抗菌肽LL-1对沙门氏菌的抗菌机制

摘要/Abstract

摘要： 为了探究LL-1对沙门氏菌的抗菌效果及机制，首先通过倍比稀释法测定LL-1对沙门氏菌的最小抑菌浓度（MIC），并以抗菌曲线评价LL-1对沙门氏菌的抗菌效果；然后，使用扫描和透射电子显微镜观察细菌形态，再通过检测核酸、蛋白质、碱性磷酸酶（ALP）的泄露情况以及PI染色实验，评价LL-1对沙门氏菌细胞壁和细胞膜的影响，又通过核酸凝胶电泳观察LL-1与沙门氏菌DNA结合情况；最后，通过检测胞内琥珀酸脱氢酶（SDH）、NADP-苹果酸脱氢酶（NADP-MDH）活性以及ATP含量，评价LL-1对沙门氏菌能量代谢的影响。结果表明：LL-1对沙门氏菌的MIC为6.25 μg/mL，有良好的抗菌效果，且呈浓度和时间依赖性；经LL-1处理的沙门氏菌出现菌体皱缩、胞膜溶解和质壁分离等形态变化；沙门氏菌经LL-1处理后，胞内核酸、蛋白质、ALP发生泄漏，并且经PI染色后胞内荧光强度增强，同时LL-1与DNA发生结合；随着LL-1浓度的升高，胞内ATP含量下降、SDH及NADP-MDH的活性降低。综上，LL-1能增大沙门氏菌细胞膜和细胞壁的通透性引起胞内容物泄漏，结合其DNA，并影响细菌能量代谢等方式抗菌，为后续深入研究LL-1的抗菌机制和应用奠定基础。

关键词: 沙门氏菌, 抗菌肽, 抗菌机制, 食源性致病菌, 细胞膜, 能量代谢

Abstract: To explore the antibacterial effect and mechanism of LL-1 against Salmonella. Firstly, the minimum inhibitory concentration (MIC) of LL-1 against Salmonella was determined by the doubling dilution method, and the antibacterial effect of LL-1 on Salmonella was evaluated by the antibacterial curve. Then, the bacterial morphology was observed using scanning and transmission electron microscopy. The effects of LL-1 on the cell wall and cell membrane of Salmonella were evaluated by detecting the leakage of nucleic acid, protein and alkaline phosphatase (ALP) and PI staining experiments. The binding of LL-1 to Salmonella DNA was detected by nucleic acid gel electrophoresis. Finally, the effects of LL-1 on the energy metabolism of Salmonella were evaluated by measuring the activities of intracellular succinate dehydrogenase (SDH), NADP-malate dehydrogenase (NADP-MDH) and ATP levels. The results showed that the MIC of LL-1 against Salmonella was 6.25 μg/mL, and LL-1 had a good antibacterial effect and presented dose and time dependence. Salmonella treated with LL-1 showed morphological changes such as shrinkage, cell membrane dissolution, and parenchymal separation. LL-1 resulted in the leakage of intracellular nucleic acids, proteins, ALP and the increase of intracellular fluorescence intensity after PI staining in Salmonella. Additionally, LL-1 could bound with DNA. With the increase of LL-1 concentration, it was decreasing for the intracellular ATP content, SDH and NADP-MDH activities in Salmonella. In conclusion, LL-1 could increase the permeability of Salmonella cell membrane and cell wall, cause content leakage, bind to DNA, and affect bacterial energy metabolism, which lays a foundation for further research on the antibacterial mechanism and application of LL-1.

Key words: Salmonella, antimicrobial peptides, antimicrobial mechanisms, foodborne pathogens, cell membrane, energy metabolism

中图分类号:

TS201.3

王宇航周玲玲周瑶玲撒俊梦张元臣马增军连凯琪. 抗菌肽LL-1对沙门氏菌的抗菌机制[J]. 食品科学.

Yu-Hang WANG. Antimicrobial Mechanism of Antimicrobial Peptide LL-1 against Salmonella[J]. FOOD SCIENCE.

参考文献

[1]施怡茹, 赵锦江, 卢晓芸, 等.沙门菌血清型多态性与分型及其相关性研究[J].检验医学与临床, 2020, 17(14):2017-2019 [2]Knodler LA, Elfenbein JR.Salmonella enterica[J].Trends Microbiol, 2019, 27(11):964-965 [3]Bumann D, Schothorst J.Intracellular Salmonella metabolism[J].Cell Microbiol, 2017, 19(10):e12766-e12766 [4]朱奇, 陆斌兴, 覃有泉, 等.沙门氏菌生物学研究进展[J].疾病监测与控制, 2015, 9(07):474-478 [5]吕奎营, 赵秋香, 赵媛媛, 等.天然食品防腐剂及其在食品中的应用研究[J].中国调味品, 2020, 45(04):186-188 [6]Wang G, Li X, Wang Z.APD3: the antimicrobial peptide database as a tool for research and education[J].Nucleic Acids Res, 2016, 44(D1):D1087-93 [7]Lazzaro BP, Zasloff M, Rolff J.Antimicrobial peptides: Application informed by evolution[J].Science, 2020, 368(6490):e-a [8]Eduardo Guaní-Guerra, Santos-Mendoza T, Saúl O Lugo-Reyes, et al.Antimicrobial peptides: General overview and clinical implications in human health and disease[J].Clinical Immunology, 2010, 135(1):1-11 [9]龙彪, 彭志英, 赵谋明.新型生物防腐剂-昆虫抗菌肽[J].中国食品添加剂, 2004, (05):32-36+96 [10]连凯琪, 王宇航, 曾雅静, 等.抗菌肽LL-1对肺炎克雷伯菌ATCC 700603的抗菌机制[J].中国畜牧兽医, 2024, (05):2237-2244 [11]连凯琪, 孟蕊, 王梦婷, 等.萜烯醇对沙门菌的抗菌机制[J].中国兽医报, 2023, 43(08):1684-1689 [12]毛婉, 熊梦霞, 李湘, 等.高酶活枯草芽孢杆菌碱性磷酸酶phoA基因克隆表达及降解内毒素的应用[J].微生物学通报:1-15[2024-05-18]. [13]汪中兴, 侯永清, 窦茂鑫, 等.3种饲用植物精油及其主要成分对猪源致病菌的抑菌作用研究[J].饲料研究, 2014(13):42-45. [14]Soubeiga AP, Kpoda DS, Compaoré MKA, et al.Molecular Characterization and the Antimicrobial Resistance Profile of Salmonella spp. Isolated from Ready-to-Eat Foods in Ouagadougou, Burkina Faso[J]. International journal of microbiology.2022:9640828. [15]Schultz BM, Paduro CA, Salazar GA, et al.A Potential Role of Salmonella Infection in the Onset of Inflammatory Bowel Diseases[J]. Frontiers in immunology. 2017, 8:191. [16]Teshome, E., Forsido, S. F., Rupasinghe, H. P. V., et al. Potentials of Natural Preservatives to Enhance Food Safety and Shelf Life: A Review[J]. TheScientificWorldJournal, 2022, 9901018. [17]段旭彤, 李浩杨, 张嵘, 等.昆虫抗菌肽的研究进展[J].沈阳药科大学学报, 2023, 40(10):1401-1408 [18]孟素香, 曹健, 张慧茹, 等.绞股蓝内生真菌对金黄色葡萄球菌的抗菌机制[J].中国抗生素杂志, 2015, 40(04):268-273 [19]邢晨.雷公藤红素阳离子脂质体的制备及其对MRSA的抗菌作用研究[D].哈尔滨:东北农业大学, 2022. [20]杨智源, 金日天, 梁铎, 等.源自蜡样芽孢杆菌抗菌肽DB16的筛选及其对金黄色葡萄球菌的抑菌机制[J].食品科学, 1-15[2024-05-26]. [21]王铭遥, 郑明静, 任中阳, 等.凡纳滨对虾抗菌肽的筛选及其抑菌活性和DNA结合机制研究[J].中国食品学报:1-12[2024-05-26]. [22]Fernie AR, Carrari F, Sweetlove LJ.Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport[J]. Curr Opin Plant Biol. 2004, 7(3):254-61. [23]计红芳, 张令文, 宋瑞清.绒白乳菇发酵液提取物对杨树叶枯病菌几种重要酶活性的影响[J].林业大学学报, 2009, 31(04):51-54 [24]李海贤, 马艳玲, 曾荣.丁香酚对金黄色葡萄球菌和酶活力的影响[J].食品科技, 2018, 43(03):213-217 [25]康世墨.乳糖酸对耐甲氧西林金黄色葡萄球菌抑菌机理的研究[D].沈阳:沈阳农业大学, 2021. [26]Tian L, Wang X, Zhang D, et al.Evaluation of the membrane damage mechanism of protocatechualdehyde against Yersinia enterocolitica and simulation of growth inhibition in pork[J]. Food Chem. 2021; 363:130340. DOI:https://doi.org/10.1016/j.foodchem.2021.130340. [27]武淑娟.短小芽孢杆菌HN-10抗菌肽P-1对粉红单端孢的抑菌机理研究[D].兰州:甘肃农业大学, 2022. [28]张会会, 昝亚楠, 金明洁, 等.趋化因子端截短肽的抗菌活性及抗生物被膜作用的研究[J].中国预防兽医学报, 2022, 44(06):642-647