酶促褐变对菊花品质形成的影响及其机制研究

For fresh flowering head of Chrysanthemum morifolium cv. Bo ju (BJ), enzymatic browning is one of the worst problems during the harvesting, handling and storing process. Browning will exert an important influence on the appearance quality, the active metabolites and antioxidant activities. Undesirable oxidative enzymatic browning occurs in the damaged tissues during drying processes and other technological operations. Here we discussed the browning reason of BJ，and the effects of different drying processing methods on the quality of BJ. For each method, different parameters of browning enzymatic oxidases activity (PPO/POD/PAL), parenchyma cell ultrastructure, active metabolites contents and antioxidant activities were studied. The correlations of oxidases activity and the quality of BJ was evaluated by the means of statistical methods. The characteristic of the key browning enzymes were studied by molecular biological methods. The results reported here will not only help us to provide fundamental understanding of the enzyme in flower of C. morifolium also give theoretical basis to prevent the enzymatic browning during processing. It will provide a theoretical basis and technical support for the drying processing of BJ.

“夫药采取不知时节，不以阴干曝干，虽有药名，终无药实”，采收加工是形成药材品质和药性形成重要环节，直接影响中药产品安全与疗效。本项目围绕影响菊花药材品相与质量形成的重要限制因素——“酶促褐变”开展研究。运用分子生物学、生物化学、中药化学、药理学、化学计量学理论和技术，系统评价菊花酶促褐变过程中褐变酶活性、细胞超微结构等生理生化指标的动态变化规律，及其对药材外观-次生代谢产物-活性等的影响，剖析酶促褐变导致品质裂变的原因，明确导致褐变的关键酶，酶促褐变最敏感成分类型。进而探讨不同控酶处理，对酶活性变化及其与药材品质改善的相关性，进一步筛选褐变酶关键基因，并对影响其表达及活性的限制性因素进行系统评价，阐明酶促褐变对药材品质形成的机理和分子机制。在此基础上，科学评价不同加工过程对菊花品相和质量形成的影响，为规范菊花加工工艺提供理论依据，同时也为其它药材酶促褐变的控制提供研究思路。

本项目以菊花为研究对象，基于“酶促褐变对花类药材品相和质量形成至关重要”的假说，深入开展酶与菊花药材外观-多酚类次生代谢产物-品质的相关性研究。首先，观察并检测了菊花褐变过程中外观性状、酶活变化以及化学成分变化，锁定酶促褐变的关键酶为多酚氧化酶（PPO）和过氧化物酶（POD），关键底物为酚酸类成分；重点研究了褐变相关关键酶的提取检测及反应温度、pH值等酶学特性，探讨了热风干燥、水蒸气杀青、微波干燥、阴干、硫磺熏蒸等几种不同干燥工艺，对菊花褐变酶活力和化学成分的影响，发现蒸汽杀青和微波干燥可以快速杀酶，可以完全抑制酶促褐变，而热风干燥随着温度升高，灭酶效果越显著；通过设计绿原酸与PPO酶和POD酶的体外模拟化学反应，检测并分离纯化鉴定反应底物结构为绿原酸二聚体，揭示了菊花酶促褐变在酶的作用下，酚酸类化合物发生聚合反应，生成二聚体，初步揭示了其化学反应机理；进一步对采摘后0h、5h的菊花进行cDNA文库的构建和Illumina HiSeq 2500测序，得到的转录组测序数据，并筛选菊花中具有差异表达的基因，成功挖掘并通过实时荧光定量PCR技术验证了在菊花酶促褐变过程中发生差异表达的PPO、POD、SOD、CHS等相关基因，其中，显著上调的为与POD、PPO、SOD有关的基因，显著下调的为与查耳酮合酶（CHS）有关的基因，初步探讨了菊花酶促褐变的分子机制。