6种葡萄籽中水解氨基酸和游离氨基酸含量测定及比较
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摘要
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(206020,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.
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(206020,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.
引文
[1]任继波,李彦奎,张晶莹,等.葡萄籽油的热榨法制备工艺研究与脂肪酸GC-MS分析[J].中外葡萄与葡萄酒,2017,41(1):27-30.
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[15]FERNANDEZFIGARES I,RODRGUEZ L C,GONZLEZCASADO A.Effect of different matrices on physiological amino acids analysis by liquid chromatography:evaluation and correction of the matrix effect[J].Journal of Chromatography B,2004,799(1):73-79.
[16]郭俊国,解孝锋,毕宏生,等.PITC柱前衍生HPLC法测定茺蔚子中游离和水解氨基酸的含量[J].中华中医药杂志,2015,30(1):91-94.
[17]全红娜,金松子,雷勇胜,等.反相高效液相色谱中流动相选择与优化的研究进展[J].现代药物与临床,2014,29(10):1 190-1 194.
[18]黄蕴芝,祁龙凯,林励,等.蜂蜜中氨基酸类成分高效液相指纹图谱研究[J].广州中医药大学学报,2015,32(4):745-750.
[19]王菲菲,张聿梅,马双成,等.高效薄层色谱、柱前衍生-超高效液相色谱和离子色谱分析疏血通注射液中游离氨基酸的比较[J].药学学报,2016,51(3):439-443.
[20]FAO/WHO.Energy and protein requirements[R].Geneva:World Health Organization,1973:63.
[2]王伏超,任育萱,张磊.葡萄籽的成分开发利用与研究进展[J].中国食品添加剂,2015,22(7):151-155.
[3]郑亚蕾,刘叶,隋银强,等.4个葡萄品种葡萄籽冷榨油的性质与体外抗氧化活性[J].食品科学,2016,36(3):27-32.
[4]李保应,高海青.葡萄多酚的抗肿瘤作用[J].中华老年病研究电子杂志,2015,2(2):28-31.
[5]吴涛,张倩,刘锐,等.儿茶素、槲皮素和葡萄籽原花青素的协同抗辐射作用[J].天津科技大学学报,2018,33(1):9-13.
[6]王红珊,曹毅敏,杨晋,等.葡萄籽原花青素对大鼠心肌缺血再灌注损伤的保护作用[J].中国生化药物杂志,2012,33(2):161-162.
[7]赫欣睿,武中庸,叶永丽,等.高效液相色谱法测定氨基酸的研究进展[J].分析测试学报,2016,35(7):922-928.
[8]张苏平,邱伟强,卢祺,等.全自动氨基酸分析仪法测定4种贝类肌肉中谷胱甘肽和游离氨基酸含量[J].食品科学,2017,38(4):170-176.
[9]刘李婷,葛武鹏,杨丽娜,等.牛乳粉中掺入大豆蛋白的特异氨基酸分析研究[J].食品工业,2018,39(3):215-217.
[10]缪潇瑶,于静,安叡,等.不同品种沙蚕中氨基酸含量的HPLC测定及多元统计分析[J].药物分析杂志,2016,36(1):53-58.
[11]陈蓉,张超,顾倩,等.柱前衍生-HPLC法同时测定不同产地茯苓中18种氨基酸含量[J].药物分析杂志,2017,37(2):297-303.
[12]许申鸿,杭瑚,郝晓丽.葡萄籽化学成分分析及其抗氧化性质的研究[J].食品工业科技,2000,21(2):18-20.
[13]周建华.葡萄籽中提取油和蛋白质的研究[J].粮食与饲料工业,2000,23(10):48-49.
[14]李想.纳豆葡萄籽胶囊中氨基酸组成及含量的测定[J].中国食物与营养,2009,15(5):57-58.
[15]FERNANDEZFIGARES I,RODRGUEZ L C,GONZLEZCASADO A.Effect of different matrices on physiological amino acids analysis by liquid chromatography:evaluation and correction of the matrix effect[J].Journal of Chromatography B,2004,799(1):73-79.
[16]郭俊国,解孝锋,毕宏生,等.PITC柱前衍生HPLC法测定茺蔚子中游离和水解氨基酸的含量[J].中华中医药杂志,2015,30(1):91-94.
[17]全红娜,金松子,雷勇胜,等.反相高效液相色谱中流动相选择与优化的研究进展[J].现代药物与临床,2014,29(10):1 190-1 194.
[18]黄蕴芝,祁龙凯,林励,等.蜂蜜中氨基酸类成分高效液相指纹图谱研究[J].广州中医药大学学报,2015,32(4):745-750.
[19]王菲菲,张聿梅,马双成,等.高效薄层色谱、柱前衍生-超高效液相色谱和离子色谱分析疏血通注射液中游离氨基酸的比较[J].药学学报,2016,51(3):439-443.
[20]FAO/WHO.Energy and protein requirements[R].Geneva:World Health Organization,1973:63.