丝状海洋真菌杀线虫代谢物的性质及发酵条件研究
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摘要
海洋真菌因其种类的多样性和遗传背景的特殊性而具有产生新型生物活性物质的巨大潜力。本文从渤海水域分离得到并证实具有杀线活性的3株丝状真菌MTJ5-3-1、MTJ4-2-1、MTJ5-1-4出发,选择杀线活性最高的MTJ5-3-1为试验菌；利用线虫致死率作为指标,通过发酵条件优化实验对发酵影响较大的pH值、接种量、培养温度、培养时间等培养条件和碳氮源及其含量进行优化,确立了优化培养条件；并对海洋真菌MTJ5-3-1的杀线虫活性代谢物质进行了理化性质分析,研究结果如下：1) MTJ5-3-1、MTJ4-2-1、MTJ5-1-43株海洋真菌的杀线活性测定
测定结果显示,MTJ5-3-1菌株杀线活性最高,杀线活性达到A级。2)MTJ5-3-1菌的发酵条件优化
通过优化试验,得到改进后培养条件为：去皮马铃薯150g/L煮汁,葡萄糖10g/L,酵母膏1g/L,海水含量600m1/L。接种量2×106的孢子悬液,转速120r/min,初始pH6.5～pH7.0,24～26℃发酵96h。3)MTJ5-3-1菌杀线虫活性代谢产物定性分析
MTJ5-3-1菌的10倍浓缩发酵液,超滤去掉6000dalton以上的大分子,采用生物活性跟踪检测的方法,对水饱和乙酸乙酯及水饱和正丁醇萃取后的水相组分,结果显示其杀线虫活性达到97.85%；理化性质分析结果表明,该产物为中性耐热活性物质,进一步的薄层层析、可见-紫外光谱分析、红外检测,以及液相色谱分析结果显示,MTJ5-3-1菌杀线虫活性代谢产物包含4种组分,具有含氮糖类或含氮糖类衍生物的分子特征。4)菌种鉴定
通过对MTJ5-3-1菌株经典分类指标的观察鉴定,将其初步鉴定为枝顶孢霉。进一步的分子生物学鉴定结果显示,该菌株ITS序列与Acremonium sp. NJM0672和Acremonium sp. NJM 0567的同源性为100%,形态学鉴定结果与Acremonium sp. NJM 0567一致,可确定为Acremonium sp.。
结论：通过研究获得一株能够产生杀线虫活性代谢物的菌株MTJ5-3-1,经鉴定该菌株为枝顶孢霉,其活性代谢物具有含氮糖类或含氮糖类衍生物的分子特征,且具有较好的耐热性和相对宽泛的、与大多土壤环境相符的pH适应范围。此外,枝顶孢霉在线虫防治领域还处于探索性研究阶段,同类报道鲜见,为抗线虫海洋真菌的研究提供了新的菌种来源和代谢产物类型,显示出该菌良好的线虫生防制剂开发前景。
Marine fungi were now highly concerned about for their potential bioactive substances based on their variety and genetic background, as well as their special habitat. Three marine filamentous fungal strains of MTJ5-3-1, MTJ4-2-1, MTJ5-1-4 from Bohai Gulf were sought for the nematicide. Among them, MTJ5-3-1 was proved the most efficiency of nematicidal effect. Based on this, its optimum condition including pH, temperature and nutrition were evaluated. Moreover, physico-chemical properties of the nematicidal components from the metabolites were detected based on lethal rate on pinewood nematode (Bursaphelenchus xylophilus). The results were summarized as follows:
a. Nematicidal effects of MTJ5-3-1,MTJ4-2-1,MTJ5-1-4
Preliminary studied of the nematieidal bioactive substances obtained from MTJ5-3-1 MTJ4-2-1 MTJ5-1-4 showed that all the three filamentous strains were strong efficiency in against pinewood nematode separately with 93.85% 85.37% and 92.25%. Among them, MTJ5-3-1 was the most efficiency of nematicidal effect (level A, lethal rate=90%). b. Optimum condition for the fermentation of MTJ5-3-1 strain
According to the results of single factor and statistical analysis, the optimum marine aquatic medium was composed of glucose 10g/L, peeled potato 150g/L and yeast extract 2g/L, sea-water 500-600ml/L with nature pH. The optimum inoculate quantity was 2×106 spores. Fermentation was performed under the conditions of 120r/min,26℃for 4 days. c. Qualitative analysis of MTJ5-3-1 nematicidal activity metabolites
First of all, the fermentation broth of MTJ5-3-1 was hyper-filtrated in order to remove the macromolecules that over 6000 dalton, substances remained were extracted subsequently by water-saturated ethyl acetate and water-saturated n-butanol. The water-soluble component was then collected and determined with a lethal rate of 97.85% in nematicidal activity. Instrumental analysis including spectro-comparator and chromatography shown the product was characterized by nitrogenous saccharides or its derivants, involving 4 heat-stable components and neutral properties. d. Identification and classification of MTJ5-3-1 strain
Morphological characteristics of MTJ5-3-1 strain suggest that the MTJ5-3-1 strain might belong to the genus Acremonium. The results of molecular biological assay on ITS sequences showed 100% homology with Acremonium sp. NJM 0672 and Acremonium sp. NJM 0567. According to the results, MTJ5-3-1 strain should be Acremonium sp MTJ5-3-1.
测定结果显示,MTJ5-3-1菌株杀线活性最高,杀线活性达到A级。2)MTJ5-3-1菌的发酵条件优化
通过优化试验,得到改进后培养条件为：去皮马铃薯150g/L煮汁,葡萄糖10g/L,酵母膏1g/L,海水含量600m1/L。接种量2×106的孢子悬液,转速120r/min,初始pH6.5～pH7.0,24～26℃发酵96h。3)MTJ5-3-1菌杀线虫活性代谢产物定性分析
MTJ5-3-1菌的10倍浓缩发酵液,超滤去掉6000dalton以上的大分子,采用生物活性跟踪检测的方法,对水饱和乙酸乙酯及水饱和正丁醇萃取后的水相组分,结果显示其杀线虫活性达到97.85%；理化性质分析结果表明,该产物为中性耐热活性物质,进一步的薄层层析、可见-紫外光谱分析、红外检测,以及液相色谱分析结果显示,MTJ5-3-1菌杀线虫活性代谢产物包含4种组分,具有含氮糖类或含氮糖类衍生物的分子特征。4)菌种鉴定
通过对MTJ5-3-1菌株经典分类指标的观察鉴定,将其初步鉴定为枝顶孢霉。进一步的分子生物学鉴定结果显示,该菌株ITS序列与Acremonium sp. NJM0672和Acremonium sp. NJM 0567的同源性为100%,形态学鉴定结果与Acremonium sp. NJM 0567一致,可确定为Acremonium sp.。
结论：通过研究获得一株能够产生杀线虫活性代谢物的菌株MTJ5-3-1,经鉴定该菌株为枝顶孢霉,其活性代谢物具有含氮糖类或含氮糖类衍生物的分子特征,且具有较好的耐热性和相对宽泛的、与大多土壤环境相符的pH适应范围。此外,枝顶孢霉在线虫防治领域还处于探索性研究阶段,同类报道鲜见,为抗线虫海洋真菌的研究提供了新的菌种来源和代谢产物类型,显示出该菌良好的线虫生防制剂开发前景。
Marine fungi were now highly concerned about for their potential bioactive substances based on their variety and genetic background, as well as their special habitat. Three marine filamentous fungal strains of MTJ5-3-1, MTJ4-2-1, MTJ5-1-4 from Bohai Gulf were sought for the nematicide. Among them, MTJ5-3-1 was proved the most efficiency of nematicidal effect. Based on this, its optimum condition including pH, temperature and nutrition were evaluated. Moreover, physico-chemical properties of the nematicidal components from the metabolites were detected based on lethal rate on pinewood nematode (Bursaphelenchus xylophilus). The results were summarized as follows:
a. Nematicidal effects of MTJ5-3-1,MTJ4-2-1,MTJ5-1-4
Preliminary studied of the nematieidal bioactive substances obtained from MTJ5-3-1 MTJ4-2-1 MTJ5-1-4 showed that all the three filamentous strains were strong efficiency in against pinewood nematode separately with 93.85% 85.37% and 92.25%. Among them, MTJ5-3-1 was the most efficiency of nematicidal effect (level A, lethal rate=90%). b. Optimum condition for the fermentation of MTJ5-3-1 strain
According to the results of single factor and statistical analysis, the optimum marine aquatic medium was composed of glucose 10g/L, peeled potato 150g/L and yeast extract 2g/L, sea-water 500-600ml/L with nature pH. The optimum inoculate quantity was 2×106 spores. Fermentation was performed under the conditions of 120r/min,26℃for 4 days. c. Qualitative analysis of MTJ5-3-1 nematicidal activity metabolites
First of all, the fermentation broth of MTJ5-3-1 was hyper-filtrated in order to remove the macromolecules that over 6000 dalton, substances remained were extracted subsequently by water-saturated ethyl acetate and water-saturated n-butanol. The water-soluble component was then collected and determined with a lethal rate of 97.85% in nematicidal activity. Instrumental analysis including spectro-comparator and chromatography shown the product was characterized by nitrogenous saccharides or its derivants, involving 4 heat-stable components and neutral properties. d. Identification and classification of MTJ5-3-1 strain
Morphological characteristics of MTJ5-3-1 strain suggest that the MTJ5-3-1 strain might belong to the genus Acremonium. The results of molecular biological assay on ITS sequences showed 100% homology with Acremonium sp. NJM 0672 and Acremonium sp. NJM 0567. According to the results, MTJ5-3-1 strain should be Acremonium sp MTJ5-3-1.
引文
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