脱植酸叶绿素标准品的制备效率低,过程复杂,这阻碍了叶绿素的测定和相关的生物活性特性研究。在本文中,来自四种植酸叶绿素的叶绿素衍生物(叶绿素a,叶绿素b,脱茶素a,和pheophytinb)酶促反应前后的定性和定量表征通过UPLC-DAD-MS。提出了一个简单的指标,通过紫外可见光谱中叶绿素衍生物典型峰的λmax及其信号强度比来表征其氧化对应物中的叶绿素色素。优化了4种叶绿素色素酶促反应的最佳反应条件,并拟合了动力学模型。结果表明,叶绿素a酶促反应的最佳温度,叶绿素b,脱茶素a,脱茶素b分别为30、30、60和60°C,分别,它们的最佳反应时间为2、3、1和3h,分别。在最佳反应条件下拟合动力学模型以研究酶促反应的Km和Vm值。实际应用:去植酸叶绿素,如叶绿素和山梨酯,在食品工业中经常被确定,并且总是需要在实验室中用植物组织中的丙酮粉末制备。此外,叶绿素色素容易氧化,这使得脱脂叶绿素色素的表征更加复杂和困难。在本文中,用柑橘丙酮粉分别研究了四种植酸叶绿素的脱植酸过程,用UPLC-DAD-MS分析反应混合物,为相关叶绿素测定研究和叶绿素鉴定方案的制定提供重要参考。
The preparation of dephytylated chlorophyll standards is inefficient and the process is complicated, which hinders chlorophyll determination and related bioactive property investigation. In this paper, chlorophyll derivatives from four phytylated chlorophylls (chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b) before and after the enzymatic reaction were qualitatively and quantitatively characterized by UPLC-DAD-MS. A simple index was proposed to characterize chlorophyll pigments from their oxidized counterparts by the λmax of the typical peak of chlorophyll derivatives in UV-visible spectrum and their signal intensity ratios. The optimal reaction conditions for the enzymatic reaction of four chlorophyll pigments were optimized, and kinetic models were fitted. The results showed that the optimal temperatures for the enzymatic reactions of chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b were 30, 30, 60, and 60°C, respectively, and their optimal reaction time was 2, 3, 1, and 3 h, respectively. Kinetic models were fitted under optimal reaction conditions to study the Km and Vm values of the enzymatic reactions. PRACTICAL APPLICATION: Dephytylated chlorophylls, such as chlorophyllide and pheophorbide, are frequently determined in food industry and are always required to be prepared in lab with acetone powder from plant tissue. Moreover, chlorophyll pigments are easy to undergo oxidations, which make the characterization of dephytylated chlorophyll pigments more complicated and difficult. In this paper, four types of phytylated chlorophylls were investigated respectively about the dephytinization process with the citrus acetone powder, and the reaction mixture was analyzed with UPLC-DAD-MS, which can provide an important reference for relevant chlorophyll determination studies and the development of chlorophyll identification protocols.