富马酸钠对荧光假单胞菌群体感应现象及其腐败活性的抑制作用
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摘要
以报告菌株紫色杆菌CV026为检测模型,探究富马酸钠对荧光假单胞菌群体感应现象及其腐败特性的影响。采用酶标法及光学显微镜观察富马酸钠对荧光假单胞菌生物被膜的抑制效果,利用气相色谱-质谱分析富马酸钠对荧光假单胞菌分泌信号分子的影响。结果表明:在亚抑菌浓度下,富马酸钠可以显著降低紫色杆菌CV026紫色菌素的产生量(P<0.05);当质量浓度为2.0 mg/mL时,富马酸钠对紫色菌素的抑制率达50.95%,且不影响CV026菌株的生长。富马酸钠能有效抑制荧光假单胞菌生物被膜形成、群集与泳动、胞外蛋白酶活性等相关腐败特性,且质量浓度与其抑制作用呈正相关。另外,由气相色谱-质谱检测结果可知,富马酸钠对荧光假单胞菌N-酰基高丝氨酸内酯信号分子的分泌有较好的抑制效果。因此,富马酸钠具有较好的群体感应抑制活性,可作为新型群体感应抑制剂用于水产品贮藏保鲜。
In this paper, Chromobacterium violaceum CV026 strain was used as a test model to investigate the effect of sodium fumarate on quorum sensing and spoilage capacity of Pseudomonas fluorescens. The inhibitory effect of sodium fumarate on biofilm formation was evaluated with a microplate reader and optical microscopy, and its effect on the secretion of signal molecules in Pseudomonas fluorescens was determined by gas chromatography-mass spectrometry(GC-MS). The results showed that sodium fumarate could significantly reduce the production of violacein in strain CV026 at sub-minimum inhibitory concentrations. Violacein production was inhibited 50.95% by sodium fumarate at 2.0 mg/mL while not affecting the growth of CV026. Sodium fumarate could effectively inhibit spoilage characteristics of Pseudomonas fluorescens as reflected by biofilm formation, swarming and swimming motility, and extracellular protease activity and this effect was positively correlated with its concentration. By GC-MS analysis, we also found that sodium fumarate significantly inhibited the secretion of the signal molecules N-acylhomoserine lactones(AHLs). Therefore,sodium fumarate has great inhibitory activity and can be developed as a quorum sensing inhibitor for refrigerated storage and preservation of aquatic products.
In this paper, Chromobacterium violaceum CV026 strain was used as a test model to investigate the effect of sodium fumarate on quorum sensing and spoilage capacity of Pseudomonas fluorescens. The inhibitory effect of sodium fumarate on biofilm formation was evaluated with a microplate reader and optical microscopy, and its effect on the secretion of signal molecules in Pseudomonas fluorescens was determined by gas chromatography-mass spectrometry(GC-MS). The results showed that sodium fumarate could significantly reduce the production of violacein in strain CV026 at sub-minimum inhibitory concentrations. Violacein production was inhibited 50.95% by sodium fumarate at 2.0 mg/mL while not affecting the growth of CV026. Sodium fumarate could effectively inhibit spoilage characteristics of Pseudomonas fluorescens as reflected by biofilm formation, swarming and swimming motility, and extracellular protease activity and this effect was positively correlated with its concentration. By GC-MS analysis, we also found that sodium fumarate significantly inhibited the secretion of the signal molecules N-acylhomoserine lactones(AHLs). Therefore,sodium fumarate has great inhibitory activity and can be developed as a quorum sensing inhibitor for refrigerated storage and preservation of aquatic products.
引文
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[19]PONNUSWAMY V.A simple method for the detection of protease activity on agar plates using bromocresolgreen dye[J].Journal of Biochemical Technology,2013,4(3):628-630.
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[28]PRATT L A,KOLTER R.Genetic analysis of Escherichia coli biofilm formation:roles of flagella,motility,chemotaxis and type I pili[J].Molecular Microbiology,1998,30(2):285-293.DOI:10.1046/j.1365-2958.1998.01061.x.
[29]LI T T,WANG D F,LIU N,et al.Inhibition of quorum sensing-controlled virulence factors and biofilm formation in Pseudomonas fluorescens,by cinnamaldehyde[J].International Journal of Food Microbiology,2018,269:98-106.DOI:10.1016/j.ijfoodmicro.2018.01.023.
[30]LOU Zaixiang,WANG Hongxin,TANG Yuxia.The effect of burdock leaf fraction on adhesion,biofilm formation,quorum sensing and virulence factors of P.aeruginosa[J].Journal of Applied Microbiology,2017,122(3):615-624.DOI:10.1111/jam.13348.
[31]MYSZKA K,SCHMIDT M T,MAJCHER M,et al.Inhibition of quorum sensing-related biofilm of Pseudomonas fluorescens,KM121by Thymus vulgare,essential oil and its major bioactive compounds[J].International Biodeterioration&Biodegradation,2016,114:252-259.
[32]崔方超,李婷婷,刘明爽,等.大菱鲆荧光假单胞菌的群体感应现象及不同碳源培养下的腐败特性研究[J].现代食品科技,2015,31(12):49-55.DOI:10.13982/j.mfst.1673-9078.2015.12.008.
[33]LOKENDER K,SANJAY C,RAJNISH K,et al.Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa[J].Fitoterapia,2015,102:84-95.DOI:10.1016/j.fitote.2015.02.002.
[2]SOLANO C,ECHEVERZ M,LASA I.Biofilm dispersion and quorum sensing[J].Current Opinion in Microbiology,2014,18(4):96-104.DOI:10.1016/j.mib.2014.02.008.
[3]崔方超,李婷婷,刘明爽,等.大菱鲆荧光假单胞菌的群体感应现象及不同碳源培养下的腐败特性研究[J].现代食品科技,2015,31(12):49-55.DOI:10.13982/j.mfst.1673-9078.2015.12.008.
[4]AMMOR M S,MICHAELIDIS C,NYCHAS G E.Insights into the role of quorum sensing in food spoilage[J].Journal of Food Protection,2008,71(7):1510-1525.DOI:10.4315/0362-028X-71.7.1510.
[5]BANERJEE G,RAY A K.The talking language in some major Gramnegative bacteria[J].Archives of Microbiology,2016,198(6):489-499.DOI:10.1007/s00203-016-1220-x.
[6]王鸿,平夏婷,陈苏,等.革兰氏阴性菌群体感应抑制剂的研究进展[J].浙江化工,2016,47(11):15-21.DOI:10.3969/j.issn.1006-4184.2016.11.005.
[7]CHEN F,GAO Y X,CHEN X Y,et al.Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection[J].International Journal of Molecular Sciences,2013,14(9):17477-17500.DOI:10.3390/ijms140917477.
[8]DU Y F,LI T,WAN Y F,et al.Signal molecule-dependent quorumsensing and quorum-quenching enzymes in bacteria[J].Critical Reviews in Eukaryotic Gene Expression,2014,24(2):117-132.DOI:10.1615/CritRevEukaryotGeneExpr.2014008034.
[9]KALIA V C,PUROHIT H J.Quenching the quorum sensing system:potential antibacterial drug targets[J].Critical Reviews in Microbiology,2011,37(2):121-140.DOI:10.3109/1040841X.2010.532479.
[10]王锦利,周爱莲,林其洋,等.阿魏植物精油对紫色杆菌群体感应的影响[J].现代食品科技,2016,32(10):90-95.DOI:10.13982/j.mfst.1673-9078.2016.10.015.
[11]CHOW S,GU K,JIANG L,et al.Salicylic acid affects swimming,twitching and swarming motility in Pseudomonas aeruginosa,resulting in decreased biofilm formation[J].Mccarthy,2011,15:22-29.
[12]周秀琴.日本开发防止鱼贝类罐头食品黑变的添加剂[J].水产科技情报,2001,28(6):267.DOI:10.3969/j.issn.1001-1994.2001.06.027.
[13]肖尔卿,潘见,孟飞龙,等.富马酸钠协同超高压抑制冻藏草莓果汁中H2S研究[J].食品工业科技,2014,35(17):80-83.DOI:10.13386/j.issn1002-0306.2014.17.008.
[14]孙磊.腌制蔬菜中复合防腐剂的开发和应用研究[D].合肥:合肥工业大学,2010:28-29.DOI:10.7666/d.y1700498.
[15]DIAO Wenrui,ZHANG Liangliang,FENG Saisai,et al.Chemical composition,antibacterial activity,and mechanism of action of the essential oil from Amomum kravanh[J].Journal of Food Protection,2014,77(10):1740-1746.DOI:10.4315/0362-028X.JFP-14-014.
[16]KATO N,TANAKA T,NAKAGAWA S,et al.Control of virulence factor expression in opportunistic pathogens using cyclodextrin immobilized gel[J].Journal of Inclusion Phenomena&Macrocyclic Chemistry,2007,57(1/2/3/4):419-423.DOI:10.1007/s10847-006-9228-5.
[17]RODE T,LANGSRUD S,HOLCK A,et al.Different patterns of biofilm formation in Staphylococcus aureus under food-related stress conditions[J].International Journal of Food Microbiology,2007,116(3):372-383.DOI:10.1016/j.ijfoodmicro.2007.02.017.
[18]ZHANG Juanmei,RUI Xin,WANG Li,et al.Polyphenolic extract from Rosa rugosa tea inhibits bacterial quorum sensing and biofilm formation[J].Food Control,2014,42(2):125-131.DOI:10.1016/j.foodcont.2014.02.001.
[19]PONNUSWAMY V.A simple method for the detection of protease activity on agar plates using bromocresolgreen dye[J].Journal of Biochemical Technology,2013,4(3):628-630.
[20]赵爱飞,黄旭镇,叶晓锋,等.气相色谱-质谱定量检测水产品腐败菌群体感应DKPs信号分子[J].微生物学通报,2016,43(2):343-350.DOI:10.13344/j.microbiol.china.150385.
[23]BLANCHETTE K A,PRABHAKARA R,SHIRTLIFF M E,et al.Inhibition of fracture healing in the presence of contamination by Staphylococcus aureus:effects of growth state and immune response[J].Journal of Orthopaedic Research,2017,35(9):1845-1854.DOI:10.1002/jor.23573.
[22]MA L Y,WANG J,WANG S W,et al.Synthesis of multiple Pseudomonas aeruginosa biofilm matrix exopolysaccharides is posttranscriptionally regulated[J].Environmental Microbiology,2012,14(8):1995-2005.DOI:10.1111/j.1462-2920.2012.02753.x.
[23]HOUDT R V,MICHIELS C W.Biofilm formation and the food industry,a focus on the bacterial outer surface[J].Journal of Applied Microbiology,2010,109(4):1117-1131.DOI:10.1111/j.1365-2672.2010.04756.x.
[24]YUNOS N Y M,TAN W S,KOH C L,et al.Pseudomonas cremoricolorata strain ND07 produces N-acyl homoserine lactones as quorum sensing molecules[J].Sensors,2014,14(7):11595-11604.DOI:10.3390/s140711595.
[25]ZHANG Ying,KONG Jie,XIE Yunfei,et al.Essential oil components inhibit biofilm formation in Erwinia carotovora,and Pseudomonas fluorescens,via anti-quorum sensing activity[J].LWT-Food Science and Technology,2018,92:133-139.DOI:10.1016/j.lwt.2018.02.027.
[26]SWIECICKI J M,SLIUSARENKO O,WEIBEL D B.From swimming to swarming:Escherichia coli cell motility in two-dimensions[J].Integrative Biology,2013,5(12):1490-1494.DOI:10.1039/c3ib40130h.
[27]SERVANT A,QIU F,MAZZA M,et al.Controlled in vivo swimming of a swarm of bacteria-like microrobotic flagella[J].Advanced Materials,2015,27(19):2981-2988.DOI:10.1002/adma.201404444.
[28]PRATT L A,KOLTER R.Genetic analysis of Escherichia coli biofilm formation:roles of flagella,motility,chemotaxis and type I pili[J].Molecular Microbiology,1998,30(2):285-293.DOI:10.1046/j.1365-2958.1998.01061.x.
[29]LI T T,WANG D F,LIU N,et al.Inhibition of quorum sensing-controlled virulence factors and biofilm formation in Pseudomonas fluorescens,by cinnamaldehyde[J].International Journal of Food Microbiology,2018,269:98-106.DOI:10.1016/j.ijfoodmicro.2018.01.023.
[30]LOU Zaixiang,WANG Hongxin,TANG Yuxia.The effect of burdock leaf fraction on adhesion,biofilm formation,quorum sensing and virulence factors of P.aeruginosa[J].Journal of Applied Microbiology,2017,122(3):615-624.DOI:10.1111/jam.13348.
[31]MYSZKA K,SCHMIDT M T,MAJCHER M,et al.Inhibition of quorum sensing-related biofilm of Pseudomonas fluorescens,KM121by Thymus vulgare,essential oil and its major bioactive compounds[J].International Biodeterioration&Biodegradation,2016,114:252-259.
[32]崔方超,李婷婷,刘明爽,等.大菱鲆荧光假单胞菌的群体感应现象及不同碳源培养下的腐败特性研究[J].现代食品科技,2015,31(12):49-55.DOI:10.13982/j.mfst.1673-9078.2015.12.008.
[33]LOKENDER K,SANJAY C,RAJNISH K,et al.Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa[J].Fitoterapia,2015,102:84-95.DOI:10.1016/j.fitote.2015.02.002.
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