五谷虫蛋白粗提液对牛源致病性Escherichia coli O_1和E.coli O_(78)体外抑菌作用
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摘要
为研究五谷虫蛋白粗提液对Escherichia coli O_1和E.coli O_(78)的体外抑菌作用,采用试剂盒测定五谷虫粗提液蛋白含量;牛津杯法测定蛋白粗提液对牛源病源性E.coli O_1和E.coli O_(78)的抑菌圈直径;通过二倍稀释法测定蛋白粗提液对2种细菌的最低抑菌浓度(MIC);平板法测定最低杀菌浓度(MBC);酶标比浊法测定粗提液对2种细菌的24h生长曲线、细胞膜通透性以及碱性磷酸酶(AKP)的含量。研究表明:1)五谷虫蛋白粗提液蛋白质量浓度为0.680mg/mL;粗提液对E.coli O_1和E.coli O_(78)的抑菌圈直径分别为(25.09±0.62)和(20.96±0.48)mm,MIC分别为15.625和31.250mg/mL,MBC为62.5和125mg/mL,传统中药水煎剂和提取缓冲液没有体外抑菌效果。2)粗提液能影响E.coli O_1和E.coli O_(78)的生长曲线,增加细菌细胞膜和细胞壁通透性。由此可见,蛋白粗提液对E.coli O_1和E.coli O_(78)均有体外抑菌效果,且对E.coli O_1的体外抑菌效果优于E.coli O_(78)。
To study the antibacterial mechanism of crude protein extract,which is aquired from Chinese medicine,on bovine pathogenic Escherichia coli O_1 and E.coli O_(78).The content of crude protein extract from maggot was determined by enhanced BCA Protein Assay Kit.The diameters of inhibition zone on bovine E.coli O_1 and E.coli O_(78) were determined by Oxford cup method;The MIC of crude protein extract for two bacteria was determined by two dilution methods.The MBC was determined by plate method.To investigate the antibacterial effects of crude protein extract to against the two bacteria,turbidimetry was adopted to measure 24 h growth curve,cell membrane permeability and the content of AKP.The result showed that:1)The concentration of crude protein extract from Maggot was 0.680mg/mL;The diameters of inhibition zone of E.coli O_1 and E.coli O_(78) were(25.09±0.62)and(20.96±0.48)mm respectively;Their MICs were 15.625 and 31.250mg/mL respectively;MBCs were 62.5and 125mg/mL;The traditional Chinese medicine decoction and the extracting buffer didn't display any antibacterial effects in vitro;2)The crude extract effected the growth curve of both E.coli O_1 and E.coli O_(78),also increased the permeability of cellmembrane and cell wall.In conclusion,the crude protein extract of maggot have antibacterial effects on both E.coli O_1 and E.coli O_(78),and the antibacterial effects in vitro on E.coli O_1 is better than E.coli O_(78).
To study the antibacterial mechanism of crude protein extract,which is aquired from Chinese medicine,on bovine pathogenic Escherichia coli O_1 and E.coli O_(78).The content of crude protein extract from maggot was determined by enhanced BCA Protein Assay Kit.The diameters of inhibition zone on bovine E.coli O_1 and E.coli O_(78) were determined by Oxford cup method;The MIC of crude protein extract for two bacteria was determined by two dilution methods.The MBC was determined by plate method.To investigate the antibacterial effects of crude protein extract to against the two bacteria,turbidimetry was adopted to measure 24 h growth curve,cell membrane permeability and the content of AKP.The result showed that:1)The concentration of crude protein extract from Maggot was 0.680mg/mL;The diameters of inhibition zone of E.coli O_1 and E.coli O_(78) were(25.09±0.62)and(20.96±0.48)mm respectively;Their MICs were 15.625 and 31.250mg/mL respectively;MBCs were 62.5and 125mg/mL;The traditional Chinese medicine decoction and the extracting buffer didn't display any antibacterial effects in vitro;2)The crude extract effected the growth curve of both E.coli O_1 and E.coli O_(78),also increased the permeability of cellmembrane and cell wall.In conclusion,the crude protein extract of maggot have antibacterial effects on both E.coli O_1 and E.coli O_(78),and the antibacterial effects in vitro on E.coli O_1 is better than E.coli O_(78).
引文
[1]胡建和,杭柏林,徐彦召,孙亚伟.动物抗菌肽[M].北京:科学出版社,2015Hu J H,Hang B L,Xu Y Z,Sun Y W.Animal Antibacterial Peptide[M].Beijing:Science Press,2015(in Chinese)
[2]马明.大肠杆菌知识漫谈[J].生物学教学,2007,32(1):67-68Ma M.The knowledge on Escherichia coli[J].Biology Teaching,2007,32(1):67-68(in Chinese)
[3]Onda T,Yanagida F,Tsuji M,Shinohara T,Yokotsuka K.Production and purification of a bacteriocin peptide produced by Lactococcus sp strain,GM005,isolated from Miso-paste[J].International Journal of Food Microbiology,2003,87(1/2):153
[4]安春菊,耿华,郝友进,李德森,杜荣骞.同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响[J]昆虫学报2004,47(5):691-694An C J,Geng H,Hao Y J,Li D S,Du R J.The influence of different factors in extraction on antibacterial activity of protein crude extract in housefly larvae[J].Acta Entomologica Sinica,2004,47(5):691-694(in Chinese)
[5]张姝,江加进,戴鼎震.五谷虫粗提物的抗炎效果及对小鼠免疫调节的影响[J].金陵科技学院学报,2013,29(4):85-89Zhang S,Jiang J J,Dai D Z.The anti-inflammatory potency of the crude extracts of maggot and its effect on mouse immune functions[J].Journal of JinLing Institute of Technology,2013,29(4):85-89(in Chinese)
[6]张振,洪亮,王寿宇,吕德成.五谷虫抗金黄色葡萄球菌物质的纯化及抗菌机制[J]中国组织工程研究,2013,17(15):2755-2760Zhang Z,Hong L,Wang S Y,Lu D C.Anti-Staphylococcus aureus substance purification from Wugu Chong and its related antibacterial mechanisms[J].Chinese Journal of Tissue Engineering Reseach,2013,17(15):2755-2760(in Chinese)
[7]朴范泽,夏成.当前牛病的发生特点和流行趋势[J].兽医导刊,2001(2):33-35Piao F Z,Xia C.The characteristics and trend about the current cow disease[J]Veterinary Tribune,2001(2):33-35(in Chinese)
[8]张振,王寿宇,刁云鹏,张厚利,黄珊珊,吕德成.五谷虫及活体蛆虫治疗慢性感染创面的研究进展[J].中国中药杂志,2009,34(24):3162-3164Zhang Z,Wang S Y,Diao Y P,Zhang H L,Huang S S,Lu D C.The research progress of maggot and live maggot about chronic wound infection[J].China Journal of Chinese Materia Medica,2009,34(24):3162-3164(in Chinese)
[9]江苏新医学院.中药大辞典[M].上海:上海科学技术出版社,1977:384Jiangsu New Medical College.Traditional Chinese Medicine Dictionary[M].Shanghai:Shanghai Science and Technology Press,1977:384(in Chinese)
[10]郁建生,杨冰,周芳英,黄雪飞,姚红燕,裴庆元.20种中草药制剂抑菌试验初报[J].中兽医学杂志,1995(1):2-5Yu J S,Yang B,Zhou F Y,Huang X F,Yao H Y,Pei Q Y.The report of 20 kinds of Chinese herbal medicine bacteriostatic[J].Chinese veterinary medicine journal,1995(1):2-5(in Chinese)
[11]杨斯琴,敖日格乐,王纯洁,斯木吉德,包永光.蒙药对牛源致病性大肠杆菌的体外抑菌效果研究[J].中国农业大学学报,2015,20(1):124-128Yang S Q,Aorigele,Wang C J,Simujide,BAO Y G.Antibacterial effects of Mongolian medicines on bovine pathogenic Escherichia coli in vitro[J].Journal of China Agricultural University,2015,20(1):124-128(in Chinese)
[12]屈军梅,陈平洁,李文平,屈孝初,唐保国,黄庭汝.莫家蝇抗菌肽提取及对鸡大肠杆菌病药效试验[J].中国畜牧兽医,2006,33(3):56-58Qu J M,Chen P J,Li W P,Qu X C,Tang B G,Huang T R.Extraction and efficacy colibacillosis in vivo an in vitro of antibacterial peptides of musca domestica[J].Chinese Animal Husbandry and Veterinary Medicine,2006,33(3):56-58(in Chinese)
[13]宫霞,王兰芳,乐国伟.家蝇幼虫抗菌肽MDL-2对细菌细胞渗透性及代谢功能的影响[J].氨基酸和生物资源,2011,33(3):1-5Gong X,Wang L F,Le G W,Effect of antibacterial peptide MDL-2 from musca domestica larvae on permeability metabolism of bacteria[J].Amino Acids&Biotic Resources,2011,33(3):1-5(in Chinese)
[14]陈玉洁,敖日格乐,王纯洁,斯木吉德.酸马奶提取Kluyveromyces marxianus代谢抗菌复合物对致病性大肠杆菌的抑菌和细胞表面特性的影响[J].微生物学通报,2015,42(4):683-689Chen Y J,Aorigele,Wang C J,Simujide.Effects of antimicrobial compounds of Kluyveromyces marxianus in Koumiss on pathogenic Escherichia coli and its cell surface characteristics[J].Microbiology China,2015,42(4):683-689(in Chinese)
[15]李凡,刘晶星,徐志凯.医学微生物学[M].北京:人民卫生出版社,2008Li F,Liu J X,Xu Z K.Medical Microbiology[M].Beijing:People’s Health Publishing House,2008(in Chinese)
[16]陈玉洁.酸马奶源酵母菌代谢物对致病性大肠杆菌的抑菌作用机理研究[D].呼和浩特:内蒙古农业大学2015Chen Y J.Study of Antibacterial Mechanism of Yeasts Metabolites from Koumiss on Pathogenic Escherichia coli[D].Hohhot:Inner Mongolia Agricultural University 2015(in Chinese)
[17]宋越.致犊牛腹泻病E.coli分离鉴定和治疗其蒙药抑菌成分筛选及作用的研究[D].呼和浩特:内蒙古农业大学,2015Song Y.The study on the isolation and identification of E.coli of calf diarrhea and the treatment of Mongolian medicine antibacteriao components selection and the functioni[D].Hohhot:Inner Mongolia Agricultural University 2015(in Chinese)
[2]马明.大肠杆菌知识漫谈[J].生物学教学,2007,32(1):67-68Ma M.The knowledge on Escherichia coli[J].Biology Teaching,2007,32(1):67-68(in Chinese)
[3]Onda T,Yanagida F,Tsuji M,Shinohara T,Yokotsuka K.Production and purification of a bacteriocin peptide produced by Lactococcus sp strain,GM005,isolated from Miso-paste[J].International Journal of Food Microbiology,2003,87(1/2):153
[4]安春菊,耿华,郝友进,李德森,杜荣骞.同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响[J]昆虫学报2004,47(5):691-694An C J,Geng H,Hao Y J,Li D S,Du R J.The influence of different factors in extraction on antibacterial activity of protein crude extract in housefly larvae[J].Acta Entomologica Sinica,2004,47(5):691-694(in Chinese)
[5]张姝,江加进,戴鼎震.五谷虫粗提物的抗炎效果及对小鼠免疫调节的影响[J].金陵科技学院学报,2013,29(4):85-89Zhang S,Jiang J J,Dai D Z.The anti-inflammatory potency of the crude extracts of maggot and its effect on mouse immune functions[J].Journal of JinLing Institute of Technology,2013,29(4):85-89(in Chinese)
[6]张振,洪亮,王寿宇,吕德成.五谷虫抗金黄色葡萄球菌物质的纯化及抗菌机制[J]中国组织工程研究,2013,17(15):2755-2760Zhang Z,Hong L,Wang S Y,Lu D C.Anti-Staphylococcus aureus substance purification from Wugu Chong and its related antibacterial mechanisms[J].Chinese Journal of Tissue Engineering Reseach,2013,17(15):2755-2760(in Chinese)
[7]朴范泽,夏成.当前牛病的发生特点和流行趋势[J].兽医导刊,2001(2):33-35Piao F Z,Xia C.The characteristics and trend about the current cow disease[J]Veterinary Tribune,2001(2):33-35(in Chinese)
[8]张振,王寿宇,刁云鹏,张厚利,黄珊珊,吕德成.五谷虫及活体蛆虫治疗慢性感染创面的研究进展[J].中国中药杂志,2009,34(24):3162-3164Zhang Z,Wang S Y,Diao Y P,Zhang H L,Huang S S,Lu D C.The research progress of maggot and live maggot about chronic wound infection[J].China Journal of Chinese Materia Medica,2009,34(24):3162-3164(in Chinese)
[9]江苏新医学院.中药大辞典[M].上海:上海科学技术出版社,1977:384Jiangsu New Medical College.Traditional Chinese Medicine Dictionary[M].Shanghai:Shanghai Science and Technology Press,1977:384(in Chinese)
[10]郁建生,杨冰,周芳英,黄雪飞,姚红燕,裴庆元.20种中草药制剂抑菌试验初报[J].中兽医学杂志,1995(1):2-5Yu J S,Yang B,Zhou F Y,Huang X F,Yao H Y,Pei Q Y.The report of 20 kinds of Chinese herbal medicine bacteriostatic[J].Chinese veterinary medicine journal,1995(1):2-5(in Chinese)
[11]杨斯琴,敖日格乐,王纯洁,斯木吉德,包永光.蒙药对牛源致病性大肠杆菌的体外抑菌效果研究[J].中国农业大学学报,2015,20(1):124-128Yang S Q,Aorigele,Wang C J,Simujide,BAO Y G.Antibacterial effects of Mongolian medicines on bovine pathogenic Escherichia coli in vitro[J].Journal of China Agricultural University,2015,20(1):124-128(in Chinese)
[12]屈军梅,陈平洁,李文平,屈孝初,唐保国,黄庭汝.莫家蝇抗菌肽提取及对鸡大肠杆菌病药效试验[J].中国畜牧兽医,2006,33(3):56-58Qu J M,Chen P J,Li W P,Qu X C,Tang B G,Huang T R.Extraction and efficacy colibacillosis in vivo an in vitro of antibacterial peptides of musca domestica[J].Chinese Animal Husbandry and Veterinary Medicine,2006,33(3):56-58(in Chinese)
[13]宫霞,王兰芳,乐国伟.家蝇幼虫抗菌肽MDL-2对细菌细胞渗透性及代谢功能的影响[J].氨基酸和生物资源,2011,33(3):1-5Gong X,Wang L F,Le G W,Effect of antibacterial peptide MDL-2 from musca domestica larvae on permeability metabolism of bacteria[J].Amino Acids&Biotic Resources,2011,33(3):1-5(in Chinese)
[14]陈玉洁,敖日格乐,王纯洁,斯木吉德.酸马奶提取Kluyveromyces marxianus代谢抗菌复合物对致病性大肠杆菌的抑菌和细胞表面特性的影响[J].微生物学通报,2015,42(4):683-689Chen Y J,Aorigele,Wang C J,Simujide.Effects of antimicrobial compounds of Kluyveromyces marxianus in Koumiss on pathogenic Escherichia coli and its cell surface characteristics[J].Microbiology China,2015,42(4):683-689(in Chinese)
[15]李凡,刘晶星,徐志凯.医学微生物学[M].北京:人民卫生出版社,2008Li F,Liu J X,Xu Z K.Medical Microbiology[M].Beijing:People’s Health Publishing House,2008(in Chinese)
[16]陈玉洁.酸马奶源酵母菌代谢物对致病性大肠杆菌的抑菌作用机理研究[D].呼和浩特:内蒙古农业大学2015Chen Y J.Study of Antibacterial Mechanism of Yeasts Metabolites from Koumiss on Pathogenic Escherichia coli[D].Hohhot:Inner Mongolia Agricultural University 2015(in Chinese)
[17]宋越.致犊牛腹泻病E.coli分离鉴定和治疗其蒙药抑菌成分筛选及作用的研究[D].呼和浩特:内蒙古农业大学,2015Song Y.The study on the isolation and identification of E.coli of calf diarrhea and the treatment of Mongolian medicine antibacteriao components selection and the functioni[D].Hohhot:Inner Mongolia Agricultural University 2015(in Chinese)