皱边石杉内生菌分离纯化及其产物四草酸钾的发酵条件研究
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摘要
本研究在建立皱边石杉组织培养体系时发现,尽管采用了各种灭菌方法,植株总是带菌。这提示皱边石杉健康植株中生活着内生菌。特定内生真菌可能在与宿主植物长期协同进化中由于遗传信息的传递获得了宿主植物的特定代谢途径,从而使内生真菌产生与宿主相同或相似的生理活性成分。本研究从皱边石杉内生菌株分离纯化入手,用以皱边石杉氯仿提取物为唯一能源的压力筛选手段对所分离的菌株做针对性筛选。发现菌株J 060918在适宜的发酵培养条件下可生产四草酸钾。并研究了生物发酵生产四草酸钾的关键工艺技术参数。得到了以下结论:
1皱边石杉拥有丰富的内生菌群
为获得皱边石杉及根际土壤中丰富的真菌群,筛选具有合成有益功能产物的菌种,本研究采用经典PDA固体培养基对皱边石杉内生菌进行了初步分离纯化,共得到了32种菌株,个体之间外观形态、培养特征差异明显。经初步镜检,可以确定为绵霉、毛霉、曲霉、青霉、红酵母等种属。
2菌株J 060918具有生产四草酸钾的功能
为对皱边石杉中分离得到的32种菌株进行定向筛选,本研究以皱边石杉的氯仿提取物作为唯一碳源进行压力筛选,发现仅有6种能相对较好地生长,菌株J 060918在适宜的发酵培养条件下可合成四草酸钾,产量在1.758g/L以上;菌株经初步形态学鉴定为黑曲霉(Aspergillus niger)。
3优化得到四草酸钾生物发酵工艺技术参数
为充分研究从皱边石杉外植体上分离纯化得到的菌株J 060918的生物发酵功能,探索其较适工艺技术参数。在单因素试验基础上,设计了正交试验,进行了四草酸钾的发酵工艺研究。研究结果表明:以马铃薯液体培养基为基本培养基,接种孢子浓度为1×10~5个/mL的孢子悬液4 mL到1 L培养基中,添加蔗糖10 g,初始pH值为自然值,120 r/min,40℃恒温培养60小时。每升培养体系,至少可生产四草酸钾粗品1.758g。
When we attempt to establish the plant tissue culture system,We find that the explant always infected no matter how strict to disinfect,so we speculate on the probability that endophyte is exist in the living plant tissure.It seems to be probable that some special endophyte fungus can produce some bioactive components which are similar to the components produced by its host plant as a result of inheritance information exchange between endophyte and host plants when they evolve cooperated with each other.We use the extraction of Huperzia crispata(Ching) Ching with chloroform exclusively to filter the fungus starting with isolation and purification of endophytic fungus.When we study on the endophyte variety of Huperzia crispata(Ching) Ching and bioactive metabolltes producing by these endophytes,We find out a kind of fungi,it can produce Potassium Hydrogen Oxalate Hydrate under appropriate fermentation condition.At the same time,We has explored the optimized fermentation condition to producing Potassium Hydrogen Oxalate Hydrate and obtained the following conclusion.
1 Huperzia crispata(Ching) Ching holds abundant endophyte community
We have obtained 32 kinds of endophytes separated from Huperzia crispata (Ching)Ching by using classical PDA solid culture medium,there are obvious contrast on the aspect of appearance,form and culture character among these 32 kinds of endophyte.These endophytes has been preliminary identified to be Achlya,Mucor, Aspergillus,Penicillium,Rhodotorula and so on.
2 Fungi J 060918 possesses the capability to produce Potassium Hydrogen Oxalate Hydrate
We have found that only 6 kinds fungi can grow up well on the hungriness culture medium which contains the Huperzia crispata(Ching) Ching chloroform extraction exclusively.We have found the fungi J 060918,It can produce Potassium Hydrogen Oxalate Hydrate with high yield(＞1.758g).This fungi has been preliminary identified to be Aspergillus niger.
3 Optimized fermentation technics to produce Potassium Hydrogen Oxalate Hydrate
We have optimized the fermentation condition from the aspects of carbon source variety,dosage of carbon source,culture time,primary pH,dosage of spore and so on. And designed orthogonal optimization experiment based on these experiment.The result indicate that the best fermentation condition is the following condition:PDA is the basal medium,the best concentration of fungi spore is 1×10~5/mL,used as 4 mL per 1 L to the medium.Dosage of sucrose is 10 g/L.nature pH,And the best culture time and temperature is 60 h and 40℃.120 r/min is the best Rolling rate.We can obtain crude Potassium Hydrogen Oxalate Hydrate sample 1.758 g/L at least under this optimized fermentation condition.
1皱边石杉拥有丰富的内生菌群
为获得皱边石杉及根际土壤中丰富的真菌群,筛选具有合成有益功能产物的菌种,本研究采用经典PDA固体培养基对皱边石杉内生菌进行了初步分离纯化,共得到了32种菌株,个体之间外观形态、培养特征差异明显。经初步镜检,可以确定为绵霉、毛霉、曲霉、青霉、红酵母等种属。
2菌株J 060918具有生产四草酸钾的功能
为对皱边石杉中分离得到的32种菌株进行定向筛选,本研究以皱边石杉的氯仿提取物作为唯一碳源进行压力筛选,发现仅有6种能相对较好地生长,菌株J 060918在适宜的发酵培养条件下可合成四草酸钾,产量在1.758g/L以上;菌株经初步形态学鉴定为黑曲霉(Aspergillus niger)。
3优化得到四草酸钾生物发酵工艺技术参数
为充分研究从皱边石杉外植体上分离纯化得到的菌株J 060918的生物发酵功能,探索其较适工艺技术参数。在单因素试验基础上,设计了正交试验,进行了四草酸钾的发酵工艺研究。研究结果表明:以马铃薯液体培养基为基本培养基,接种孢子浓度为1×10~5个/mL的孢子悬液4 mL到1 L培养基中,添加蔗糖10 g,初始pH值为自然值,120 r/min,40℃恒温培养60小时。每升培养体系,至少可生产四草酸钾粗品1.758g。
When we attempt to establish the plant tissue culture system,We find that the explant always infected no matter how strict to disinfect,so we speculate on the probability that endophyte is exist in the living plant tissure.It seems to be probable that some special endophyte fungus can produce some bioactive components which are similar to the components produced by its host plant as a result of inheritance information exchange between endophyte and host plants when they evolve cooperated with each other.We use the extraction of Huperzia crispata(Ching) Ching with chloroform exclusively to filter the fungus starting with isolation and purification of endophytic fungus.When we study on the endophyte variety of Huperzia crispata(Ching) Ching and bioactive metabolltes producing by these endophytes,We find out a kind of fungi,it can produce Potassium Hydrogen Oxalate Hydrate under appropriate fermentation condition.At the same time,We has explored the optimized fermentation condition to producing Potassium Hydrogen Oxalate Hydrate and obtained the following conclusion.
1 Huperzia crispata(Ching) Ching holds abundant endophyte community
We have obtained 32 kinds of endophytes separated from Huperzia crispata (Ching)Ching by using classical PDA solid culture medium,there are obvious contrast on the aspect of appearance,form and culture character among these 32 kinds of endophyte.These endophytes has been preliminary identified to be Achlya,Mucor, Aspergillus,Penicillium,Rhodotorula and so on.
2 Fungi J 060918 possesses the capability to produce Potassium Hydrogen Oxalate Hydrate
We have found that only 6 kinds fungi can grow up well on the hungriness culture medium which contains the Huperzia crispata(Ching) Ching chloroform extraction exclusively.We have found the fungi J 060918,It can produce Potassium Hydrogen Oxalate Hydrate with high yield(＞1.758g).This fungi has been preliminary identified to be Aspergillus niger.
3 Optimized fermentation technics to produce Potassium Hydrogen Oxalate Hydrate
We have optimized the fermentation condition from the aspects of carbon source variety,dosage of carbon source,culture time,primary pH,dosage of spore and so on. And designed orthogonal optimization experiment based on these experiment.The result indicate that the best fermentation condition is the following condition:PDA is the basal medium,the best concentration of fungi spore is 1×10~5/mL,used as 4 mL per 1 L to the medium.Dosage of sucrose is 10 g/L.nature pH,And the best culture time and temperature is 60 h and 40℃.120 r/min is the best Rolling rate.We can obtain crude Potassium Hydrogen Oxalate Hydrate sample 1.758 g/L at least under this optimized fermentation condition.
引文
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