高效液相色谱法测定莲子酶解液中果糖、葡萄糖、蔗糖和麦芽糖含量

Determination of Fructose, Glucose, Sucrose and Maltose in Lotus-Seeds Enzymatic Hydrolysates by HPLC

摘要: 目的：建立高效液相色谱–示差折光检测器法测定莲子酶解液中葡萄糖、果糖、蔗糖和麦芽糖的分析方法。方法：莲子粉末溶于水后，经超声波提取，加入α-淀粉酶、糖化酶、纤维素酶、半纤维素酶发生酶解，提取液经离心、过滤后，进行上机测定。采用Prevail Carbohydrate ES色谱柱(5 μm, 250 mm × 4.6 mm)，以乙腈–水(体积比为75:25)为流动相，流速为1.0 mL/min，柱温25℃，检测器池温度35℃。结果：本方法有较好的线性关系(r = 0.999)，精密度高(RSD < 4%)，准确度好(回收率97.3%~101.7%)，适用于莲子酶解液中葡萄糖、果糖、蔗糖和麦芽糖的定量分析。采用该方法确定了莲子酶解最优混合酶比例为α-淀粉酶:糖化酶:纤维素酶:半纤维素酶 = 1.1%:1.4%:0.9%:0.9%，在该条件下，莲子酶解液中果糖、葡萄糖、蔗糖和麦芽糖含量分别为2.74 mg/mL，21.58 mg/mL，0.26 mg/mL，3.62 mg/mL。该方法前处理简单，分析时间短，操作步骤简单，重复性好，定量准确，可对莲子酶解液中的果糖、葡萄糖、蔗糖、麦芽糖进行准确测定。

Abstract: Objective: A method for the quantitative determination of glucose, fructose, sucrose and maltose in Lotus-Seeds enzymatic hydrolysates was established by high-performance liquid chromatography-refractive index detector (HPLC-RID). Methods: Lotus-Seeds powder was dissolved in pure water and extracted by ultrasonic wave. After adding α-amylase-glucoamylase-cellulase-hemicellulase system, the enzymatic extraction was then centrifuged, filtered and determined by HPLC-RID using Prevail Carbohydrate ES column (5 μm, 250 mm × 4.6 mm). The mobile phase is 75% acetonitrile-25% water solution at the flow rate of 1.0 mL/min. The column temperature and the detection pool temperature were controlled at 25˚C and 35˚C. Results: The calibration curves exhibited good linearity (r = 0.999), precision (RSD < 4%) and accuracy (recoveries range from 97.3% to 101.7%). This method met the needs of quantification of glucose, fructose, sucrose and maltose in Lotus-Seeds enzymatic hydrolysates. The optimized enzyme system addition in lotus seeds was α-amylase 1.1%, glucoamylase 1.4%, cellulose 0.9%, hemicellulose 0.9%. Under this experimental condition, the contents of fructose, glucose, sucrose and maltose in the lotus-seed hydrolysates were 2.74 mg/mL, 21.58 mg/mL, 0.26 mg/mL and 3.62 mg/mL, respectively. Conclusion: This method has simple pretreatment, short analysis time, simple operation steps, good repeatability and high accuracy. It can provide accurate quantitative results for the determination of fructose, glucose, sucrose and maltose in the lotus-seed enzymatic hydrolysates solution.