乳酸乳球菌表达重组牛乳铁蛋白肽的抑菌活性分析

牛乳铁蛋白肽是由牛乳铁蛋白经消化酶水解产生的一类具有广谱抑菌活性的短肽;乳酸乳球菌作为食品级微生物,既有天然的益生作用,又是理想的表达牛乳铁蛋白肽的载体。【目的】探究重组乳酸乳球菌pAMJ399-LFcinBA/MG1363表达牛乳铁蛋白肽的抑菌活性。【方法】利用牛乳铁蛋白肽标准品绘制定量标准曲线来确定重组牛乳铁蛋白肽的含量,利用牛津杯法及微量肉汤稀释法测定重组牛乳铁蛋白肽对大肠杆菌、金黄色葡萄球菌等35株细菌的抑菌活性及最小抑菌浓度,利用扫描电镜、透射电镜、荧光显微镜、凝胶阻滞试验、黏附试验来探究重组牛乳铁蛋白肽对菌体结构、细菌DNA及黏附力的影响,利用CCK-8检测其对RAW 264.7细胞的毒性作用,并对小鼠红细胞溶血率进行测定。【结果】重组乳酸乳球菌上清中牛乳铁蛋白肽的浓度为24.39μg/mL,重组牛乳铁蛋白肽对测试的25株致病菌均有不同程度的抑制作用,抑菌浓度范围在16–128μg/mL,但对9株乳酸菌以及粪肠球菌没有明显的抑制作用,对大肠杆菌、金黄色葡萄球菌、多杀性巴氏杆菌、鸡白痢沙门菌的菌体完整性具有不同程度的破坏作用,其主要作用靶点为细菌的细胞膜,可以与细菌DNA结合并抑制细菌对Caco-2、IPEC细胞的黏附作用,重组牛乳铁蛋白肽对小鼠红细胞及RAW 264.7细胞没有明显的细胞毒性。【结论】乳酸乳球菌表达重组牛乳铁蛋白肽的抑菌活性与牛乳铁蛋白肽标准品相一致,通过直接作用于细菌细胞膜、胞内核酸或抑制细菌对正常细胞的黏附作用等多方面实现抑制或杀死细菌,发挥广谱的抗菌活性,且对真核细胞没有明显的细胞毒性作用。

Antibacterial activity of the recombinant bovine lactoferrin peptide expressed by Lactococcus lactis

In order to evaluate the weed control effect and wheat safety of the new herbicide quinotrione for weed control in wheat field, the combined action of quinotrione and bromoxynil octanoate was determined with co-toxicity coefficient (CTC) method in the greenhouse, and the efficacy of the mixture of quinotrione, bromoxynil octanoate and florasulam on weeds in wheat field and the safety of wheat were verified. The results of greenhouse experiment showed that the efficacy of quinotrione was excellent on Veronica persica, and killed all of the weeds under the experimental dose. The efficacy of quinotrione on Descurainia sophia was higher than that on Lithospermum arvense with a GR₅₀ 12.67 g (a.i.)/hm² and 152.99 g (a.i.)/hm², respectively while the GR₅₀ of bromoxynil octanoate was 26.22, 13.36, and 85.12 g (a.i.)/hm² on D. sophia, L. arvense and V. persica, respectively. When quinotrione and bromoxynil octanoate were mixed in a ratio of 1:1-1:2.5, all of the V. persica weeds died. The co-toxicity coefficient of quinotrione+bromoxynil octanoate on D. sophia were between 140.34 and 202.77, and more than 700 on L. arvense, showing obvious synergistic effect. The results of field experiment showed that quinotrione was highly effective on V. persica, but less effective on Galium aparine and D. sophia, the bromoxynil octanoate had a quick effect, but some weeds revived in the later period. The efficacy of quinotrione+bromoxynil octanoate was excellent with an 88.54%~93.83% control on the fresh weight, and the action speed was fast. The fresh weight control efficacy was over 96.12% when it was mixed with florasulam. No influence on the wheat growth was detected when quinptrine was used alone or mixed with bromoxynil octanoate and florasulam.