The Investigation Of Tricholoma Matsutake And Cordyceps Militaris Co-cultivating And The Activities Of Polysaccharides

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.