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 perfor-mance liquid chromatography-diode array detector

中文关键词:  γ-淀粉酶残留量  蜂蜜掺假鉴定  高效液相色谱-二极管阵列检测法

英文关键词:γ-amylase activity  honey adulteration detection  high performance liquid chromatography- diode array detector

中文摘要:

      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3.6 U/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。

英文摘要:

      Objective To develop a new method for determination of exogenous γ-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 of γ-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 acetonitrile/water (15:85, v:v). By identifying the content of enzymatic hydrolysate at 310 nm, the residue of γ-amylase in honey could be determined. Results The method showed a good linearity between the concentration and peak area with the correlation coefficient over than 0.999. The linear range of γ-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 of γ-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 effectively identify honey adulteration by starch syrups from the perspective of enzymology.