[Objective] Phloridzin oxidation product 2 (POP2) is a final polyphenol oxidation product generated by enzymatic oxidation in the process of fruit juice,and it has an important impact on the browning and deterioration of fruit juice.In order to detect POP2 effects during process and storage of apple juice,the phloridzin polyphenol oxidase reaction system was established,POP2 was separated and purified,structural characterization was analyzed.[Method] The three reaction factors of pH,temperature and substrate concentration were tested first,and then enzyme reaction in ethanol solution were studied,and content changes of phloridzin,intermediate products,POP2 during oxidation process were detected by HPLC method.The POP2 was isolated and purified by ethyl acetate extraction,anhydrous ethanol desalting and recrystallization,the structure of POP2 was characterized by the methods of ESI-MS and IR.[Result] The optimal conditions for the enzymatic oxidation ofphloridzin were pH 6.5,35℃,substrate concentration 3 mg·mL-1.The addition of different concentrations of absolute ethanol had a significant increase in the rate of enzymatic oxidation of phloridzin,the highest reaction conversion rate was 2.4 times at 20％ ethanol concentration.The color of the phloridzin reaction fluid changes from slightly yellow,bright yellow,orange to orange-red at the beginning,middle and final reaction stage.The HPLC results showed that the conversion rate of phlorizin was 90.2％ when the substrate concentration was 3 mg·mL-1 and the reaction time was 48 h.Intermediates X1 and POP1 were almost close to trace,the accumulated POP2 was stable.Theresidual phloridzin in the reaction solution could be removed by ethyl acetate extraction.The purity of 96.8％ POP2 was obtained by anhydrous ethanol desalting and recrystallization from methanol.The molecular weight of POP2 was 467 by positive mode ESI-MS analysis,molecular formula is C21H22O12.IR results showed that POP2 had characteristic ether,ketone,benzene ring and carboxyl functional groups,and it was consistent with the structure of the predicted phlorizin oxidation product structure.[Conclusion] The major oxidation products of phloridzin in enzymatic system is POP2,POP2 content can be established a landmark test indicators in apple juice browning and deterioration.%[目的]根皮苷氧化物POP2是苹果汁加工过程中由酶促氧化生成的一种多酚氧化物,对果汁的褐变和劣变有着重要影响.通过多酚氧化酶氧化根皮苷反应体系的建立,POP2的分离纯化与结构表征,探讨根皮苷氧化物POP2合成条件与反应历程,为苹果汁中POP2的检测分析,及弄清其对果汁褐变和劣变的影响奠定基础.[方法]通过pH、温度、底物浓度3个单因素试验建立根皮苷酶促氧化体系的基础上,研究乙醇溶液对酶促反应的促进作用和反应历程,通过高效液相色谱法检测根皮苷酶促氧化过程中根皮苷、中间产物及POP2含量的变化,通过乙酸乙酯萃取、无水乙醇除盐、重结晶等方法对氧化产物POP2进行分离纯化,并采用质谱(ESI-MS)和红外光谱(IR)对POP2进行结构表征.[结果]根皮苷酶促氧化反应的最佳条件为pH 6.5、35℃、底物浓度3 mg·mL-1,添加不同浓度的无水乙醇均能显著提高根皮苷酶促氧化速率,当添加浓度为20％时,可提高反应转化率,最大为2.4倍;根皮苷反应液随时间的变化颜色也发生改变,反应刚开始溶液颜色呈微黄色,随着反应的进行颜色逐渐从亮黄色变为橙黄色,最终产物的颜色为橙红色;高效液相色谱法分析根皮苷酶促氧化反应历程发现,当底物浓度为3 mg·mL-1,反应48 h时,根皮苷的转化率最大为90.2％,中间产物X1和POP1也基本接近痕量,POP2的积累量基本不再发生变化;通过乙酸乙酯萃取可以将反应液中残留的根皮苷除去,用无水乙醇除盐并经甲醇重结晶后,获得纯度为96.8％的POP2;经正离子模式ESI-MS分析,POP2的m/z为467,分子式为C21H22012;IR结果发现POP2具有特征性的醚、氧化羧基、酮基及苯环官能团,与推测的根皮苷氧化物结构式吻合.[结论]根皮苷酶促氧化体系中的主要氧化物为POP2,可以根据POP2的含量高低建立苹果汁褐变和劣变的标志性检测指标.
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