Studies On The Major Substrates For Polyphenol Oxidases In Pericarp Tissues Of Litchi Fruit

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.