基于细胞膜损伤的蔗糖月桂酸酯对金黄色葡萄球菌的作用机制
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摘要
研究蔗糖月桂酸酯对金黄色葡萄球菌的抑菌活性及其对细胞膜的损伤机制。首先采用二倍稀释法考察了蔗糖月桂酸酯对金黄色葡萄球菌的最小抑菌浓度,用平板计数法绘制了时间-杀菌曲线;通过DiSC_3(5)探针标记荧光分光光度法考察了蔗糖月桂酸酯对菌体细胞膜电势差的影响,使用荧光显微镜结合流式细胞术研究了菌体细胞膜渗透性的影响,使用紫外分光光度法考察了大分子物质的泄漏情况,使用PBFI钾离子探针测定了胞内钾离子泄漏量,最后通过透射电子显微镜观察了蔗糖月桂酸酯处理前后菌体的超微结构。结果表明:蔗糖月桂酸酯对金黄色葡萄球菌的最小抑菌浓度(minimum inhibitory concentration,MIC)为0.312 5 mg/mL;经蔗糖月桂酸酯作用的金黄色葡萄球菌与对照组相比,Di SC_3(5)探针的荧光强度呈现剂量依赖性增大;PI探针对菌体的沾染率增加,MIC处理30 min菌体PI沾染率达到84.7%;260 nm波长处的吸光度随作用时间的延长逐渐增大,但增大量较小;蔗糖月桂酸酯会导致金黄色葡萄球菌细胞钾离子泄漏,且泄漏量与添加的蔗糖月桂酸酯质量浓度呈正相关;经MIC蔗糖月桂酸酯作用1 h后,金黄色葡萄球菌体表面变粗糙,边缘模糊。结论:蔗糖月桂酸酯可以通过破坏细胞膜渗透性消散细胞膜电势,从而导致胞内物质发生轻微泄漏,最终达到抑菌作用,本研究可为多功能性糖酯抑菌产品的开发提供理论依据。
The antibacterial activity of sucrose laurate against Staphylococcus aureus was determined, and the mechanism of cell membrane damage by sucrose laurate was investigated. The minimum inhibitory concentration(MIC) against S. aureus was determined by the two-fold dilution method, and the time-killing curve was assayed by the plate counting method. We studied the effect of sucrose laurate on the membrane potential of S. aureus was studied by fluorescence spectroscopy using DiSC_3(5) as the probe, the effect on the membrane permeability by fluorescence microscopy and flow cytometry, the effect on the leakage of macromolecular substances by ultraviolet spectrophotometry, the effect on the leakage of intracellular potassium ions using the fluorescent probe PBFI, and the effect on the ultrastructure by transmission electron microscopy.The results showed that the MIC of sucrose laurate against S. aureus was 0.312 5 mg/mL. Compared with the control group, the fluorescence intensity of S. aureus with the probe DiSC_3(5) increased in a concentration-dependent manner after treatment with sucrose laurate; The percentage of S. aureus stained with PI was 84.7% after the treatment of sucrose laurate at MIC for 30 min. The absorbance at 260 nm gradually increased with increasing treatment time at each concentration of sucrose laurate, but the increment was small. Sucrose laurate caused leakage of intracellular potassium ions and this effect was positively correlated with its concentration. After treatment with sucrose laurate at MIC for 1 h, the surface of bacterial cells became rough with blurred edges. Accordingly we concluded that sucrose laurate could dissipate cell membrane potential and destroy membrane permeability, leading to slight leakage of intracellular substances and consequently exerting antibacterial activity. This investigation can provide a theoretical basis for the development of versatile antibacterial products with glycolipids.
The antibacterial activity of sucrose laurate against Staphylococcus aureus was determined, and the mechanism of cell membrane damage by sucrose laurate was investigated. The minimum inhibitory concentration(MIC) against S. aureus was determined by the two-fold dilution method, and the time-killing curve was assayed by the plate counting method. We studied the effect of sucrose laurate on the membrane potential of S. aureus was studied by fluorescence spectroscopy using DiSC_3(5) as the probe, the effect on the membrane permeability by fluorescence microscopy and flow cytometry, the effect on the leakage of macromolecular substances by ultraviolet spectrophotometry, the effect on the leakage of intracellular potassium ions using the fluorescent probe PBFI, and the effect on the ultrastructure by transmission electron microscopy.The results showed that the MIC of sucrose laurate against S. aureus was 0.312 5 mg/mL. Compared with the control group, the fluorescence intensity of S. aureus with the probe DiSC_3(5) increased in a concentration-dependent manner after treatment with sucrose laurate; The percentage of S. aureus stained with PI was 84.7% after the treatment of sucrose laurate at MIC for 30 min. The absorbance at 260 nm gradually increased with increasing treatment time at each concentration of sucrose laurate, but the increment was small. Sucrose laurate caused leakage of intracellular potassium ions and this effect was positively correlated with its concentration. After treatment with sucrose laurate at MIC for 1 h, the surface of bacterial cells became rough with blurred edges. Accordingly we concluded that sucrose laurate could dissipate cell membrane potential and destroy membrane permeability, leading to slight leakage of intracellular substances and consequently exerting antibacterial activity. This investigation can provide a theoretical basis for the development of versatile antibacterial products with glycolipids.
引文
[1]SARAH D,LAURENCE D,NIA Y,et al.Food-borne outbreak investigation and molecular typing:high diversity of Staphylococcus aureus strains and importance of toxin detection[J].Toxins,2017,9(12):1-10.
[2]SCALLAN E,HOEKSTRA R M,ANGULO F J,et al.Foodborne illness acquired in the United States:major pathogens[J].Emerging Infectious Diseases,2011,17(1):7-15.DOI:10.3201/eid1701.P11101.
[3]STARO?J,DABROWSKI J M,CICHO?E,et al.Lactose esters:synthesis and biotechnological applications[J].Critical Reviews in Biotechnology,2018,38(2):245-258.DOI:10.1080/07388551.2017.1332571.
[4]CHENG J J,CUI J,MA Y,et al.Effects of soy-to-milk protein ratio and sucrose fatty acid ester addition on the stability of ice cream emulsions[J].Food Hydrocolloids,2016,60:425-436.DOI:10.1016/j.foodhyd.2016.04.002.
[5]CHOI S J,DECKER E A,HENSON L,et al.Formulation and properties of model beverage emulsions stabilized by sucrose monopalmitate:influence of pH and lyso-lecithin addition[J].Food Research International,2011,44(9):3006-3012.DOI:10.1016/j.foodres.2011.07.007.
[6]SANDRA G,HEIKE B.Preparation of lipid nanoemulsions by premix membrane emulsification with disposable materials[J].International Journal of Pharmaceutics,2016,511(2):741-744.DOI:10.1016/j.ijpharm.2016.07.067.
[7]CHANSANROJ K,BETZ G.Sucrose esters with various hydrophiliclipophilic properties:novel controlled release agents for oral drug delivery matrix tablets prepared by direct compaction[J].Acta Biomaterialia,2010,6(8):3101-3109.DOI:10.1016/j.actbio.2010.01.044.
[8]Markets and Markets.Sucrose esters market by application(food,detergents&cleaners,and personal care)form(powder,liquid,and pellet)&by region(North America,Europe,Asia-Pacific,Latin America,and rest of the world)-global trends&forecast to 2020[EB/OL].(2016-02-08)[2018-02-26].https://www.giiresearch.com/report/mama352126-sucrose-esters-market-by-application-food.html.
[9]PERINELLIA D R,LUCARINIB S,FAGIOLIB L,et al.Lactose oleate as new biocompatible surfactant for pharmaceutical applications[J].European Journal of Pharmaceutics and Biopharmaceutics,2018,124:55-62.DOI:10.1016/j.ejpb.2017.12.008.
[10]WAGH A,SHEN S J,SHEN F A,et al.Effect of lactose monolaurate on pathogenic and nonpathogenic bacteria[J].Applied and Environmental Microbiology,2012,78(9):3465-3468.DOI:10.1128/AEM.07701-11.
[11]DEVULAPALLE K S,GóMEZ D S A,FERRER M,et al.Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity[J].Carbohydrate Research,2004,339(6):1029-1034.DOI:10.1016/j.carres.2004.01.007.
[12]PPEUM L K,KWOUN K H.Antibacterial effect of fructose laurate synthesized by Candida antarctica B lipase-mediated transesterification[J].Journal of Microbiology and Biotechnology,2016,26(9):1579-1585.
[13]ZHAO Lei,ZHANG Heyan,HAO Tianyang,et al.In vitro antibacterial activities and mechanism of sugar fatty acid esters against five food-related bacteria[J].Food Chemistry,2015,187:370-377.DOI:10.1016/j.foodchem.2015.04.108.
[14]赵磊,张鹤龑,郝添阳,等.蔗糖癸酸酯对沙门氏菌的抗菌作用及其机理初探[J].现代食品科技,2016,32(5):46-51.DOI:10.13982/j.mfst.1673-9078.2016.5.08.
[15]曾哲灵,赵存洋,罗春燕,等.单癸月桂酸甘油酯的抑菌作用及机理研究[J].食品科学,2013,34(3):70-74.
[16]WIEGAND I,HILPERT K,HANCOCK R E W.Agar and broth dilution methods to determine the minimal inhibitory concentration(MIC)of antimicrobial substances[J].Nature Protocols,2008,3(2):163-175.DOI:10.1371/journal.pone.0045818.
[17]康联娣.生物电子显微技术[M].北京:中国科学技术大学出版社,2003:47-95.
[18]GARCIA-GONZALEZ L,GEERAERD A H,MAST J,et al.Membrane permeabilization and cellular death of Escherichia coli,Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment[J].Food Microbiology,2010,27(4):541-549.DOI:10.1016/j.fm.2009.12.004.
[19]WEEKS M E G,CARON N,JAMES D C,et al.Monitoring changes in nisin susceptibility of Listeria monocytogenes Scott a as an indicator of growth phase using FACS[J].Journal of Microbiological Methods,2006,66(1):43-55.DOI:10.1016/j.mimet.2005.10.008.
[20]LI Lirong,SHI Yonghui,SU Guanfang,et al.Selectivity for and destruction of Salmonella typhimurium via a membrane damage mechanism of a cell-penetrating peptide Pptg20 analogue[J].International Journal of Antimicrobial Agents,2012,40(4):337-343.DOI:10.1016/j.ijantimicag.2012.05.026.
[21]李瑞胜.乳酸菌素LLC518对单核增生李斯特氏菌作用机理的研究[D].合肥:安徽农业大学,2012:25.
[22]LIU G R,SONG Z Q,YANG X L,et al.Antibacterial mechanism of bifidocin A,a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04[J].Food Control,2016,62:309-316.DOI:10.1016/j.foodcont.2015.10.033.
[23]YANG Xu,HUANG En,YOUSEF A E.Brevibacillin,a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus[J].Microbiological Research,2017,195:18-23.DOI:10.1016/j.micres.2016.11.002.
[24]LYU Y F,YANG Y,LYU X T,et al.Antimicrobial activity,improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida[J].Scientific Reports,2016,6:1-7.DOI:10.1038/srep27258.
[25]胡静,赵小慧,朱春玉,等.槐糖脂对金黄色葡萄球菌的抑菌机理[J].食品科学,2012,33(5):33-36.
[26]ANARJAN N,TAN C.Effects of Selected polysorbate and sucrose ester emulsifiers on the physicochemical properties of astaxanthin nanodispersions[J].Molecules,2013,18(1):768-777.DOI:10.3390/molecules18010768.
[27]AMALINI A N,NORZIAH M H,KHAN I,et al.Exploring the properties of modified fish gelatin films incorporated with different fatty acid sucrose esters[J].Food Packaging and Shelf Life,2018,15:105-112.DOI:10.1016/j.fpsl.2017.12.003.
[28]NAKAMURA S,ISHII N,NAKASHIMA N,et al.Evaluation of sucrose fatty acid esters as lubricants in tablet manufacturing[J].Chemical and Pharmaceutical Bulletin,2017,65(5):432-441.DOI:10.1248/cpb.c16-00745.
[29]FURUKAWA S,AKIYOSHI Y,O’TOOLE G A,et al.Sugar fatty acid esters inhibit biofilm formation by food-borne pathogenic bacteria[J].International Journal of Food Microbiology,2010,138(1):176-180.DOI:10.1016/j.ijfoodmicro.2009.12.026.
[30]MAJA H,SASA S,ZELJKO K,et al.Enzymatic synthesis of sugar fatty acid esters in organic solvent and in supercritical carbon dioxide and their antimicrobial activity[J].Journal of Supercritical Fluids,2008,45(3):338-345.DOI:10.1016/j.supflu.2008.01.002.
[2]SCALLAN E,HOEKSTRA R M,ANGULO F J,et al.Foodborne illness acquired in the United States:major pathogens[J].Emerging Infectious Diseases,2011,17(1):7-15.DOI:10.3201/eid1701.P11101.
[3]STARO?J,DABROWSKI J M,CICHO?E,et al.Lactose esters:synthesis and biotechnological applications[J].Critical Reviews in Biotechnology,2018,38(2):245-258.DOI:10.1080/07388551.2017.1332571.
[4]CHENG J J,CUI J,MA Y,et al.Effects of soy-to-milk protein ratio and sucrose fatty acid ester addition on the stability of ice cream emulsions[J].Food Hydrocolloids,2016,60:425-436.DOI:10.1016/j.foodhyd.2016.04.002.
[5]CHOI S J,DECKER E A,HENSON L,et al.Formulation and properties of model beverage emulsions stabilized by sucrose monopalmitate:influence of pH and lyso-lecithin addition[J].Food Research International,2011,44(9):3006-3012.DOI:10.1016/j.foodres.2011.07.007.
[6]SANDRA G,HEIKE B.Preparation of lipid nanoemulsions by premix membrane emulsification with disposable materials[J].International Journal of Pharmaceutics,2016,511(2):741-744.DOI:10.1016/j.ijpharm.2016.07.067.
[7]CHANSANROJ K,BETZ G.Sucrose esters with various hydrophiliclipophilic properties:novel controlled release agents for oral drug delivery matrix tablets prepared by direct compaction[J].Acta Biomaterialia,2010,6(8):3101-3109.DOI:10.1016/j.actbio.2010.01.044.
[8]Markets and Markets.Sucrose esters market by application(food,detergents&cleaners,and personal care)form(powder,liquid,and pellet)&by region(North America,Europe,Asia-Pacific,Latin America,and rest of the world)-global trends&forecast to 2020[EB/OL].(2016-02-08)[2018-02-26].https://www.giiresearch.com/report/mama352126-sucrose-esters-market-by-application-food.html.
[9]PERINELLIA D R,LUCARINIB S,FAGIOLIB L,et al.Lactose oleate as new biocompatible surfactant for pharmaceutical applications[J].European Journal of Pharmaceutics and Biopharmaceutics,2018,124:55-62.DOI:10.1016/j.ejpb.2017.12.008.
[10]WAGH A,SHEN S J,SHEN F A,et al.Effect of lactose monolaurate on pathogenic and nonpathogenic bacteria[J].Applied and Environmental Microbiology,2012,78(9):3465-3468.DOI:10.1128/AEM.07701-11.
[11]DEVULAPALLE K S,GóMEZ D S A,FERRER M,et al.Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity[J].Carbohydrate Research,2004,339(6):1029-1034.DOI:10.1016/j.carres.2004.01.007.
[12]PPEUM L K,KWOUN K H.Antibacterial effect of fructose laurate synthesized by Candida antarctica B lipase-mediated transesterification[J].Journal of Microbiology and Biotechnology,2016,26(9):1579-1585.
[13]ZHAO Lei,ZHANG Heyan,HAO Tianyang,et al.In vitro antibacterial activities and mechanism of sugar fatty acid esters against five food-related bacteria[J].Food Chemistry,2015,187:370-377.DOI:10.1016/j.foodchem.2015.04.108.
[14]赵磊,张鹤龑,郝添阳,等.蔗糖癸酸酯对沙门氏菌的抗菌作用及其机理初探[J].现代食品科技,2016,32(5):46-51.DOI:10.13982/j.mfst.1673-9078.2016.5.08.
[15]曾哲灵,赵存洋,罗春燕,等.单癸月桂酸甘油酯的抑菌作用及机理研究[J].食品科学,2013,34(3):70-74.
[16]WIEGAND I,HILPERT K,HANCOCK R E W.Agar and broth dilution methods to determine the minimal inhibitory concentration(MIC)of antimicrobial substances[J].Nature Protocols,2008,3(2):163-175.DOI:10.1371/journal.pone.0045818.
[17]康联娣.生物电子显微技术[M].北京:中国科学技术大学出版社,2003:47-95.
[18]GARCIA-GONZALEZ L,GEERAERD A H,MAST J,et al.Membrane permeabilization and cellular death of Escherichia coli,Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment[J].Food Microbiology,2010,27(4):541-549.DOI:10.1016/j.fm.2009.12.004.
[19]WEEKS M E G,CARON N,JAMES D C,et al.Monitoring changes in nisin susceptibility of Listeria monocytogenes Scott a as an indicator of growth phase using FACS[J].Journal of Microbiological Methods,2006,66(1):43-55.DOI:10.1016/j.mimet.2005.10.008.
[20]LI Lirong,SHI Yonghui,SU Guanfang,et al.Selectivity for and destruction of Salmonella typhimurium via a membrane damage mechanism of a cell-penetrating peptide Pptg20 analogue[J].International Journal of Antimicrobial Agents,2012,40(4):337-343.DOI:10.1016/j.ijantimicag.2012.05.026.
[21]李瑞胜.乳酸菌素LLC518对单核增生李斯特氏菌作用机理的研究[D].合肥:安徽农业大学,2012:25.
[22]LIU G R,SONG Z Q,YANG X L,et al.Antibacterial mechanism of bifidocin A,a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04[J].Food Control,2016,62:309-316.DOI:10.1016/j.foodcont.2015.10.033.
[23]YANG Xu,HUANG En,YOUSEF A E.Brevibacillin,a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus[J].Microbiological Research,2017,195:18-23.DOI:10.1016/j.micres.2016.11.002.
[24]LYU Y F,YANG Y,LYU X T,et al.Antimicrobial activity,improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida[J].Scientific Reports,2016,6:1-7.DOI:10.1038/srep27258.
[25]胡静,赵小慧,朱春玉,等.槐糖脂对金黄色葡萄球菌的抑菌机理[J].食品科学,2012,33(5):33-36.
[26]ANARJAN N,TAN C.Effects of Selected polysorbate and sucrose ester emulsifiers on the physicochemical properties of astaxanthin nanodispersions[J].Molecules,2013,18(1):768-777.DOI:10.3390/molecules18010768.
[27]AMALINI A N,NORZIAH M H,KHAN I,et al.Exploring the properties of modified fish gelatin films incorporated with different fatty acid sucrose esters[J].Food Packaging and Shelf Life,2018,15:105-112.DOI:10.1016/j.fpsl.2017.12.003.
[28]NAKAMURA S,ISHII N,NAKASHIMA N,et al.Evaluation of sucrose fatty acid esters as lubricants in tablet manufacturing[J].Chemical and Pharmaceutical Bulletin,2017,65(5):432-441.DOI:10.1248/cpb.c16-00745.
[29]FURUKAWA S,AKIYOSHI Y,O’TOOLE G A,et al.Sugar fatty acid esters inhibit biofilm formation by food-borne pathogenic bacteria[J].International Journal of Food Microbiology,2010,138(1):176-180.DOI:10.1016/j.ijfoodmicro.2009.12.026.
[30]MAJA H,SASA S,ZELJKO K,et al.Enzymatic synthesis of sugar fatty acid esters in organic solvent and in supercritical carbon dioxide and their antimicrobial activity[J].Journal of Supercritical Fluids,2008,45(3):338-345.DOI:10.1016/j.supflu.2008.01.002.