Study On Regulation And Purification Technology In Biotransformation Of Natural 2-phenylethanol

2-Phenylethanol (2-PE) is an important aromatic alcohol with rose scent. In this study, L-phenylalanine was used as the precursor for 2-PE production through the Ehrlich pathway using active dry Saccharomyces cerevisiae. Alternative strategy of glucose metabolism and ethanol oxidation is the basic system of 2-phenylethanol production.This system can not only solve the 2-PE and ethanol synergistic inhibition of biotransformation system, but also improve carbon substrate utilization efficiency. Extraction method of cofactors NAD+ and NADH in yeast cells was optimized by Plackett-Burman experimental design and response-surface analysis. The determination method for cofactor NAD+ and NADH concentrations was established. Therefore, the relationship between NADH/NAD+ ratio and 2-PE production rate was explored based on the cofactor regulation. According to the results, the average production rates of 2-PE in glucose metabolism (stage Ⅰ) and ethanol oxidation (stage Ⅱ) were 0.38 g/(L·h) and 0.14 g/(L·h), respectively. The NADH/NAD+ ratio in stage Ⅰ was 2 to 7 times higher than that in stage Ⅱ, in which the reducing power is limited. Keeping the NADH/NAD+ ratio in a relative high degree was beneficial to 2-PE accumulation during 2-PE production by batch addition of glucose. Furthermore, butylated hydroxytoluene and sodium peroxide could promote the NADH/NAD+ ratios in stage land stage Ⅱ, respectively. Then, the production rate of 2-PE was consequently increased. Total 2-PE concentration reached 15.1 g/L in 85 hours.2-PE separation and purification techniques are major bottleneck in natural 2-PE industrialization. Costs, quality and purity were the key for separation of natural 2-PE product. In this study, separation and purification steps included screening of macroporous resin, byproducts control and enhancement of the 2-PE aroma quality. Firstly, adomestic resin CZ90 was chosen for the experiments, because the price was 10 times lower than the imported resin. The adsorption quality was evaluated by adsorption equilibrium, dynamic adsorption, desorption and resin wear experiments. The result showed that resin adsorption capacity was 133 mg/g. After the wear experiments conducted over 200h, the adsorption capacity could reach 125mg/g. Because the resin adsorption capacity and easy desorption, it can be used in large-scale production. Secondly, ascorbic acid was added into 2-PE biotransformation system to improve the redox reaction and suppress the generation of by-products. The sample that exhibited the best similarity to the standard 2-PE sample (99.19%) was the sample to which ascorbic acid had been added during glucose metabolism. Finally, the main impurity of 2-PE raw material was analyzed and determined. The purity of natural 2-PE reached 99.9% from 96.5% by the twin towers distillation using aspen plus.