抗真菌农用抗生素分离及发酵工艺优化的初步研究
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摘要
本实验室从土壤中筛选得到利迪链霉菌变种AS 4.2501(Streptomyces lydicus AS 4.2501)。该菌能产生抗植物真菌病害的拮抗物,对棉花立枯病、小麦赤霉病、番茄早疫病等18种病原菌都有明显的抑制作用,具有较高的生物活性和良好的工业化产品开发潜力。本文在摇瓶水平上,对利迪链霉菌的发酵培养基进行优化,并将其应用到2L发酵罐水平上,考察了利迪链霉菌AS 4.2501的发酵过程规律;综合利用吸附法、溶媒萃取法和硅胶柱层析法对抗生素进行了纯化。
以立枯丝核菌(Rhizoctonia solani)作为生物活性检验指示菌,在摇瓶水平上运用利用单因子实验和正交实验对发酵培养基配方进行了优化。结果表明优化后的培养基使发酵液的抑菌率从原来的65.9%提高到78.6%,相对增长19.2%。
通过测定还原糖含量、菌浓、pH值、溶氧、抑菌率等参数,考察了生产菌株在2L发酵罐中的发酵过程规律。结果表明优化培养基明显缩短菌体的延滞期,在30h菌浓达到最高,13.3mg/mL,较原始培养基提高了44.6%;发酵液抑菌率最大值为83.1%,较原始培养基的82.4%略有提高,而抑菌率的最大峰值出现的时间从54h提前到了36h,缩短了生产周期。
经预处理的发酵液分别经活性炭和Diaion HP-20大孔树脂吸附,洗脱液浓缩后再用等体积的正丁醇萃取,取有机相浓缩后,上样到预先制好的硅胶层析柱,以氯仿:甲醇:29%氨水(5:3:1)为流动相进行分离,得到了组分较为简单且具有很强抑菌活性的样品。经质谱分析,发现经活性炭分离的样品中能检测到m/z为432.2,579.2和574.0的三个质谱峰,而经Diaion HP-20大孔树脂的样品中能检测到m/z为654.3和625.4的两个质谱峰,这两组样品都具有较高抗真菌活性,由此推测发酵液中含有多种抗真菌抗生素。
A strain of Streptomyces lydicus AS 4.2501 had been obtained from the soil and reserved in our lab. It was able to produce some antibiotics, which could inhibit cotton Rhizoctonia solani, wheet scab, Alternaria solani and other 15 pathogens. Therefore, these antibiotics have enormous potentiality to be developed as industrial products thanks to their strong bioactivity. In this paper, firstly the medium composition was optimized in flask scale, and then applied it in 2L fermentor to study the fermention kinetics of Streptomyces lydicus AS 4.2501. Finally, the antibiotics were purified by adsorption, solvent extraction and silica gel column chromatography.
Firstly, with Rhizoctonia solani as the indicator microorganism, we optimized the medium composition in flask scale through uniform design and orthogonal experiments. The result showed that the percentage of fungistasis rose from 65.9% to 78.6% with the optimized medium, which was 19.2% higher than the original one.
Secondly, the fermention process in 2L fermentor scale was studied through monitoring reducing sugar, cell dry weight, pH and dissolved oxygen. The result showed the lag phase of the strain growed with the optimized medium was remarkably shortened compared with that with the original one. After 30 hours of fermentation, the cell dry weight reached the peak value of 13.3mg/mL, which was 44.6% higher than the control. The maximum percentage of fungistasis was 83.1%, which was slightly higher than that of the original medium. However, the culture time was reduced from 54h to 36h.
Finally, the pretreated fermention broth was adsorbed by activated carbon and Diaion HP-20 macroporous adsorption resin respectively. The concentrated eluant was extracted by equal-volume n-butyl alcohol, and the organic layer was concentrated and chromatographed on a silica gel column with chloroform-methanol-29% ammonia water (5:3:1). Purified active fractions were obtained from the corresponding eluate. The MS analysis result showed three peaks with m/z of 432.2, 579.2 and 574.0 were detected in the samples separated by activated carbon, while two peaks with m/z of 654.3 and 625.4 were detected in the samples separated by Diaion HP-20 macroporous adsorption resin. Both groups had strong antifungal activity, thus it was concluded that there were multiple antifungal antibiotics in the broth.
以立枯丝核菌(Rhizoctonia solani)作为生物活性检验指示菌,在摇瓶水平上运用利用单因子实验和正交实验对发酵培养基配方进行了优化。结果表明优化后的培养基使发酵液的抑菌率从原来的65.9%提高到78.6%,相对增长19.2%。
通过测定还原糖含量、菌浓、pH值、溶氧、抑菌率等参数,考察了生产菌株在2L发酵罐中的发酵过程规律。结果表明优化培养基明显缩短菌体的延滞期,在30h菌浓达到最高,13.3mg/mL,较原始培养基提高了44.6%;发酵液抑菌率最大值为83.1%,较原始培养基的82.4%略有提高,而抑菌率的最大峰值出现的时间从54h提前到了36h,缩短了生产周期。
经预处理的发酵液分别经活性炭和Diaion HP-20大孔树脂吸附,洗脱液浓缩后再用等体积的正丁醇萃取,取有机相浓缩后,上样到预先制好的硅胶层析柱,以氯仿:甲醇:29%氨水(5:3:1)为流动相进行分离,得到了组分较为简单且具有很强抑菌活性的样品。经质谱分析,发现经活性炭分离的样品中能检测到m/z为432.2,579.2和574.0的三个质谱峰,而经Diaion HP-20大孔树脂的样品中能检测到m/z为654.3和625.4的两个质谱峰,这两组样品都具有较高抗真菌活性,由此推测发酵液中含有多种抗真菌抗生素。
A strain of Streptomyces lydicus AS 4.2501 had been obtained from the soil and reserved in our lab. It was able to produce some antibiotics, which could inhibit cotton Rhizoctonia solani, wheet scab, Alternaria solani and other 15 pathogens. Therefore, these antibiotics have enormous potentiality to be developed as industrial products thanks to their strong bioactivity. In this paper, firstly the medium composition was optimized in flask scale, and then applied it in 2L fermentor to study the fermention kinetics of Streptomyces lydicus AS 4.2501. Finally, the antibiotics were purified by adsorption, solvent extraction and silica gel column chromatography.
Firstly, with Rhizoctonia solani as the indicator microorganism, we optimized the medium composition in flask scale through uniform design and orthogonal experiments. The result showed that the percentage of fungistasis rose from 65.9% to 78.6% with the optimized medium, which was 19.2% higher than the original one.
Secondly, the fermention process in 2L fermentor scale was studied through monitoring reducing sugar, cell dry weight, pH and dissolved oxygen. The result showed the lag phase of the strain growed with the optimized medium was remarkably shortened compared with that with the original one. After 30 hours of fermentation, the cell dry weight reached the peak value of 13.3mg/mL, which was 44.6% higher than the control. The maximum percentage of fungistasis was 83.1%, which was slightly higher than that of the original medium. However, the culture time was reduced from 54h to 36h.
Finally, the pretreated fermention broth was adsorbed by activated carbon and Diaion HP-20 macroporous adsorption resin respectively. The concentrated eluant was extracted by equal-volume n-butyl alcohol, and the organic layer was concentrated and chromatographed on a silica gel column with chloroform-methanol-29% ammonia water (5:3:1). Purified active fractions were obtained from the corresponding eluate. The MS analysis result showed three peaks with m/z of 432.2, 579.2 and 574.0 were detected in the samples separated by activated carbon, while two peaks with m/z of 654.3 and 625.4 were detected in the samples separated by Diaion HP-20 macroporous adsorption resin. Both groups had strong antifungal activity, thus it was concluded that there were multiple antifungal antibiotics in the broth.
引文
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[6]朱昌雄等,农抗120防治西瓜枯萎病菌的室内药效实验,生物防治通报,6(3): 124~127
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[8]朱昌雄等,农抗120对苹果树腐烂病菌的作用研究.生物防治通报,8(4):163~167
[9].朱昌雄等,农抗120对水稻纹枯病菌抗生活性试验.生物防治通报,9 (1):15~18.
[10]顾惠儿等,农抗120中脂溶性主要组份A的结构鉴别,生物防治通报,10(4):166~168
[11]林永年等,多效霉素在农业应用上研究,全国抗生素学术会议论文集,318
[12]尹萃耘,荀培琪,多效霉素在农业上应用的研究(摘要),第四次全国抗生素学术会议论文集(下〕[C].上海:上海科学出版社,1985,320
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[14] Boeck L. D., ClemG. M.: WilsonM.M. 1973. A9145, a new adenine containing antirngal antibiotic Antimicrob Agents Chemother.3:49~56
[15]上海植物生理研究所微生物室农抗组,庆丰祥素的研究,II.分离、纯化及理化性质,微生物学报,1975,15(2):101
[16]施跃峰,桑金隆,竺利红,新徽生物农药抑菌素的研究,核农学报2004, 18 (1):68~71
[17]宋普球,魏玉玲,庄名扬,核苷N9705的抗病毒、抗菌杀虫活性研究,中国病毒学,杀虫嫩生物专刊,2000(15):180~183
[18]宋普球等,核昔类化合物产生曲的筛选及其活性组分研究,天然产物研究与开发,1999,11(5):15~19
[19]抗真菌药物研究进展,制药原料及中间体信息,2006(7),28~31
[20]陈昭容,向固西等.农用抗生素16A-6产生菌的鉴定,微生物学报,1983, 23(4):305~308.
[21]向固西,胡厚芝等,一种所的农用抗生素——宁南祥素,微生物学报, 1995, 35 (5):368~374.
[22]林祖纶,王大杰等.应用反向离子对色带研究农用抗生索16A-6在各类农作物中的残留,成都大学学报自然科学版,1988,(2):47~51.
[23]樊连梅,陈洁敬,刘更森,宁南霉素素(16A-6)性质的初步鉴定,莱阳农学院学报2002,19(4):279~281
[24]张国生,甲氧基丙烯酸酯类杀菌剂的应用、开发现状及展望,农药科学与管理,2003(12):30~34
[25]盛春泉,季海涛.新型抗真菌药物的研究进展,国外医学药学分册,2001,28(6):347~351
[26]顾觉奋,张绍谭,β-1,3-葡聚糖合成酶抑制剂类抗真菌抗生素,中国医药情报,2003, 9(6):10~17
[27] Cabib E, Drgon T, Krgonova J.The yeast cell wall, a dynamic structure engaged in growth and morphogenesis, Biochem Soc Trans, 1997, 25: 200~204
[28] Cabib E,Drgon.T,Krgonova.J,et a1.The yeast cell wall,adynamicstructure engagedin growth and morphogenesis,Biochem. Soe. Trans 1997, 25: 200~204
[29] Turner.W W. & Current W L. Echinocandin antifungal agents. Drugs Pharm, Sci.1997; 82: 315~334
[30] Fujie.A, MuramatsuH, Okudaira, et a1. FR901-469, a novel antifungal antibiotic from an unidentified fungus NO.11243: Taxonomy, fermentation, isolation, physicochemical properties and biological properties. J. Antibiotics, 2000, 53: 912~919
[31] McCreath KJ, Specht CA, Liu Y. Molecular cloning of a third chitinase gene from Candida albicans. Yeast, 1996, 12 (5): 501~504
[32]徐锡权,抗真菌药物研究进展,中国新药杂志,1994, 3(6):10~20
[33]孙青典,刘超美,抗真菌药物的研究与开发.药学进展,2001,25 (4): 213~220
[34] Ueki T, Nunata K, SawadaY Studies on the mode of antifungal action of pradimicin antibiotics.II. D-mannopyranoside-binding site and calcium-binding site.Antibiot, 1993, 46 (3): 455~464
[35] Takeuchi T, Hara T, Naganawa H.New antifungal antibiotics, benanomicins A and B from an actinomycete.Antibiot, 1988, 41 (6):807~811
[36] Oki, KakushimaM, Hirano M. In vitro and in vivo antifungal activites of BMS-181184.Antibiot, 1992, 45 (9): 1512~1517
[37] Oakley K L, Moore C B, Denning D W. Activity of pradimicin BMS-181184 against Aspergillus spp.Antimicrob Agents, 1999, 12 (3):267-269
[38] Lopez SN, Castelli MV, Zacchino SA, et al. In vitro antifungal evaluation and structure activity relationships of a new series of chalcone derivatives and synthetic analogues with inhibtory properties against polymer of the fungal cell wall [J]. Bioorg Med Chem, 2001, 9 (8):1999.
[39]叶丽娟,王辂,朱辉,国外医药抗生素分册,2006,27(5):221~227
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