6种葡萄籽中水解氨基酸和游离氨基酸含量测定及比较

Determination and comparison of hydrolyzed amino acids and free amino acids in six kinds of grape seeds

以异硫氰酸苯酯为柱前衍生试剂,采用Thermo Acclaim^(TM) 120 C_(18)色谱柱(250mm×4.6mm,5μm),流动相A为pH 6.5的乙酸钠缓冲溶液,流动相B为甲醇—乙腈—水(体积比20:60:20),在流速1.0mL/min,梯度洗脱,柱温30℃,检测波长254nm的条件下,建立了高效液相色谱法测定葡萄籽中水解氨基酸和游离氨基酸的含量。结果表明,在试验浓度范围内,17种氨基酸的R^2均>0.999 0;水解氨基酸的加样回收率为92.0%～104.6%,RSD为0.83%～4.07%;游离氨基酸的加样回收率为95.2%～102.4%,RSD为1.06%～3.98%。同时,6种葡萄籽中水解氨基酸的含量为130.46～163.24 mg/g,游离氨基酸的含量为2.04～5.13mg/g;用于酿造白葡萄酒的雷司令、赛美容和贵人香3种葡萄籽中游离氨基酸含量较高,用于酿造红葡萄酒的黑皮诺、蛇龙珠和赤霞珠3种葡萄籽中水解氨基酸含量较高。该方法稳定可靠,可用于葡萄籽中水解氨基酸和游离氨基酸的含量测定。

High performance liquid chromatography（HPLC） was developed to detect the hydrolyzed amino acids and free amino acids in grape seeds with phenyl isothiocyanate（PITC） as the derivative reagent.For chromatographic analysis,a Thermo Acclaim^（TM） 120 C_（18） column（250 mm × 4.6 mm,5μm） was used.The mobile phase consisted of solvent A and B with the flow rate of 1.0 mL/min by gradient elution.Solvent A was sodium acetate buffer solution（pH6.5） and solvent B was methanol-acetonitrile-water（206020,v/v/v）.The temperature of column was maintained at 30 ℃.The wavelength was set at 254 nm.The results showed that the R2 of 17 kinds of amino acids were more than 0.999 0 in the range of experimental concentration.The average recoveries of hydrolyzed amino acids were 92.0%～104.6%,and RSDs were 0.83%～4.07%.The average recoveries of free amino acids were 95.2% ～102.4%,and RSDs were 1.06%～3.98%.The contents of hydrolyzed amino acids in 6 kinds of grape seeds were 130.46～163.24 mg/g.The contents of free amino acids were 2.04～5.13 mg/g.More free amino acids were contained in the grape seeds from Leisiling,Saimeirong and Guirenxiang which three kinds of grape seeds were usually used to brew the white wine.The contents of hydrolyzed amino acids were higher in the grape seeds from Heipinuo,Shelongzhu and Chixiazhu which were always used for the red wine brewing.The method was stable and reliable,can be used for the determination of hydrolyzed amino acids and free amino acids in grape seeds.