| Zinc, an abundant essential trace element in the body, is involved in the structure and function of over 300 enzymes and responsible for numerous important physiological functions. Zinc deficiency could lead to a series of clinical symptoms, such as stunted growth, depressed immunity, decreased antioxidant activity, reduced learning and memory capability and skin incomplete keratosis. However, epidemiological studies have shown that approximately 50% of the world’s population is at risk of inadequate zinc intake. Edible and medicinal fungi polysaccharides are high molecular weight polymers isolated from fruiting bodies, fungal mycelia and fermentation broth, which have been demonstrated to have a variety of biological activities, such as immunoregulatory, antioxidant, antihyperlipidemic, hypoglycaemic, hepatoprotective and antitumour activities.Pholiota nameko, which belongs to Cortinariaceae family, is a widely cultivated edible mushroom in China. In the present study, P. nameko SW-03 was used as a vector of zinc biotransformation with the purpose of producing mycelia zinc polysaccharide(MZPS). MZPS has properties of both organic zinc and polysaccharide. Therefore, MZPS could be developed as a zinc-enriched product or a functional food.(1) Protoplast mutagenesis breeding technology was used to screen mycelia polysaccharide(MPS) high-yield strains. The results showed that using mycelia of 7d mycelial age as testing material, 0.6mol/L magnesium sulfate as osmotic stabilizer, enzymolysis with complex enzyme(containing 2% snailase and 2% cellulase) for 3h at 30°C under p H 6.5 was the optimum conditions for P. nameko protoplast preparation. Compared to the starting strain, the MPS yield of mutant strain 16 creased by 33.43%. In addition, mutant strain 16 showed hereditary stability, and mutant strain 16 was designated as SW-03.(2) Zinc enrichment capability and endurance ability were analyzed. The results showed that 80mg/L of zinc concentration in liquid medium could be chosen to produce MZPS. After fermenting for 168 h in the fermentation tank, the dry biomass was 2.28g/L, the MZPS yield was 41.31mg/L. In addition, the zinc content of MZPS(16.39±0.72mg/g) was markedly higher than that of MPS(10.76±0.41mg/g)(P<0.01).(3) Extraction process for MZPS was optimized by Plackett-Burman experimental design and response surface experimental design. The optimum conditions of MZPS extraction were water multiple of 25.00, p H value of 8.00 and precipitation time of 22.00 h. The optimized extraction yield of MZPS was 2.98%.(4) The MZPS was purified by DEAE-cellulose anion-exchange chromatography and Sephadex G-100 gel permeation chromatography. As a result, the MZPS was fractioned into three independent elution fractions(MZPS-1a, MZPS-2a and MZPS-3a), and MZPS-2a was the main fraction. By the analysis of high-performance gel permeation chromatography(HPGPC), the weight-average molecular weight(Mw) of MZPS-2a was 1.36×104Da, and it was a homogeneous polysaccharide. Fourier transform infrared spectroscopy(FT-IR) spectrum of MZPS-2a showed typical structure of fungi polysaccharides. The monosaccharide composition was analyzed by high performance liquid chromatography(HPLC) after pre-column derivatization. The results showed that MZPS-2a was composed of glucose, mannose, glucuronic acid, galactose, galacturonic acid and arabinose in a molar ratio of 172.59:5.29:4.61:4.20:1.01:1.00.(5) The anti-ageing activity of MZPS was analyzed by antioxidant testing in vitro and in vivo. For antioxidant testing in vitro, MZPS(crude and its fractions) had considerable antioxidant activity. In particularly, MZPS-2a showed the highest antioxidant capacity, which might be attributed to its much higher content of uronic acid, a lower ratio of glucose in monosaccharide composition and the relatively low molecular weight. For antioxidant testing in vivo, we found that supplementation of MZPS could evoke a dose-dependent increased activities of superoxide dismutase(SOD) and total antioxidant capability(T-AOC), which was associated with reduction of lipid peroxide(LPO) and malondialdehyde(MDA) contents in D-gal-induced ageing mice. MZPS showed better inhibitory effects than MPS on the levels of LPO and MDA, and this biological activity might be related to the antioxidant properties of zinc. This study suggested that MZPS might be used as a natural antioxidant to slow the progression of ageing.(6) The antihyperlipidemic and hepatoprotective activities of MZPS were studied by building hyperlipidaemic mice models. The results showed that the administration of MZPS could significantly reduce the levels of total cholesterol(TC), triacylglycerols(TG), low-density lipoprotein cholesterol(LDL-C) and very low-density lipoprotein cholesterol(VLDL-C) and improve the level of high-density lipoprotein cholesterol(HDL-C) in serum. Moreover, MZPS also could reduce the levels of TC and TG in liver. Simultaneously, we found that MZPS could evidently improve the activities of SOD and T-AOC and decrease the levels of LPO and MDA in liver and serum. In addition, the histopathological observations of mice livers indicated that the MZPS could attenuate liver injury. These results demonstrated that the MZPS might be effective in lowering lipid and protecting against high-fat diet-induced hyperlipidemia and non-alcoholic fatty liver. |
