荔枝採收後快速褐變反應機制之探討

Abstract

Polyphenol oxidase（PPO）又稱Tyrosinase（E.C. 1.14.18.1），為一種內含銅離子之酵素，使用氧分子催化兩種不同的反應，首先將monophenols經脫氫氧化作用形成o-diphenols ，再於含氧的環境下氧化o-diphenols 形成o-quinones。繼續進行非酵素性聚合反應產生非水溶性之褐色物質。此酵素廣泛的存在於微生物、動物、以及植物中，並且會造成動物形成黑色素(melanian) 以及蔬菜水果褐變反應(browning reaction)。我們研發出新的方式”PAGE-blot assay”可以快速鑒別及分析PPO之存在及分子量，而此技術更提供了一個初步的研究方法來找出潛在於蔬果中的天然抑制劑，並利用此方法研究分析荔枝皮PPO。 本實驗利用HPLC及SDS-PAGE萃取，成功的純化荔枝皮PPO，其分子量分別為86及66 kDa，使用catechol當 PPO的substrate定義出86 kDa的 PPO，其Km及Vmax分別為66 mM及382 □M/min；而66 kDa PPO之值分別為102 mM及290 □M/min。有趣的是86 kDa PPO可使用，3,3’-diaminobenzidine 及 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)當為substrate，但是它們通常只會被雙氧水氧化產生的自由基所氧化反應，故荔枝PPO除了氧化酵素的活性外，同時具有超氧化酵素之活性，即可解釋荔枝之快速褐變的現象。此外，我們證明荔枝內含揮發性之PPO抑制劑，且藉由掃瞄式電子顯微鏡觀察荔枝皮表面結構，其結構隨時間的增長由平滑緊密漸漸地變為粗糙孔洞變大，促使PPO抑制劑揮發愈快速，降低對PPO之制劑效果，加速了褐變現象。這些新發現解釋了荔枝採收後果皮快速褐變現象之反應機制。此外，PPO於人類中又稱為Tyrosinase會造成皮膚形成黑斑，發現此揮發性的PPO抑制劑，可以用於發展新的化妝、保養品。

Polyphenol oxidase (PPO) is a copper-containing enzyme that catalyzes the chain-oxidation from monophenol or polyphenols to o-diphenols and subsequent o-quinones. The enzyme reflects the browning reaction in fruits. In the present study, we investigated the oxidation activity of PPO in litchi pericarp and the mechanism by which PPO instantly makes pericarp browning. PPO of litchi pericarp was initially extracted and isolated through gel filtration chromatography and then eluted directly from SDS-PAGE. Two molecular forms of litchi PPO were identified as 86 and 66 kDa, and thereafter named as PPO-86 and PPO-66, respectively. The Km and Vmax were determined as 66 mM and 382 mM/min for PPO-86 and 102 mM and 290 mM/min for PPO-66, respectively. Most importantly, PPO-86 could trigger the 3,3’-diaminobenzidine and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation, which was not found in other plant PPO. Thus, the unique PPO activity of litchi might account for its superior rapid-browning reaction. In addition, we show the presence of a potent volatile inhibitor(s) for PPO in litchi pericarp. The surface of post-harvesting litchi pericarp revealed an opening ultra structure under the scanning electron macroscopic examination, therefore allowing an instant evaporation of PPO inhibitor. As such, the PPO oxidation was proceeded. The novel finding clarifies the mechanism involved in the rapid browning phenomenon of post-harvesting litchi pericarp. Since the PPO is also know existed in human as tyrosinase responsible for the formation of “darkening spots” on skin, the finding of evaporation of potent PPO inhibitor may be potentially used as a strategy in developing a novel cosmetic product.