ε-聚赖氨酸对阪崎克罗诺杆菌细胞结构与生物被膜形成的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0396

摘要/Abstract

摘要：

ε-聚赖氨酸（ε-polylysine,ε-PL）作为一种抗菌活性强、安全性高、热稳定性好的阳离子型天然多肽,已作为安全的食品防腐剂受到关注。以阪崎克罗诺杆菌（Cronobacter sakazakii）为供试菌株,揭示ε-PL的抑菌机理,为细菌耐药性和食品防腐措施研究提供理论支持。研究了ε-PL作用下对阪崎克罗诺杆菌的最小抑菌浓度（minimum inhibitory concentration,MIC）、细胞膜壁通透性、细胞表面疏水性及运动性等生理特性的影响,并利用透射电子显微镜对ε-PL作用下的细菌细胞形态变化进行了观察,在此基础上,探究了ε-PL对阪崎克罗诺杆菌生物被膜（biofilm,BF）的抑制和清除作用效果,并利用荧光染色显微观察清除后被膜菌通透性的改变。ε-PL对阪崎克罗诺杆菌的抑菌活性具有浓度依赖性,ε-PL对阪崎克罗诺杆菌ATCC51329的MIC为256 μg/mL。ε-PL能够增强细胞膜壁通透性,使其细胞内容物如核酸、碱性磷酸酶等大量渗出,从而表现对阪崎克罗诺杆菌的杀菌作用。同时ε-PL能够降低阪崎克罗诺杆菌的表面疏水性和运动性,进而影响阪崎克罗诺杆菌生物被膜的形成。ε-PL对阪崎克罗诺杆菌生物被膜抑制和清除的作用效果显著,结合物理振荡,可大大提高生物被膜清除效率。ε-PL能够破坏阪崎克罗诺杆菌的细胞结构,从而达到抑菌效果。ε-PL能够降低阪崎克罗诺杆菌细胞表面的疏水性、运动性,进而ε-PL能够抑制生物被膜的形成,对成熟生物被膜也具有清除作用。

关键词: ε-聚赖氨酸, 阪崎克罗诺杆菌, 细胞结构, 抑菌, 生物被膜

Abstract:

ε-polylysine（ε-PL）,as a cationic natural peptide with strong antibacterial activity,high safety and good thermal stability,has been applied as a food preservative. Having Cronobacter sakazakii as the test strain,the bacteriostatic mechanism of ε-PL was revealed,which may provide theoretical support for the study of bacterial drug resistance and food preservative measures. The effects of ε-PL on the physiological characteristics of C. sakazakii,such as the minimum inhibitory concentration（MIC）,membrane wall permeability,cell surface hydrophobicity and motility,were studied. The morphological changes of C. sakazakii cells treated with ε-PL were observed by transmission electron microscope,and the scavenging and inhibitory effects on the biofilm（BF）of C. sakazakii were investigated,and the permeability of the cell in the biofilm was also observed by fluorescence staining. ε-PL had positively concentration-dependent antibacterial activity against C. sakazakii,the MIC of ε-PL to C.sakazakii ATCC51329 was 256 μg/mL. Physiological studies on bacteria have found that ε-PL enhanced the permeability of cell membrane wall,and transuded cell contents such as nucleic acid and alkaline phosphatase,thus presented the bactericidal effect on C.sakazakii. Meanwhile,ε-PL reduced the surface hydrophobicity and motility of C. sakazakii,and then affected the formation of the biofilm. ε-PL demonstrated a significant effect on the inhibition and eradication of biofilm,and the removal efficiency of biofilm can be greatly improved when combined with physical oscillation. ε-PL destroyed the cell structure of C. sakazakii and the bacteriostatic effect was achieved. By reducing the cell surface hydrophobicity and motility of C. sakazakii,ε -PL also inhibited the formation of biofilm,and removed the mature biofilm.

Key words: ε-polylysine, C. sakazakii, cell structure, bacteriostasis, biofilm

石成龙, 汪锡武, 李安琪, 钱森和, 王洲, 赵世光, 刘艳, 薛正莲. ε-聚赖氨酸对阪崎克罗诺杆菌细胞结构与生物被膜形成的影响[J]. 生物技术通报, 2022, 38(9): 147-157.

SHI Cheng-long, WANG Xi-wu, LI An-qi, QIAN Sen-he, WANG Zhou, ZHAO Shi-guang, LIU Yan, XUE Zheng-lian. Effect of ε-Polylysine on the Cell Structure and Biofilm Formation of Cronobacter sakazakii[J]. Biotechnology Bulletin, 2022, 38(9): 147-157.

图/表 8

图1 ε-PL对阪崎克罗诺杆菌的抑菌评价 A：最小抑菌浓度测定;B：生长曲线。图中误差线表示标准偏差,*表示与CK组相比差异显著（P<0.05）,下同

Fig. 1 Bacteriostatic evaluation of ε-PL against C. sakazakii A：Determination of minimum inhibitory concentration; B：growth curve. The error line in the figure refers to the standard deviation,* indicates that there is significant difference compared with CK group（P<0.05）,the same below

图2 ε-PL对阪崎克罗诺杆菌膜壁特性的影响 A：核酸紫外吸收;B：碱性磷酸酶浓度

Fig. 2 Effect of ε-PL on membrane wall properties of C. sakazakii A：Nucleic acid UV absorption. B：AKP concentration

图3 ε-PL处理后阪崎克罗诺杆菌的透射电镜图像（×15.0 k） A：无药物处理对照组;B：1×MIC药物浓度处理组;C：2 ×MIC药物浓度处理组

Fig. 3 Transmission electron microscope image of C. saka-zakii after ε-PL treatment（×15.0 k） A：Control group without treatment. B：1×MIC treatment group. C：2×MIC treatment group

图4 ε-PL对阪崎克罗诺杆菌表面疏水性的影响

Fig. 4 Effect of ε-PL on the surface hydrophobicity of C. sakazakii

图5 ε-PL对阪崎克罗诺杆菌泳动性的影响

Fig. 5 Effect of ε-PL on the motility of C. sakazakii

图6 ε-PL对阪崎克罗诺杆菌生物被膜形成量的影响

Fig. 6 Effect of ε-PL on the amount of C. sakazakii biofilm formation

图7 ε-PL对阪崎克罗诺杆菌生物被膜清除率的影响 A：静置处理;B：振荡处理

Fig. 7 Effect of ε-PL on the biofilm clearance of C. sakazakii A：Static treatment. B：Shaking treatment

图8 不同处理方式对生物被膜菌存活的影响（×200） A：静置20 h的对照组;B：静置20 h的1×MIC组;C：静置20 h的2×MIC组;D：振荡3 h的对照组;E：振荡3 h的1×MIC组;F：振荡3 h的2×MIC组

Fig. 8 Effects of different treatment methods on the living of biofilms（×200） A：Control group with 20 h rest. B：1×MIC group with 20 h rest. C：2×MIC group with 20 h rest. D：Control group with oscillation for 3 h. E：1×MIC group with oscillation for 3 h. F：2×MIC group with oscillation for 3 h

参考文献 41

[1]	柴阿丽, 韩云, 武军, 等. 基于FDA-PI双荧光复染法的茄病镰刀菌孢子活性检测[J]. 中国农业科学, 2015, 48(14):2757-2766.
	Chai AL, Han Y, Wu J, et al. Determination of spore viability of Fusarium solani based on dual fluorescence assay[J]. Sci Agric Sin, 2015, 48(14):2757-2766.
[2]	Norberg S, Stanton C, Ross RP, et al. Cronobacter spp. in powdered infant formula[J]. J Food Prot, 2012, 75(3):607-620. doi: 10.4315/0362-028X.JFP-11-285 URL
[3]	Yan QQ, Condell O, Power K, et al. Cronobacter species(formerly known as Enterobacter sakazakii)in powdered infant formula:a review of our current understanding of the biology of this bacterium[J]. J Appl Microbiol, 2012, 113(1):1-15. doi: 10.1111/j.1365-2672.2012.05281.x pmid: 22420458
[4]	Fang RY, Wang QN, Yang BW, et al. Prevalence and subtyping of Cronobacter species in goat milk powder factories in Shaanxi Province, China[J]. J Dairy Sci, 2015, 98(11):7552-7559. doi: 10.3168/jds.2015-9661 URL
[5]	Kragh KN, Hutchison JB, Melaugh G, et al. Role of multicellular aggregates in biofilm formation[J]. mBio, 2016, 7(2):e00237.
[6]	齐子琦, 秦子晋, 黄永震, 等. 生物防腐剂ε-聚赖氨酸的研究进展[J]. 食品工业, 2019, 40(10):289-293.
	Qi ZQ, Qin ZJ, Huang YZ, et al. Research progress of ε-polylysine as a biological preservative[J]. Food Ind, 2019, 40(10):289-293.
[7]	Hiraki J, Ichikawa T, Ninomiya SI, et al. Use of ADME studies to confirm the safety of Epsilon-polylysine as a preservative in food[J]. Regul Toxicol Pharmacol, 2003, 37(2):328-340. doi: 10.1016/S0273-2300(03)00029-1 URL
[8]	You XM, Einson JE, Lopez-Pena CL, et al. Food-grade cationic antimicrobial ε-polylysine transiently alters the gut microbial community and predicted metagenome function in CD-1 mice[J]. Npj Sci Food, 2017, 1:8. doi: 10.1038/s41538-017-0006-0 pmid: 31304250
[9]	卢绪志, 王伟, 金维忠. 天然生物防腐剂在肉制品中的应用[J]. 肉类工业, 2020(6):46-49.
	Lu XZ, Wang W, Jin WZ. Application of natural biological preservatives in meat products[J]. Meat Ind, 2020(6):46-49.
[10]	巴良杰, 罗冬兰, 吉宁, 等. 生物保鲜纸对李子贮藏期品质的影响[J]. 食品与机械, 2020, 36(7):140-143, 226.
	Ba LJ, Luo DL, Ji N, et al. Effect of biological preservative paper on the storage quality of plum fruit[J]. Food Mach, 2020, 36(7):140-143, 226.
[11]	Badaoui Najjar M, Kashtanov D, Chikindas ML. Epsilon-poly-L-lysine and nisin A act synergistically against Gram-positive food-borne pathogens Bacillus cereus and Listeria monocytogenes[J]. Lett Appl Microbiol, 2007, 45(1):13-18. doi: 10.1111/j.1472-765X.2007.02157.x pmid: 17594454
[12]	Mizan MFR, Ashrafudoulla M, Hossain MI, et al. Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains[J]. Biofouling, 2020, 36(4):467-478. doi: 10.1080/08927014.2020.1772243 URL
[13]	郑丽平, 陆兆新, 孔梁宇, 等. 鼠伤寒沙门氏菌生物被膜的清除效果研究[J]. 食品工业科技, 2021, 42(22):140-152.
	Zheng LP, Lu ZX, Kong LY, et al. Study on the removal effect of Salmonella typhimurium biofilm[J]. Sci Technol Food Ind, 2021, 42(22):140-152.
[14]	鲍佳佳, 李倩, 孙铭艳, 等. ε-聚赖氨酸对铜绿假单胞菌生长及生物膜形成的影响[J]. 中国病原生物学杂志, 2021, 16(3):258-260, 265.
	Bao JJ, Li Q, Sun MY, et al. The effect of ε-polylysine on the growth of and biofilm formation by Pseudomonas aeruginosa[J]. J Pathog Biol, 2021, 16(3):258-260, 265.
[15]	Shen CK, Islam MT, Masuda Y, et al. Transcriptional changes involved in inhibition of biofilm formation by ε-polylysine in Salmonella Typhimurium[J]. Appl Microbiol Biotechnol, 2020, 104(12):5427-5436. doi: 10.1007/s00253-020-10575-2 URL
[16]	Ning HQ, Lin H, Wang JX. Synergistic effects of endolysin Lysqdvp001 and ε-poly-lysine in controlling Vibrio parahaemolyticus and its biofilms[J]. Int J Food Microbiol, 2021, 343:109112. doi: 10.1016/j.ijfoodmicro.2021.109112 URL
[17]	陈晓青, 李可可, 余甜, 等. ε-聚赖氨酸对金黄色葡萄球菌生长及生物膜形成的影响[J]. 中国抗生素杂志, 2018, 43(1):91-95.
	Chen XQ, Li KK, Yu T, et al. Effects of ε-PL on growth and biofilm formation of Staphylococcus aureus[J]. Chin J Antibiot, 2018, 43(1):91-95.
[18]	Long M, Wang J, Zhuang H, et al. Performance and mechanism of standard nano-TiO₂(P-25)in photocatalytic disinfection of foodborne microorganisms - Salmonella typhimurium and Listeria monocytogenes[J]. Food Control, 2014, 39:68-74. doi: 10.1016/j.foodcont.2013.10.033 URL
[19]	Liu KW, Zhou XJ, Fu MR. Inhibiting effects of Epsilon-poly-lysine(ε-PL)on Pencillium digitatum and its involved mechanism[J]. Postharvest Biol Technol, 2017, 123:94-101. doi: 10.1016/j.postharvbio.2016.08.015 URL
[20]	Huang JF, Yang LY, Zou Y, et al. Antibacterial activity and mechanism of three isomeric terpineols of Cinnamomum longepaniculatum leaf oil[J]. Folia Microbiol(Praha), 2021, 66(1):59-67.
[21]	Lan WQ, Zhang NN, Liu SC, et al. Ε-polylysine inhibits Shewanella putrefaciens with membrane disruption and cell damage[J]. Molecules, 2019, 24(20):3727. doi: 10.3390/molecules24203727 URL
[22]	王梓源, 李欣颖, 吕俊阁, 等. ε-聚赖氨酸对大肠杆菌的抑菌机制[J]. 食品与发酵工业, 2020, 46(21):34-41.
	Wang ZY, Li XY, Lyu JG, et al. The antimicrobial mechanism of ε-poly-L-lysine against Escherichia coli[J]. Food Ferment Ind, 2020, 46(21):34-41.
[23]	吴晨奇, 高以任, 宋京城, 等. ε-聚赖氨酸在肉制品保鲜中的应用[J]. 食品安全导刊, 2021(33):183-185, 189.
	Wu CQ, Gao YR, Song JC, et al. Application of ε-polylysine in preservation of meat products[J]. China Food Saf Mag, 2021(33):183-185, 189.
[24]	赵昇, 吴学妍, 张硕, 等. ε-聚赖氨酸对重要食源性致病菌的作用效果研究进展[J]. 食品与发酵工业, 2021. DOI: 10.13995/j. cnki. 11-1802/ts. 029852. doi: 10.13995/j. cnki. 11-1802/ts. 029852
	Zhao S, Wu XY, Zhang S, et al. Research progress on the effect of ε-polylysine on important foodborne pathogens[J]. Food Ferment Ind, 2021. DOI: 10.13995/j. cnki. 11-1802/ts. 029852. doi: 10.13995/j. cnki. 11-1802/ts. 029852
[25]	Hyldgaard M, Mygind T, Vad BS, et al. The antimicrobial mechanism of action of Epsilon-poly-l-lysine[J]. Appl Environ Microbiol, 2014, 80(24):7758-7770. doi: 10.1128/AEM.02204-14 URL
[26]	Gupta P, Sarkar S, Das B, et al. Biofilm, pathogenesis and prevention-a journey to break the wall:a review[J]. Arch Microbiol, 2016, 198(1):1-15. doi: 10.1007/s00203-015-1148-6 URL
[27]	Bowler PG. Antibiotic resistance and biofilm tolerance:a combined threat in the treatment of chronic infections[J]. J Wound Care, 2018, 27(5):273-277. doi: 10.12968/jowc.2018.27.5.273 URL
[28]	李芬, 范玉堂, 王丽娟, 等. 牛乳源大肠杆菌生物被膜形成及抗生素的作用[J]. 西北农业学报, 2020, 29(7):983-989.
	Li F, Fan YT, Wang LJ, et al. Biofilm formation and antibiotics resistance of Escherichia coli isolated from milk[J]. Acta Agric Boreali Occidentalis Sin, 2020, 29(7):983-989.
[29]	Damrongsaktrakul P, Ruengvisesh S, Rahothan A, et al. Removal of Salmonella typhimurium biofilm from food contact surfaces using Quercus infectoria gall extract in combination with a surfactant[J]. J Microbiol Biotechnol, 2021, 31(3):439-446. doi: 10.4014/jmb.2101.01014 URL
[30]	Packiavathy IASV, Priya S, Pandian SK, et al. Inhibition of biofilm development of uropathogens by curcumin - an anti-quorum sensing agent from Curcuma longa[J]. Food Chem, 2014, 148:453-460. doi: 10.1016/j.foodchem.2012.08.002 pmid: 24262582
[31]	杨昆, 王欢, 高洁, 等. 抗菌肽BCp12对大肠杆菌壁膜及DNA损伤的作用机制[J]. 食品科学, 2021, 42(19):114-121.
	Yang K, Wang H, Gao J, et al. Mechanism by which antimicrobial peptide BCp12 acts on the cell wall and membrane of Escherichia coli cells and induces DNA damage[J]. Food Sci, 2021, 42(19):114-121.
[32]	景春娥, 李萍, 杜欣军, 等. 黄芩苷对阪崎克罗诺杆菌生物膜的抑制作用[J]. 微生物学通报, 2016, 43(8):1774-1784.
	Jing CE, Li P, Du XJ, et al. Inhibition of Cronobacter sakazaki biofilms by baicalin[J]. Microbiol China, 2016, 43(8):1774-1784.
[33]	郭都, 赵宇阳, 王瑞霞, 等. 硫辛酸对阪崎克罗诺杆菌感染能力的抑制作用[J]. 食品科学, 2020, 41(17):188-195.
	Guo D, Zhao YY, Wang RX, et al. Anti-infective activity of lipoic acid against Cronobacter sakazakii[J]. Food Sci, 2020, 41(17):188-195.
[34]	Topa SH, Subramoni S, Palombo EA, et al. Cinnamaldehyde disrupts biofilm formation and swarming motility of Pseudomonas aeruginosa[J]. Microbiology, 2018, 164(9):1087-1097. doi: 10.1099/mic.0.000692 URL
[35]	Robertson J, McGoverin C, White JR, et al. Rapid detection of Escherichia coli antibiotic susceptibility using live/dead spectrometry for lytic agents[J]. Microorganisms, 2021, 9(5):924. doi: 10.3390/microorganisms9050924 URL
[36]	Huang ZW, Kuang X, Chen ZX, et al. Comparative studies of tri- and hexavalent chromium cytotoxicity and their effects on oxidative state of Saccharomyces cerevisiae cells[J]. Curr Microbiol, 2014, 68(4):448-456. doi: 10.1007/s00284-013-0496-1 URL
[37]	刘志恬, 董玉鹏, 李永才, 等. ε-聚赖氨酸对梨果实黑斑病菌Alternaria alternata的抑制作用及其机理[J]. 食品科学, 2021, 42(11):213-220.
	Liu ZT, Dong YP, Li YC, et al. Antifungal effect of ε-polylysine on Alternaria alternata isolated from pears with black spot and its possible mechanism[J]. Food Sci, 2021, 42(11):213-220.
[38]	顾玉卿, 殷成芮, 潘凤好, 等. 冬凌草甲素纳米脂质体对嗜水气单胞菌生物膜的抑制活性研究[J]. 食品研究与开发, 2022, 43(5):21-27.
	Gu YQ, Yin CR, Pan FH, et al. Inhibitory activity of oridonin nanoliposomes against Aeromonas hydrophila biofilm[J]. Food Res Dev, 2022, 43(5):21-27.
[39]	袁帅, 王秀红, 白春美, 等. ε-聚赖氨酸在果蔬采后贮藏保鲜中的应用研究进展[J]. 食品研究与开发, 2021, 42(17):196-203.
	Yuan S, Wang XH, Bai CM, et al. Recent progress in the application of ε-polylysine in postharvest storage and preservation of fruits and vegetables[J]. Food Res Dev, 2021, 42(17):196-203.
[40]	蔡芝玲, 莫梓童, 郑诗倩, 等. 黄绵马酸BB联合红霉素抑制表皮葡萄球菌及其生物被膜的形成研究[J]. 中草药, 2022, 53(8):2417-2427.
	Cai ZL, Mo ZT, Zheng SQ, et al. Antibacterial and antibiofilm effects of flavaspidic acid BB combined with erythromycin on Staphylococcus epidermidis[J]. Chin Tradit Herb Drugs, 2022, 53(8):2417-2427.
[41]	Li AQ, Shi CL, Qian SH, et al. Evaluation of antibiotic combination of Litsea cubeba essential oil on Vibrio parahaemolyticus inhibition mechanism and anti-biofilm ability[J]. Microb Pathog, 2022, 168:105574. doi: 10.1016/j.micpath.2022.105574 URL