摘要
松茸是一种珍稀食、药用真菌,在国际市场上享有“蘑菇之王”的赞誉,其多糖含量为食用蕈菌之首,抗肿瘤作用位居抗癌真菌首位。蛹虫草亦是我国名贵的中药材和高级补品,它与传统冬虫夏草具有十分相似的食用和药用价值,其有效成分蛹虫草多糖的药用价值已得到世界医药界的证实。
本论文是在对松茸(T)、蛹虫草(C)大量实验研究的基础上,确定以松茸、蛹虫草作为混菌发酵体系(T-C混菌)与单菌进行比较研究。本实验利用工农业废料玉米浆和豆饼粉通过单因素和响应面分析法分别确定了松茸、蛹虫草和T-C混菌培养获得胞外多糖产量最高的培养基配方,并对菌丝体胞内多糖的热水浸提工艺进行了优化,分别得到了利于胞外多糖产量的最优发酵条件和利于胞内多糖得率的最优提取条件。比较最优发酵条件下的胞外多糖产率和胞内多糖提取率,混菌均优于单菌。同时对松茸、蛹虫草和T-C混菌胞外、胞内多糖对B16黑色素瘤体外增殖的抑制作用进行了比较研究,实验结果表明:不同浓度混菌胞外多糖在不同作用时间均表现出优于其它5种多糖的显著的抗肿瘤活性,多糖作用时间达到72h时,混菌胞外胞内多糖的抗肿瘤活性均优于单菌。
Tricholoma matsutake and Cordyceps Militaris are two kinds of most valu-able and high-class edible fungus. However, Tricholoma matsutake is typically difficult to cultivate, so far there is no successful cultivation in artificial pure cul-ture of fruitbody. And the natural resources of Cordyceps Militaris due to over-harvesting caused natural resources very tense. Due to its limited supply from natural sources and the long growth period in some artificial specific solid media, the submerged culture on a large scale using artificial media has recently been developed as a resource of mushrooms alternative. Using cheap and abun-dant source of industrial and agricultural waste to replace the experimental re-agents as a medium for cultivating mushrooms was low-cost, and it has an impor-tant theoretical and practical meaning to the realization of the new biotechnology industry. This study evaluate the capability of industrial and agricultural waste being low cost substrate by co-cultivating Tricholoma matsutake and Cordyceps Militaris in view of getting better growth, higher extraction rate of exopolysac-charides and endopolysaccharides, stronger in vitro anti-tumor activity.
The concentration of corn steep liquor and soybean meal in the medium was investigated by means of single factor tests and Response Surface Methodology (RSM) based on a five-level two-factor central composite design. Optimized val-ues of the factors in co-cultivating fermentation were determined to be corn steep liquor 9.17% and soybean meal 13.05% and a maximum exopolysaccharides pro-duction of 4.09g/L was obtained. Optimized values of the factors were determined to be corn steep liquor 9.07% and soybean meal 10.43% and a maximum exopolysaccharides production of 4.02g/L was obtained in Tricholoma matsutake fermentation. While in Cordyceps Militaris fermentation, the maximum produc-tion was 3.47g/L when the concentrations of these two factors were 16.40% and 14.34% respectively. Using cheap and abundant source of industrial and agricul-tural waste to replace the experimental reagents as a medium for cultivating mushrooms was feasible and the exopolysaccharides production can be increased by 1 to 5 times. T-C co-cultivating exopolysaccharides production was a little higher than single-cultivating ones.
One-factor tests and Response Surface Methodology (RSM) were applied to optimize the water extraction conditions of co-cultivating and single cultivating mycelium polysaccharides. Experiment factors and levels were first selected by one-factor tests. On the basis of this, the ratio of sample to water , extracting tem-perature, extracting time were chosen as the factors in RSM and each factor in the design was studied at three different levels. The optimum extraction conditions were confirmed as follows: co-cultivating mycelium polysaccharides—extracting time 3h, the ratio of water to sample 38:1, extracting temperature 63℃, the ratio of water to sample has the most important effect on polysaccharides production followed by extracting time; Tricholoma matsutake mycelium polysaccharides—4h, 29:1, 94℃, sequence of each factor : extracting temperature> extracting time> the ratio of water to sample; Cordyceps Militaris mycelium polysaccharides—2h, 30:1, 74℃, sequence of each factor: extracting temperature> extracting time> the ratio of water to sample. The extraction rate of polysaccharides under the opti-mum conditions could reach the highest value of 23.47%, 21.63% and 6.15% re-spectively with two-times extraction. Therefore it can be seen from the compari-son that the extraction rate of co-cultivating mycelium polysaccharides can be higher than single-cultivating mycelium even under low extracting temperature, thereby saving energy.
MTT assay was used to test the extracted co-cultivating and sin-gle-cultivating exopolysaccharides and endopolysaccharides against B16 mouse melanoma in different concentrations and different time. All solvent extracts showed a significant concentration and time-dependent inhibitory effect on the proliferation of B16 mouse melanoma. Co-cultivating exopolysaccharides showed much higher inhibition in vitro anti-tumor activity at any concentrations and any times. And in 72h of treatment, both co-cultivating exopolysaccharides and endo-polysaccharides had higher anti-tumor activity than single-cultivating ones.
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