| Schizophyllan(SPG),a kind of neutral exopolysaccharide,was obtained by deep fermentation of Schizophyllum commune.Natural SPG with anti-tumor,moisturizing and other biological activity,had a wide range of applications in medicine,cosmetics and health care products and other fields.However,natural SPG udually have a large molecular weight,resulting in poor water solubility,high viscosity,which might affect its application.Thereby,is important to regulate the molecular weight of SPG and improve the yield of SPG.In order to determine the predominant regulatory factors,the culture conditions and regularities of SPG and ?-1,3-glucanase production,as well as the relationship between polysaccharide yield,molecular weight and enzyme yield were studied systematically for the first time.Theoretical theoretical basis were provided for the regulation of enzymatic hydrolysis under the premise of ensuring the yield of polysaccharides and improving the enzyme activity.The main research contents and conclusions were as follows:1.Scelection of Schizophyllum commune and optimization of culture mediumTaking schizophyllan yield and beta-1,3-endoglucanase and total enzyme activity as the indexes,using congo red agar staining method and flask culture,the strain GIM5.43 was selected which had relatively high yied of polysaccharide and enzyme activity from stain GIM5.42,GIM5.43,GIM5.44,Sc1,for further study to optimize the addition of yeast extract and NH4 Cl.The results showed that: when yeast extract was 1.00 g/L,getting the small particles of mycelium,the polysaccharide yield was 5.31 g/L,but the total enzyme activity was relatively low.When the yeast extract was above 7.00 g/L,getting flocculent villous mycelium,the polysaccharide yield was 0.50 g/L,the total enzyme activity was 0.23 U/m L.In order to ensure the yield of polysaccharide,the concentration of 1.00 g/L was added to the initial yeast extract,0.10 g/L yeast extract was added to the sixth day of fermentation,and the yield of polysaccharide was 4.16 g/L,which was higher than that of the non-optimized 61.24%,the enzyme activity was 0.34 U/mL,an increase of 142.86%.When the content of yeast extract was low(1.00 g/L),the addition of NH4 Cl had no effect on the enzyme activity.When the content was 3.00 g/L,NH4 Cl could increase the enzyme activity by 51.20%.2.Effects of growth factors and polymers on fermentationThe growth factors such as L-glutamic acid,vitamin B1,citric acid and high polymers were added to the fermentation system to study the effects of these environmental factors on the fermentation process and polysaccharide production and enzyme activity.The results showed that:(1)content of L-glutamic acid and vitamin B1 were 1.5 mg/L which have the highest total activity,respectively 0.27 U/mL,0.24 U/m L,compared with not adding growth factors increased by 35.00%,33.33%,and the polysaccharide yield were increased by 7.52% and 13.68%.When the content of citric acid was 100 mg/L,the yield of polysaccharide and total activity of enzyme were the highest,which were 9.34% and 15.79% higher than those without adding them.In order to ensure the polysaccharide yield and increase the activity of the enzyme,the initial contents of L-glutamic acid,vitamin B1 and citric acid were 1.5 mg/L,1.5 mg/L and 100 mg/L respectively.(2)The addition of polymer SPG,gel polysaccharide,sodium carboxymethyl cellulose(CMC)and hyaluronic acid in the initial medium can increase the viscosity of the culture medium,change the morphological characteristics of the mycelium and make the mycelium more dispersed.The results showed that the yield of polysaccharides were increased by 45.74%,49.61%,37.03% and 27.45% respectively,and the activity of enzyme were increased by 40.57%,37.14%,71.00% and 72.58% respectively.Adding SPG,gel polysaccharide,CMC could get small mycelium,small particles,feather-like floc.3.Study on deep fermentation technology of Schizophyllum communeStudy on liquid deep fermentation of Schizophyllum commune on 7 L fermentor.The effects of dissolved oxygen and pH value on the fermentation process and polysaccharide and enzyme production were studied respectively.The optimum dissolved oxygen and pH were obtained.On this basis,the three-stage fermentation control strategy and batch feeding strategy were studied.The results showed that:(1)In the early stage of fermentation(0-36 h),pH was natural,dissolved oxygen(DO)was 40.00%(36-84 h);in the middle of fermentation,pH was 3.80,DO was 30.00%;at the end of fermentation(84-108 h),pH was 5.40,DO was 45%.The yield of polysaccharide and the activity of glucanase were 4.11 g/L and 0.26 U/mL,respectively,which were 13.50% and 107.98% higher than those before optimization.(2)Through a single patch method with 15.00 g/L glucose and intermittent three times every 12 h adding 5.00 g/L glucose at 48 h,the results show that the synthesized more intermittent feeding way for polysaccharide and enzyme,respectively feeding group increased by 44.20%,176.92%.The results showed that the method of intermittent addition was more favorable for the synthesis of polysaccharides and enzymes by adding 15.00 g/L glucose and intermittent supplementation of 5.00 g/L glucose three times every 12 h at 48 h.Compared with the non-fed group increased by 44.20%,176.92%.4.Preliminary study on the control strategy of polysaccharide molecular weight in latestage of fermentation.The effects of exogenous endo-?-1,3-glucanase and the endogenous ?-1,3-glucanase on the molecular weight of polysaccharides were studied.The exogenous ?-1,3-glucanase was added to the flasks on the 7th day.Enzymes could significantly degrade macromolecular polysaccharides.But the molecular weight of the hydrolyzate was 7.96 × 103 Da,which indicated that the enzyme was not suitable for the regulation of molecular weight of SPG.After the fermentation broth was cultured for 4 days,the mycelia were separated and removed to allow the endogenous ?-1,3-glucanase to be digested.The results showed that the endogenous enzyme had a certain degradation effect on the polysaccharides,And the molecular weight of the polysaccharide was decreased to some extent,which indicated that the molecular weight of the polysaccharide was regulated by the endogenous enzyme regulation system. |
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