| Lycoperdon pyriforme Schaeff.:Pers., belonging to Agaricomycetidae, Agaricales, Lycoperdaceae, is considered one of macro-fungus, which has become attractive as a source for the development of drugs and nutraceuticals. Up to now, polysaccharides are mainly extracted from the fruiting body of macro-fungi,which are generally produced in solid culture using composts or lingocellulosis waste such straws or wood and it is always time-consuming to culture their fruiting bodies and the product quality control is difficult. Submerged culture is viewed as a promising alternative approach to produce a large amount of mycelial biomass and macro-fungi polysaccharides. There are several advantages of the submerged culture over solid culture on polysaccharides (EPS) production:low fermentation period, low costs, availability of convenient control of the product quality during its cultivation, high-quality and concentration of products and easy downstream processing. Currently, lots of researches have been focusing on increasing the production of mycelial growth and metabolite production in macro-fungi submerged culture. For centries the fungi have been used in traditional Asian medicines for the prevention and treatment of various types of diseases, such as antitumor, bacteriostasis, anti-inflammatory, febrifugal, antitussive and arresting hemorrhage. Because of their novel properties, the bioactive microbial polysaccharides β-D-glucans have been used for immune modulation and tumouristasis. Furthermore, most of these bioactive polysaccharides are (1-3)-β-D-glucans with (1-6)-β-linked side branches. Modern studies show that the polysaccharides from L. pyriforme Schaeff.:Pers. and Calvatia gigantea have various boiological functions such as immunomodulating and anticancer effects.Thus, EPSs from puffballs have attracted more attentions from scientific and industrial communities.To fully use L. pyriforme Schaeff.:Pers., this study was focusing on screening the optimum ingredient of submerged culture medium, extraction, isolation and purification technology and antioxidative activity for the polysaccharide from L. pyriforme Schaeff: Pers.. The main results were as follows:1. Carbon source tests showed that the glucose was the best carbon for producing mycelia biomass in all the tested carbon sources, the yeild of dry cell weight and EPS was9.03g/L、0.82g/L.Nitrogen source tests showed that the glutamic acid is superior to the other nitrogen sources,the yeild of dry cell weight and EPS was62.49g/L、1.82g/L.Inorganic salts and growth factors tests showed0.15%KH2PO4,0.1%MgSO4·7H2O can promote the yield of CDW and EPS.The optimal medium was determined was glucose2.0%, glutamic acid at the same nitrogenous content with0.5%NH4NO3, KH2PO40.15%, MgSO4·7H2O0.1%through the one-factor-once-time method.And then,the effect of medium components on the performance of exopolysaccharide (EPS) production with response surface methodology (RSM) by L. pyriforme Schaeff was investigated in submerged culture. The optimal medium of produce CDW was determined as follows (%):maltose7.14, glutamic acid0.87, VB12.81×10-4, yeast extract0.5, KH2PO40.15, MgSO4·7H2O0.10,initial pH value5.5. The optimal medium of produce EPS was determined as follows (%):maltose6.11, glutamic acid0.70, VB12.81×10-4, yeast extract0.5, KH2PO40.15, MgSO4·7H2O0.10,initial pH value5.5. The response equation was as follows:YCDw=-163.624+31.599X+173.827Y+39.174Z-1.652X2-59.195Y2+1.89Z2-8.61YY-1.711XZ-32.078YZYEPS=0.87156+0.08858X-3.60919Y+3.61938Z-0.03512X2+0.66526Y2-1.60012Z2+0.54955XY-0.16320XZ-2.44860YZYCDW:dry cell weight, YEps:exopolysaccharide, X:maltose, Y:glutamic acid, Z: VB1At last, the optimal culture condition of produce EPS were obtained as follows (%):maltose6.11, glutamic acid0.70, VB12.81×10-4, yeast extract0.5, KH2PO40.15, MgSO4·7H2O0.10, culture temperature28℃rotating speed160rpm. the culture duration6days. After the confirmatory experiment, the yield of EPS produced by L. pyriforme in the optimal medium increased4.6times compared with that in the initial medium of0.82±0.09g/L and reached3.71±0.16g/L2. It was studied in this paper to obtain the optimum extraction conditions of exopolysaccharides from L. pyriforme Schaeff:Pers. by submerged culture. The optimum extraction conditions of exopolysaccharides were determined according to the result of one-factor-a-time and Box-Benhken design. The optimum conditions of extracting were obtained as follows:extraction times is8hours, ethanol proportion is95℃,the initial pH value was8.0. The Minitab16.0was employed to design response surface and the yield of polysaccharides was used as the responsive values. The optimum extraction conditions were as follows:extraction times is14hours, ethanol proportion is97%,the initial pH value was7.4, and the predict yield of EPS was0.58g, which was1.45,1.57and1.57times than before.3. In order to comprehensively utilize the submerged culture products, the methods to remove the protein from the fermentation liquid of L. pyriforme Schaeff.:Pers. were studied.The different methods of removing protein (NH4)2SO4and trichloroacetic acid method were investigated,the latter destroyed polysaccharide structure, so (NH4)2SO4was used in this experiment,and the optimum concentration is36%.4. Through studied on the polysaccharide by infrared spectroscopy, there was characteristic absorption peak of exopolysaccharides between spectrum of4000～400cm-1, it existed amic (-NH-CO-) and hydroxyl and polysaccharide configuration wasβ-conformation.5. For determine the antioxidation, a minisize method was developed for determination of the hydroxyl radical in fenton reaction by spectrophotometry. According to the experiment, when the total volume was177μL, CV(2×10-5mol/L):20μL; Fe2+(5×10-3mol/L):18μL;1%H2O2:9μL; the volume of buffer solution:136μL, and Na2HPO4·2H2O (0.2mol/L):61.82μL; C6H8O7·H2O (0.1mol/L):74.18μL stabilization time:10min.The study found that the polysaccharide of L. pyriforme Schaeff:Pers. had reduction ability and antioxidant activity. It can effectively eliminate hydroxyl radical, superoxide anion. As the polysaccharide concentration increased from0.01%-0.5%, the polysaccharide with concentration of1%has better antioxidant activity than others. |
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