Application of Physical Mutagenesis and High Throughput Screening Technology in the Selection of Probiotics
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摘要: 野生型菌株活性较低，难以满足工业化需求，通过使用物理诱变方法可改善菌种性能，进而获得高产、优质菌株。同时需寻找快速、合适的筛选方法从突变文库中获得理想目标菌株。传统人工筛选及摇瓶培养成本高、耗时耗力，高通量筛选技术解决了这一难题。本文探讨了传统及新型物理诱变技术原理，对比了两类诱变技术的区别及阐述了其在益生菌选育中的应用。同时总结了各高通量筛选技术（微量滴定板筛选、荧光激活细胞分选、生物传感器的筛选、液滴微流控平台筛选及模式动物平台的筛选）的特点及其在益生菌筛选的相关应用。本文为后续降低筛选成本、提高筛选效率、获得高产理想目标菌株提供重要参考。Abstract: Wild-type strains hardly meet the current industrial demands due to low stability. The performance of microbial strains (high-yielding, high-quality strains) can be improved by using physical mutagenesis techniques. At the same time, high throughput screening methods and techniques can be used to quickly obtain ideal strains from the mutation library. However, traditional manual screening and shaker culture are high-cost, time-consuming, and laborious, and high-throughput screening technology can solve this problem. In this review, the applications of traditional and novel mutagenesis used for the improvement of probiotics are summarized, the principles of traditional and novel physical mutagenesis techniques are discussed, the differences between the two kinds of mutagenesis techniques are compared and their applications in the improvement of probiotics are described. At the same time, the characteristics and relevant applications of various high-throughput screening technologies (microtitration plate screening, fluorescence activated cell sorting, biosensors screening, droplet microfluidic platform screening and model animal platform screening) are summarized. This review provides important reference for reducing screening cost, improving screening efficiency and obtaining high yield ideal target strains.
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Key words:
- physical mutagenesis /
- high-throughput screening /
- probiotics /
- application
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表 2 物理诱变在益生菌育种中的应用
Table 2. Application of physics mutagenesis in probiotics breeding
诱变菌株 诱变方法 改善性状 参考文献 提高产物产率/产量 乳酸片球L15 紫外诱变 改良菌株胞外多糖产量可达232.34 mg/L，比出发菌株提高56.46 mg/L [10] 乳酸菌 L--SZ303 紫外诱变 目标菌株发酵液中 γ-氨基丁酸产量14.646 g/L，比出发菌株提高了1.271 g/L [37] 枯草芽孢杆菌HDBF-DJ3N7 激光诱变 突变菌株纤溶酶活力提升18.40%，其酶活力值达到了429.89±5.74 IU/mL [38] 乳酸杆菌 微波诱变 目标菌株产共轭亚油酸多达48.85 μg/mL，与诱变前相比提高了41.4% [17] 植物乳杆菌 ARTP 与初始菌株相比，目标菌株产酸能力提高了50%，产酸量为0.015 mol/L [22] 干酪乳杆菌 HIB 目标菌株乳酸产量较原始菌株提高41.6%~83.3% [39] 嗜热乳酸菌 HIB 突变体高产L（+）-乳酸，产量为23.24±0.66 g/L，与野生型相比有显著增加 [27] 干酪乳杆菌CICC6028 LEIP 突变株高产L-（+）-乳酸，产量为136 g/L，比原菌株提高了38.8% [36] 提高菌株环境耐受力 嗜酸乳杆菌FZU-LA1301 UV 目标菌株能耐受45 ℃，比出发菌株（37 ℃）提高8 ℃ [11] 嗜酸乳杆菌 ARTP 突变体在pH为2、3的条件下培养3 h，其乳酸胁迫耐受性分别提高了75.67%和25.78%，
且与亲本菌株（76.2%）相比，具有更高的疏水性（87.2%）[24] 鼠李糖乳杆菌JF12-1 HIB 8株改良菌株的体外抑菌性比野生菌株提高15%以上 [31] 提高底物利用率 干酪乳杆菌NRRL-B-1922 激光诱变 突变菌株发酵乳制品后抗氧化及蛋白水解能力分别显著提高41%和14% [40] 
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表 3 高通量筛选技术对比
Table 3. Comparison of high-throughput screening techniques
类型 测定指标 筛选设备 筛选方法 检测指标 微量滴定板的筛选 吸光度值 紫外/可见光谱 直接筛选 番茄红素[41]、β-胡萝卜素[42]、对香豆酸[43] 间接筛选 L-乳酸[44] 荧光激活细胞分选 荧光强度 流式细胞仪 直接筛选 核黄素[46] 间接筛选 L-多巴[48] 生物传感器的筛选 电信号强度 生物传感器 蛋白质生物传感器 黄酮类化合物[51]（转录因子） 核酸生物传感器 维生素B2[54]（RNA核糖体开关） 液滴微流控平台筛选 荧光信号 液滴微流控筛选系统 表型筛选 α-淀粉酶[55] 组学筛选 多种代谢物 模式动物平台筛选 荧光强度 高内涵细胞成像系统、荧光倒置显微镜 表型筛选 生存、行为能力；果蝇眼睛发育、
斑马鱼胚胎发育[59]等表型益生菌体内筛选 益生菌定植
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