Study On The Breeding Of Bacillus Thuringiensis And Its Polysaccharides Fermentation And Performance

Polysaccharide is a kind of natural macromolecular organic active substance in nature.It is the main macromolecular organic compound that composes life in addition to biological macromolecules such as nucleic acid and protein.Microbial exopolysaccharides（EPS）is a kind of carbohydrate polymer produced by adapting to the culture environment.It is not only a metabolite,but also a nutrient that can be used as an energy source.In recent years,research on EPS has found that it has a variety of biological activities and high safety,and has been applied in many fields.Compared with polysaccharides derived from plants and animals,EPS produced from microorganisms has fewer limitations,controllable production,and potential industrial production value.Based on this,this study screened out Bacillus thuringiensis with high EPS production from specialty foods,and found that there are few studies on EPS of this strain.Therefore,this paper focused on optimizing the fermentation process,and carring out purification optimization and structural characterization of the new EPS,and finally exploring the EPS activity study,the main research content is as follows:（1）Screening of target strains.73 strains were obtained from specialty fermented foods.Based on multiple indicators such as biomass optical density value（OD）value of the strain,p H value,extracellular heteropolysaccharide content,and fermentation liquid viscosity of the strain.Strain IX17 with high EPS production was obtained after preliminary screening and re-screening.Through morphological analysis,physiological and biochemical identification,and16S r DNA sequencing,it was confirmed that the strain was Bacillus thuringiensis and was named BT-IX17.（2）Optimization of fermentation process.At the shake flask level,single-factor optimization of the fermentation medium components and fermentation culture conditions were carried out,through single-factor experiments on the components of the fermentation medium and fermentation culture conditions,and then the Plackett-Burman（PB）design is used to determine the importance of the 9 factors Judging,three important factors were selected for Box-Benhnken experimental design optimization to obtain the final optimal culture plan:glucose 22.6 g·L^-1,soybean meal 9.3 g·L^-1,corn syrup 2.0 g·L^-1.K₂HPO₄ 0.5 g·L^-1,Na H₂PO₄1.5 g·L^-1,Mg SO₄ 1.5 g·L^-1,Mn Cl₂·4H₂O 0.05 g·L^-1,initial p H 7.5,filling volume 70 m L,temperature set to 34℃,the amount of inoculation is 6%.Compared with the initial fermentation process,the output of EPS has increased by 133.3%.After a 7-liter fermentation tank,the strategy of p H control and two-stage regulation of stirring speed was adopted,and the final EPS output was increased by 153.40%compared with the optimum process conditions at the shake flask level.（3）EPS purification and structural characterization.Crude EPS were subjected to 6 times the volume of ethanol and alcohol precipitation,Sevage reagent removes protein,passes through DEAE Fast Flow anion exchange chromatography column and Sephadex G100separation column,and finally obtains a neutral polysaccharide EPS-A.After structural characterization,the EPS-A has a molecular weight of about 3.356×10⁴ Da,contains the characteristic functional groups of polysaccharides and hasβ-type glycosidic bonds.Its monosaccharide composition and proportion are fucose:rhamnose:arabinose:glucosamine:Glucose:Xylose:Fructose=6.46:0.46:5.2:5.98:1.39:16.98:2.62.The surface microstructure of EPS-A is a stacked layered structure,with a smooth surface and rich pore structure.The thermogravimetric analysis（TGA）analysis shows that the mass loss rate of EPS-A is 60%,and it has good thermal stability.Through the Congo red experiment,it can be known that the spatial structure of EPS-A may be irregularly coiled and does not contain a three-strand helix structure.（4）Application of EPS activity.（1）Take VC as a positive control,then compare and analyze the scavenging ability of EPS-A on ABTS⁺free radicals,hydroxyl free radicals and DPPH free radicals.The results show that EPS-A has a certain degree of antioxidant capacity,which is dose-dependent.When the mass concentration of EPS-A is low,the antioxidant activity is not obvious.When the EPS-A mass concentration is large,the antioxidant activity reaches the maximum value.The scavenging ability of EPS-A is in the order of hydroxyl radical scavenging ability>DPPH radical scavenging ability>ABTS⁺radical scavenging ability.（2）Study on rheological properties.The effects of different sugar concentrations,p H values,salt ions and temperature on the apparent viscosity of EPS-A were investigated.It was found that EPS-A has good stability on the changes in the external environment.The potential of a food additive.（3）Emulsification stability study.Commercial grade chitosan,xanthan gum,thermogel,pullulan and locust bean gum were used as reference controls to investigate the emulsification stability of EPS-A.The results show that EPS-A has good emulsification.The stability is second only to pullulan.（4）The study of flocculation ability characteristics,with chitosan as a reference control,the results show that EPS-A has good and stable flocculation ability,which is expected to become a natural polysaccharide flocculant.