Study On Submerged Fermentation, Isolation And Purification Of Lentinan

Lentinula edobes is one of the most important edible and medicinal mushrooms, which has attracted much attention due to its immunomodulatory, antitumor and antioxidant function. In this dissertation, research of submerged fermentation, extraction, isolation, purification and physicochemical properties of alkali-polysaccharide from Lentinula edobes mycelium were made, a comparision of in vitro DPPH scavenging activity was also employed between three fractions with different molecular weight. Aimed at application aspect and scale-up, in this thesis, the study was mainly focused on to develop the extraction technology and purification of intracellular polysaccharide obtained from its mycelium with tangential flow-ultrafiltration method based on the optimization of submerged fermentation conditions of Lentinula edobes strain by using the method of PB design and response surface analysis, and then DPPH scavenging activity of different fractions of lentinan were conducted after measurement of their physicochemical properties with HPLC and IR. The research expressed that the design of process route was reasonable, the method was proper and the main contents and conclusions are listed as following:1. An optimal Lentinula edobes strain L0010 was screened out in term of the biomass weight and exopolysaccharide contents. The experimental data obtained were fitted to a second-order polynomial equation with analysis of multiple regressions. And the optimal culture conditions of strain L0010 by submerged fermentation were determined: bean powder (A) 3.676g/L, yeast extract powder (B) 3.892g/L, glucose (D) 26.961g/L and FeSO4.7H2O(C)=0.632g/L. At this condition, the predicted yield of biomass was 5.224g/L.2. The ultrasonic extracting water soluble polysaccharide and the alkaline soluble polysaccharide under nitrogen atmosphere from fermented mycelium of Lentinula edodes were studied. The yield of water soluble polysaccharide is 6.14 mg/g, and the yield of alkaline soluble polysaccharide is 23.134 mg/g. The optimal extracting alkaline soluble polysaccharide under nitrogen atmosphere conditions are as follows: temperature 70℃, extraction time 3h and 1:40. By DPPH determination, the antioxidant activity of alkaline soluble polysaccharide is 61.32%, which lower slightly than that of water soluble polysaccharide(67.16%). In this way, it proves that the antioxidant activity of the extracted alkaline soluble polysaccharide under nitrogen atmosphere can be protected well.3. The tangential flow-ultrafiltration technology was carried out to isolate alkali intracellular polysaccharide obtained from strain L0010 mycelium. Four different fractions were collected after being used two kinds of membrane of molecular weight cut off 100KDa and 8KDa, named LNTS-1,LNTS-2,LNTJ-1 and LNTJ-2. Those white powders were soluble andβ-pyran type glucan determined by IR spectroscophy. Among them, LNTS-1 was a purified fraction with 1.84×106 molecular weight after HPLC detection. while LNTJ-1 were consisted of two different sugars with 1.64×106 and 1.37×106 molecular weight respectively. From the results of DPPH scavenging ability, it suggested that the alkali intracellular polysaccharides of Lentinula edobes strain L0010 with nitrogen protection presented a higher scavenging activity comparing with water-soluble polysaccharides of Lentinula edobes strain L0010. Furthermore, the ability of them had a positive relationship with their protein contents.