and then decreased.