家蝇抗菌肽MDC对鼠伤寒沙门氏菌的抑菌稳定性研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0847

摘要/Abstract

摘要：

以鼠伤寒沙门氏菌（Salmonella typhimurium）作为研究对象,探讨家蝇抗菌肽（Musca domestica cecropin,MDC）在不同条件下的抑菌稳定性。采用牛津杯法研究pH值、温度、光照、蛋白酶、金属离子、表面活性剂及有机试剂对MDC抑菌活性（抑菌圈大小）的影响。在酸性条件下MDC的抑菌活性稍有下降,保持在85%左右,但在碱性条件下的抑菌活性无明显改变,稳定性较好。经低温和高温处理后,抑菌活性无显著差异。MDC在黑暗、正常光及紫外线照射处理30 min,其抑菌活性仍保持稳定。MDC对胰蛋白酶敏感,抑菌活性完全消失,而对胃蛋白酶具有良好的稳定性。MDC对Ca²⁺、K⁺有良好的稳定性,但对Mg²⁺敏感,在Fe³⁺作用下其抑菌活性较对照组有所升高（P<0.05）。表面活性剂和有机试剂处理MDC后,其抑菌活性均明显降低。家蝇抗菌肽MDC能显著抑制鼠伤寒沙门氏菌生长繁殖,其活性几乎不受pH值,温度和光照变化、胃蛋白酶和某些金属离子的影响,且Fe³⁺能够协同增强MDC的抑菌活性,但Mg²⁺、表面活性剂、有机试剂以及胰蛋白酶水解作用可分别使其抑菌活性部分或完全丧失。

关键词: 家蝇抗菌肽, 稳定性, 鼠伤寒沙门氏菌, 抑菌活性

Abstract:

This work aims to investigate the antibacterial stability of the housefly antimicrobial peptide（Musca domestica cecropin,MDC）against Salmonella typhimurium under different conditions. The Oxford cup method was used to determine the influences of pH,temperature,light,protease,metal ions,surfactants and organic reagents on the antibacterial activity（bacteriostatic ring size）of MDC against S. typhimurium. The results showed that the activity of MDC decreased slightly under acidic condition（remained 85% of that under the normal condition）,but not changed under alkaline condition with a good stability. Under low or high temperature treatment,no obvious difference was observed in its antibacterial activity. In addition,the activity of MDC still remained stable after exposed to darkness,normal light or ultraviolet for 30 min. MDC was sensitive to trypsin and its activity completely disappeared,but good stability to pepsin. MDC was still stable in the Ca²⁺ and K⁺,but it was sensitive to Mg²⁺. Interestingly,the antibacterial activity of MDC under Fe³⁺increased compared with the control group（P<0.05）. Moreover,the antimicrobial activities of MDC treated with surfactant and organic reagents significantly reduced but still partially preserved. To sum up,MDC can significantly inhibit the growth and reproduction of S. typhimurium,and its activity is almost not affected by the change of pH value/temperature/light and different pepsin and metal ions. Ferric ion can synergistically enhance the antibacterial activity of MDC,but Mg²⁺,surfactant,organic reagent and trypsin hydrolysis caused its antibacterial activity partially or completely lost,respectively.

Key words: Musca domestica cecropin, stability, Salmonella typhimurium, antibacterial activity

王子琰, 王建, 张伦, 桂水清, 卢雪梅. 家蝇抗菌肽MDC对鼠伤寒沙门氏菌的抑菌稳定性研究[J]. 生物技术通报, 2022, 38(3): 149-156.

WANG Zi-yan, WANG Jian, ZHANG Lun, GUI Shui-qing, LU Xue-mei. Study on Antibacterial Stability of Musca domestica Cecropin-MDC Against Salmonella typhimurium[J]. Biotechnology Bulletin, 2022, 38(3): 149-156.

图/表 9

图1 MDC的HLPC色谱图

Fig.1 HLPC of MDC

图2 MDC的质谱图

Fig.2 MS analysis of MDC

图3 MDC经不同pH处理后对鼠伤寒沙门氏菌的抑菌活性抑菌平板图中样品均以顺时针方向编号,1、2、3、4依次为生理盐水、处理后的MDC、头孢曲松钠、未处理的MDC;统计图中其他各组均与Control组（未处理的MDC）相比,*P<0.05,**P<0.01,n=3

Fig. 3 Antibacterial activity of MDC on S. typhimurium after different pH treatment All the samples in the bacteriostatic plate are numbered clockwise. 1,2,3 and 4 represent normal saline,treated MDC,ceftriaxone sodium and untreated MDC respectively. Compared with the control group（untreated MDC）,*P < 0.05,**P < 0.01,n=3

图4 MDC经不同温度处理后对鼠伤寒沙门氏菌的抑菌活性抑菌平板图中样品均以顺时针方向编号,1、2、3依次为生理盐水、不同温度处理后的MDC和头孢曲松钠,n=3

Fig. 4 Antibacterial activity of MDC on S. typhimurium after different temperature treatment All the samples in the bacteriostatic plate are numbered clockwise. 1,2 and 3 are normal saline,MDC treated with different temperature and ceftriaxone sodium respectively,n=3

图5 MDC经不同光照条件处理后对鼠伤寒沙门氏菌的抑菌活性抑菌平板图中样品均以顺时针方向编号,1、2、3依次为生理盐水、不同光照处理后的MDC和头孢曲松钠,n=3

Fig. 5 Antibacterial activity of MDC on S. typhimurium under different light condition All the samples in the bacteriostatic plate are numbered clockwise. 1,2 and 3 are normal saline,MDC treated with different light conditions and ceftriaxone sodium respectively,n = 3

图6 MDC经不同蛋白酶处理后对鼠伤寒沙门氏菌的抑菌活性抑菌平板图中样品均以顺时针方向编号,1、2、3、4依次为生理盐水、蛋白酶处理后的MDC、头孢曲松钠、95℃处理的MDC;统计图中其他各组均与Control组（95℃处理的MDC）相比,****P<0.0001,n=3

Fig. 6 Antibacterial activity of MDC on S. typhimurium after different proteases treatment All the samples in the bacteriostatic plate are numbered clockwise. 1,2,3 and 4 represent normal saline,MDC treated with protease,ceftriaxone sodium and MDC treated at 95℃ respectively. Compared with the control group（MDC treated at 95℃）,****P < 0.0001,n = 3

图7 MDC经不同金属离子处理后对鼠伤寒沙门氏菌的抑菌活性抑菌平板图中样品均以顺时针方向编号,1、2、3、4依次为生理盐水、未处理的MDC、头孢曲松钠、金属离子处理的MDC;统计图中其他各组均与Control组（未处理的MDC）相比,***P<0.001,n=3

Fig. 7 Antibacterial activity of MDC on S. typhimurium after different metal ion treatment All the samples in the bacteriostatic plate are numbered clockwise. 1,2,3 and 4 represent normal saline,untreated MDC,ceftriaxone sodium and MDC treated with metal ion respectively. Compared with the control group（untreated MDC）,***P < 0.001,n = 3

图8 MDC经不同表面活性剂处理后对鼠伤寒沙门氏菌的抑菌活性抑菌平板图中样品均以顺时针方向编号,1、2、3、4依次为生理盐水、未处理的MDC、头孢曲松钠、表面活性剂处理的MDC;统计图中其他各组均与Control组（未处理的MDC）相比,***P<0.001,n=3

Fig. 8 Antibacterial activity of MDC on S. typhimurium after different surfactant treatment All the samples in the bacteriostatic plate are numbered clockwise. 1,2,3 and 4 represent normal saline,untreated MDC,ceftriaxone sodium and MDC treated with surfactant respectively. Compared with the control group（untreated MDC）,***P < 0.001,n = 3

图9 MDC经不同有机试剂处理后对鼠伤寒沙门氏菌的抑菌活性抑菌平板图中样品均以顺时针方向编号,1、2、3、4依次为生理盐水、未处理的MDC、头孢曲松钠、有机试剂处理的MDC;统计图中其他各组均与Control组（未处理的MDC）相比,***P<0.001,n=3

Fig. 9 Antibacterial activity of MDC on S. typhimurium after different organic solvent treatment All the samples in the bacteriostatic plate are numbered clockwise. 1,2,3 and 4 represent normal saline,untreated MDC,ceftriaxone sodium and MDC treated with organic solvent respectively. Compared with the control group（untreated MDC）,***P < 0.001,n = 3

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