基于刚果红的嗜热链球菌胞外多糖高产菌株高通量筛选方法的建立。
Development of a high-throughput screening method for exopolysaccharide-producing Streptococcus thermophilus based on Congo red.
机构信息
Shanghai Engineering Research Center of Food Microbiology, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China.
Shanghai Engineering Research Center of Food Microbiology, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China.
出版信息
Food Res Int. 2022 Dec;162(Pt B):112094. doi: 10.1016/j.foodres.2022.112094. Epub 2022 Nov 2.
As a thickening or stabilizing agent, exopolysaccharide (EPS) can significantly enhance food texture. However, the conventional analytical and screening methods of EPS have been unable to meet the current needs of industrial production, owing to low efficiency and heavy time-consumption. A simple and rapid qualitative/quantitative method is needed to accelerate the selection of high EPS-producing strains. Here, a high-throughput screening (HTS) program for EPS production was established in S. thermophilus by combining Congo red agar method (CRA, primary screening) and microplate colorimetric assay (MPC, secondary screening). The correlation coefficient (R) between CRA/MPC and the phenol-sulfuric acid assay (a classical EPS quantification method) were 0.779 and 0.862, respectively, suggesting the feasibility of this HTS method. A mutant library (>300 colonies) of S. thermophilus was constructed using ribosome engineering strategy, and EPS-producing mutants with the titer from 50 mg/L to 200 mg/L were rapidly obtained by our developed HTS method, confirming that the HTS method is simple and efficient for the isolation of EPS-producing S. thermophilus. Taken together, this HTS method was developed for rapidly screening of EPS-producing S. thermophilus, which could be a valuable tool not only for the rapid detection of EPS production capabilities, but also for the screening of strain libraries from genetic engineering with desirable characteristics.
作为一种增稠或稳定剂，胞外多糖（EPS）可以显著改善食品质地。然而，由于效率低、耗时耗力，传统的 EPS 分析和筛选方法已经无法满足当前工业生产的需求。因此，需要一种简单、快速的定性/定量方法来加速高 EPS 产生菌株的选择。本研究通过结合刚果红琼脂法（CRA，初筛）和微孔板比色法（MPC，复筛），在嗜热链球菌中建立了一种 EPS 生产高通量筛选（HTS）程序。CRA/MPC 与苯酚-硫酸法（一种经典的 EPS 定量方法）之间的相关系数（R）分别为 0.779 和 0.862，表明该 HTS 方法具有可行性。利用核糖体工程策略构建了嗜热链球菌突变体文库（>300 个菌落），通过我们开发的 HTS 方法，快速获得了滴度为 50mg/L 至 200mg/L 的 EPS 产生突变体，证实了 HTS 方法用于分离 EPS 产生的嗜热链球菌是简单、高效的。总之，本研究开发了一种用于快速筛选 EPS 产生的嗜热链球菌的 HTS 方法，该方法不仅可用于快速检测 EPS 的产生能力，还可用于从具有理想特性的遗传工程菌株文库中进行筛选。
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