ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)是一种具有广谱抑菌性的聚阳离子多肽,目前已作为生物防腐剂被广泛应用。为揭示ε-PL的抑菌机理,以大肠杆菌(Escherichia coli)为模式菌株,研究了ε-PL作用下E. coli的生长曲线、存活率以及ε-PL对E. coli细胞表面疏水性、内外膜穿透活性的影响,并且利用扫描电子显微镜观察了E. coli细胞形态在ε-PL作用下的变化,探究了ε-PL处理后E. coli聚团黏连现象。结果表明,ε-PL对E. coli的抑菌活性具有浓度依赖性,与ε-PL浓度呈正相关,当ε-PL质量浓度达到100 μg/mL时,有明显抑菌效果。研究还发现,ε-PL能够增强E. coli细胞表面疏水性及细胞内、外膜的通透性,并且改变E. coli细胞膜内外电势,使其细胞内容物如核酸、蛋白质等大量渗出,从而实现对E. coli的杀菌作用。基于上述实验结果推测,ε-PL可能是通过毡毯模型中描述的作用模式将E. coli细胞杀死。
ε-Poly-L-lysine (ε-PL), a poly cationic peptide with broad-spectrum antimicrobial activity, has been widely used as a natural preservative. In order to reveal the antimicrobial mechanism of ε-PL, its antibacterial properties against Escherichia coli were investigated. and the effects of ε-PL on the morphology, the growth curve, survival rate, cell-surface hydrophobicity, inner and outer membrane penetrating activity were studied. Results showed that ε-PL inhibited the growth of E. coli in a dose-dependent manner and the inhibition effect was positively correlated with ε-PL concentration, 100 μg/mL of ε-PL achieved significant antibacterial effect. In addition, ε-PL enhanced the hydrophobicity and permeability of E. coli membrane which led to the leakage of nucleic acid, protein and electrolyte and finally the cell death. The holes and micelles appeared on the cell membrane of E. coli and the cells became aggregative and adhesive after ε-PL treatment and was further confirmed by the dynamic light scattering. In conclusion, ε-PL may achieve the bacteriostatic effect by destroying the cell structure of E. coli and the antibacterial mechanism of ε-PL against E. coli is similar to the way described by the carpet model. These findings give insights for better application of ε-PL in the field of food preservation, and also have a reference value for the study of bacteriostatic mechanism of ε-PL against other bacteria.
[1] LIN L,GU Y L,LI C Z,et al.Antibacterial mechanism of epsilon-poly-lysine against Listeria monocytogenes and its application on cheese[J].Food Control,2018,91:76-84.
[2] 吴光耀, 陈旭升,王靓,等.核糖体工程技术选育ε-聚赖氨酸高产菌株[J].微生物学通报,2016,43(12):2 744-2 751.
[3] 李南臻, 王刚,万玉军,等.ε-聚-L-赖氨酸高产菌株诱变选育[J].食品与发酵科技,2018,54(6):1-4.
[4] 王军华, 王易芬,裘纪莹,等.ε-聚赖氨酸对牡蛎的防腐抗菌效果[J].食品工业科技,2019,40(24):270-275.
[5] 刘璐, 鲁晓翔,陈绍慧,等.ε-聚赖氨酸采后处理对樱桃冰温贮藏期间品质的影响[J].食品工业科技,2015,36(12):319-323.
[6] BO T,LIU M,ZHONG C,et al.Metabolomic analysis of antimicrobial mechanisms of ε-poly-L-lysine on Saccharomyces cerevisiae [J].Agriculture Food Chemistry,2014,62(19):4 454-4 465.
[7] BO T,HAN P P,SU Q Z,et al.Antimicrobial ε-poly-L-lysine induced changes in cell membrane compositions and properties of Saccharomyces cerevisiae [J].Food Control,2016,61:123-134.
[8] 程雅文. ε-聚-L-赖氨酸诱导酿酒酵母活性氧偶联的细胞凋亡[D].天津:天津科技大学,2017.
[9] YANG X Q,WANG H,HE A N,et al.Microbial efficacy and impact on the population of Escherichia coli of a routine sanitation process for the fabrication facility of a beef packing plant[J].Food Control,2017,71:353-357.
[10] RANDALL L P,LODGE M P,ELVISS N C,et al.Evaluation of meat,fruit and vegetables from retail stores in five united kingdom regions as sources of extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant Escherichia coli [J].International Journal of Food Microbiology,2017,241:283-290.
[11] YANG S R,PEI X Y,WANG G,et al.Prevalence of food-borne pathogens in ready-to-eat meat products in seven different chinese regions[J].Food Control,2016,65:92-98.
[12] SKOCKOVA A,KOLACKOVA I,BOGDANOVICOVA K,et al.Characteristic and antimicrobial resistance in Escherichia coli from retail meats purchased in the czech republic[J].Food Control,2015,47:401-406.
[13] ZHANG X W,SHI C,LIU F G,et al.Antibacterial activity and mode of action of ε-polylysine against Escherichia coli O157∶H7[J].Journal of Medical Microbiology,2018,67(6):838-845.
[14] 施庆珊, 陈仪本,欧阳友生.ε-聚赖氨酸的抑菌特性及应用前景[J].食品与发酵工业,2005(6):76-79.
[15] MUKHERJEE R M,MAITRA T K,HALDAR D P,et al.Adherence of entamoeba histolytica to hydrophobic matrices:a simple method for measuring cell surface hydrophobicity[J].Transactions of the Royal Society of Tropical Medicine and Hygiene,1993,87(4):492-493.
[16] KONG M,CHEN X G,LIU C S,et al.Antibacterial mechanism of chitosan microspheres in a solid dispersing system against E.coli colloids and surfaces B[J].Colloids and Surfaces B-Biointerfaces,2008,65(2):197-202.
[17] JE J Y,KIM S K.Antimicrobial action of novel chitin derivative[J].Biochim Biophys Acta,2006,1760(1):104-109.
[18] SHIMADA S,ANDOU M,NAITO N,et al.Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae [J].Applied Microbiology and Biotechnology,1993,40(1):123-131.
[19] 薄涛, 杨萍,宋萌,等.ε-聚-L-赖氨酸对酿酒酵母抑菌机制的初步研究[J].中国食品添加剂,2015(6):53-59.
[20] 何静如, 刘雪,陈文学,等.(-)-β-蒎烯对沙门氏菌的抑菌机制[J].食品科学,2019,40(1):44-49.
[21] 蒋佳佳, 卢小菊,孟鸳,等.氧化石墨烯纳米银复合材料的制备及对大肠杆菌抑菌性能的研究[J].化工新型材料,2019,47(12):121-126.
[22] 马鹏, 胡晓清,陈久洲,等.大肠杆菌细胞外膜渗透性与脂多糖结构的关系[J].微生物学通报,2011,38(8):1 307-1 315.
[23] LONG M,WANG J M,ZHUANG H.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.
[24] SONG R,WEI R B,LUO H Y,et al.Isolation and characterization of an antibacterial peptide fraction from the pepsin hydrolysate of half-fin anchovy (Setipinna taty)[J].Molecules,2012,17(3):2 980-2 991.
[25] BREUKINK E,KRUIJFF B D.The lantibiotic nisin,a special case or not?[J].Biochimica et Biophysica Acta,1999,1462(1-2):223-234.
[26] POUNY Y,RAPAPORT D,MOR A,et al.Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes[J].Biochemistry,1992,31(49):12 416-12 423.
[27] LADOKHIN A S,WHITE S H.‘Detergent-like' permeabilization of anionic lipid vesicles by melittin[J].Biochimica et Biophysica Acta,2001,1514(2):253-260.
[28] SHAI Y.Mechanism of the binding,insertion and destabilization of phospholipid bilayer membranes by alphahelical antimicrobial and cell non-selective membrane-lytic peptides[J].Biochimica et Biophysica Acta,1999,1462(1-2):55-70.
[29] 何国庆, 贾英民,丁立孝.食品微生物学(第2版) [M].北京:中国农业大学出版社,2009:20-21.
[30] HYLDGAARD M,MYGIND T,VAD B S,et al.The antimicrobial mechanism of action of epsilon-poly-l-lysine[J].Applied and Environmental Microbiology,2014,80(24):7 758-7 770.
[31] VAARA M,VAARA T.Polycations as outer membrane-disorganizing agents[J].Antimicrobial Agents and Chemotherapy,1983,24(1):114-122.
