Study On Preparation Of 5'-nucleotides By Waste Beer Yeast

Preparing waste beer yeast with high RNA content, the processing of the RNA and 5'-nucleotides production from waste beer yeast were studied in this paper.Optimization of the culture components for preparing waste beer yeast with high ribonucleic acid content was studied. In the first step, two level factorial design of Plackett-Burman was used to evaluate the influence of nine related factors. It showed that three important factors in the medium were ammonium sulfate, yeast extract and NaCl. The path of steepest ascent was used to approach the optimal region of the fermentation conditions subsequently. In the third step, the concentrations of those three main factors were further optimized by using Box-Behnken and Response Surface Analysis. By solving the quadratic regression model equation, the optimal concentrations of the variables were determined. The results as follows: 30% of yeast mass ratio was added in the 100mL of medium which contained glucose 4 g, (NH₄)₂SO₄ 5.8 g, yeast extract 0.28 g, beef extract 0.3 g, KH₂PO₄ 0.3 g, MgSO₄ 0.03 g, ZnSO₄ 0.001 g, NaCl 4.77 g. And the process conditions were as follows: concentration of yeast 8%, liquid volume 50 mL/250mL bottle, rotational speed 150r/min, processing temperature 20℃, and processing temperature 7 h. As the result, the content of RNA in waste beer yeast increased from 4.175% to 7.723%. The content was increased by 85.0%.The RNA extraction conditions were studied. The results of orthogonal experiments showed that the optimum conditions were as follows:extraction time 5 h,extraction temperature 100℃, concentration of sodium chloride 8%, and concentration of yeast 8%. Under the conditions, the extraction rate of ribonucleic acid was 6.68(g/100g dry yeast), the yield was 94.08%, and OD260/OD280 was 2.01.The 5'-phosphodiesterase was extracted from the barley malt root. Enzymolysis conditions for four kinds of 5'-nucleotide from RNA were: the RNA concentration 1.8%, 5'-phosphodiesterase 2000 U/g RNA, pH 6.8, reacted for 2.0 h at 70℃. The total yield of four kinds of the mononucleotides was 87.3%.The separations of crude 5'-nucleotides by anion ion exchange resin 201×7 were investigated. Separating conditions including solution pH, nucleotides loaded amount and the velocity were optimized. The four nucleotides were separated thoroughly and the average yield was over 80%.