论文部分内容阅读
柚苷酶能水解多种糖苷,在食品工业中具有重要的用途。相关文献报道的柚苷酶测定方法不能检测和监控食品糖苷的酶水解过程。本文利用液相色谱测定柚苷酶水解柚皮苷、熊果苷及杨梅苷等糖苷的底物和产物,从而探究柚苷酶在应用过程中的化学反应并测定其活力。采用的色谱分析条件:Symmetry C18反相柱,检测波长280 nm,柱温35℃,进样20μL,流速0.4 mL/min;流动相为水(A)、甲醇(B)及乙腈(C),溶剂的梯度洗脱程序是:0~7 min,A/B/C(v/v/v)=62/12/26;7~9 min,A/B/C(v/v/v)=15/35/50;9~15 min,A/B/C(v/v/v)=15/35/50;15~17min,A/B/C(v/v/v)=62/12/26;17~20 min,A/B/C(v/v/v)=62/12/26。该方法能有效分离柚皮苷、杨梅苷、熊果苷及水杨苷等底物及其水解产物;用该方法测定柚皮苷和柚皮素的相对标准偏差均小于1%,回收率分别在100.4%~105.2%,94.5%~117.3%。在50℃,pH 4.0的条件下,柚苷酶水解柚皮苷的米氏常数Km为0.10 mmol/L,最大反应速率Vmax为256 U/mg,Kcat为1 487/s。该液相色谱方法可检测柚苷酶对多种糖苷水解过程,能准确测定柚皮苷水解柚皮苷的酶活力及酶水解动力学,对于深入研究柚苷酶在食品中的应用具有重要作用。
Naringinase can hydrolyze various glycosides and has important uses in the food industry. Naringin assays reported in the literature can not detect and monitor enzymatic hydrolysis of food glycosides. In this paper, naringinase was used to determine the substrate and product of naringin, arbutin and myricitrin by liquid chromatography, so as to explore the chemical reaction of naringinase and determine its activity. The chromatographic conditions were as follows: Symmetry C18 reversed-phase column, detection wavelength 280 nm, column temperature 35 ℃, injection 20μL, flow rate 0.4 mL / min; mobile phase was water, methanol and acetonitrile; The gradient elution program of the solvent was: 0-7 min, A / B / C (v / v / v) = 62/12/26; 15/35/50; 9-15 min, A / B / C (v / v / v) = 15/35/50; 15-17 min, A / B / C / 26; 17-20 min, A / B / C (v / v / v) = 62/12/26. The method can effectively separate naringin, myricitrin, arbutin and salicin and other substrates and their hydrolyzates; the relative standard deviations of naringin and naringenin determined by the method are less than 1% In 100.4% ~ 105.2%, 94.5% ~ 117.3%. Under the conditions of 50 ℃ and pH 4.0, the Michaelis constant Km of naringin hydrolysis was 0.10 mmol / L and the maximum reaction rate Vmax was 256 U / mg and Kcat was 1 487 / s. This liquid chromatographic method can detect the hydrolysis process of naringin against various glycosides, and can accurately determine the enzyme activity and enzymatic hydrolysis kinetics of naringin hydrolyzing naringin, which plays an important role in the further study of the application of naringin in food .