【作者】 陈宏伟；

【导师】 樊美珍； 李增智；

【作者基本信息】 安徽农业大学 ， 微生物学， 2008， 硕士

【摘要】 蛹虫草（Cordyceps militaris（L.）Link）是虫草属真菌的模式种,含有多种活性物质,具有增强免疫力、消炎、抗疲劳、抗氧化、抗衰老、抗突变、抗肿瘤等多种功效,已有研究表明其成分和功效不亚于著名的冬虫夏草,而且某些成分含量还高于冬虫夏草。另有研究证实蛹虫草无性型液体发酵的菌丝体成分与野生冬虫夏草的成分相近,具有与野生冬虫夏草相似的生理功效。在野生资源日益匮乏的情况下,研究开发人工虫草,选育优良虫草菌株,探讨液体发酵条件、研究菌丝体活性成分、含量及生理功能对进一步利用和开发虫草资源具有重要的理论意义和深远的现实意义。本研究以蛹虫草（Cordyceps militaris（L.）Link）无性型——蛹拟青霉（Paecilomyces militaris（Kob.）Liang）为材料,利用正交实验研究了蛹虫草无性型液体发酵产胞、内外多糖和生物量的最佳条件;利用蚕豆根尖细胞微核实验探讨了虫草胞、内外多糖的抗突变特性;采用多糖喂食果蝇并利用试剂盒测定其体内SOD、MDA的含量,分析了乙醇分级多糖对果蝇体内SOD和MDA含量的影响,同时通过测定多糖对羟自由基、超氧自由基和有机自由基DPPH的清除能力,确定乙醇分级多糖的抗氧化性能;研究了蛹虫草原生质体的形成和再生条件,并以原生质体诱变的方法对多糖高产菌株进行了选育,为进一步开发利用虫草资源奠定了理论基础。研究结果表明:1、蛹虫草液体发酵条件蛹虫草菌丝体液体深层发酵产胞内、外多糖及菌丝体产量最佳培养基为:蔗糖5.0%,玉米浆2.0%,酵母浸出汁0.03%,KNO₃ 0.08%,KH₂PO₄ 0.25%,NH₄NO₃ 0.04%,FeSO₄·7H₂O 0.08%,MgSO₄·7H₂O 0.15%;最佳培养条件为:温度22℃;接种量10%-15%,pH 6.0,深层发酵5 d。每100 mL发酵液得到胞内多糖为170.3 mg,胞外多糖为114.8 mg,菌丝体干重为546 mg。2、蛹虫草胞内、外多糖的组成成分均为:葡萄糖、半乳糖和甘露糖。3、蛹虫草胞内、胞外多糖对化学因素的抗突变作用（1）蛹虫草胞内、胞外多糖均可有效抑制丝裂霉素诱导的蚕豆根尖细胞微核的产生,且在实验浓度内有正向剂量-效应关系。在胞内、胞外多糖实验浓度为100μg/mL时抑制微核产生的效果最佳,其蚕豆根尖细胞微核抑制率分别为60.48%和62.20%。（2）蛹虫草胞内、外多糖对环磷酰胺诱导的蚕豆根尖细胞微核具有显著的抑制作用,其抑制作用随着多糖浓度的升高而增强,当胞内、外多糖实验浓度为100μg/mL,对环磷酰胺诱导的蚕豆根尖细胞微核的抑制率分别为53.52%和52.11%,虫草多糖具有显著的抑制突变作用。4、蛹虫草胞内、胞外多糖对物理因素的抗突变作用蛹虫草胞内、外多糖均可有效抑制由紫外线照射所诱发的蚕豆根尖细胞微核的产生,且在实验浓度范围内存在一定的正向剂量-效应关系。当蛹虫草胞内、外多糖浓度为100μg/mL时对蚕豆根尖细胞微核的抑制率分别达到了47.68%和43.04%。5、蛹虫草醇沉分级多糖的抗氧化作用（1）70%的乙醇醇沉虫草多糖量最多,可使胞内、外多糖的得率分别为1.513 mg/mL和0.483 mg/mL。（2）不同浓度的乙醇醇沉虫草胞外多糖均具有较好的抗氧化性。可以提高果蝇体内的SOD含量,降低MDA含量,70%的乙醇醇沉虫草胞外多糖可以使果蝇体内SOD含量比对照组高13.13%;使果蝇体内的MDA含量比对照组低67.2%,且多糖添加量为2%的比1%的抗氧化效果好。（3）50%乙醇醇沉虫草胞内多糖具有较好的DPPH清除作用,当多糖浓度为1mg/mL时,37℃,20 min,DPPH清除率可达93.4%;虫草胞内多糖具有一定的清除羟自由基的能力,但效果不是很好,当多糖浓度为0.1 mg/mL,37℃,30 min,50%的醇沉多糖对羟自由基的清除率可达47.5%;虫草多糖清除超氧自由基的能力很弱,当多糖浓度为0.1 mg/mL,37℃,20 min,50%的醇沉多糖对超氧自由基的清除率为27.3%。6、蛹虫草醇沉分级多糖的抗菌性70%乙醇醇沉虫草胞内多糖抗菌性好于胞外多糖,可使黑曲霉的抑菌圈直径达8.6 mm,大肠杆菌的抑菌圈直径达11.7 mm。70%乙醇醇沉胞内、外多糖的抑菌效果均好于50%和30%的胞内外多糖。7、蛹虫草原生质体制备条件在蜗牛酶和纤维素酶混合的酶解液中,蜗牛酶酶解液的最佳浓度为1%,纤维素酶的最佳浓度为0.5%,蜗牛酶酶解液和纤维素酶酶解液的最佳体积比是1:2,最佳酶解温度为35℃,最佳酶解时间为3 h,最佳酶解pH为6.5。在以上的条件下,原生质体的制备率为1.36×10⁸个/mL。8、蛹虫草原生质体再生条件渗透压稳定剂在实验材料内甘露醇最好,甘露醇浓度为0.6 mol/L时原生质体的再生率最高0.658%;最佳酶解时间为2.5 h,原生质体再生率为5.68‰;双层平板法好于单层平板法;在最佳条件下的再生率为8.8‰。9、高生物量、高产多糖菌株的选育经紫外线诱变原生质体得到2株突变株0704号和0706号,两菌株的生物量产量和胞内、外多糖产量均较高,其生物量分别是出发菌株的2.26倍和2.19倍;这两株菌的胞内、外多糖产量也比较高,分别比出发菌株高32%、70.1%和28.1%、50.51%;0704号和0706号菌株的胞内、外多糖分别达到214.8 mg/100mL、198.7 mg/100mL和208.5 mg/100mL、175.8 mg/100mL。更多还原

【Abstract】 Cordyceps militaris （L.） Link is a kind of type specie of the cordyceps genus fungi, including many kinds of active materials with the functions of enhancing immunity, eliminating inflammation, anti-fatigue, anti-oxidation, anti-aging, anti-mutation, anti-tumor, etc. Some study results have indicated that the efficiency and the components of C. militaris were not inferior to those of the famous C. sinensis, and the contents of some components were higher than the latter. Some other researches have proved that the liquid fermentative mycelium components of anamorph of C. militaris are similar to those of natural C. sinensis which have the same physiological functions. On the condition of increasing scarcity of the wild caterpillar fungus resources, researching and developing artificial caterpillar fungus, breeding good strains of caterpillar fungus, discussing its liquid fermentation conditions, and studying the active components, contents and physiological functions have an important theoretical and practical significance to the further development and utilization of caterpillar fungus resources.In this study, the optimum conditions of internal and external polysaccharides and biomass of liquid fermentation were studied by orthogonal experiment using Paecilomyces militaris （Kob.） Liang, that the anamorph of C. militaris as materials. The anti-mutation characteristics of internal and external polysaccharides were investigated by the test of cell micronucleus of bean root tips. The effect of polysaccharides sedimented by different concentrations ethanol to the contents of SOD and MDA was studied and anti-oxidation was discussed through measuring the amounts of SOD and MDA by kits in fruit fly which were feed by polysaccharides. Anti-oxidation characteristics of polysaccharides sedimented by different concentrations ethanol were also measured by the determination of DPPH scavenging abilities of polysaccharides to hydroxyl free radical, super-oxide radical, organic free radical. Strains producing polysaccharides highly were bred by the mutagenesis of protoplast. The results as follows:1 The optimum conditions of liquid submerged fermentation of mycelia of C. militaris producing internal and external polysaccharides and high yield of mycelia were: sucrose 5.0 %, maize liquid 2.0 %, yeast extraction juice 0.03 %, KNO₃ 0.08 %,KH₂PO₄ 0.25 %, NH₄NO₃ 0.04 %,FeSO₄·7H₂O 0.08%,MgSO₄·7H₂O 0.15%. The optimum culture conditions were: temperature 22℃,inoculation amount10 %-15 %, pH controlled at 6 during the 5 days’ submerged fermentation. 170.3 mg internal polysaccharides and 114.8 mg external polysaccharides and 546 mg dried mycelia were gained from each 100 mL fermentation broth.2 The components of internal and external polysaccharides of C.militaris both were glucose, galactose and mannose.3 The anti-mutation effect of internal and external polysaccharides of C. militaris to chemical factors showed that, （1） The internal and external polysaccharides both could inhibit effectively the production of cell micronucleus induced by mitomycin of bean root tips. （2） The internal and external polysaccharides both had a significant effect on inhibiting cell micronucleus of bean root tip induced by cyclophosphamide, and inhibitory effect is enhancing with the increasing of the polysaccharides concentration. When the experimental consistence of internal and external polysaccharides were 100μg/mL,the inhibitory rates to cell micronucleus of bean root tip induced by cyclophosphamide were 53.52 % and 52.11 %, respectively. Caterpillar fungus polysaccharides had a remarkable effect on inhibiting mutation.4 The anti-mutation effect of internal and external polysaccharides of C. militaris to physical factors indicated that both the internal and external polysaccharides of C. militaris could effectively inhibit the production of cell micronucleus of bean root tip induced by UV, and had a positive dose-effect relation in the scale of experiment consistences. When the consistences of internal and external polysaccharides both were 100μg/mL, the inhibitory effects to cell micronucleus of bean root tip were 47.68 % and 43.04 %, respectively.5 Anti-oxidation functions of C. militaris polysaccharides sedimented by different contents ethanol were showed as follows:（1） Most polysaccharides can be sedimentated by 70% ethanol and the producing rates of internal and external are 1.513 mg/mL and 0.483 mg/mL, respectively.（2） The external polysaccharides of C. militaris sedimented by different consistences ethanol all had a good anti-oxidation ability. It increased the activity of SOD and decreased the amount of MDA in fruit flies. The polysaccharides sedimentated by 70 % ethanol increased the value of SOD by 13.13 % and decreased MDA by 67.2 % compared with the controls, and 2% addition amount had a better anti-oxidation effect than 1 %.（3） The internal polysaccharides of C. militaris sedimented by 50 % ethanol can be scavenging DPPH better, when the concentration of internal polysaccharides was 1mg/mL, the temperature was 37℃,and effect time was 20 min, DPPH scavenging rate can be up to 93.4 %; the internal polysaccharides had some scavenging abilities of hydroxyl free radical, when the concentration of internal polysaccharides sedimented by 50% ethanol was 0.1 mg/mL, the temperature was 37℃,and effect time was 30 min, hydroxyl free radical scavenging rate was 47.5 %; the internal polysaccharides had less scavenging abilities of super-oxide radical, when the concentration of internal polysaccharides sedimented by 50% ethanol was 0.1 mg/mL, the temperature was 37℃,and effect time was 30 min, super-oxide radical scavenging rate was 27.3 %.6 Antibacterial functions of C. militaris polysaccharides sedimented by different contents ethanol showed that the internal polysaccharides had stronger ability than externalpolysaccharides both sedimentated by 70 % ethanol, making the diameter of antibacterial circle of Aspergillus niger to 8.6 mm and of E.coli to 11.7 mm. The internal and external polysaccharides sedimentated by 70% ethanol both had better antibacterial effect than those sedimentated by 50 % and 30 % ethanol.7 The preparation conditions of protoplast of C. militaris as follows: in the mixed enzymolysis liquid of helicase and cellulase, the optimum concentration of helicase was 1 %,cellulase was 0.5 %, the optimum volume ratio of two enzymes was 1:2, and the optimum temperature, time, pH were 35℃,3 h, 6.5, respectively. In this condition, the producing rate of protoplast was 1.36×10⁸/mL.8 The regeneration conditions of protoplast of C. militarisMannitol was the best osmotic stabilizer of experimental materials. The highest regeneration rate of protoplast was 0.658 % in 0.6 mol/L mannitol solution. The optimum enzymolysis time was 2.5 h and regeneration rate was 5.68 %o. Double-plate method was better than monolayer method. In the optimum conditions, the regeneration rate was 8.8‰.9 The breeding of strains with high biomass and polysaccharides showed that two mutant strains of 0704 and 0706 with higher production of biomass and polysaccharides were gained via mutagenesis of protoplast by UV.The biomass of these two mutant strains increased 126% and 119%,respectively. Compared with the original strains, the internal and external polysaccharides increased 32%,and 70.1%,and 28.1%,and 50.51%,even reaching to 214.8 mg/100mL,198.7 mg/100mL and 208.5 mg/100mL,175.8 mg/100mL.更多还原