柚皮苷-羟丙基-β-环糊精包合物酶解制备柚皮素工艺的研究

Preparation of naringenin by enzymolysis of naringin-HP-β-CD

目的:确定羟丙基-β-环糊精包合柚皮苷后酶解制备柚皮素的主要因素,并优选出高转化效率、稳定、适合工业化生产的工艺条件。方法:采用逐步滴加法制备柚皮苷-羟丙基-β-环糊精包合物,并利用蜗牛酶将其水解制备柚皮素,以转化率为指标,通过单因素考察pH、温度、反应时间、酶用量及底物浓度对转化率的影响,并通过正交试验优化其制备工艺;采用差示扫描量热法验证柚皮苷是否被羟丙基-β-环糊精包合,采用1H-NMR,13C-NMR鉴定水解产物。结果:柚皮苷-羟丙基-β-环糊精包合物在最佳酶解条件下(温度37℃、pH 5.0、底物质量浓度30 g.L-1,酶与底物比3∶5,反应时间12 h)的转化率为98.4%,远大于未包合柚皮苷的转化效率;反应产物相对分子质量为272.25,核磁共振谱证实产物为柚皮素。结论:柚皮苷经羟丙基-β-环糊精包合后酶解制备柚皮素,可以缩短反应时间、提高底物浓度、减少酶用量,从而显著提高转化效率。该工艺稳定,适合工业化生产。

Objective： To determine the major factors affecting the conversion efficiency of naringin-HP-β-CD that was en- zymed to prepare naringenin were determined and select the process condition with high conversion efficiency, stable and suitable for industrial production. Method： The dropping method was used to prepare naringin-HP-β-CD, which was hydrolyzed by snailase to obtain naringenin. With the bioconversion rate as the index, the effects of pH value, temperature, reaction time, dosage of enzyme and concentration of naringin-HP-β-CD on conversion rate of naringenin were detected for the purpose of optimizing the preparation condition. the conversion efficiency of naringin-HP-β-CD was verified by scanning calorimetry, and the Hydrolysis product was identified by 1H-NMR, and t3C_NMR. Result： The optimum enzymolysis of naringin-HP-β-CD with snailase was 98.4% under the conditions of 37 ℃, a pH 5.0 acetic acid- sodium acetate buffer solution for 12 hours. The substrate concentration was 30 g·L-1 and the weight ratio of enzyme and substrate was 3：5. Under the optimum enzymolysis condition, the conversion rate of naringin-HP-β-CD was higher than naringin that was not entrapped with HP-β-CD, with 272.25 reaction product relative molecules. The structure of nalingenin was con- firmed by the analysis of 1H-NMR and 13 C-NMR. Conclusion： Naringin which is entrapped with HP-β-CD to prepare naringenin can significantly improve the conversion efficiency by shortening the reaction time, increasing the concentration of the substrate and reduc- ing the amount of enzyme. Therefore, the process is stable and it was suitable for industrialization.