【作者】 王伟霞；

【导师】 陈立国；

【作者基本信息】 华中农业大学 ， 微生物学， 2003， 硕士

【摘要】 茯苓（Wolfiporia cocos）为我国的传统中药材之一，是一种极具开发价值的食（药）用菌。培养茯苓菌丝体，易出现菌丝发黑现象，从而严重影响菌种质量，故本文对茯苓菌丝体的固体培养条件进行了研究。同时进行了菌丝体液体培养条件及其富集钙和微量元素碘的相关试验。其结果如下： 1．通过对茯苓菌丝体所需营养条件的研究，得出菌丝发黑与碳源、常用矿质盐、生长因子及碳氮比无关；以有机态氮（除玉米浆外）作氮源，加入高浓度KI，或者pH值高于7.10时，菌丝体易产生黑色素。并且，筛选出适宜的碳源为松木屑和葡萄糖；适宜的氮源是玉米浆；矿质盐KH₂PO₄和MgSO₄显著促进菌丝生长；VB₁为适宜的生长因子；适宜的pH值范围是3.17～6.05；合适的碳氮比为25：1～35：1。 2．通过不同温度进行培养后，置于低温室内保存，发现经22℃培养的茯苓菌丝体保存7天，经28℃培养的保存9天，有大量黑色素产生；经25℃培养的茯苓菌丝体保存30天后，才出现菌丝发黑现象。 3．通过分离不同生长状态的菌核，观察到自生长泡松的菌核分离的茯苓菌株比从僵硬的菌核分离的菌株容易分泌黑色素。并且通过挑取菌丝尖端进行培养，发现抑制菌丝发黑效果不明显。 4．通过接种量、装液量试验，确认摇瓶培养茯苓菌丝体适宜的接种量为10％，装液量为50mL／150mL的三角瓶。通过正交试验，筛选出适宜的培养基配方为：葡萄糖25g，玉米浆10mL，KH₂PO₄ 1.5g，MgSO₄ 0.75g，VB₁ 5mg，水1000mL。并且，该培养基优于玉米浆培养基。以上述培养基配方和培养条件进行摇瓶发酵，得到发酵过程中菌丝体生物量、pH值、胞外多糖及胞内多糖含量的变化曲线。 5．通过对茯苓菌丝体耐钙研究，得出当CaCl₂浓度为2.00g／L时，菌丝生长不受抑制，当浓度大于4.00g／L时，生长速度减慢，菌丝密度较小。加入2.00g／L CaCl₂进行液体富钙培养，研究结果表明，以浓度梯度法加入CaCl₂，钙的富集率较高，为78.44％，菌丝体钙含量为94.22mg／g，其中26.83％为有机钙。 6．加入不同浓度KI进行固体培养试验，得出当KI浓度为0.25g／L时，菌丝长势较好；当KI浓度增加到0.50g／L时，菌丝生长受到轻微抑制作用；高于1.00g／L，严重抑制菌丝生长。以0.50g／LKI进行液体富碘试验，结果表明以浓度梯度法加入KI，碘的富集率较高，为1.64％，菌丝体内含碘量为2.3mg／g。更多还原

【Abstract】 Being the one of traditional Chinese medicine, Wolfiporia cocos is a kind of edible and medicinal fungi which has great value to study. When the mycelia secreted melanin, the growth of mycelia was inhibited. This paper studied the condition of solid cultivation and liquid fermentation; And this paper studied the condition of calcium-enriched and iodine-enriched of Wolfiporia cocos. The results of the study were as follows:1. Nutrition tests of Wolfiporia cocos suggested that the optimal carbon source, nitrogen source, mineral, growth factor, pH, C/N value were pinewood and glucose, corn plasm, KH2PO4and MgSO4, VB1, 3.17-6.05, 25:1-35:1 respectively. And secreting melanin of the mycelia did not relate to carbon source, mineral, growth factor, and C/N. When nitrogen source were organic nitrogen which didn’t include corn plasm, the concentration of KI was high, or pH was greater than 7.0, the mycelia of Wolfiporia cocos secreted melanin.2. Temperature test suggested that when the Wolfiporia cocos which cultured 22℃ was preserved after 7 days, and which cultured 28# was preserved after 9 days, the mycelia began to secret melanin. But the mycelia which cultured 25# only began to secret melanin after 30 days.3. The strain of Wolfiporia cocos isolated from pultaceous sclerotium secreted the more melanin than that of the strain isolated from rigid sclerotium. Culturing mycelia’s tip of Wolfiporia cocos could not inhibit that the mycelia secreted melanin well.4. Inoculation number and medium volume tests suggested that the optimal inoculation number was 10%, and the optimal medium volume was 50mL/150mL with Erlenmeyer flask. Through orthogonal test, the optimal medium was selected and it contained glucose 25g, corn plasm 10mL, KH2PO4 1.5g, MgSO4 0.75g, VBi 5mg, distillation water 1000mL. And this medium was better than corn plasm medium to Wolfiporia cocos. Under the optimal fermentation condition we got the variation curves of mycelia biomass, pH value, extracellular polysaccharide and endocellular polysaccharide content.5. The test of enduring calcium in the solid medium suggested that the mycelia growth was not inhibited when the concentration of CaCl2 was 2.00g/L. When the concentration of CaCl2 was 4.00g/L or greater than it, the mycelia growth was inhibited. When the concentration of CaCl2 was 2.00g/L and added with concentration grads, the test of calcium-enriched in the course of fermentation suggested that enriching ratio was 78.46%, calcium content of the mycelia was 94.25mg/g, and 26.83% was organic calcium.6. The test of enduring iodine suggested that the mycelia growth was not inhibitedwhen the concentration of KI was 0.25g/L, when it was 0.50g/L the mycelia growth was inhibited, when it was 1 .00g/L or greater ,the mycelia growth was bad inhibited. When the concentration of KI was 0.50g/L, the test of iodine-enriched suggested that enriching ratio was 1.64%, iodine content of the mycelia was 2.3mg/g, adding KI with concentration grads.更多还原