大蒜果聚糖的水解方法及其定量分析

Acid Hydrolysis of Garlic Fructan and Its Quantification Using HPLC Method

为了建立大蒜果聚糖含量的快速高效检测方法,本试验以乐都紫皮大蒜为试材,首先通过热水、超声、超声-热水法提取大蒜总糖,分别利用不同的水解试剂类型、pH值、浓度、水解时间将其中的果聚糖彻底水解,通过响应面分析筛选了果聚糖降解的最优条件;随后利用分光光度计和高效液相法测定水解前后果聚糖降解产物(蔗糖、葡萄糖、果糖)的含量并计算差值,得到大蒜组织中所有果聚糖的总含量;最后利用方法学验证对确立的方法进行了优化和评价。结果表明,热水法更有利于提取大蒜可溶性总糖;大蒜果聚糖最佳水解工艺条件为水解温度90℃、HCl浓度3 mol·L^(-1)、水解时间1 h,经此条件水解后大蒜果聚糖含量预测值为627.903 mg·g^(-1)FW,与实际测定值(620.470 mg·g^(-1)FW)差异不显著,所得回归模型拟合良好。方法学验证结果显示,该果聚糖定量方法精密度、重复性及稳定性良好,三者的相对标准偏差(RSD)均小于1.5%;平均回收率为98.45%~101.04%,其RSD为0.59%~1.75%。利用此方法鉴定大蒜资源果聚糖性质发现,不同品种大蒜鳞茎中果聚糖含量差异显著。本研究筛选确立的检测方法准确性较高,结果可靠,适用于大蒜果聚糖含量的测定分析。本研究结果为大蒜资源果聚糖性状的深入评价和开发利用提供了理论依据。

To establish a quick and efficient method of fructan content measurement from garlic,the total sugar of garlic was extracted through hot water,ultrasonic and ultrasonic pretreatment-hot water firstly.Le Du purple skin galic was used as the raw material,and the fructan was hydrolyzed using different hydrolytic reagntes under varied pH,concentrations and time,and its optimal degradation condition was obtained with a response surface analysis.The content of degradation products(sucrose,glucose and fructose)before and after fructan degradation was measured by spectrophotometry and high performance liquid chromatography(HPLC),and the difference value represented the total content in all tissues of garlic.Finally,the establishment method was optimized and evaluated through methodology validation.The results showed that the hot water method was the most conducive to the extraction of garlic soluble total sugar.The optimum hydrolysis conditions of garlic fructan were hydrolysis temperature of 90℃,HCl concentration of 3 mol·L^(-1)and hydrolysis time of 1 h.The predicted value of garlic fructan content after hydrolysis under these conditions was 627.903 mg·g^(-1)FW,which was not significantly different from the actual measured value(620.470 mg·g^(-1)FW),and the regression model fitted well.The relative standard deviations(RSD)of precision,reproducibility and stability were less than 1.5%.The recoveries ranged from 98.45%~101.04%with RSD 0.59%~1.75%.It was found that the fructan content in the cloves of different varieties of garlic was significantly different from the fructan contents using this method.It was suggested that the detection system established in this study was viable for garlic fructan content determination with high accuracy.The results of this study provides a theoretical basis for deep evaluation of fructan trait of garlic resources.