Fermentation Process And Bioactivity Of Water-soluble β-1,3-glucan With Glycerol As Substrate

Water-solubleβ-1,3-glucan（WSBG）is a water-soluble polymers composed of glucose formed byβ-1,3-bonds,which have unique physiological and biochemical activities.Water-solubleβ-1,3-glucan is widely used in the fields of pharmaceutical,daily chemical raw materials,agricultural and livestock additives,has become a hotspot of research in recent years.But the methods of preparation of water-solubleβ-1,3-glucan still have problems such as high production cost,damage to pollution,and unstable process.This paper based on the strain of Agrobacterium sp.WS-8E3T,which can directly produce the water-soluble?-1,3-glucan by single bacterial fermentation.The fermentation process of this strain was studied to improve the water-soluble?-1,3-glucan production by using glycerol as carbon source in the shake flask and 7-Lfermentor.The structure and bioactivity of the product was preliminarily studied.The main research contents and conclusions of this paper are as follows:（1）In the shake flask experiment the carbon source concentration,nitrogen source concentration,cultivation conditions,carbon source supplementation method,and nitrogen source supplementation method was optimized,and the structure of the product was characterized.The most suitable initial substrate is glycerol 40 g·L^-1,yeast extract 4 g·L^-1,（NH₄）₂SO₄3 g·L^-1,and Ca CO₃2 g·L^-1.The cultivation conditions was inoculation ratio 10%,liquid volume 75 m L,and rotational speed 220 r·min^-1.The supplementation method was 60 g·L^-1glucose with 30 g·L^-1glycerol added at 24 hours.At the same time,based on the 4 g·L^-1yeast extract then add 3 g·L^-1（NH₄）₂SO₄at 4 hours.The production has increased from 1.6 g·L^-1to 3.9 g·L^-1.The results of MALDI-TOF-MS,FT-IR,and ion chromatography proved that the product is water-solubleβ-1,3-glucan with a DP of 18-22.（2）Multiple two-stage fermentation strategies were carried out in a 7-L fermentor for increasing production of WSBG.The two-stage control strategy of p H was proposed:During the0～24 h,p H was set to 7,and p H was set to 6.3 during 24～96 h,the expression of ATP-binding cassette transporters on cell membrane was up-regulated,which is more conducive to cell sugar production,and the WSBG production has increased to 8.0 g·L^-1.The two-stage compound carbon source supplementation process showed that the WSBG production was increased to 11.8 g·L^-1by maintaining a concentration of 30 g·L^-1with glucose during the 0～24 hours and 30 g·L^-1with glycerol during the 24～96 h.The two-stage compound carbon source supplementation process was used to couple with the compound nitrogen source supplementation process:Set the time for adding（NH₄）₂SO₄to 4 hours after fermentation,the WSBG production has reached to 13.1 g·L^-1.（3）The bioactivity of WSBG in the field of daily chemicals was analyzed.When the concentration of WSBG was 5 g·L^-1,the inhibition rates of tyrosinase monophenolase and diphenolase activity were 56.69%and 35.78%,the inhibition rate of hyaluronidase activity reached71.76%,respectively.The scavenging rate of hydroxyl radical was 94.93%when the concentration of WSBG was 3 g·L^-1,the scavenging rate of superoxide anion and DPPH radical were 41.75%and53.04%when the concentration of WSBG was 5 g·L^-1.Then the results of moisturizing experiments indicate that WSBG has certain moisture absorption and moisturizing bioactivity.（4）The potential prebiotic bioactivity and cholesterol inhibitory effects of WSBG were analyzed.WSBG has a significant promoting effect on the proliferation of Bifidobacterium,Bifidobacterium brevis,and Lactobacillus acidophilus.It could effectively promote the production of metabolite by intestinal microorganisms,and the gas production was significantly lower than inulin.WSBG had a certain binding effect on free bile salts,and further experiments showed that WSBG had a certain inhibitory effect on cholesterol in cholesterol micelle solution.