绞股蓝内生真菌抗大肠杆菌抗菌机制的研究
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摘要
【目的】药用植物内生真菌是一种新型抗生素的微生物资源,研究绞股蓝内生真菌JY25的抗菌机制对内生真菌的研发具有重要意义。【方法】以二倍稀释法测定发酵液的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);以MIC测定发酵液对大肠杆菌生长的抑制作用;扫描电镜观察发酵液作用下的大肠杆菌形态变化;同时,以β-半乳糖酶、碱性磷酸酶和电导率测定发酵液对细胞膜和细胞壁的损伤效果;采用考马斯亮蓝法检测发酵液对蛋白质合成的影响。【结果】JY25发酵液对大肠杆菌MIC为7 g/L、MBC为14 g/L;MIC浓度的发酵液使细菌对数生长期延迟12 h,菌体形态发生严重的畸形和破损;随着抑菌作用时间的延长,β-半乳糖酶含量增加、电导率增加,同时,实验发现大肠杆菌蛋白质合成异常,未检测到碱性磷酸酶。【结论】绞股蓝内生真菌JY25主要以破坏细菌的细胞膜及影响细菌蛋白质合成而抑制细菌生长。
[Objective] Endophytic fungi of medicine plants are the new microbial resource of antibiotic medicines. These antibacterial mechanisms of endophytic fungi named JY25 from Gynostemma pentaphyllum have important significance for the research and development of endophytic fungi. [Methods] The method of double dilution has adopted to measure the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) of fermentation broth to Escherichia coli. Then, the JY25 fermentation broth with MIC concentrations was used to inhibit the growth of E. coli, and the morphology changes of E. coli were observed by scanning electron microscope. Meanwhile, β-galactosidase, alkaline phosphatase and conductivity were used to test the damaged effects of fermentation broth on E. coli cell membrane and cell wall. Finally, the effects of protein synthesis of E. coli were detected by coomassie blue method. [Results] The results showed that MIC of JY25 fermentation broth to E. coli was 7 g/L, MBC was 14 g/L, and the fermentation broth with MIC concentration could delay the logarithmic growth of E. coli up to 12 h, with severe deformity and damage of cell morphology. With the extension of treating time, both the β-galactosidase activity and conductivity increased, and the protein synthesis was abnormal. The alkaline phosphatase did not be detected in the broth. [Conclusion] JY25 endophytic fungi from Gynostemma pentaphyllum primarily inhibited the growth of E. coli by damaging the cell membranes and impacting protein synthesis.
[Objective] Endophytic fungi of medicine plants are the new microbial resource of antibiotic medicines. These antibacterial mechanisms of endophytic fungi named JY25 from Gynostemma pentaphyllum have important significance for the research and development of endophytic fungi. [Methods] The method of double dilution has adopted to measure the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) of fermentation broth to Escherichia coli. Then, the JY25 fermentation broth with MIC concentrations was used to inhibit the growth of E. coli, and the morphology changes of E. coli were observed by scanning electron microscope. Meanwhile, β-galactosidase, alkaline phosphatase and conductivity were used to test the damaged effects of fermentation broth on E. coli cell membrane and cell wall. Finally, the effects of protein synthesis of E. coli were detected by coomassie blue method. [Results] The results showed that MIC of JY25 fermentation broth to E. coli was 7 g/L, MBC was 14 g/L, and the fermentation broth with MIC concentration could delay the logarithmic growth of E. coli up to 12 h, with severe deformity and damage of cell morphology. With the extension of treating time, both the β-galactosidase activity and conductivity increased, and the protein synthesis was abnormal. The alkaline phosphatase did not be detected in the broth. [Conclusion] JY25 endophytic fungi from Gynostemma pentaphyllum primarily inhibited the growth of E. coli by damaging the cell membranes and impacting protein synthesis.
引文
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[12]Jia CH.Antibacterial selection and mechanism of pathogenic Vibrio and Shewanella to Chinese herbal compounds[D].Zhanjiang:Master?s Thesis of Guang Dong Ocean University,2013(in Chinese)贾春红.抗致病性弧菌及希瓦氏菌有效中药与方剂的筛选、抗菌机理研究[D].湛江:广东海洋大学硕士学位论文,2013
[2]Yang BS,Miao CP,Zhang JH,et al.Population distribution and antimicrobial activities of endophytes in Toddalia asiatica[J].Acta Microbiologica Sinica,2014,54(3):273-284(in Chinese)杨本寿,苗翠苹,张建华,等.飞龙斩血内生菌种群分布及抑菌活性检测[J].微生物学报,2014,54(3):273-284
[3]Xia LJ,Zhang K,Huang HR,et al.The antimicrobial activity of5 kinds of mangrove fungi in rhizosphere soil and endophytic fungi[J].Chinese Agricultural Science Bulletin,2014,30(4):259-263(in Chinese)夏丽娟,张焜,黄华荣,等.5种红树根际土壤真菌和内生真菌的分离及抑制菌活性的研究[J].中国农学通报,2014,30(4):259-263
[4]Piao XL,Wu Q.Progressive studies on Gynostemma pentaphyllum[J].Li Shi Zhen Medicine and Materia Medica Research,2010,21(7):1758-1760(in Chinese)朴香兰,吴倩.绞股蓝研究进展[J].时珍国医国药,2010,21(7):1758-1760
[5]Shang F,Wei XY,Liu Z,et al.Isolation,identification and antioxidant activity of an endophytic Fungi from Gynostemma pentaphyllum makino[J].Pharmaceutical Biotechnology,2011,18(6):519-521(in Chinese)尚菲,魏希颖,刘竹,等.具有抗氧化活性的绞股蓝内生真菌的分离及研究[J].药物生物技术,2011,18(6):519-521
[6]Zhou SL,Chen CF,Fang XT,et al.Isolating of endophytic fungi from five medicinal plants and screening of their antibacterial activities[J].Journal of Xuzhou Normal University(Natural Science Edition),2007,25(4):69-72(in Chinese)周生亮,陈才法,房兴堂,等.5种药用植物内生真菌的分离及其抑菌活性的研究[J].徐州师范大学学报:自然科学版,2007,25(4):69-72
[7]Fan WH.Preliminary study on isolation and identification and biological activities of antibacterical endophytic fungi from Gynostemma[D].Henan:Master?s Thesis of Henan University of Technology,2012(in Chinese)范文慧.抗菌性绞股蓝内生真菌的分离鉴定及生物学活性初步研究[D].河南:河南工业大学硕士学位论文,2012
[8]Zhang HR,Xu CP,He XY,et al.The use of endophytic fungi from Gynostemma pentaphyllum.Chinese patent:201310002710[P].2013-11-27(in Chinese)张慧茹,许春平,贺秀媛,等.绞股蓝内生真菌及其用途.中国专利:201310002710[P].2013-11-27
[9]Zhang HR,Yi YJ,Zhao HY,et al.The PG2 molecule identification and antimicrobial substances in taraxacum herb[J].Feed Industry,2012,33(9):30-33(in Chinese)张慧茹,伊艳杰,赵红月,等.蒲公英内生真菌PG2分子鉴定及抑菌物质的研究[J].饲料工业,2012,33(9):30-33
[10]Shi CF,Yin ZQ,Wei Q,et al.Bacteriostatic action and mechanism ofα-terpineol on Escherichia coli[J].Acta Veterinariaet Zootechnica Sinica,2013,44(5):796-801(in Chinese)石超峰,殷中琼,魏琴,等.α-松油醇对大肠杆菌的抑菌作用及其机理研究[J].畜牧兽医学报,2013,44(5):796-801
[11]Ai QJ,Yu QH,Zhang HX,et al.Studies on bacter iostatic action and antimicrobial mechanism of pyrolin against Staphylococcus aureus[J].Journal of Chinese Institute of Food Science and Technology,2007,7(2):33-36(in Chinese)艾启俊,于庆华,张红星,等.鹿蹄草素对金黄色葡萄球菌的抑制作用及其机理研究[J].中国食品学报,2007,7(2):33-36
[12]Jia CH.Antibacterial selection and mechanism of pathogenic Vibrio and Shewanella to Chinese herbal compounds[D].Zhanjiang:Master?s Thesis of Guang Dong Ocean University,2013(in Chinese)贾春红.抗致病性弧菌及希瓦氏菌有效中药与方剂的筛选、抗菌机理研究[D].湛江:广东海洋大学硕士学位论文,2013