【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， 2010， 博士

【摘要】 本研究利用蛹虫草无性型——蛹拟青霉(Paecilomyces militaris)为载体,采用液体深层发酵法对硒、锌、锗三种重要微量元素进行富集和有机转化,探索了蛹虫草菌丝体对三种微量元素的富集、有机转化情况、蛹虫草菌丝体活性成分变化情况、微量元素与蛹虫草菌丝体代谢物的结合形式、微量元素在菌丝体内的分布以及有机转化物的生物活性和功能等问题,为更好地研制和开发对人体有益,且安全、高效,既能补充微量元素又具有生理活性的功能性食品和医用药物奠定基础。1、以生物量和微量元素硒、锌、锗的富集能力为指标,对本实验室已有的30株蛹拟青霉进行了筛选。实验结果表明,不同菌株对硒、锌、锗的富集能力各不相同,富集能力的高低基本上与菌丝体生物量和硒、锌、锗有机转化率有关。菌丝体的耐硒能力最弱,耐锌能力较好,耐锗能力最强；从有机转化率来看,硒最高,锌次之,锗最低。(1)富硒能力最强的菌株为PM14号菌株,在Na2Se03浓度为20μg/mL条件下,有机硒转化率达到31.6%。其生物量在相同浓度下也最高,达到17.0 mg/mL。(2)富锌能力较好的菌株为PM28号和PM14号菌株,在ZnS04浓度为200μg/mL条件下,有机锌转化率分别达到6.65%和5.78%。在相同浓度下,其生物量达到最大分别为19.5 mg/mL,17.6 mg/mL(3)富锗最佳菌株为PM28号菌株和PM14号菌株,在锗浓度为500μg/mL条件,有机锗转化率分别达到2.3%和1.9%。在相同浓度下,其生物量分别为15.1 mg/mL，13.8 mg/mL。从总体上衡量,由于PM14号菌株富集硒、锌、锗的能力都很强,因此,确定PM14号菌株为进一步研究的菌株。2、研究了有机硒、锌、锗在蛹拟青霉菌丝体内蛋白质、多糖和核酸等大分子物质中的含量分布情况。实验结果表明,有机硒、锌、锗含量在蛋白质中最多,其次是多糖,最少的是核酸。硒、锌、锗浓度对其含量有影响,当浓度过高或过低时其含量均降低。(1)当培养基中Na2Se03浓度为20μg/mL时,蛋白质、多糖、核酸中有机硒含量均达到最多,分别为221.5μg/g、86.2μg/g和0.6μg/g;当Na2Se03浓度为10μg/mL时,蛋白硒、多糖硒和核酸硒分别占菌丝体总有机硒的比例最大,分别为菌丝体总有机硒的65.8%、28.1%和0.2%,分别是空白的2.7倍、3.9倍和3.8倍。蛋白、多糖、核酸中总的有机硒含量在培养基Na2Se03浓度为20μg/mL时最多为308.4μg/g。在培养基Na2Se03浓度为10μg/mL时,蛋白硒、多糖硒、核酸硒在菌丝体总有机硒中占的比例最大,达到94.1%。(2)在实验锌浓度范围内,随着培养基中锌浓度的增加,蛋白质、多糖、核酸中有机锌的含量不断增加,蛋白质、多糖中有机锌占菌丝体总有机锌的比例都随着培养基中锌浓度的增加而增加,而核酸中有机锌比例却不断减少,但变化不明显。当ZnSO4浓度为200μg/mL时,有机锌含量最多,其在体内的分布情况是,蛋白质最多占菌丝体总有机锌的65.6%,其次是多糖有机锌占7.2%,核酸中有机锌最少,只占0.8%。此时蛋白锌和多糖锌分别是空白的2.2倍和1.1倍,而核酸锌却比空白少了33.9%。由此可见锌对蛋白质和多糖中有机锌合成的促进作用比较明显。(3)实验表明,低浓度锗对蛋白质、多糖、核酸中有机锗的含量有促进作用,高浓度锗对有机锗的形成有一定的抑制作用。当Ge02浓度为500μg/mL时,蛋白质、多糖、核酸中有机锗含量均达到最大,分别达到284.0μg/g、261.5μg/g和137.6μg/g,分别是空白组的4.4倍、5.4倍和8.3倍。蛋白锗和多糖锗所占菌丝体总有机锗比例相似。当Ge02浓度在100μg/mL时,蛋白质、多糖和核酸中的有机锗含量占菌丝体总有机锗的比例最高,分别达到39.8%,36.8%和18.2%。蛋白、多糖和核酸中有机锗之和在Ge02浓度为100μg/mL时,所占菌丝体总有机锗比例最大,达到94.8%。3、探讨了硒、锌、锗浓度对蛹拟青霉菌丝体的生物量和主要活性成分(胞内多糖、微量元素含量、虫草素、菌丝体SOD、蛋白质含量、氨基酸含量等)的影响。结果表明,硒、锌、锗在适宜浓度范围内,对菌丝体的主要活性成分有促进作用,浓度过大,对相关活性成分有抑制作用,不同的生物学指标所需的硒、锌、锗浓度各不相同。硒、锌、锗的适宜浓度分别为：10-20μg/mL,100μg/mL和200μg/mL-300μg/mL。4、抗氧化作用实验表明,富硒、锌、锗多糖均具有较好的清除超氧自由基、羟自由基和有机自由基DPPH的能力,硒、锌多糖清除自由基的能力与空白多糖相比具有显著差异。从清除自由基的种类来看,它们清除DPPH的能力最强,其次是羟自由基,最后是氧自由基。总体来看,硒多糖清除自由基能力最好,其次是锌多糖,最后是锗多糖。5、果蝇寿命实验表明,硒、锌、锗多糖对果蝇的半数死亡时间、平均寿命和最高寿命都有显著影响,对果蝇具有明显的延缓衰老作用。多糖对半数死亡时间的延长最好,而且是雄性好于雌性；硒多糖对最高寿命和平均寿命的影响无显著差异,锌、锗多糖对之的影响是最高寿命好于平均寿命,从性别差异来看,基本上是雌性优于雄性。硒多糖对于增加体弱果蝇的寿命效果显著,锌、锗多糖对于延长体魄强壮果蝇的寿命较好。6、小鼠负重游泳力竭实验、血乳酸含量和血尿素氮含量测定以及小鼠常压耐缺氧实验结果表明,富硒、锌、锗这三种蛹虫草菌丝体不同浓度对小鼠不同生理时期具有不同的耐疲劳能力。它们可以显著延长小鼠负重游泳力竭实验时间,具有明显减少小鼠在疲劳状态下体内产生乳酸的作用,对疲劳过程中产生的血尿素氮也有显著的减少或清除作用。然而,它们的耐缺氧效果不够明显。7、蚕豆微核实验表明,富硒、锌、锗多糖没有致突变作用,对抑制丝裂霉素和紫外线诱发的蚕豆根尖细胞微核的产生具有显著作用,多糖浓度与微核抑制率有明显的剂量-效应关系,微核抑制率随着多糖浓度的增加而增加。当多糖浓度为100μg/mL时,富硒、锌、锗多糖对丝裂霉素和紫外线诱发微核的抑制率分别为46.5%、37.2%、34.1%和53.3%、48.6%、43.8%。对微核的抑制效果是富硒多糖>富锌多糖>富锗多糖。8、体外抗肿瘤实验表明,硒多糖、锌多糖对肺腺癌A549细胞株生长的抑制作用与空白对照具有极显著差异,可以显著提高对肺腺癌A549细胞株生长的抑制作用,当多糖浓度为4 mg/mL时,硒多糖和锌多糖对肺腺癌A549细胞株生长的抑制率分别为54.2%和53.9%,分别比空白菌丝体多糖的抑制率提高38.8%和38.0%。硒、锌、锗多糖对鼻咽癌CNE-1细胞株生长的抑制作用与空白对照均具有极显著差异,当多糖在浓度为4 mg/mL时,硒、锌、锗多糖对鼻咽癌CNE-1细胞生长的抑制率分别为40.2%,32.1%和39.56%,分别比空白对照提高128.8%、82.4%和125.2%。硒、锌、锗多糖可以显著提高鼻咽癌CNE-1细胞株生长的抑制作用。9、小鼠急性经口毒理实验表明,蛹拟青霉富硒、锌、锗菌丝体属于无毒级产品。本研究表明,蛹拟青霉液体培养菌丝体具有较好的富集硒、锌、锗的能力,在适当的微量元素浓度时,富集微量元素后的菌丝体主要活性成分含量有明显提高,富硒、锌、锗菌丝体及微量元素有机物的抗氧化、抗疲劳、抗突变和抗肿瘤能力有显著增强,小鼠急性经口毒理实验和蚕豆根尖细胞微核实验表明,富硒、锌、锗蛹拟青霉菌丝体没有致突变作用,对小鼠无毒性。更多还原

【Abstract】 In this study, anamorphic strains of Cordyceps militaris, Paecilomyces militaris were used as carrier for accumulation and transform of 3 trace elements, selenium, zinc and germanium, and enrichment and organic transform of the three trace elements, change of P. militaris mycelium composition, combination form of trace elements and P. militaris mycelium metabolites, distribution of trace elements in mycelium and bioactive and function of organic transformant were investigated for the development of healthy functional food and medical drugs of bioactivities and as an effective supplement of trace element.1. With biomass and enrichment capacity of Se, Zn and Ge as estimates,30 strains of P. militaris were screened. The result showed that different strains had different enrichment capability. The capacity was generally related with mycelium biomass and organic conversion rate of Se, Zn and Ge. Their mycelia were poorly tolerant to Se, tolerant to Zn and strongly tolerant to Ge. Se had the highest transformation rate, followed by Zn and Ge.(1) The strain PM14 showed the strongest Se-enrichment capacity. With sodium selenite concentration at 20μg/mL, the transformation rate of organic Se was up to 31.6%. At these concentration, the biomass was the highest up to 17.0 mg/mL.(2) Strains PM28 and PM14 showed strong Zn-enrichment capacity. With ZnSO4 concentration at 200μg/mL, the transformation rates of organic Se reached 6.65% and 5.78%, respectively. At these concentrations, the biomasses were the highest up to 19.5 mg/mL and 17.6 mg/mL, respectively.(3) Strains PM28 and PM14 showed the strongest Ge-enrichment capacity. With GeO2 concentration at 500μg/mL, the transformation rates of organic Ge reached 2.3% and 1.9%, respectively. At these concentrations, the biomasses were up to 15.1 mg/mL and 13.8 mg/mL, respectively.In general, because strain PM14 was chosen for further study due to its strong capacity of enrichment of Se, Zn and Ge.2. The distribution of the organic Se, Zn and Ge in the macromolecules such as protein, polysaccharide and nucleic acid was investigated. The results showed that protein, polysaccharide and nucleic acid were the main vectors and existing forms of organic Se, Zn and Ge. In addition, small portions of organic Se, Zn and Ge were combined with other substances. The content of organic Se, Zn and Ge was mainly in the protein, followed by polysaccharide and the nucleic acid. The concentration of Se, Zn and Ge had some influence on the content of the 3 elements. When the concentration was too high or too low, the content of the elements decreased.(1) As the sodium selenite concentration was at 20μg/mL in the culture medium, the organic Se content in protein, polysaccharide and nucleic acid reached the the peak,221.5μg/g,86.3μg/g and 0.6μg/g, respectively. When sodium selenite concentration was 10μg/mL, the protein-Se, polysaccharide-Se and nucleic acid-Se of the total mycelium took the highest proportion, up to 65.8%,28.1% and 0.2% of organic Se, respectively. They were 2.7,3.9 and 3.8 times of the corresponding controls. The whole organic Se content in the protein, polysaccharide and nucleic acid was 308.4μg/g when the sodium selenite concentration was 20μg/mL. As the sodium selenite concentration decreased to 10μg/g, of the total mycelium organic Se, protein Se, polysaccharide Se and nucleic acid Se showed the highest proportion, up to 94.1%.(2) Within the experiment range of Zn concentration, the organic Zn content in protein, polysaccharide and nucleic acid rose with increase of the Zn concentration in the culture medium, organic Zn proportion in protein and polysaccharide rose with increase of Zn concentration, while the proportion in nucleic acid delined, but with the change not obvious. When ZnSO4 concentration was 200μg/mL, the content of organic Zn was the highest. The distribution of the organic Zn in the mycelium was as following:the proportion of protein-Zn was the highest up to 65.6%, followed by polysaccharide Zn (7.2 %) and nucleic acid Zn (0.8%). The protein-Zn and polysaccharide-Zn were 2.2 and 1.1 times of the corresponding control, respectively, but nucleic acid Zn was decreased 33.9% compared with the control. This indicating that the Zn has promotive effect on synthesis of organic Zn in protein and polysaccharide.(3) Low concentration Ge had promotive effect on the organic Ge content in protein, polysaccharide and nucleic acid, while high concentration Ge had inhibitive effect on organic Ge formation. When GeO2 concentration was 500μg/mL, the organic Ge content in the protein, polysaccharide and nucleic acid reached the highest up to 284.0μg/g,261.5μg/g and 137.6μg/g, respectively. They were 4.4,5.4 and 8.3 folds of the control group. The proportion of the protein-Ge to total organic Ge in mycelium was similar with polysaccharide-Ge. When the GeO2 concentration was 100μg/mL, of the total organic Ge in the mycelium, protein-Ge, polysaccharide-Ge and nucleic acid-Ge showed highest proportion, which were 39.8%,36.8% and 18.2%, respectively. When GeO2 concentration was 100μg/mL, the proportion of the sum of protein-Ge, polysaccharide-Ge and nucleic acid-Ge to total organic Ge was the highest (94.8%).3. The effects of Se, Zn and Ge concentration on biomass and the main active ingredients (intracellular polysaccharide, trace elements content, cordycepin, mycelium SOD, protein content, amino acids content and so on) in mycelium were investigated. The results showed that the Se, Zn or Ge at an appropriate concentration had promotive effect on the main ingredients of mycelium. However, when the concentrations were excessive, they had inhibitive effect on the related ingredients. Different biological directors needed different Se, Zn and Ge concentrations. The most appropriate concentration of Se, Zn, and Ge were 10-20μg/mL,100μg/mL and 200μg/mL-300μg/mL, respectively.4. Antioxidation test indicated that Se, Zn or Ge-enriched polysaccharide had substantial function of scavenging superoxide radical, hydroxyl radical and DPPH. Compared with the control, the capability of scavenging radical was significantly stronger for Se-olysaccharide and Zn-polysaccharide. From the point of the radical type, the scavenging capability of DPPH was the highest, followed by those of hydroxyl radical and superoxide radical. Generally, for the capability of scavenging radical, Se-polysaccharide was the strongest, followed by Zn- and Ge-polysaccharide.5. The drosophila life-span experiment showed that the Se, Zn or Ge-polysaccharides all had significant effect on the half death time, average and maximum life span, suggesting obvious antisenescence effect. The polysaccharides dramatically elongated half death time, particularly to the males. Se-polysaccharide had no obvious effect on maximum and average life-span, but with Zn- and Ge-polysaccharide, the effect on maximum life-span was stronger than that on average life-span. In general, the effect on the males was more obvious than that on the females. The elongating effect on weak drosophila was significant with Se-polysaccharide, while that on healthy drosophila was with Zn- or Ge- polysaccharide.6. The exhaustive swimming experiments of mice and tests on contents of blood lactic acid and blood urea nitrogen, hypoxia-tolerance under normal air pressure showed that different concentrations of mycelium enriched with Se, Zn or Ge had different fatigue tolerance effect on the mice at different physiological periods. They significantly elongated exhaustive swimming time of the mice and showed the effects of reducing lactic acid production at the status of fatigue and reducing or removing blood urea nitrogen. However, the effect of hypoxia-tolerance was not obvious.7. Micronucleus test of broad bean showed that the polysaccharides enriched with Se, Zn or Ge had no mutagenic effects. Instead, they significantly inhibited the production of cell micronucleus in broad beans induced by mitomycin and ultraviolet radiation. Concentration of polysaccharide and the inhibition rate had obvious dose-effect relationship. The micronucleus inhibition rate rose with increase of the polysaccharide concentration. When concentration of the polysaccharide enriched with Se, Zn or Ge was at 100μg/mL, the inhibition rates induced by mitomycin and UV radiation were 46.5%, 37.2%,34.1% and 53.3%,48.6%,43.8%, respectively. The Se-polysaccharide had the highest inhibition, followed by Zn-polysaccharide and Ge-polysaccharide.8. In vitro antitumor experiments showed that Se-polysaccharide or Zn-polysaccharide had significant inhibition effect on lung cancer cell line, as comppared with control group. They significantly inhibited the growth of the lung cancer cell A594. When the polysaccharide concentration was 4 mg/mL, the inhibition rates of Se-polysaccharide and Zn-polysaccharide were 54.2% and 53.9%, respectively. Compared with the control of mycelium without the trace elements added, the inhibition rates increased by 38.8% and 38.0%, respectively.Se-polysaccharide, Zn-polysaccharide or Ge-polysaccharide all displayed significant inhibitionx on cell line CNE-1 of nasopharyngeal carcinoma, as compared to the control group. At concentration of 4 mg/mL, the inhibition rate of Se-polysaccharide, Zn-polysaccharide and Ge-polysaccharide were 40.2%,32.1% and 39.6%, respectively. As compared with the control of mycelium without the trace elements added, the inhibition rates increased by 128.8%,82.4% and 125.2%, respectively, suggesting strong inhibition of the growth of CNE-1.9. Acute oral toxicity test of mice showed that the mycelium of P. militaris enriched with Se, Zn and Ge belonged to nontoxic products.It is concluded that the mycelium of P. militaris produced in liquid culture enriched Se, Zn and Ge. At appropriate concentration of trace elements, the content of main active ingredients in the mycelium enriched with trace elements increased significantly and the antioxidant, antimutagenic and anti-tumor functions of the enriched mycelium rose significantly. The mycelium enriched with Se, Zn and Ge had no mutagenic effect and displayed no toxicity to mice.更多还原