提高盾叶薯蓣内生真菌Dzf12中螺二萘类化合物palmarumycins C_(12)和C_(13)产量的策略
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摘要
药用植物内生真菌是新型天然活性物质的重要来源,其在农业、医药以及食品工业等领域具有潜在的应用前景。利用内生真菌发酵来实现活性物质的工业化生产,可以提高其产量,同时对减少野生药用植物多样性的破坏具有重要意义。螺二萘化合物是一类具有抗菌、抗癌、抗细胞毒和抗寄生虫等活性的萘醌类衍生物。前期研究发现,盾叶薯蓣内生真菌Dzfl2是一株螺二萘类化合物的高产菌株,具有良好的研究价值。本文以盾叶薯蓣内生真菌Dzf12为研究对象,分别制备了其宿主植物的多糖和寡糖,研究了多糖和寡糖对内生真菌Dzf12菌丝生长和螺二萘类化合物palmarumycins C12和C13产量的影响,评价了多糖的抗氧化活性,优化了水提多糖的提取制备工艺,同时探讨了表面活性剂和氧载体对内生真菌Dzf12菌丝生长和螺二萘类化合物palmarumycins C12和C13产量的影响。通过实验研究,以期为宿主植物与内生真菌之间相互关系的研究提供依据,为Dzf12菌株中活性产物palmarumycins C12和C13的大规模工业化生产打下基础,为盾叶薯蓣抗氧化活性多糖和寡糖的开发利用奠定基础。本研究获得的主要结果如下：
1、通过制备盾叶薯蓣水提多糖(WEP)、碱提多糖(SEP)和酸提多糖(AEP)分别与内生真菌Dzfl2发酵共培养,发现WEP对内生真菌Dzf12的菌丝生物量和螺二萘类化合物palmarumycins C12和C13产量具有显著的促进作用。当添加400mg/L的WEP到培养了3d的培养液中,培养15d后可使palmarumycins C12和C13的产量分别达67.73和350.76mg/L,分别是对照的8.76和2.69倍。
2、研究了寡糖对内生真菌Dzf12菌丝生长及次生代谢产物的影响。通过制备水提多糖(WEP)、碱提多糖(SEP)和酸提多糖(AEP)相应的寡糖(OW, OS和OA)与内生真菌Dzfl2共培养。在三种寡糖中,发现OW对内生真菌Dzfl2的菌丝生物量和螺二萘类化合物palmarumycins C12和C13产量的促进作用最显著。当在第3d添加300mg/L的OW到Dzfl2培养液中,培养15d后可使palmarumycins C12和C13的产量分别达87.96和422.28mg/L,分别是对照的9.83和3.24倍。
3、对WEP、SEP和AEP的体外抗氧化活性进行了评价。发现盾叶薯蓣水提多糖WEP在DPPH自由基清除模型、Fe3+还原模型、Fe2+螯合模型和.OH自由基清除模型均表现出较好的活性,其EC50值分别为661.19、413.86、22.36和135.27μg/mL；SEP在Fe2+螯合模型和-OH自由基清除模型中的EC50分别为885.44和412.36μg/mL；AEP在Fe2+螯合模型和-OH自由基清除模型中的EC50分别为771.37和600.08μg/mL。
4、对盾叶薯蓣水提多糖WEP的提取工艺过程进行了优化。通过单因素实验,确定了盾叶薯蓣水提多糖WEP得率有显著影响的取值范围,分别为：料液比1：20～1：60g：mL、提取温度60～100℃、提取时间60～180min。并且采用中心组合设计(CCD)和响应面分析方法(RSM)进一步优化了研究变量,建立了WEP得率与研究变量之间的二次多项回归方程,确定了WEP的最佳提取条件为：料水比为1：52g：mL、提取温度为94℃、提取时间为147min时,WEP得率达到最大值10.77%。
5、研究了三种不同表面活性剂和氧载体对盾叶薯蓣内生真菌Dzf12产螺二萘类化合物的影响。结果表明,Tween-20(0.2%,v/v)和液体石蜡(4%,v/v)的效果最好,palmarumycins C12和C13的总产量分别是344.25和353.89mg/L；然后对其不同添加天数进行优化,发现在第6d添加,其总产量分别是350.71和373.69mg/L。在加入液体石蜡的同时,再加入表面活性剂Tween-20,palmarumycins C12和C13的总产量为402.09mg/L,与对照(138.50mg/L)相比,产量提高了2.90倍。
Plant endophytic fungi are an important and novel resource of natural bioactive compounds with great potential applications prospect in agriculture, medicine and food industry. The industrial productions of bioactive substances that make use of endophytic fungi are capable of increasing their yields. In the meantime, it is also very important in avoiding the damage to the diversity of wild medicinal plants. Spirobisnaphthalenes are a group of naphthaquinone derivatives with antimicrobial, antitumor, cytotoxic and antiparasite activities. Endophytic fungus Dzf12derived from Dioscorea zingiberensis C. H. Wright was found to be a high producer of spirobisnaphthalenes in our previous investigation, and it had a good development and utilization of value. This study takes Berkleasmium sp. Dzf12which were isolated from the rizhomes of D. zingiberensis as study object, polysaccharides and oligosaccharides were prepared from the rizhomes of D. zingiberensis. The effects of these saccharide elicitors on mycelia growth and palmarumycins C12and C13of Berkleasmium sp. Dzf12were investigated. The antioxidant activities of polysaccharides from D. zingiberensis were also evaluated. And the preparation process of polysaccharide possessing excellent biological activities was optimized. This study also discussed the enhancement of palmarumycins C12and C13production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12by surfactants and oxygen vector. This research could not only provide promising baseline information for the study of interaction between the host plant (D. zingiberensis) and the endophytic fungi, but also establish the foundation for the large-scale industrial production of active product(palmarumycins C12and C13) as well as for the exploitation and utilization of D. zingiberensis polysaccharides and oligosaccharides with excellent biological activities.The main results are as follows:
1. In order to study the effects of the extracts of host plant D. zingiberensis on secondary metabolites in liquid culture of Berkleasmium sp. Dzf12, Three polysaccharides, namely water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide(SEP) and acid-extracted polysaccharide(AEP) were sequentially prepared from the rhizomes of D. zingiberensis. Among them, WEP was found to be the most effective elicitor to enhance palmarumycins C12and C13production. When WEP was added to the medium at400mg/L on day3of culture, the maximal palmarumycins C12and C13yield (intracellular C13in mycelia plus extracellular C13in medium)of67.73and350.76mg/L on day15was achieved, which was about8.76-fold and2.69-fold in comparison with the control.
2. To investigate further the effects of oligosaccharides from its host plant D. zingiberensis on secondary metabolites in liquid culture of Berkleasmium sp. Dzfl2. Three crude oligosaccharides were respectively prepared by acid hydrolysis of three polysaccharides, which were water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide (SEP) and acid-extracted polysaccharide (AEP) from the rhizomes of D. zingiberensis. Among the three oligosaccharides, the crude oligosaccharide prepared by acid hydrolysis of WEP was found to be the most efficient elicitor to enhance the production of palmarumycins C12and C13in liquid culture of endophytic fungus Berkleasmium sp. Dzfl2. When OW was applied to the medium at300mg/L on day3of culture, the maximal yields of palmarumycin C12(87.96mg/L) and palmarumycin C13(422.28mg/L) were achieved on day15of culture, which were9.83and3.24-fold in comparison with control, respectively.
3. The in vitro antioxidant acitivities of WEP、SEP and AEP were respectively evaluated. WEP from D. zingiberensis showed excellent activities in the DPPH radical scavenging activity, reducing Fe3+power, chelating Fe2+ability, and hydroxyl radical scavenging activity assays, with the corresponding EC50as661.19、413.86、22.36and135.27μg/mL. SEP also exhibited antioxidant activities in chelating Fe2+ability and hydroxyl radical scavenging activity assays, with the corresponding EC50values as885.44and412.36μg/mL; AEP also exhibited antioxidant activities in chelating Fe2+ability and hydroxyl radical scavenging activity assays, with the corresponding EC50values as771.37and600.08μg/mL.
4. The extraction process of WEP from D. zingiberensis were optimized. The factors and their ranges which could significantly affect the yield of WEP were determined by single-factor experiments, which were respectively extraction ratio of water to raw materials1:20-60(g:mL), extraction temperature60-100℃, extraction time60～180min,. And then central composite design (CCD) and response surface methodology (RSM) were employed to optimize the tested variables and establish the second-order polynomial regression equation between the WEP yiled and the tested variables. The optimal extraction parameters for WEP extraction were determined as1:52(g:mL)for ratio of raw material weight (g) to water volume (mL),147min for extraction time and'94℃for extraction temperature, when the WEP yield arrived at the maximum10.77%.
5. In order to investigate the effects of surfactants and oxygen vectors on mycelia growth and palmarumycins C12and C13in liquid culture of Berkleasmium sp. Dzfl2. While all organic liquids showed a positive effect, Tween-20(0.2%, v/v) and liquid paraffin (4%, v/v) was proved to be the most beneficial for the mycelia growth and palmarumycins C12and C13production, the maximal yield of palmarumycins344.25and353.89mg/L, respectively. And then, the addition time to the mycelial liquid culture of endophytic fungus Dzfl2were further evaluated and optimized. When the0.2%Tween-20and4%liquid paraffin were added to medium in each flask on day6, the palmarumycins yield was increased to350.71and373.69mg/L, respectively. The total palmarumycins yield(402.09mg/L) were2.90-fold in comparison with control (138.50mg/L) when Tween-20and liquid paraffin were used at the same time.
1、通过制备盾叶薯蓣水提多糖(WEP)、碱提多糖(SEP)和酸提多糖(AEP)分别与内生真菌Dzfl2发酵共培养,发现WEP对内生真菌Dzf12的菌丝生物量和螺二萘类化合物palmarumycins C12和C13产量具有显著的促进作用。当添加400mg/L的WEP到培养了3d的培养液中,培养15d后可使palmarumycins C12和C13的产量分别达67.73和350.76mg/L,分别是对照的8.76和2.69倍。
2、研究了寡糖对内生真菌Dzf12菌丝生长及次生代谢产物的影响。通过制备水提多糖(WEP)、碱提多糖(SEP)和酸提多糖(AEP)相应的寡糖(OW, OS和OA)与内生真菌Dzfl2共培养。在三种寡糖中,发现OW对内生真菌Dzfl2的菌丝生物量和螺二萘类化合物palmarumycins C12和C13产量的促进作用最显著。当在第3d添加300mg/L的OW到Dzfl2培养液中,培养15d后可使palmarumycins C12和C13的产量分别达87.96和422.28mg/L,分别是对照的9.83和3.24倍。
3、对WEP、SEP和AEP的体外抗氧化活性进行了评价。发现盾叶薯蓣水提多糖WEP在DPPH自由基清除模型、Fe3+还原模型、Fe2+螯合模型和.OH自由基清除模型均表现出较好的活性,其EC50值分别为661.19、413.86、22.36和135.27μg/mL；SEP在Fe2+螯合模型和-OH自由基清除模型中的EC50分别为885.44和412.36μg/mL；AEP在Fe2+螯合模型和-OH自由基清除模型中的EC50分别为771.37和600.08μg/mL。
4、对盾叶薯蓣水提多糖WEP的提取工艺过程进行了优化。通过单因素实验,确定了盾叶薯蓣水提多糖WEP得率有显著影响的取值范围,分别为：料液比1：20～1：60g：mL、提取温度60～100℃、提取时间60～180min。并且采用中心组合设计(CCD)和响应面分析方法(RSM)进一步优化了研究变量,建立了WEP得率与研究变量之间的二次多项回归方程,确定了WEP的最佳提取条件为：料水比为1：52g：mL、提取温度为94℃、提取时间为147min时,WEP得率达到最大值10.77%。
5、研究了三种不同表面活性剂和氧载体对盾叶薯蓣内生真菌Dzf12产螺二萘类化合物的影响。结果表明,Tween-20(0.2%,v/v)和液体石蜡(4%,v/v)的效果最好,palmarumycins C12和C13的总产量分别是344.25和353.89mg/L；然后对其不同添加天数进行优化,发现在第6d添加,其总产量分别是350.71和373.69mg/L。在加入液体石蜡的同时,再加入表面活性剂Tween-20,palmarumycins C12和C13的总产量为402.09mg/L,与对照(138.50mg/L)相比,产量提高了2.90倍。
Plant endophytic fungi are an important and novel resource of natural bioactive compounds with great potential applications prospect in agriculture, medicine and food industry. The industrial productions of bioactive substances that make use of endophytic fungi are capable of increasing their yields. In the meantime, it is also very important in avoiding the damage to the diversity of wild medicinal plants. Spirobisnaphthalenes are a group of naphthaquinone derivatives with antimicrobial, antitumor, cytotoxic and antiparasite activities. Endophytic fungus Dzf12derived from Dioscorea zingiberensis C. H. Wright was found to be a high producer of spirobisnaphthalenes in our previous investigation, and it had a good development and utilization of value. This study takes Berkleasmium sp. Dzf12which were isolated from the rizhomes of D. zingiberensis as study object, polysaccharides and oligosaccharides were prepared from the rizhomes of D. zingiberensis. The effects of these saccharide elicitors on mycelia growth and palmarumycins C12and C13of Berkleasmium sp. Dzf12were investigated. The antioxidant activities of polysaccharides from D. zingiberensis were also evaluated. And the preparation process of polysaccharide possessing excellent biological activities was optimized. This study also discussed the enhancement of palmarumycins C12and C13production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12by surfactants and oxygen vector. This research could not only provide promising baseline information for the study of interaction between the host plant (D. zingiberensis) and the endophytic fungi, but also establish the foundation for the large-scale industrial production of active product(palmarumycins C12and C13) as well as for the exploitation and utilization of D. zingiberensis polysaccharides and oligosaccharides with excellent biological activities.The main results are as follows:
1. In order to study the effects of the extracts of host plant D. zingiberensis on secondary metabolites in liquid culture of Berkleasmium sp. Dzf12, Three polysaccharides, namely water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide(SEP) and acid-extracted polysaccharide(AEP) were sequentially prepared from the rhizomes of D. zingiberensis. Among them, WEP was found to be the most effective elicitor to enhance palmarumycins C12and C13production. When WEP was added to the medium at400mg/L on day3of culture, the maximal palmarumycins C12and C13yield (intracellular C13in mycelia plus extracellular C13in medium)of67.73and350.76mg/L on day15was achieved, which was about8.76-fold and2.69-fold in comparison with the control.
2. To investigate further the effects of oligosaccharides from its host plant D. zingiberensis on secondary metabolites in liquid culture of Berkleasmium sp. Dzfl2. Three crude oligosaccharides were respectively prepared by acid hydrolysis of three polysaccharides, which were water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide (SEP) and acid-extracted polysaccharide (AEP) from the rhizomes of D. zingiberensis. Among the three oligosaccharides, the crude oligosaccharide prepared by acid hydrolysis of WEP was found to be the most efficient elicitor to enhance the production of palmarumycins C12and C13in liquid culture of endophytic fungus Berkleasmium sp. Dzfl2. When OW was applied to the medium at300mg/L on day3of culture, the maximal yields of palmarumycin C12(87.96mg/L) and palmarumycin C13(422.28mg/L) were achieved on day15of culture, which were9.83and3.24-fold in comparison with control, respectively.
3. The in vitro antioxidant acitivities of WEP、SEP and AEP were respectively evaluated. WEP from D. zingiberensis showed excellent activities in the DPPH radical scavenging activity, reducing Fe3+power, chelating Fe2+ability, and hydroxyl radical scavenging activity assays, with the corresponding EC50as661.19、413.86、22.36and135.27μg/mL. SEP also exhibited antioxidant activities in chelating Fe2+ability and hydroxyl radical scavenging activity assays, with the corresponding EC50values as885.44and412.36μg/mL; AEP also exhibited antioxidant activities in chelating Fe2+ability and hydroxyl radical scavenging activity assays, with the corresponding EC50values as771.37and600.08μg/mL.
4. The extraction process of WEP from D. zingiberensis were optimized. The factors and their ranges which could significantly affect the yield of WEP were determined by single-factor experiments, which were respectively extraction ratio of water to raw materials1:20-60(g:mL), extraction temperature60-100℃, extraction time60～180min,. And then central composite design (CCD) and response surface methodology (RSM) were employed to optimize the tested variables and establish the second-order polynomial regression equation between the WEP yiled and the tested variables. The optimal extraction parameters for WEP extraction were determined as1:52(g:mL)for ratio of raw material weight (g) to water volume (mL),147min for extraction time and'94℃for extraction temperature, when the WEP yield arrived at the maximum10.77%.
5. In order to investigate the effects of surfactants and oxygen vectors on mycelia growth and palmarumycins C12and C13in liquid culture of Berkleasmium sp. Dzfl2. While all organic liquids showed a positive effect, Tween-20(0.2%, v/v) and liquid paraffin (4%, v/v) was proved to be the most beneficial for the mycelia growth and palmarumycins C12and C13production, the maximal yield of palmarumycins344.25and353.89mg/L, respectively. And then, the addition time to the mycelial liquid culture of endophytic fungus Dzfl2were further evaluated and optimized. When the0.2%Tween-20and4%liquid paraffin were added to medium in each flask on day6, the palmarumycins yield was increased to350.71and373.69mg/L, respectively. The total palmarumycins yield(402.09mg/L) were2.90-fold in comparison with control (138.50mg/L) when Tween-20and liquid paraffin were used at the same time.
引文
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