水菖蒲内生菌的分离及其抑菌活性的研究
摘要
水菖蒲(Acorus calamus Linn.)作为一种传统的民间药,长期生长在污泥这种特殊的环境中,国内外文献研究表明具有很好的抑菌杀虫活性。
植物内生菌能产生大量新的天然产物,有些内生菌还可以产生与宿主植物相同或相近的代谢产物,已成为天然产物潜在的重要资源。本研究对水菖蒲内生菌进行了分离,得到113株内生菌,以大肠杆菌和金黄色葡萄球菌为指示菌对其抗菌活性进行筛选,研究了菌株S9,S15,S24和S70的抗菌活性。发现S9具有广谱的抑菌活性,对其进行了抑菌活性物质与菌株鉴定、生理条件与稳定性,培养基条件的优化以及水菖蒲抑菌活性的相关研究。本研究旨在探索从水菖蒲内生菌中发现抑菌活性成分的可能性,为研究新的天然产物抑菌药物提供思路。主要研究内容如下:
(1)采用75%乙醇、次氯酸钠溶液浸泡和无菌水洗涤、培养、分离的方法从水菖蒲内分离内生菌。研究表明:应同时设置超净工作台无菌状态检测对照、漂洗液检测对照和组织印迹检测对照等3种对照处理,确保内生菌分离准确性。水菖蒲体内存在内生菌且种类繁多、十分丰富,共分离到113株内生菌,根茎、叶中均有分布,叶中的内生菌数量明显少于茎中。
(2)用大肠杆菌、金黄色葡萄球菌和稻瘟霉作为初筛指示菌,分别代表了G~+,G~-菌,真菌,对分离得到的113株水菖蒲内生菌进行了抗菌活性初筛,有50株菌对稻瘟霉P-2b具有抑制作用,46株对大肠杆菌或金黄色葡萄球菌有抗菌活性的菌株,分别占所有菌株的44.2%和40.7%,表明水菖蒲中有丰富的有抑菌活性的内生菌资源。对抑菌效果最强的四株菌—S9、S15、S24、S70,进行了进一步的抗菌能力研究。结果表明,S9对多种指示菌都有很好的抑制和杀灭效果。
(3)研究了活性菌株S9发酵1-14天的生长特性,并检测了生物量,pH值,残糖量和对指示菌的最小抑菌浓度。结果显示发酵液在1-3天进入对数增长期,第4-6d为稳定期,从第7d开始进入衰亡期,在整个过程中,生物量、pH、发酵液中的残糖量和抑菌活性变化显著。
(4)研究了S9发酵液对温度、光照、pH值和传代等的稳定性。结果表明,发酵液对温度、光照有较好的稳定性,1-7代内菌株抑菌活性无明显改变。但pH值对发酵液的活性影响较大,在偏酸的环境下,活性较好,在碱性环境下无活性。并初步研究了S9发酵液的活性部位,结果表明S9发酵液的活性成分存在于胞外产物,胞内物质基本无抑菌活性;同时,发酵液中的多糖蛋白等大分子物质无抑菌活性。
(5)通过单因素试验,Plackett-Burman试验和响应面法对水菖蒲内生菌S9的发酵条件进行了优化,结果表明水菖蒲内生菌S9的最佳发酵条件为温度28℃,蔗糖用量26 g·L~(-1),pH值为6.7,培养基装液量50mL,转速140 r/min,KH_2PO_4为1mmol/L,马铃薯加入量为250 g·L~(-1),酵母膏的含量20 g·L~(-1)。
(6)采用五部分离法,研究了水菖蒲内生菌S9发酵产物各极性部位的抑菌活性,初步判定其最强的抑菌活性部位为石油醚相,对各种细菌、丝状真菌和酵母菌均有不同程度的抑制作用,对各种指示细菌的最小抑菌浓度(MIC)为15-30μg·mL~(-1),对指示细菌的最小杀菌浓度为30-60μg·mL~(-1);对四种酵母样真菌的最小抑菌浓度为12.5-50μg·mL~(-1):对六种丝状真菌的最小抑菌活性为15-500μg·mL~(-1)。并通过形态鉴定和ITS序列分析,鉴定水菖蒲内生菌S9为木霉属,其与Hypocrea lixii sp.属中的各菌株具有极高的同源性高达98%。通过GC-MS分析,结果表明其石油醚相的主要成分为2-(2,2-二甲基亚丙基)环己烷-1,3-二酮。
(7)研究了S9内生菌的宿主植物水菖蒲各极性部位的抑菌活性,其中石油醚相对各种指示细菌和丝状真菌表现出了较好的抑菌效果,并对其相进行的GC-MS分析,结果表明其主要成分为1-甲氧基-4-乙炔基苯。
As Traditional Chinese medicine,Acorus calamus Linn.grows in sullage and has a strong antimicrobial activity.
Some endophytic fungi could produce large new natural products,some metabolite produced by endophytic fungi were identical or semblable with host plant.It now appears that endophytes is an enormous,relatively untapped source of diversity the microbial.In this paper the endophytes from Acorus calamus Linn.were isolated for the first time.With high throughput microdilution method,the endophytes of antifungal,antimitotic and antimicrobial activity were preliminary screened by model of PyricuLaria oryzae P-2b and bacterium,through detecting deformation of mycelia germinated from conidia of P. Oryzae P-2b.S9 strain possessed strongest antimicrobial activity to broad-spectrum bacterium,yeast and mycelial fungi.
The further research was made for the activity substance,classification,stability, Optimization of fermentation conditions and the plant for antimicrobial activity.This research is for the purpose of exploring for endophytes from Acorus calamus Linn.to produce antimicrobial activity constituent possibility,providing the idea for studies the new resource to search new antimicrobial compounds.
Endophyte is a new resource of antimicrobial activity and potential for application.
Main contents of this thesis as follows:
1.The plant materials are thoroughly surface treated with 75%ethanol,sodium hypochlorite solution and sterilizated water.Controlled experiments that detecting of biologic safe cabinet,rinse water and tissue blotting were set simultaneously were extremely essential.There are a great variety of endophytes in Acorus calamus Linn. 113 strains were isolated from stem and leaf.
2.With high throughput microdilution method and screening model of P.Oryzae P-2b and bacteria,50 strains against P.Oryzae P-2b,46 strains against bacteria were screened from 113 strains.S9、S15、S24 and S70 had intense anti-plant pathogenic fungi and anti-human pathogenic fungi ability,and S9 have a broad-spectrum antimicrobial activity.
3.Growth characteristic test have been examine by the changes of saccharide value,biomass,pH value and minimum inhibitory concentration.we find Logarithmic phase are in the first to third day and Stable phase are in the fourth to sixth day and from the seventh day is Decline phase.During the whole Growth cycle,there are big changes in saccharide value,biomass,pH value and minimum inhibitory concentration.
4.The stability of S9 have been studied by temperature、illumination、pH and existing from generation to generation.S9 have a good stability on temperature、illumination and generation.However,pH has great effect on antimicrobial activity of S9,which has greater antimicrobial activity in acidic environment than in alkaline environment.The activity components are outside cell but not the Macromolecules such as Polysaccharide and Protein.
5.Optimize fermentation conditions by Single factor test,Plackett-Burman and Response Surface Method,the optimization of fermentation conditions for S9 is 28℃, 140 r/min,saccharose 26 g·L~(-1),KH_2PO_4 1mmol·L~(-1),potato 250 g·L~(-1),Yeast extract 20 g·L~(-1).
6.Active systematic separation portions of six strains of S9 were traced in order to determined active intensity with indicator and found the Petroleum ether has the strongest antimicrobial activity.The activity components are analyzed by GC-MS.Strain identification by ITS sequence analysis and Colony morphology,S9 belong to Trichoderma sp.and is similar with Hypocrea lixii sp.The key activity components analyzed by GC-MS are 2-(2,2-dimethylpropylidene)cyclohexane-1,3-dione.
7.Active systematic separation portions of six strains of Acorus calamus Linn. were traced in order to determined active intensity with indicator and found the Petroleum ether has the strongest antimicrobial activity.The key activity components analyzed by GC-MS are 1-methoxy-4-phenylethynyl-Benzene.
植物内生菌能产生大量新的天然产物,有些内生菌还可以产生与宿主植物相同或相近的代谢产物,已成为天然产物潜在的重要资源。本研究对水菖蒲内生菌进行了分离,得到113株内生菌,以大肠杆菌和金黄色葡萄球菌为指示菌对其抗菌活性进行筛选,研究了菌株S9,S15,S24和S70的抗菌活性。发现S9具有广谱的抑菌活性,对其进行了抑菌活性物质与菌株鉴定、生理条件与稳定性,培养基条件的优化以及水菖蒲抑菌活性的相关研究。本研究旨在探索从水菖蒲内生菌中发现抑菌活性成分的可能性,为研究新的天然产物抑菌药物提供思路。主要研究内容如下:
(1)采用75%乙醇、次氯酸钠溶液浸泡和无菌水洗涤、培养、分离的方法从水菖蒲内分离内生菌。研究表明:应同时设置超净工作台无菌状态检测对照、漂洗液检测对照和组织印迹检测对照等3种对照处理,确保内生菌分离准确性。水菖蒲体内存在内生菌且种类繁多、十分丰富,共分离到113株内生菌,根茎、叶中均有分布,叶中的内生菌数量明显少于茎中。
(2)用大肠杆菌、金黄色葡萄球菌和稻瘟霉作为初筛指示菌,分别代表了G~+,G~-菌,真菌,对分离得到的113株水菖蒲内生菌进行了抗菌活性初筛,有50株菌对稻瘟霉P-2b具有抑制作用,46株对大肠杆菌或金黄色葡萄球菌有抗菌活性的菌株,分别占所有菌株的44.2%和40.7%,表明水菖蒲中有丰富的有抑菌活性的内生菌资源。对抑菌效果最强的四株菌—S9、S15、S24、S70,进行了进一步的抗菌能力研究。结果表明,S9对多种指示菌都有很好的抑制和杀灭效果。
(3)研究了活性菌株S9发酵1-14天的生长特性,并检测了生物量,pH值,残糖量和对指示菌的最小抑菌浓度。结果显示发酵液在1-3天进入对数增长期,第4-6d为稳定期,从第7d开始进入衰亡期,在整个过程中,生物量、pH、发酵液中的残糖量和抑菌活性变化显著。
(4)研究了S9发酵液对温度、光照、pH值和传代等的稳定性。结果表明,发酵液对温度、光照有较好的稳定性,1-7代内菌株抑菌活性无明显改变。但pH值对发酵液的活性影响较大,在偏酸的环境下,活性较好,在碱性环境下无活性。并初步研究了S9发酵液的活性部位,结果表明S9发酵液的活性成分存在于胞外产物,胞内物质基本无抑菌活性;同时,发酵液中的多糖蛋白等大分子物质无抑菌活性。
(5)通过单因素试验,Plackett-Burman试验和响应面法对水菖蒲内生菌S9的发酵条件进行了优化,结果表明水菖蒲内生菌S9的最佳发酵条件为温度28℃,蔗糖用量26 g·L~(-1),pH值为6.7,培养基装液量50mL,转速140 r/min,KH_2PO_4为1mmol/L,马铃薯加入量为250 g·L~(-1),酵母膏的含量20 g·L~(-1)。
(6)采用五部分离法,研究了水菖蒲内生菌S9发酵产物各极性部位的抑菌活性,初步判定其最强的抑菌活性部位为石油醚相,对各种细菌、丝状真菌和酵母菌均有不同程度的抑制作用,对各种指示细菌的最小抑菌浓度(MIC)为15-30μg·mL~(-1),对指示细菌的最小杀菌浓度为30-60μg·mL~(-1);对四种酵母样真菌的最小抑菌浓度为12.5-50μg·mL~(-1):对六种丝状真菌的最小抑菌活性为15-500μg·mL~(-1)。并通过形态鉴定和ITS序列分析,鉴定水菖蒲内生菌S9为木霉属,其与Hypocrea lixii sp.属中的各菌株具有极高的同源性高达98%。通过GC-MS分析,结果表明其石油醚相的主要成分为2-(2,2-二甲基亚丙基)环己烷-1,3-二酮。
(7)研究了S9内生菌的宿主植物水菖蒲各极性部位的抑菌活性,其中石油醚相对各种指示细菌和丝状真菌表现出了较好的抑菌效果,并对其相进行的GC-MS分析,结果表明其主要成分为1-甲氧基-4-乙炔基苯。
As Traditional Chinese medicine,Acorus calamus Linn.grows in sullage and has a strong antimicrobial activity.
Some endophytic fungi could produce large new natural products,some metabolite produced by endophytic fungi were identical or semblable with host plant.It now appears that endophytes is an enormous,relatively untapped source of diversity the microbial.In this paper the endophytes from Acorus calamus Linn.were isolated for the first time.With high throughput microdilution method,the endophytes of antifungal,antimitotic and antimicrobial activity were preliminary screened by model of PyricuLaria oryzae P-2b and bacterium,through detecting deformation of mycelia germinated from conidia of P. Oryzae P-2b.S9 strain possessed strongest antimicrobial activity to broad-spectrum bacterium,yeast and mycelial fungi.
The further research was made for the activity substance,classification,stability, Optimization of fermentation conditions and the plant for antimicrobial activity.This research is for the purpose of exploring for endophytes from Acorus calamus Linn.to produce antimicrobial activity constituent possibility,providing the idea for studies the new resource to search new antimicrobial compounds.
Endophyte is a new resource of antimicrobial activity and potential for application.
Main contents of this thesis as follows:
1.The plant materials are thoroughly surface treated with 75%ethanol,sodium hypochlorite solution and sterilizated water.Controlled experiments that detecting of biologic safe cabinet,rinse water and tissue blotting were set simultaneously were extremely essential.There are a great variety of endophytes in Acorus calamus Linn. 113 strains were isolated from stem and leaf.
2.With high throughput microdilution method and screening model of P.Oryzae P-2b and bacteria,50 strains against P.Oryzae P-2b,46 strains against bacteria were screened from 113 strains.S9、S15、S24 and S70 had intense anti-plant pathogenic fungi and anti-human pathogenic fungi ability,and S9 have a broad-spectrum antimicrobial activity.
3.Growth characteristic test have been examine by the changes of saccharide value,biomass,pH value and minimum inhibitory concentration.we find Logarithmic phase are in the first to third day and Stable phase are in the fourth to sixth day and from the seventh day is Decline phase.During the whole Growth cycle,there are big changes in saccharide value,biomass,pH value and minimum inhibitory concentration.
4.The stability of S9 have been studied by temperature、illumination、pH and existing from generation to generation.S9 have a good stability on temperature、illumination and generation.However,pH has great effect on antimicrobial activity of S9,which has greater antimicrobial activity in acidic environment than in alkaline environment.The activity components are outside cell but not the Macromolecules such as Polysaccharide and Protein.
5.Optimize fermentation conditions by Single factor test,Plackett-Burman and Response Surface Method,the optimization of fermentation conditions for S9 is 28℃, 140 r/min,saccharose 26 g·L~(-1),KH_2PO_4 1mmol·L~(-1),potato 250 g·L~(-1),Yeast extract 20 g·L~(-1).
6.Active systematic separation portions of six strains of S9 were traced in order to determined active intensity with indicator and found the Petroleum ether has the strongest antimicrobial activity.The activity components are analyzed by GC-MS.Strain identification by ITS sequence analysis and Colony morphology,S9 belong to Trichoderma sp.and is similar with Hypocrea lixii sp.The key activity components analyzed by GC-MS are 2-(2,2-dimethylpropylidene)cyclohexane-1,3-dione.
7.Active systematic separation portions of six strains of Acorus calamus Linn. were traced in order to determined active intensity with indicator and found the Petroleum ether has the strongest antimicrobial activity.The key activity components analyzed by GC-MS are 1-methoxy-4-phenylethynyl-Benzene.
引文
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