抗菌肽BSN-37的抑菌活性及其稳定性分析
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摘要
试验旨在研究抗菌肽BSN-37的抑菌活性和稳定性。采用微量倍比稀释法测定抗菌肽BSN-37的最小抑菌浓度(MIC),菌落计数法分析抗菌肽BSN-37对大肠杆菌CVCC1568的杀菌动力学特征,扩散法测定抗菌肽BSN-37的盐离子稳定性、酸碱稳定性、血清稳定性、胰酶稳定性、热稳定性、反复冻融稳定性、室温保持稳定性、药效保持时间稳定性。结果显示,抗菌肽BSN-37对革兰氏阴性菌(大肠杆菌和沙门氏菌)的MIC为1.5625～25μg/mL,对革兰氏阳性菌(枯草芽孢杆菌和短小芽孢杆菌)的MIC为1.5625～12.5μg/mL,对真菌(白色念珠菌)没有活性;抗菌肽BSN-37可在90min内完全杀灭大肠杆菌CVCC1568;除了Fe2+和胰酶会影响抗菌肽BSN-37的抑菌活性外,在75～100℃的高温、150～250mmol/L高浓度盐离子(Na+、K+、Ca2+和Mg2+)、20%～25%饱和浓度的Cu2+、pH 4～10、20%～25%的血清、10～12次的反复冻融、10～12d室温保存等条件下仍能保持较好的抑菌活性,药效保持时间可达7.5d。本试验结果表明,抗菌肽BSN-37具有替代抗生素成为新抗菌药物的潜力,为抗菌肽BSN-37的临床应用提供了理论依据。
The aim of the experiment was to study the bacteriostatic activity and stability of the antimicrobial peptide BSN-37.The minimum inhibitory concentration(MIC)of BSN-37 against several bacteria were detected with serial dilution method.The bactericidal dynamics of BSN-37 against Escherichia coli(E.coli)CVCC1568were measured by colony counting method.The salt ion stability,acid-alkali stability,serum stability,pancreatic enzyme stability,thermal stability,repeated freeze-thaw stability,maintain stability at room temperature,and efficacy stabilization stability were analyzed through diffusion method.The results showed that the MIC of the antimicrobial peptide BSN-37 against Gram negative bacteria(E.coli and Salmonella)was 1.5625to25μg/mL,and the MIC of BSN-37 against Gram positive bacteria(Bacillus subtilis and Bacillus pumilus)were 1.5625 to 12.5μg/mL.But BSN-37 had no bacteriostatic activity against fungi(Candida albicans).The antimicrobial peptide BSN-37 could completely kill E.coli CVCC1568 within 90min.In addition to the Fe2+and pancreatic enzymes could affect the bacteriostatic activity of antimicrobial peptide BSN-37,at other conditions including the high temperature(75to100℃),150 to 250mmol/L high concentration of salt ions(Na+,K+,Ca2+and Mg2+)and 20%to 25% of saturation concentration of Cu2+,pH 4to 10,20%to 25% of serum,10 to 12times of repeated freezing and thawing,preservation for 10 to 12days at room temperature,BSN-37 could still maintain good bacteriostatic activity.Efficacy maintain time of BSN-37 was more than 7.5days.The results provided a theoretical basis for the clinical application of antimicrobial peptide BSN-37 which had a potential to replace antibiotics as new antimicrobial agents.
The aim of the experiment was to study the bacteriostatic activity and stability of the antimicrobial peptide BSN-37.The minimum inhibitory concentration(MIC)of BSN-37 against several bacteria were detected with serial dilution method.The bactericidal dynamics of BSN-37 against Escherichia coli(E.coli)CVCC1568were measured by colony counting method.The salt ion stability,acid-alkali stability,serum stability,pancreatic enzyme stability,thermal stability,repeated freeze-thaw stability,maintain stability at room temperature,and efficacy stabilization stability were analyzed through diffusion method.The results showed that the MIC of the antimicrobial peptide BSN-37 against Gram negative bacteria(E.coli and Salmonella)was 1.5625to25μg/mL,and the MIC of BSN-37 against Gram positive bacteria(Bacillus subtilis and Bacillus pumilus)were 1.5625 to 12.5μg/mL.But BSN-37 had no bacteriostatic activity against fungi(Candida albicans).The antimicrobial peptide BSN-37 could completely kill E.coli CVCC1568 within 90min.In addition to the Fe2+and pancreatic enzymes could affect the bacteriostatic activity of antimicrobial peptide BSN-37,at other conditions including the high temperature(75to100℃),150 to 250mmol/L high concentration of salt ions(Na+,K+,Ca2+and Mg2+)and 20%to 25% of saturation concentration of Cu2+,pH 4to 10,20%to 25% of serum,10 to 12times of repeated freezing and thawing,preservation for 10 to 12days at room temperature,BSN-37 could still maintain good bacteriostatic activity.Efficacy maintain time of BSN-37 was more than 7.5days.The results provided a theoretical basis for the clinical application of antimicrobial peptide BSN-37 which had a potential to replace antibiotics as new antimicrobial agents.
引文
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[21]申艳敏,李荣誉,张国祖,等.抗菌肽LL-37的抑菌作用及其稳定性研究[J].畜牧与兽医,2013,45(6):75-77.SHEN Y M,LI R Y,ZHANG G Z,et al.Bacteriostatic action and stability of LL-37antibacterial peptide[J].Animal Husbandry&Veterinary Medicine,2013,45(6):75-77.(in Chinese)
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[23]刘晓燕,李大鹏.米酒乳杆菌素C2生物稳定性的研究[J].中国食品添加剂,2010,4:66-70.LIU X Y,LI D P.Biological stability of the sakacin C2[J].China Food Additives,2010,4:66-70.(in Chinese)
[24]江汉湖.食品微生物学[M].第2版.北京:中国农业出版社,2005.JIANG H H.Food Microbiology[M].2nd Edition.Beijing:China Agriculture Press,2005.(in Chinese)
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[26]杭柏林,张炜,李杰,等.抗菌肽BSN-37的生物信息学分析[J].河南科技学院学报(自然科学版),2018,46(1):34-38.HANG B L,ZHANG W,LI J,et al.Bioinformatics analysis of antimicrobial peptide BSN-37[J].Journal of Henan Institute of Science and Technology(Natural Science Edition),2018,46(1):34-38.(in Chinese)
[27]郑文官,张捷,杨刚齐,等.抗菌肽[L6,K11]-IsCT抑菌性能及其稳定性研究[J].中国畜牧兽医,2013,40(4):153-158.ZHENG W G,ZHANG J,YANG G Q,et al.Antibacterial activity and stability study of antimicrobial peptides[L6,K11]-IsCT[J].China Animal Husbandry&Veterinary Medicine,2013,40(4):153-158.(in Chinese)
[2]LEONG K,COOLEY L A,ANDERSON T L,et al.Emergence of vancomycin-resistant Enterococcus faecium at an Australian hospital:A whole genome sequencing analysis[J].Scientific Reports,2018,8(1):6274.
[3]KUMAR R,CHANDAR B,PARANI M.Use of succinic&oxalic acid in reducing the dosage of colistin against New Delhi metallo-β-lactamase-1bacteria[J].Indian Journal of Medical Research,2018,147(1):97-101.
[4]KOWALCZYK E,PATYRA E,KWIATEK K.Organic acids and their importance in animal husbandry[J].Medycyna Weterynaryjna,2013,69(5):269-273.
[5]PAKPOUR S,JABAJI S,CHNIER M R.Frequency of antibiotic resistance in a swine facility 2.5years after a ban on antibiotics[J].Microbial Ecology,2012,63(1):41-50.
[6]KIM K H,CHO E S,KIM K S,et al.Investigation on changes in pig farm productivity after ban of antibiotics growth promoter in commercial mixed feed[J].Korean Journal of Agriculturalence,2015,42(3):223-229.
[7]LIU Q,YAO S,CHEN Y,et al.Use of antimicrobial peptides as a feed additive for juvenile goats[J].Scientific Reports,2017,7(1):12254.
[8]赵飞,腾达,杨娜,等.抗菌肽NZ2114对多重耐药2型猪链球菌感染小鼠的治疗效果评价[J].中国畜牧兽医,2018,45(4):1089-1097.ZHAO F,TENG D,YANG N,et al.Evaluation of the therapeutic efficacy of antimicrobacterial peptide NZ2114in mice infected with Streptococcus suis serotype 2[J].China Animal Husbandry&Veterinary Medicine,2018,45(4):1089-1097.(in Chinese)
[9]靳纯嘏,叶耿坪,唐新仁.反刍动物饲用抗生素替代物研究进展[J].中国畜牧兽医,2018,45(1):77-85.JIN C J,YE G P,TANG X R.Research advances on alternatives to antibiotic feed additives in ruminant production[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):77-85.(in Chinese)
[10]胡建和,杭柏林,徐彦召,等.动物源抗菌肽[M].北京:科学出版社,2015.HU J H,HANG B L,XU Y Z,et al.Antimicrobial Peptides from Animal[M].Beijing:Science Press,2015.(in Chinese)
[11]祁丽,姜宁,张爱忠,等.抗菌肽研发现状及其改造策略[J].中国畜牧兽医,2016,43(2):450-456.QI L,JIANG N,ZHANG A Z,et al.Research present status and reform strategy of antimicrobial peptides[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):450-456.(in Chinese)
[12]汪吴晶,高金燕,佟平,等.抗菌肽的作用机制、应用及改良策略[J].动物营养学报,2017,29(11):3885-3892.WANG W J,GAO J Y,TONG P,et al.Antimicrobial peptides:Action mechanism,application and improvement strategy[J].Chinese Journal of Animal Nutrition,2017,29(11):3885-3892.(in Chinese)
[13]单安山,田昊天,邵长轩,等.抗菌肽抗细菌机理研究进展[J].东北农业大学学报,2018,49(3):84-94.SHAN A S,TIAN H T,SHAO C X,et al.Research advance on antimicrobial mechanism of antimicrobial peptides[J].Journal of Northeast Agricultural University,2018,49(3):84-94.(in Chinese)
[14]WU G,YANG Q,LONG M,et al.Evaluation of agar dilution and broth microdilution methods to determine the disinfectant susceptibility[J].Journal of Antibiotics,2015,68(11):661-665.
[15]ZHANG X,JIANG A,YU H,et al.Human lysozyme synergistically enhances bactericidal dynamics and lowers the resistant mutant prevention concentration for metronidazole to Helicobacter pylori by increasing cell permeability[J].Molecules,2016,21(11):E1435.
[16]潘晓倩,成晓瑜,张顺亮,等.不同检测方法在抗菌肽抑菌效果评价的比较研究[J].肉类研究,2014,28(12):17-20.PAN X Q,CHENG X Y,ZHANG S L,et al.Comparison of activity assays for antimicrobial peptides[J].Meat Research,2014,28(12):17-20.(in Chinese)
[17]GENNARO R,SKERLAVAJ B,ROMEO D.Purification,composition,and activity of two bactenecins,antibacterial peptides of bovine neutrophils[J].Infection&Immunity,1989,57(10):3142-3146.
[18]RAJ P A,MARCUS E,EDGERTON M.Delineation of an active fragment and poly(L-proline)Ⅱconformation for candidacidal activity of bactenecin 5[J].Biochemistry,1996,35(14):4314-4325.
[19]董柱,钟亨任,罗文杰,等.海南产沼蛙皮肤temporin家族抗菌肽抗菌活性及稳定性研究[J].海南大学学报(自然科学版),2016,34(3):250-256.DONG Z,ZHONG H R,LUO W J,et al.Antimicrobial activities and influential factors of temporin family antimicrobial peptides derived from Hylarana guentheri of Hainan[J].Nature Science Journal of Hainan University,2016,34(3):250-256.(in Chinese)
[20]许兵红,曾莉萍,赵春澎,等.丝光绿蝇抗菌肽的热稳定性研究[J].中国媒介生物学及控制杂志,2005,16(4):254-257.XU B H,ZENG L P,ZHAO C P,et al.A study on antibacterial activity of antibacterial peptide fromLucilia sericatu after treatment at high temperature[J].Chinese Journal of Vector Biology and Control,2005,16(4):254-257.(in Chinese)
[21]申艳敏,李荣誉,张国祖,等.抗菌肽LL-37的抑菌作用及其稳定性研究[J].畜牧与兽医,2013,45(6):75-77.SHEN Y M,LI R Y,ZHANG G Z,et al.Bacteriostatic action and stability of LL-37antibacterial peptide[J].Animal Husbandry&Veterinary Medicine,2013,45(6):75-77.(in Chinese)
[22]夏嫱,赵启凤,廖业,等.黑水虻抗菌肽粗提物生物学稳定性观察[J].山东医药,2013,53(36):91-92.XIA Q,ZHAO Q F,LIAO Y,et al.Observation on biological stability of antibacterial peptide crude extract of Tabanus[J].Shandong Medical Journal,2013,53(36):91-92.(in Chinese)
[23]刘晓燕,李大鹏.米酒乳杆菌素C2生物稳定性的研究[J].中国食品添加剂,2010,4:66-70.LIU X Y,LI D P.Biological stability of the sakacin C2[J].China Food Additives,2010,4:66-70.(in Chinese)
[24]江汉湖.食品微生物学[M].第2版.北京:中国农业出版社,2005.JIANG H H.Food Microbiology[M].2nd Edition.Beijing:China Agriculture Press,2005.(in Chinese)
[25]夏国良.动物生理学[M].北京:高等教育出版社,2013.XIA G L.Animal Physiology[M].Beijing:Higher Education Press,2013.(in Chinese)
[26]杭柏林,张炜,李杰,等.抗菌肽BSN-37的生物信息学分析[J].河南科技学院学报(自然科学版),2018,46(1):34-38.HANG B L,ZHANG W,LI J,et al.Bioinformatics analysis of antimicrobial peptide BSN-37[J].Journal of Henan Institute of Science and Technology(Natural Science Edition),2018,46(1):34-38.(in Chinese)
[27]郑文官,张捷,杨刚齐,等.抗菌肽[L6,K11]-IsCT抑菌性能及其稳定性研究[J].中国畜牧兽医,2013,40(4):153-158.ZHENG W G,ZHANG J,YANG G Q,et al.Antibacterial activity and stability study of antimicrobial peptides[L6,K11]-IsCT[J].China Animal Husbandry&Veterinary Medicine,2013,40(4):153-158.(in Chinese)
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