| Tricholoma matsutake is known as one of the rarest and the most valuable natural medicinal mushroom in the world, as well as the second class endangered spice in China. There are various of bioactive compounds in Tricholoma matsutake. And it has been proven by a large number of pharmacological and clinical studies that Tricholoma matsutake has several Physiological and Medicinal functions such as Immune regulation, anti-cancer activity, control blood sugar, cholesterol, blood fat. Furthermore, recent investments home and abroad show the Anti-oxidative, Anti-fatigue, Anti-anoxia activities of Tricholoma matsutake, which presents great potential for application.In this study, mutation technique was used to rapidly improve the fermentation level of Tricholoma matsutake: mutation by NTG. An improved mutant, labeled as T35, was obtained from NTG mutation. The biomass, the content of polysaccharide, and the Ergosterol were 12.1640 g/L, 1.3180 g/L and 0.1474 g/L, which were enhanced by 41.75%, 33.86% and 19.25% compared with the wild strain, respectively.In this study, in order to comprehensively considerate of the biomass and the production of polysaccharide, and the Ergosterol at the same time, desirability function was employed in the optimization of the fermentation medium and condition. Based on single-factor optimization strategy, the suitable carbon sources, nitrogen sources and inorganic salts were obtained. Then key medium components were identified by Plackett-Burman design(PBD) and further optimized by Box-Behnken design(BBD). Finally, response surface methodology(RSM) and artificial neural network – genetic algorithm(ANN-GA) were further used to model and optimize the experimental results obtained from BBD. The optimum components of nutrient medium comprised(g/L): glucose 23.65 g/L, Yeast Extract 13.27 g/L, soya peptone 12.78 g/L, KH2PO4 0.50 g/L, Ca Cl2 0.52g/L, Mg SO4?7H2O 0.47 g/L, VB1 0.15 g/L. In a word, a mean value of desirability values Dv = 0.5963 was obtained, which was 26.09% higher than the value achieved by the basal medium. The biomass, the production of the polysaccharide and the yields Ergosterol were enhanced by10.47%, 33.65% and 21.69%, respectively. On the basis of medium optimization study, the suitable culture condition was further optimized using PBD and BBD. The suitable culture condition was: initial p H 5.87, temperature 27.31℃, culture time 4.65 d, seed age 4d, rotating speed 150r/min, inoculation amount 6%, liquid volume in 250 m L flask 80 m L. The best desirability value was 0.6935, which was 16.31% higher than that before optimization. The biomass, the production the polysaccharide and the Ergosterol yields were enhanced by 14.45%, 20.75% and 13.21% respectively.The experimental mice were orally treated with high, medium, low-dose Tricholoma matsutake suspension, with the Tricholoma matsutake body fruit micropowder as positive control group. The results showed that, after 14-day-treatment, the anti-fatigue performance has been significantly enhanced, compared to control group. The dose-dependently enhanced exercise endurance of T35-treated mice in weigh-loaded swimming, forced running and rotating rod test revealed the anti-fatigue activities of Tricholoma matsutake. Further, the micropowder of Tricholoma matsutake fruit body treatment could significantly reduce the levels of MDA and elevate the activities of SOD and GSH-Px in plasma and tissue of mice, and show higher level of ATP and glucogen comparing with control group. The expressions of P-AMPK, T-AMPK, PFK-1, and PGC-1α were detected to further analyze the preliminary molecular mechanisms under TM-mediated anti-fatigue effects. After 30-min swimming, over two folds enhancement of P-AMPK expression was observed in liver tissue of TM or ABM-treated mice. Furthermore, TM dose-dependently enhanced the expressions of PFK-1 and PGC-1α in the liver of exercised mice, and the similar results were noted in ABM-treated group. It demonstrated that orally treatment with TM could successfully promote cell survival by inhibiting free radical accumulation and accelerate the metabolism of free radical, protect body from the damage of exercise, accelerate the decomposition of lactic acid, and effectively alleviate the oxidation of normal mice. |
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