Mutation Breeding And Fermenting Of Curdlan Producing Strain

Curdlan is a water-insoluble polysaccharide composed of D-glucose monomers connected byβ-1,3-D-glycosidic linkages.It can form a high-set thermal nonreversible gel while heated.As a food additive,it is widely used in food industry to improve the water holding,viscoelasticity and stability of food.In order to improve the production of curdlan,the UV mutation and NTG mutation method were established in this paper.The high throughput screening method was established to improve screening efficiency.The optimization of the culture medium was also tried to further improve the production of curdlan.These were foundamental work for large-scale industrial production of curdlan.The main results were as follows(1)Mutation and screening of curdlan producing strainStrains CA03 was mutated.The optimized mutation conditions were got.Mutation time was 30s.The distance was 30cm.UV lamp was 15w.The fatality rate was 99.7%.The morphological positive mutation rate was 0.23%.One high yield strain UV150824-02 was screened from 67200 strains whose crude yield of curdlan was 45.5g/L,8.3%higer than CA03NTG mutation conditions were got.The NTG concentration was 0.7mg/mL.The reaction time was 30min.The pH was 8.5.Three high yield strains were screened from 105000 strains.One of them was NTG 160615-05 whose yield of curdlan was 45.5g/L,16.9%higer than UV150824-02 and 26.7%higer than CA03(2)Characteristics of curdlan from high-yielding strains and optimization of fermentation mediumThe properties of the high-yield strains were as follows:The structure of 3 high-yield strains were consistent with commercial samples and had good gel properties.The gel from curdlan of strain NTG 160615-05 was more brittle than the gel from commercial curdlanThe fermentation medium was optimized.The optimum carbon source,nitrogen source and phosphate was determined by single factor experiment.The optimum components included 7%sucrose,0.162%NH4Cl,0.1%KH2PO4,0.345%K2HPO4,0.1MgSO4·7H2O,0.4%corn pulp,0.2%CaCO3 by orthogonaltest.The production of the strain NTG 160615-05 was improved by 23.4%in the optimized medium(3)Establishment of high throughput screening methodA microscale analysis method of curdlan was established.The analysis conditions were as follows:1.Omol/L NaOH,2.0mg/mL aniline blue,0.5mol/L phosphate buffer(pH=7),90 min reaction time,550nm detection wavelength.The correlation between the curdlan content measured by this method and the production measured by weight method was 0.8629.This method can be used to replace the traditional method to quantify the content of the strain.48-well plate fermentation system was established.The fermentation conditions:1mL culture volume,culture 48h at 30℃ with 250 rpm.There was a good correlation between the result of the 48-well plate fermentation system and result of shake flask system,and the variation of 48-well plate fermentation system was only 10.3%.This new system can replace the traditional fermentation system for the initial fermentation of mutagenic strains.