绿色合成纳米银材料的制备及其对水产病原菌的抗菌效果测定
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摘要
近年来随着水产养殖规模不断扩大,渔药安全问题备受重视。为积极寻找抗生素替代品,本研究采用银杏叶提取物和硝酸银反应合成纳米银材料,并通过紫外-可见光全波段扫描、透射电镜以及X射线衍射对其结构表征进行鉴定。在此基础上,以金黄色葡萄球菌、大肠杆菌,以及水产病原菌迟缓爱德华氏菌和嗜水气单胞菌为测试对象,采用杯碟法、常量肉汤稀释法以及抑菌动力学实验测定合成的纳米银抗菌效果。结果显示,在波长450 nm处有纳米银等离子共振体形成的吸收峰;透射电镜观察到纳米银颗粒平均直径小于10 nm;X射线衍射图谱验证了金属银的生成;本研究所合成的纳米银材料对4种病原菌均有良好的抗菌效果。研究表明,采用银杏叶绿色合成纳米银,不仅方便、快捷,而且成本低廉、环境友好,在水产病害防控中具有良好的前景。
With the development of aquaculture, far more attention has been paid to safety in fish drugs usage. In order to find alternatives to antibiotics, silver nanoparticles(AgNPs) were synthesized using extracts of Ginkgo leaves with AgNO_3 in this study. The synthesized silver nanoparticles were characterized by UV-Visible spectrophotometer, transmission electron microscope and X ray diffraction spectrum. The antibacterial efficacy was assessed against Staphylococcus aurous, Escherichia coli and common bacterial pathogens in aquacuture including Edwardsiella tarda and Aeromonas hydrophila, by using cylinder plate method, broth macrodilution method and bacteriostasis kinetic experiments. The results showed that the absorbance maximum was observed at460 nm corresponding to the plasmon absorbance of the AgNPs. Transmission electron microscope revealed that the AgNPs had an average size below 10 nm.The X-Ray Diffraction(XRD) spectrum of the nanoparticles confirmed the formation of metallic silver.The AgNPs synthesised in this research exhibited good antibacterial efficacy against the test pathogens. This investigation demonstrated that the green synthesis of AgNps with leaves of Ginkgo biloba was not only fast and convenient but also cost effective and ecofriendly, which has good prospects in control of aquaculture diseases.
With the development of aquaculture, far more attention has been paid to safety in fish drugs usage. In order to find alternatives to antibiotics, silver nanoparticles(AgNPs) were synthesized using extracts of Ginkgo leaves with AgNO_3 in this study. The synthesized silver nanoparticles were characterized by UV-Visible spectrophotometer, transmission electron microscope and X ray diffraction spectrum. The antibacterial efficacy was assessed against Staphylococcus aurous, Escherichia coli and common bacterial pathogens in aquacuture including Edwardsiella tarda and Aeromonas hydrophila, by using cylinder plate method, broth macrodilution method and bacteriostasis kinetic experiments. The results showed that the absorbance maximum was observed at460 nm corresponding to the plasmon absorbance of the AgNPs. Transmission electron microscope revealed that the AgNPs had an average size below 10 nm.The X-Ray Diffraction(XRD) spectrum of the nanoparticles confirmed the formation of metallic silver.The AgNPs synthesised in this research exhibited good antibacterial efficacy against the test pathogens. This investigation demonstrated that the green synthesis of AgNps with leaves of Ginkgo biloba was not only fast and convenient but also cost effective and ecofriendly, which has good prospects in control of aquaculture diseases.
引文
[1]曾涛.水产养殖中几种抗生素替代物的应用[J].北京农业,2012,(21):95.Zeng T.Aquaculture several antibiotics in the application of substitutes[J].Beijing Agriculture,2012,(21):95(in Chinese).
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[3]Kim J S,Kuk E,Yu K N,et al.Antimicrobial effects of silver nanoparticles[J].Nanomedicine:Nanotechnology,Biology and Medicine,2007,3(1):95-101.
[4]El-Sheekh M M,El-Kassas H Y.Application of biosynthesized silver nanoparticles against a cancer promoter cyanobacterium,Microcystis aeruginosa[J].Asian Pacific Journal of Cancer Prevention,2014,15(16):6773-6779.
[5]汪江节.纳米改性植物灭螺剂的制备及其灭螺增效性能的研究[D].合肥:安徽大学,2011.Wang J J.Study on preparation of nano-modified plant molluscicide and its molluscicidal synergistic properties[D].Hefei:Anhui University,2011(in Chinese).
[6]Kowalska-góralska M,Senze M,Polechoński R,et al.Biocidal properties of silver-nanoparticles in water environments[J].Polish Journal of Environmental Studies,2015,24(4):1641-1647.
[7]王权,王茜.制备银纳米线专利技术分析[J].信息记录材料,2016,17(5):62-65.Wang Q,Wang X.Patent analysis of the synthesis of silver nanowires[J].Information Recording Materials,2016,17(5):62-65(in Chinese).
[8]Iravani S,Korbekandi H,Mirmohammadi S,et al.Synthesis of silver nanoparticles:chemical,physical and biological methods[J].Research in Pharmaceutical Sciences,2014,9(6):385-406.
[9]Ahmed S,Ahmad M,Swami B L,et al.A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications:a green expertise[J].Journal of Advanced Research,2016,7(1):17-28.
[10]王盟盟,钱君超,张玉珠,等.生物模板法制取微纳米多孔材料[J].材料导报,2013,27(S2):197-200,204.Wang M M,Qian J C,Zhang Y Z,et al.Synthesis of micro-nano hollow materials by biological templates[J].Materials Review,2013,27(S2):197-200,204(in Chinese).
[11]Murugesan S,Elumalai M,Dhamotharan R.Green synthesis of silver nano particles from marine alga Gracillaria edulis[J].Bioscience Biotechnology Research Communications,2011,4(1):105-110.
[12]Mahanty A,Mishra S,Bosu R,et al.Phytoextractssynthesized silver nanoparticles inhibit bacterial fish pathogen Aeromonas hydrophila[J].Indian Journal of Microbiology,2013,53(4):438-446.
[13]李焰,杨小燕,林跃鑫,等.银杏叶有效成分提取与抑菌效果研究[J].中国畜牧杂志,2006,42(15):54-56.Li Y,Yang X Y,Lin Y X,et al.Study on extraction and antibacterial effects of active ingredients in leaves of Ginkgo biloba[J].Chinese Journal of Animal Science,2006,42(15):54-56(in Chinese).
[14]曾献,龚玉子,王焕姣.银杏叶的药理作用[J].湖南林业科技,2008,35(1):6-8.Zeng X,Gong Y Z,Wang H J.Pharmacological action of Ginkgo biloba leaves[J].Hunan Forestry Science&Technology,2008,35(1):6-8(in Chinese).
[15]韦琮智,丛新奇,叶淑红.银杏多糖的抑菌性研究[C]//中国食品科学技术学会第十三届年会论文集.北京:中国食品科学技术学会,2016:169-170.Wei C Z,Cong X Q,Ye S H.Research on the bacteriostatic activities of polysaccharides from leaves of Ginkgo biloba and endophytic bacterium[C]//Abstracts of the13th Annual Meeting of Chinese Institute of Food Science and Technology.Beijing:Chinese Institute of Food Science and Technology,2016:169-170(in Chinese).
[16]张庆红,王洪星,谷洪燕.银杏叶化学成分及提取分离研究概况[C]//山东省药学会第一届学术年会论文集(上).济南:山东省药学会,2005:364-367.Zhang Q H,Wang H X,Gu H Y.Review of chemical composition anlysis and extraction techniques for Ginkgo biloba leaves[C]//Proceedings of the 1st Annual Meeting of Shandong Pharmaceutical Association.Ji'nan:Shandong Pharmaceutical Association,2005:364 -367(in Chinese).
[17]Ahmed K,Ahmed N,Siddiqui M T,et al.Green synthesis of silver nano particles by plant leaf extract[J].Fuuast Journal of Biology,2016,6(1):61-64.
[18]Song J Y,Kim B S.Rapid biological synthesis of silver nanoparticles using plant leaf extracts[J].Bioprocess and Biosystems Engineering,2009,32(1):79-84.
[19]孙彩华,何佳奇,余朝辉.不同形态纳米银的制备及其杀菌性能的研究[J].中国医药导报,2015,12(34):15-19.Sun C H,He J Q,Yu C H.Study of different forms of nanometer silver preparation and bactericidal performance[J].China Medical Herald,2015,12(34):15-19(in Chinese).
[20]熊旭华.不同形态纳米银的制备及其杀菌性能研究[D].广州:华南理工大学,2014.Xiong X H.Preparation of silver nanostructures with different shapes and its antibacterial activity[D].Guangzhou:South China University of Technology,2014(in Chinese).
[21]姜宇,李福艳,刘冲冲,等.山楂提取物生物合成纳米银对四种常见水产病原菌的抑制作用[J].海洋与湖沼,2016,47(1):253-260.Jiang Y,Li F Y,Liu C C,et al.Biosynthezied silver nanopaticles using hawthorn fruit extract and their antibacterial activity against four common aquatic pathogens[J].Oceanologia et Limnologia Sinica,2016,47(1):253-260(in Chinese).
[22]Jena J,Pradhana N,Dash B P,et al.Biosynthesis and characterization of silver nanoparticles using microalga Chlorococcum humicola and its antibacterial activity[J].International Journal of Nanomaterials and Biostructures,2013,3(1):1-8.
[23]谢小保,李文茹,曾海燕,等.纳米银对大肠杆菌的抗菌作用及其机制[J].材料工程,2008(10):106-109.Xie X B,Li W R,Zeng H Y,et al.Study of antimicrobial activity and mechanism of silver nanoparticles on Escherichia coli[J].Journal of Materials Engineering,2008(10):106-109(in Chinese).
[24]魏殿军,王剑飞,于晓旭.纳米银抗菌机制的研究[C]//第六届中国临床微生物学大会暨微生物学与免疫学论坛论文汇编.长沙:中国微生物学会临床微生物学专业委员会,2015:252.Wei D J,Wang J F,Yu X X.Antibacterial mechanism of silver nanoparticles on bacteria[C]//Proceedings of Microbiology and Immunology Symposium in the 6th China Congress of Clinical Microbiology.Changsha:Clinical Microbiology Division of Chinese Society for Microbiology,2015:252(in Chinese).
[25]段晓杰,杜晓丹,张蓓蓓.纳米银对金黄色葡萄球菌的抗菌作用及其机制研究[J].生物医学工程与临床,2015,19(3):237-240.Duan X J,Du X D,Zhang B B.Antibacterial effect of silver nanoparticles on Staphylococcus aureus and the underlying mechanism[J].Biomedical Engineering and Clinical Medicine,2015,19(3):237-240(in Chinese).
[26]Velmurugan P,Iydroose M,Lee SM,et al.Synthesis of silver and gold nanoparticles using cashew nut shell liquid and its antibacterial activity against fish pathogens[J].Indian Journal of Microbiology,2014,54(2):196-202.
[27]El-Sheekh M M,El-Kassas H Y.Algal production of nano-silver and gold:their antimicrobial and cytotoxic activities:a review[J].Journal of Genetic Engineering and Biotechnology,2016,14(2):299-310.
[28]谢小保,李文茹,袁英姿,等.纳米银对金黄色葡萄球菌的抑制及其作用机制[C]//雪莲杯第10届功能性纺织品及纳米技术应用研讨会论文集.常州:北京纺织工程学会,2010:520-524.Xie X B,Li W R,Yuan Y Z,et al.Study of the antimicrobial effect and mechanism of silver nanoparticles on Staphylococcus aureus[C]//Proceedings of the10th Symposium of Functional Textiles and Nanotechnology Applications.Changzhou:Beijing Textile Engineering Institute,2010:520-524(in Chinese).
[29]Mathew P P,Thankachen N,Abraham E.Green synthesis and applications of silver nano particles[J].European Journal of Pharmaceutical and Medical Research,2016,3(5):233-240.
[30]Sarkar B,Mahanty A,Netam S P,et al.Inhibitory role of silver nanoparticles against important fish pathogen,Aeromonas hydrophila[J].International Journal of Nanomaterials and Biostructures,2012,2(4):70-74.
[2]姚晓东.抗生素在水产养殖中应用存在的问题及对策[J].农业与技术,2016,36(24):103.Yao X D.Problems and solutions of antibiotics used in aquaculture[J].Agriculture and Technology,2016,36(24):103(in Chinese).
[3]Kim J S,Kuk E,Yu K N,et al.Antimicrobial effects of silver nanoparticles[J].Nanomedicine:Nanotechnology,Biology and Medicine,2007,3(1):95-101.
[4]El-Sheekh M M,El-Kassas H Y.Application of biosynthesized silver nanoparticles against a cancer promoter cyanobacterium,Microcystis aeruginosa[J].Asian Pacific Journal of Cancer Prevention,2014,15(16):6773-6779.
[5]汪江节.纳米改性植物灭螺剂的制备及其灭螺增效性能的研究[D].合肥:安徽大学,2011.Wang J J.Study on preparation of nano-modified plant molluscicide and its molluscicidal synergistic properties[D].Hefei:Anhui University,2011(in Chinese).
[6]Kowalska-góralska M,Senze M,Polechoński R,et al.Biocidal properties of silver-nanoparticles in water environments[J].Polish Journal of Environmental Studies,2015,24(4):1641-1647.
[7]王权,王茜.制备银纳米线专利技术分析[J].信息记录材料,2016,17(5):62-65.Wang Q,Wang X.Patent analysis of the synthesis of silver nanowires[J].Information Recording Materials,2016,17(5):62-65(in Chinese).
[8]Iravani S,Korbekandi H,Mirmohammadi S,et al.Synthesis of silver nanoparticles:chemical,physical and biological methods[J].Research in Pharmaceutical Sciences,2014,9(6):385-406.
[9]Ahmed S,Ahmad M,Swami B L,et al.A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications:a green expertise[J].Journal of Advanced Research,2016,7(1):17-28.
[10]王盟盟,钱君超,张玉珠,等.生物模板法制取微纳米多孔材料[J].材料导报,2013,27(S2):197-200,204.Wang M M,Qian J C,Zhang Y Z,et al.Synthesis of micro-nano hollow materials by biological templates[J].Materials Review,2013,27(S2):197-200,204(in Chinese).
[11]Murugesan S,Elumalai M,Dhamotharan R.Green synthesis of silver nano particles from marine alga Gracillaria edulis[J].Bioscience Biotechnology Research Communications,2011,4(1):105-110.
[12]Mahanty A,Mishra S,Bosu R,et al.Phytoextractssynthesized silver nanoparticles inhibit bacterial fish pathogen Aeromonas hydrophila[J].Indian Journal of Microbiology,2013,53(4):438-446.
[13]李焰,杨小燕,林跃鑫,等.银杏叶有效成分提取与抑菌效果研究[J].中国畜牧杂志,2006,42(15):54-56.Li Y,Yang X Y,Lin Y X,et al.Study on extraction and antibacterial effects of active ingredients in leaves of Ginkgo biloba[J].Chinese Journal of Animal Science,2006,42(15):54-56(in Chinese).
[14]曾献,龚玉子,王焕姣.银杏叶的药理作用[J].湖南林业科技,2008,35(1):6-8.Zeng X,Gong Y Z,Wang H J.Pharmacological action of Ginkgo biloba leaves[J].Hunan Forestry Science&Technology,2008,35(1):6-8(in Chinese).
[15]韦琮智,丛新奇,叶淑红.银杏多糖的抑菌性研究[C]//中国食品科学技术学会第十三届年会论文集.北京:中国食品科学技术学会,2016:169-170.Wei C Z,Cong X Q,Ye S H.Research on the bacteriostatic activities of polysaccharides from leaves of Ginkgo biloba and endophytic bacterium[C]//Abstracts of the13th Annual Meeting of Chinese Institute of Food Science and Technology.Beijing:Chinese Institute of Food Science and Technology,2016:169-170(in Chinese).
[16]张庆红,王洪星,谷洪燕.银杏叶化学成分及提取分离研究概况[C]//山东省药学会第一届学术年会论文集(上).济南:山东省药学会,2005:364-367.Zhang Q H,Wang H X,Gu H Y.Review of chemical composition anlysis and extraction techniques for Ginkgo biloba leaves[C]//Proceedings of the 1st Annual Meeting of Shandong Pharmaceutical Association.Ji'nan:Shandong Pharmaceutical Association,2005:364 -367(in Chinese).
[17]Ahmed K,Ahmed N,Siddiqui M T,et al.Green synthesis of silver nano particles by plant leaf extract[J].Fuuast Journal of Biology,2016,6(1):61-64.
[18]Song J Y,Kim B S.Rapid biological synthesis of silver nanoparticles using plant leaf extracts[J].Bioprocess and Biosystems Engineering,2009,32(1):79-84.
[19]孙彩华,何佳奇,余朝辉.不同形态纳米银的制备及其杀菌性能的研究[J].中国医药导报,2015,12(34):15-19.Sun C H,He J Q,Yu C H.Study of different forms of nanometer silver preparation and bactericidal performance[J].China Medical Herald,2015,12(34):15-19(in Chinese).
[20]熊旭华.不同形态纳米银的制备及其杀菌性能研究[D].广州:华南理工大学,2014.Xiong X H.Preparation of silver nanostructures with different shapes and its antibacterial activity[D].Guangzhou:South China University of Technology,2014(in Chinese).
[21]姜宇,李福艳,刘冲冲,等.山楂提取物生物合成纳米银对四种常见水产病原菌的抑制作用[J].海洋与湖沼,2016,47(1):253-260.Jiang Y,Li F Y,Liu C C,et al.Biosynthezied silver nanopaticles using hawthorn fruit extract and their antibacterial activity against four common aquatic pathogens[J].Oceanologia et Limnologia Sinica,2016,47(1):253-260(in Chinese).
[22]Jena J,Pradhana N,Dash B P,et al.Biosynthesis and characterization of silver nanoparticles using microalga Chlorococcum humicola and its antibacterial activity[J].International Journal of Nanomaterials and Biostructures,2013,3(1):1-8.
[23]谢小保,李文茹,曾海燕,等.纳米银对大肠杆菌的抗菌作用及其机制[J].材料工程,2008(10):106-109.Xie X B,Li W R,Zeng H Y,et al.Study of antimicrobial activity and mechanism of silver nanoparticles on Escherichia coli[J].Journal of Materials Engineering,2008(10):106-109(in Chinese).
[24]魏殿军,王剑飞,于晓旭.纳米银抗菌机制的研究[C]//第六届中国临床微生物学大会暨微生物学与免疫学论坛论文汇编.长沙:中国微生物学会临床微生物学专业委员会,2015:252.Wei D J,Wang J F,Yu X X.Antibacterial mechanism of silver nanoparticles on bacteria[C]//Proceedings of Microbiology and Immunology Symposium in the 6th China Congress of Clinical Microbiology.Changsha:Clinical Microbiology Division of Chinese Society for Microbiology,2015:252(in Chinese).
[25]段晓杰,杜晓丹,张蓓蓓.纳米银对金黄色葡萄球菌的抗菌作用及其机制研究[J].生物医学工程与临床,2015,19(3):237-240.Duan X J,Du X D,Zhang B B.Antibacterial effect of silver nanoparticles on Staphylococcus aureus and the underlying mechanism[J].Biomedical Engineering and Clinical Medicine,2015,19(3):237-240(in Chinese).
[26]Velmurugan P,Iydroose M,Lee SM,et al.Synthesis of silver and gold nanoparticles using cashew nut shell liquid and its antibacterial activity against fish pathogens[J].Indian Journal of Microbiology,2014,54(2):196-202.
[27]El-Sheekh M M,El-Kassas H Y.Algal production of nano-silver and gold:their antimicrobial and cytotoxic activities:a review[J].Journal of Genetic Engineering and Biotechnology,2016,14(2):299-310.
[28]谢小保,李文茹,袁英姿,等.纳米银对金黄色葡萄球菌的抑制及其作用机制[C]//雪莲杯第10届功能性纺织品及纳米技术应用研讨会论文集.常州:北京纺织工程学会,2010:520-524.Xie X B,Li W R,Yuan Y Z,et al.Study of the antimicrobial effect and mechanism of silver nanoparticles on Staphylococcus aureus[C]//Proceedings of the10th Symposium of Functional Textiles and Nanotechnology Applications.Changzhou:Beijing Textile Engineering Institute,2010:520-524(in Chinese).
[29]Mathew P P,Thankachen N,Abraham E.Green synthesis and applications of silver nano particles[J].European Journal of Pharmaceutical and Medical Research,2016,3(5):233-240.
[30]Sarkar B,Mahanty A,Netam S P,et al.Inhibitory role of silver nanoparticles against important fish pathogen,Aeromonas hydrophila[J].International Journal of Nanomaterials and Biostructures,2012,2(4):70-74.