根皮苷氧化物POP2的酶促反应体系及其结构表征

doi:10.3864/j.issn.0578-1752.2018.01.017

摘要/Abstract

摘要： 【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C₂₁H₂₂O₁₂；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。

关键词: 果汁, 根皮苷, 氧化物POP2, 酶促反应, 结构表征

Abstract: 【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 of phloridzin 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. The residual 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.

Key words: fruit juice, phloridzin, oxidation products POP2, enzymatic reaction, structure characterization

王娟，邓红，刘芸，郭玉蓉，孟永宏. 根皮苷氧化物POP2的酶促反应体系及其结构表征[J]. 中国农业科学, 2018, 51(1): 182-190.

WANG Juan, DENG Hong, LIU Yun, GUO YuRong, MENG YongHong. Enzymatic Reaction System and Structural Characterization of Phloridzin Oxidation Products POP2[J]. Scientia Agricultura Sinica, 2018, 51(1): 182-190.

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参考文献

[1] Février H, Quéré J M L, Bail G L, Guyot S. Polyphenol profile, PPO activity and pH variation in relation to colour changes in a series of red-fleshed apple juices. LWT-Food Science and Technology, 2016, 11(6): 1-10.

[2] Álvarez S, Riera F A, Álvarez R, Coca J, Cuperus, F P, Bouwer S T. A new integrated membrane process for producing clarified apple juice and apple juice aroma concentrate. Journal of Food Engineering, 2000, 46(2): 109-125.

[3] WANG X, LI C, LIANG D, ZOU Y, LI P, MA F. Phenolic compounds and antioxidant activity in red-fleshed apples. Journal of Functional Foods, 2015, 18: 1086-1094.

[4] QUEIROZ C, SILVA A J R D, LOPES M L M, FIALHO E, VALENTEMESQUITA V L. Polyphenol oxidase activity, phenolic acid composition and browning in cashew apple (Anacardium occidentale L.) after processing. Food Chemistry, 2011, 125(1): 128-132.

[5] MISHRA B B, GAUTAM S, SHARMA A. Free phenolics and polyphenol oxidase (PPO): The factors affecting post-cut browning in eggplant (Solanum melongena). Food Chemistry, 2013, 139(1/4): 105-114.

[6] CHRISTIANE Q, MARIALUCIAMENDES L, ELIANE F, VERALUCIA V M. Polyphenol oxidase: Characteristics and mechanisms of browning control. Food Reviews International, 2008, 24(4): 361-375.

[7] FROMM M, LOOS H M, BAYHA S, CARLE R, KAMMERER D R. Recovery and characterisation of coloured phenolic preparations from apple seeds. Food Chemistry, 2013, 136(3/4): 1277.

[8] HADDOUCHE L, PHALAK A, TIKEKAR R V. Inactivation of polyphenol oxidase using 254 nm ultraviolet light in a model system. LWT-Food Science and Technology, 2015, 62(1): 97-103.

[9] 贺小化, 孟永宏, 王玉珠, 李娜，郭玉蓉，邓红. LSA-900C型果汁脱色树脂中苹果多酚的回收及纯化. 食品与发酵工业, 2014, 40(5): 142-146.

HE X H, MENG Y H, WANG Y Z, LI N, GUO Y R, DENG H. The recycle and purification technology of apple polyphenols from juice decolorization resin LSA-900C. Food and Fermentaion Industries, 2014, 40(5): 142-146. (in Chinese)

[10] QUEVEDO R, VALENCIA E, LÓPEZ P, GUNCKEL E, PEDRESCHI F, BASTIAS J. Characterizing the variability of enzymatic browning in fresh-cut apple slices. Food and Bioprocess Technology, 2014, 7(5): 1526-1532.

[11] KAHLE K, KRAUS M, RICHLING E. Polyphenol profiles of apple juices. Molecular Nutrition & Food Research, 2010, 49(8): 797-806.

[12] 宋烨, 翟衡, 刘金豹, 杜远鹏, 陈锋, 魏树伟. 苹果加工品种果实中的酚类物质与褐变研究. 中国农业科学, 2007, 40(11): 2563-2568.

SONG Y. ZHAI H, LIU J B, DU Y P, CHEN F, WEI S W. Polyphenolic compound and degree of browning in processing apple varieties. Journal of Integrative Agriculture, 2007, 40(11): 2563-2568. (in chinese)

[13] OSZMIANSKI J, LEE C Y. Enzymatic oxidation of phloretin glucoside in model system. Journal of Agricultural & Food Chemistry, 1991, 39(6): 1050-1052.

[14] AMAKI K, SAITO E, TANIGUCHI K, JOSHITA K, MURATA M. Role of chlorogenic acid quinone and interaction of chlorogenic acid quinone and catechins in the enzymatic browning of apple. Bioscience Biotechnology and Biochemistry, 2011, 75(5): 829-832.

[15] QUEVEDO R, DÍAZ O, VALENCIA E, PEDRESCHI F, BASTIAS J M, SICHE R. Differences between the order model and the Weibull Model in the modeling of the enzymatic browning. Food & Bioprocess Technology, 2016: 1-7.

[16] QUEVEDO R, PEDRESCHI F, BASTIAS J M, DIAZ O. Correlation of the fractal enzymatic browning rate with the temperature in mushroom, pear and apple slices. LWT - Food Science and Technology, 2016, 65: 406-413.

[17] 魏丽娜. 根皮素胺乙基醚化修饰及其生物活性、安全性和对三文鱼片保藏效果[D]. 西安: 陕西师范大学, 2015.

WEI L N. Modification of phloretin by aminoethyl and its biological activity, safety and preservative effect on salmon sliced [D]. Xi’an: Shaanxi Normal University, 2015. (in chinese)

[18] SCHULTZ T H, FLATH R A, BLACK D R, GUADAGNI D G, SCHULTZ W G, TERANISHI R. Volatiles from delicious apple essence-Extraction methods. Journal of Food Science, 2010, 32(3): 279-283.

[19] DENKTA? C, YILDIRIM H, SAN N. Influence of apple flavor absorption on physical and mechanical properties of poly (ethylene terephthalate) films. Journal of Applied Polymer Science, 2010, 99(4): 1802-1807.

[20] 左荣. 天然苹果香精检测方法及品质影响因素研究[D]. 杨凌: 西北农林科技大学, 2013.

ZUO R. Aroma composition determination method and quality influencing factors of natural apple essence[D]. Yangling: Northwest A&F University, 2013. (in Chinese)

[21] 马永昆, 李祥波, 蒋家奎. 基于GC-MS和嗅闻仪联用的天然苹果香精关键香气成分分析. 食品科学, 2009, 30(8): 231-234.

MA Y K, LI X B, JIANG J K. Determination of representative aroma components of natural apple essence by GC-MS combined with GC-olfactory. Food Science, 2009, 30(8): 231-234. (in Chinese)

[22] CEYMANN M, ARRIGONI E, SCHÄRER H, BOZZI N A, HURRELL R F. Identification of apples rich in health-promoting flavan-3-ols and phenolic acids by measuring the polyphenol profile. Journal of Food Composition & Analysis, 2012, 26(1/2): 128-135.

[23] KLIBANOV A M. Improving enzymes by using them in organic solvents. Nature, 2001, 409(6817): 241.

[24] MA H L, KERMASHA S, GAO J M, ROGELIO M B, YU X Z. Laccase-catalyzed oxidation of phenolic compounds in organic media. Journal of Molecular Catalysis B Enzymatic, 2009, 57(1/4): 89-95.

[25] 郭盼. 非水介质中漆酶催化酚类化合物特性及产物抗氧化活性研究[D]. 杨凌: 西北农林科技大学, 2011.

GUO P. Study on laccase catalyzed oxidation of phenolic compounds in non-aqueous media and anti-oxidation activity of its end products[D].Yangling: Northwest A&F University, 2011. (in Chinese)

[26] SULLIVAN M L. Beyond brown: polyphenol oxidases as enzymes of plant specialized metabolism. Frontiers in Plant Science, 2015, 5: 783.

[27] LOZANO J E, DRUDISBISCARRI R, IBARZRIBAS A. Enzymatic browning in apple pulps. Journal of Food Science, 2010, 59(3): 564-567.

[28] ONSA G H, SAARI N B, JINAP SELAMAT A, BAKAR J. Latent polyphenol oxidases from Sago Log (Metroxylon sagu): Partial purification, activation, and some properties. Journal of Agricultural & Food Chemistry, 2000, 48(10): 5041-5045.

[29] GUYOT S, SERRAND S, LE Q J, SANONER P, CMGC R. Enzymatic synthesis and physicochemical characterisation of phloridzin oxidation products (POP), a new water-soluble yellow dye deriving from apple. Innovative Food Science & Emerging Technologies, 2007, 8(3): 443-450.

[30] GUERNEVÉ C L, SANONER P, DRILLEAU J F, GUYOT S. New compounds obtained by enzymatic oxidation of phloridzin. Tetrahedron Letters, 2004, 45(35): 6673-6677.

[31] 王雪晖. 根皮苷酶法转化制备功能性黄色素研究[D]. 郑州: 河南农业大学, 2016.

Wang X H. Study on enzymatic conversion of phloridzin to functional yellow pigment[D]. Zhengzhou: Henan Agricultural University, 2016. (in Chinese)