【作者】 孙健；

【导师】 蒋跃明；

【作者基本信息】 中国科学院研究生院（华南植物园） ， 植物学， 2008， 博士

【摘要】 荔枝（Litchi chinensis Sonn.）原产我国南方,是具有较高经济价值的亚热带水果。荔枝果实采后果皮极易褐变,这极大地限制了其长期贮运,并降低其商品价值。一般认为荔枝采后褐变主要是由于果皮细胞中多酚氧化酶（PPO）与酚类底物区域化分布的破坏而导致酶促反应的发生,表现为酚类物质被催化氧化为醌,后者则通过聚合反应形成褐色产物。本研究首次对荔枝果皮组织中PPO催化的褐变底物进行了分离;鉴定了主要底物的种类、组成和化学结构;同时分析比较了内、外源底物与荔枝果皮PPO的酶学反应特性,果实发育和常温贮藏期间褐变底物含量的变化以及贮运条件对褐变底物理化性质的影响。结果表明:对荔枝果皮提取物采用聚酰胺柱层析、硅胶柱层析、Sephadex LH-20凝胶柱层析、制备薄层层析（PTLC）分离纯化,0.5% FeCl₃和荔枝的PPO检测,得到2个荔枝褐变底物。通过紫外（UV）、电喷雾质谱（ESI-MS）、核磁共振（NMR）等光谱分析,荔枝果皮主要的PPO褐变底物鉴定为（-）-表儿茶素和原花色素A2。比较内源底物（-）-表儿茶素和外源底物邻苯二酚与荔枝果皮PPO的酶学反应特性发现当底物不同时,PPO活性的最适pH值和最适温度不同,抑制剂和金属离子对内、外源底物与PPO酶促反应的影响也不同,此外,两个反应系统的酶促动力学参数也有差别。通过高效液相色谱（HPLC）定量分析,发现果实发育初期褐变底物积累较多,随着发育过程其含量递减;采后随着常温贮藏时间的延长,荔枝果皮褐变指数逐渐增加,果皮中褐变底物的含量逐渐降低,果皮褐变程度不断加深。此外,研究采后贮运条件对荔枝PPO底物性质的影响发现,褐变底物在光照、高温、碱性条件下结构不稳定,容易变褐;O₂加速底物的酶促褐变,而CO₂起减缓作用;氧化剂H₂O₂加速了（-）-表儿茶素的酶促褐变反应,还原剂Vc和Na₂S₂O₅则抑制底物被PPO催化氧化。K⁺、Na⁺和Zn²⁺对褐变底物的稳定性无显著影响,Fe3+、Fe²⁺、Cu²⁺和Pb²⁺的存在影响褐变底物稳定性,而Ca²⁺的存在使原花色素A2不稳定。更多还原

【Abstract】 Litchi （Litchi chinensis Sonn.）, native to South China, is subtropical fruit with a high commercial value. However, the fruit brown rapidly once harvested, which greatly restricts its storage and transporation, and reduces its commercial value. The postharvest browning of litchi fruit is mainly due to the decompartmentalization of polyphenol oxidase （PPO） and its phenolic substrates present in pericarp tissues, which results in the contact of PPO with substrates, and then initiates the enzyme-catalyzed oxidation in the presence of oxygen. In the enzymatic reaction, the browning substrates are oxidized toο-quinones and finally polymerized into the brown-colored by-products.To identify the browning substrates caused by PPO of pericarp tissues of litchi fruit, the experiments were conducted for the first time to separate, select and purify these substrates. The composition and chemical structure of the major browning substrates were identified. Litchi PPO properties were comparatively analyzed by using endogeous and exogenous substrates. The variation of substrate during fruit development was analyzed, while the effects of storage conditions on the variation in substrate contents at room temperature were also investigated.Litchi pericarp tissues were extracted and the extracts were isolated by combined column chromatography over polyamide, silica gel, Sephadex LH-20 and preparative thin layer chromatography （PTLC）. Two PPO substrates from litchi fruit were selected out by a combination of 0.5% FeCl3 and PPO solutions. Based on the spectra data of ultraviolet （UV）, electrospray ionization mass spectrometry （ESI-MS）, and nuclear magnetic resonance （NMR）, the major PPO substrates from litchi fruit were identified as （?）-epicatechin and procyanidin A2 （epicatechin-（2→O→7, 4→8）-epicatechin）.By the comparative analysis of PPO properties, it was found that the pH optima and temperature optima for litchi PPO activity were very different when the enzyme reacted with endogenous substrate （?）-epicatechin and exogenous substrate catechol. The addition of inhibitors and metal ions into the endogenous or exogenous substrate-enzyme system also exhibited the different effects on the PPO activity. Moreover, based on the kinetic analysis, litchi PPO could strongly bind endogenous substrate, but it possessed a higher catalytic efficiency to exogenous substrate.Based on the quantitative analysis by HPLC, the contents of PPO substrates of litchi fruit were higher at initial stage of fruit development, but decreased during this development. In the other hand, the storage extension at ambient temperature increased the skin browning index and reduced the contents of PPO substrates.Under the conditions of sunlight, heat and alkalescence, litchi PPO substrates were unstable in structure and were easy to be browned. Increasing oxygen concentration enhanced but increasing carbon dioxide concentration reduced browning reaction of these substrates. H2O2 increased enzymatic browning reaction of （?）-epicatechin, however, Vc and Na2S2O5 inhibited the oxidization of substrates by PPO. K⁺, Na⁺ and Zn²⁺ had little effect on substrate stability but Fe³⁺, Fe²⁺, Cu²⁺ and Pb²⁺ influenced the stability. Ca²⁺ reduced the stability of procyanidin A2 solution.更多还原