高效液相色谱-二极管阵列检测法检测蜂蜜中外源性γ-淀粉酶残留量

Determination of the exogenous γ-amylase residue in honey by high performance liquid chromatography-diode array detector

目的利用高效液相色谱-二极管阵列检测法(HPLC-DAD)测定蜂蜜中外源性γ-淀粉酶残留量。方法选取对硝基苯-β-D-麦芽三糖作为γ-淀粉酶的酶解底物,于55℃和pH 4.50的0.10 mol/L乙酸钠缓冲液中反应90 min。采用C18柱分离底物和酶解产物对硝基苯-β-D-葡萄糖,流动相为乙腈/水(15:85,v:v)。通过测定酶解产物的含量来确定γ-淀粉酶残留量。结果本方法的线性范围为1~50 U/kg,定量限为1 U/kg,回收率在94.0%~107.2%之间,相对标准偏差在3.2%~5.1%之间。采用本方法对市售蜂蜜和淀粉类糖浆共58个样本进行考察,γ-淀粉酶检出率为79.3%。采用本方法测定一个掺入5%淀粉类糖浆的蜂蜜,测得γ-淀粉酶残留量为3.6U/kg。结论本方法能够有效地从酶学的角度快速鉴定蜂蜜中淀粉类糖浆的掺假。

Objective To develop a new method for determination of exogenousγ-amylase activity in honey using high performance liquid chromatography-diode array detector (HPLC-DAD).Methods 4-nitrophenyl beta-D-maltotriose was chosen as the substrate ofγ-amylase. This enzymatic reaction was under the condition of 55℃and 0.10 mol/L sodium acetate-acetic acid buffer solution (pH 4.50) for 90 min. Sub-strate and enzymatic hydrolysate 4-nitrophenyl beta-D-gulcose were separated by high performance liquid chromatography on a C18 column. Isocratic elution was employed with a mobile phase consisting of acetoni-trile/water (15:85, v:v). By identifying the content of enzymatic hydrolysate at 310 nm, the residue ofγ-amylase in honey could be determined.ResultsThe method showed a good linearity between the concentration and peak area with the correlation coefficient over than 0.999. The linear range ofγ-amylase was 1~50 U/kg with the quantification limit 1 U/kg. Recoveries were between 94.0% and 107.2%, with relative standard deviations from 3.2% to 5.1%. This method was used to analyze 58 honey and starch syrup samples, and the detection rate ofγ-amylase was 79.3%. To further verify the detection capability of this method, an authentic honey was mixed with 5% rice syrup.γ-amylase content of this sample was 3.6 U/kg.Conclusion This method can ef-fectively identify honey adulteration by starch syrups from the perspective of enzymology.