松乳菇菌丝深层发酵及其多糖功能评价

【作者】 敖常伟；

【导师】 李忠海； 钟海雁；

【作者基本信息】 中南林学院 ， 森林培育学， 2002， 硕士

【摘要】 松乳菇是我国一种著名的野生真菌，其味鲜美，营养价值高。本研究以野生松乳菇子实体为材料，通过组织分离得其菌丝体，进而探索菌丝生长的条件，分析菌丝体与子实体的营养成分，检测了松乳菇多糖对小鼠免疫功能的影响。主要研究结果如下： 分别对培养基、温度、pH和光照强度进行单因素比较实验，研究不同因素对松乳菇菌丝体在固体平板培养基上生长量的影响。结果表明松乳菇菌丝较适宜的培养条件是PDA培养基、28℃、pH5；菌丝在黑暗环境中的生长状态比在光照环境中长势要好。 通过碳、氮源的单因素实验确定了松乳菇在深层发酵培养基上的最适碳源是葡萄糖；最适氮源是玉米浆和硝酸铵，两者间无明显差异(P＞0.05)。选择葡萄糖(A因子)、玉米浆(B因子)、硝酸铵(C因子)作3因素3水平正交实验，测定不同碳、氮营养组合对发酵菌丝干重的影响，发现碳、氮营养的最佳组合是葡萄糖2.00％，玉米浆1.50％，硝酸铵0.15％。在培养基中加0.40％的CMC(羧甲基纤维素钠)，可增加菌丝的生长点，能提高菌丝产量。选择接种量在10％～15％之间，28℃培养15天可得到最大的菌丝产量。 松乳菇子实体与菌丝体中化学分析表明，松乳菇营养素种类齐全，粗蛋白、粗脂肪及人体必需氨基酸含量相对较高，具有较高的营养价值。发酵得到的菌丝体与野生子实体相比，其所含营养成分相近，氨基酸组成相同，但多糖含量较高，脂类和粗纤维含量相对较少。通过正交实验得出松乳菇多糖提取的最佳条件为浸提温度40℃，加水倍数20倍，浸提1次，保温50min。水提取所得粗多糖，经醇析和Sevage法处理可被进一步纯化。水解后的松乳菇多糖以层析法分析表明含有葡萄糖、甘露糖、果糖等几类糖。 采用高、中、低浓度的松乳菇多糖为受试物，以小白鼠免疫器官重量法、小白鼠碳粒廓清实验法、小白鼠抗应激能力实验和小白鼠耐高温实验为研究方法，测定多糖对小白鼠非特异性免疫的效果。结果表明：高浓度组与对照组相比较，小白鼠胸腺指数和脾脏指数均有显著增加，P＜0.05，与低浓度组相比较，脾脏指数有显著增加，p＜0.05；与对照组、低浓度组相比较其吞噬系数有显著差异，p＜0.05，其它各组吞噬系数无显著差异：高浓度组与对照组在游泳时间上也存在显著差异，p＜0.05；各浓度受试物无明显提高小白鼠耐高温能力。上述表明松乳菇多糖能明显提高小白鼠的免疫能力。 综上所述，从子实体中分离的松乳菇菌丝体可通过液体深层发酵法来进行人工培养，其主要营养成分—多糖和蛋白质明显高于子实体的含量，对小白鼠进行非特异性免疫试验表明，松乳菇多糖能够提高机体的免疫功能。更多还原

【Abstract】 Lactarius deliciosus is a famous wild epiphyte in the world. It has not only delicious flavor, but also rich nutrition value. The optimum conditions for the growth of the mycelium, nutrient ingredients, and the immune functions of the extracted polysaccharides on mice were studied based on the mycelium separated from the Ldeliciosus-The results as follows:The influences of different factors to the growth of the mycelium of L.deliciosus were gained by single factor experiments. These factors are variational culture, temperature, pH, and light, respectively. The results showed that the better conditions for the growth of the L deliciosus were the PDA culture in pH5 at 28 "C; The dark environment is better than the illuminated.The optimum sources of carbon and nitrogen were established by single factor experiments, which are good for the growth of mycelium of L. deliciosus. The former was glucose; the latter were maize plasma and NH^Oa (two sources have no significantly difference, p>0.05). Orthogonal test was used to measure the effects of the varying combinations of carbon and nitrogen on the dry weight of fermented mycelium. The ingredients include glucose (factor A), maize plasma (factor B) and NH玁O3 (factor C). The optimum combination of carbon and nitrogen is glucose 2%, maize plasma 1.5% and NH4NO3 0.15%. CMC 0.4% added to the culture can increase the growing positions and the output of mycelium. Most output of mycelium can be obtained under the circumstance of inoculation volume between 10% and 15% at 28 "C for 15 days.Chemical analysis indicated that L.deliciosus has nearly all the nutrients. The crude protein, lipids, and the essential ammo acids of Ldeliciosus are richer. Compared with wild fruit body, fermented mycelium of L.Deliciosus has similar nutrients with the former, and has the same composition of amino acids. But polysaccharides are much richer, lipids and crude fibre is less. The orthogonal test shows that the optimum extraction condition for polysaccharides of L. deliciosus was as follows: adding water at a ratio of 1:20 (dried fruit body or mycelium: water), extracting for 50 min at 40癈. Using the ethanol precipitating and method of Sevage can further purify polysaccharides extracted by water. The glucose, mannose and fructose were analyzed by paper chromatography from the hydrolyzed polysaccharides of L. deliciosus.The effects of L.deliciosus Polysaccharides on immune function were investigated with mice. The methods of immune organs weight of mice, carbon clearance, swimming, and enduring high temperature were used. Compared with control, high concentration group could significantly increase the thymic and splenic index of mice (PO.05), while compared with low concentration group, high concentration group could significantly increase splenic index of mice (PO.05); compared with control and low concentration group, high concentration group could significantly increase swallow coefficient (PO.05); high concentration group could significantly prolong the time of swimming; different groups had no obvious effect on the livability of enduring high temperature. L.deliciosus Polysaccharides can improve the immune function of mice.In a word, the mycelium by separating from fruit body of L.deliciosus can be cultivated by application of submerged fermentation; its main nutrient (polysaccharides and proteins) is more richer than fruit body; The non-differential immune experiment showed that Polysaccharides of L.deliciosus can improve the immune function of organism.更多还原