Structure Characterization,Fermentation Condition Optimization And Application Of Exopolysaccharide Produced By A Thermotolerant Bacillus Sp.

Fructans can be divided into inulin,levan and mixed types according to the position of the fructose group connection.Inulin is mainly found in plants such as inulin,whose fructose group is linked by a β-(2,1)bond.Levan is mainly derived from microorganisms,but rarely from plants.Its fructoses are linked by β-(2,6)bonds.Levan fructans are widely used in food,pharmaceutical and cosmetic applications because of the properties that distinguish them from common polysaccharides.In this thesis,the fermentation,isolation,purification,structural analysis,physical and chemical properties and preliminary applications of levan-type fructans were systematically investigated with a heat-tolerant strain of Bacillus sp.SFLV-ZM107 and preliminary identification of the production strains were also carried out.This has important academic and practical significance for the production of levan fructans by the microbial method.The main research contents and conclusions are as follows:The ability of strain SFLV-ZM107 to produce extracellular polysaccharides from sucrose was determined by observing the colony morphology of strain SFLV-ZM107 on glucose and sucrose plates.The morphology of strain SFLV-ZM107 was observed by light microscopy and transmission electron microscopy,and it was determined that strain SFLV-ZM107 was a bacillus with periphytic flagella,mesophytic or paraphytic budding,and not dilated.The strain SFLV-ZM107 was identified as Bacillus licheniformis by carbon source utilization experiments,physiological and biochemical experiments and 16 S r DNA sequence analysis.The extracellular polysaccharides produced by strain SFLV-ZM107 were purified by ethanol precipitation and ion exchange.The hydrolysis products of the purified samples were analyzed by high performance liquid chromatography(HPLC),and the monosaccharide composition was mainly fructose and a small amount of glucose.The extracellular polysaccharides produced by strain SFLV-ZM107 were identified by various analytical tools:(1)The polysaccharide was confirmed to be a levan-type fructan by NMR and IR scanning techniques using standard levan as a control.(2)Molecular weight determination using high performance volume exclusion chromatography(HPSEC).The results showed that its molecular weight was heterogeneous and was a mixture of fructans of different polymerization degrees,with an average molecular weight of about 5569 Da,which was smaller than the reported molecular weight of levan produced by Bacillus.(3)Differential scanning calorimetry(DSC)and X-ray diffraction analysis showed that the fructan had a fixed melting point with a main peak of 205.8°C.The X-ray diffraction peak is sharp,indicating that it has a crystal structure.(4)Scanning electron microscopy results showed that the structure of the obtained polysaccharides showed irregular spherical shapes with variable morphology.Fermentation conditions were optimized for levan production by strain SFLV-ZM107.The results showed that the increase in initial sucrose concentration favored the production of levan,but the fermentation period was prolonged accordingly.High concentrations of sucrose inhibited the growth of the bacterium.Sugar consumption rate,product conversion and yield were better at sucrose concentration of400-450 g/L;Strain SFLV-ZM107 grew best at 37℃ and its yield was higher than that at 50℃,but its sugar consumption rate was low and its cycle was long;The fermentation process is fast at 50°C,but the yield is low.The optimal conditions were optimized by response surface experiments and determined to be an incubation temperature of 40°C,a rotation speed of 200 rpm,an incubation time of 18 h,and a levan yield of 175 g/L.Based on the shake flask fermentation,a 5L fermenter was used to scale up the experiment,and the 50°C fermentation was significantly faster than the shake flask fermentation.The physical and chemical properties of levan produced by strain SFLV-ZM107 were measured,including hygroscopicity,moisturizing property,oil-holding property and emulsifying property.A preliminary investigation into the application of levan fructans was conducted:(1)As a freeze-drying protectant,10% levan solution had a protective effect on the freeze-preservation of lactic acid bacteria,but the protective effect was not as good as glycerol and alginate.The survival rates of lactic acid bacteria after preservation without protectant,10% levan,glycerol and alginate were 0.5%,12.9%,30.1% and 63.3%,respectively.(2)Vegetable preservative-levan has a certain effect on the freshness of lettuce and parsley.(3)The addition of different concentrations of levan to milk enhanced the viscosity,acidity and water retention of the fermented milk,with the 0.2% levan addition having the highest overall score.