Breeding Of Production Strain Of Riboflavin Nutrition Yeast And Studies On Its Culture Conditions

According to the metabolic path and metabolic control theory of riboflavin in yeast, the dissertation focuses on the breeding of high riboflavin content nutritious yeast strain and its culture conditions. The main research contents and results are as follows:(1) Three determination methods were studied, included spectrophotometer, fluorometry and high performance liquid chromatogram. It was showed in the results that fluorometry was the suitable method to determine the content of intracellular riboflavin of yeast. Fluorescence was excited at the wavelength of 458nm, emission wavelength 525nm. To eliminate the effect of other fluorescent substance on determination of riboflavin, Na₂S₂O₄ was employed as quencher of riboflavin. Liner relationship between response value and content of riboflavin was obtained with high reproducibility and veracity in this method.(2) S. fragilis RY-3 as starter, its cell and protoplast were mutated with ultraviolet radiation. 19 strains of mutant with 8-AG resistance and oligomycin resistance were obtained through primary screening, compound screening, capability verifying and continuous passage cultivation. The growth circles of RY605,RY606,RY618 were shortened markedly and contents of intracellular riboflavin were increased 79.97%,69.56%,71.09% compared with their starters, respectively. Genetic character of these mutants was stable testified by passage cultivation.(3)The mutant RY605 and E. ashybii were employed as parent strains of protoplast fusion. Protoplast formation conditions of two parent strains were as follow: RY605 was treated for 60min with 2% snail enzyme at 30℃ to form protoplast, and E. ashybii was treated for 120min with 1% cellulose and 2% snail enzyme at 30℃. Deactivated protoplasts of two parent strains fused for 60min in 35% PEG solution. The riboflavin content of fusant FY309 was 87.86mg/kg, increased 15.27% compared with parent strain RY605.(4) The effect of different fermentation conditions on growth and riboflavin content of yeast were studied in this dissertation. Through single factor experiment and uniform design experiment, medium component was optimized as: Glucose 40g/L, (NH₄)₂SO₄ 3.5g/L, yeast extract 5g/L, biotin 100μg/L, thiamine 1.5mg/L, MgSO₄·7H₂O 1.1g/L, CoSO₄·7H₂O 28mg/L, CuSO₄·5H₂O 0.01 mg/L, MnCl₂ 0.02 mg/L, ZnSO₄·7H₂O 34 mg/L, FeSO₄·7H₂O 14 mg/L. The suitable batch culture conditions of RY605 in shack flask was: 60mL medium with the initial pH value of 5.5 in 500mL flask, 30℃ , 180r/min. The yeast washarvested at stationary phase.(5)The effect of fermentation conditions (concentration of soluble oxygen, pH value, concentration of glucose) on fermentation were studied, the result showed that during feeding cultivation of yeast, the suitable conditions were as follows: relative concentration of soluble oxygen 20%, pH value 5.5 and concentration of glucose lg/L⁵g/L. Yeast had high growth speed and large biomass under optimized conditions. The riboflavin content of yeast could be increased by increasing of riboflavin content of seed. By treatment of temperature shock before the end of cultivation, the riboflavin content of RY605 increased to 102.41mg/kg.The innovation points of this dissertation in the research of riboflavin nutritional yeast are that the accumulation of intracellular riboflavin of yeast was studied first time. The researches included the change of intracellular riboflavin of yeast with the growth of yeast during shack flask batch cultivation and feeding batch cultivation, and the effect of cultivating conditions on the growth of yeast and content of intracellular riboflavin. According to the theory of metabolism control, two-phase-control method was provided to adjust temperature and soluble oxygen, riboflavin content of yeast could be increased to 102.41mg/kg by temperature shock before the end of cultivation. The protoplast mutagenic technique was successfully applied to breed a mutant RY605 which had 8-AG resistance and oligomycin resistance, content of riboflavin in shack flask reached 87.86mg/kg, increased 79.97% than starter. The principles were discovered including yeast growth in batch cultivation and feeding cultivation, change of riboflavin content during yeast growth circle and effect of yeast growth on content of riboflavin were determined. Fluorometry was selected as determination method of intracellular riboflavin of yeast, and Na2S2O4 was employed as quencher of riboflavin to eliminate the effect of other fluorescent substance on determination of riboflavin.