抗生素环丙沙星胁迫植物乳杆菌ZLC-18应激反应的研究

本研究采用0.1、0.5、2.0 μg/mL的环丙沙星作为实验药物,通过研究环丙沙星主动胁迫下肠道内植物乳杆菌产生的应激反应,为开发一类具有抵御抗生素胁迫能力、保持肠道菌群微生态平衡的益生菌制剂提供理论依据。结果表明,环丙沙星胁迫下,植物乳杆菌菌体存活率整体呈下降趋势,且菌体存活率随环丙沙星浓度增大而降低;观察菌体细胞膜完整性发现,细胞膜受损程度与环丙沙星浓度呈正相关;测定菌体侧向扩散速率来反映其细胞膜流动性,结果显示,2.0 μg/mL的环丙沙星会使菌体细胞膜流动性极显著降低(P<0.01),与对照组相比降低了39.93%;菌体超微结构观察发现,环丙沙星会明显改变植物乳杆菌的菌体形态,严重时可造成细胞质外泄,甚至导致细胞死亡;菌体糖代谢关键酶活力结果表明,2.0 μg/mL环丙沙星处理后的己糖激酶、丙酮酸激酶及乳酸脱氢酶酶活力均极显著下降(P<0.01),而经0.5 μg/mL环丙沙星胁迫后,与对照组乳酸脱氢酶酶活(0.069±0.002)U/mg prot相比,其乳酸脱氢酶活力反而增加,酶活为(0.081±0.006)U/mg prot(P<0.01);反复用质量浓度为0.5 μg/mL的环丙沙星胁迫菌体后,其乳酸脱氢酶酶活为(0.126±0.004)U/mg prot(P<0.01)。说明在低浓度环丙沙星的胁迫下,植物乳杆菌会产生应激胁迫响应,启动自我保护机制,抵御抗生素不良胁迫,这为肠道益生菌制剂开发提供了重要的技术支撑。

Study on Stress Response of Lactobacillus plantarum ZLC-18 to Ciprofloxacin

In this study,0.1,0.5 and 2.0 μg/mL of ciprofloxacin were used as experimental drugs to study the stress response of Lactobacillus plantarum derived from gut under active stress of ciprofloxacin,so as to provide theoretical basis for the development of probiotics with the ability to resist antibiotic stress and maintain the microecological balance of intestinal flora. Results showed that,the overall viability of the cells showed a downward trend,and the cell viability decreased with the increase of the concentration of ciprofloxacin. By observing the cell membrane integrity of the cells,it was found that the damage of cell membrane integrity was positively correlated with the concentration of ciprofloxacin. The lateral diffusion rate of the cells was measured to reflect the cell membrane fluidity,the results showed that 2.0 μg/mL of ciprofloxacin significantly reduced the cell membrane fluidity of the cells,which was reduced by 39.93% compared with the control group(P<0.01). Observation of the ultrastructure of the cells revealed that ciprofloxacin significantly changed the cell morphology of Lactobacillus plantarum,which could cause cytoplasm leakage and even cell death in severe cases. The results of the activity of key enzymes in cell glucose metabolism showed that the activities of hexokinase,pyruvate kinase and lactate dehydrogenase significantly decreased after treatment with 2.0 μg/mL ciprofloxacin(P<0.01),and after 0.5 μg/mL ciprofloxacin stress,the lactate dehydrogenase activity increased compared with the control lactate dehydrogenase activity(0.069±0.002) U/mg prot,and the enzyme activity was(0.081±0.006) U/mg prot(P<0.01). After further repeatedly stress on the cells with ciprofloxacin at a concentration of 0.5 μg/mL,the activity of lactate dehydrogenase was determined to be(0.126±0.004) U/mg prot(P<0.01). It indicated that under the stress of low concentration of ciprofloxacin,Lactobacillus plantarum produced stress response,initiated self-protection mechanism and resisted antibiotic adverse stress,which provided important technical support for the development of intestinal probiotic preparations.