| 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. |
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