Study On Mechanism Of Blackheart Development In Pineapple Fruit

Pineapple (Ananas comosus) is native to south central America and is a subtropicaland tropical fruit with a high commercial value. However, postharvest pineapple fruit issusceptible to black heart (also known as internal browning) during the storage andtransport, leading to fruit quality deterioration and seriously affecting the value andmarketing of pineapple fruit. Blackheart is the most serious problem of postharvestpineapple in many countries. Blackheart is a physiological disorder of pineapple fruit, as aresult of the the disappearance of the regional isolation of polyphenol oxidase (PPO) andits phenolic substrates in flesh cell, which caused enzyme-catalyzed oxidation and resultedin the production of quinines that finally polymerized into the melanin by-products bynon-enzyme reaction.Postharvest pineapple fruit was used to extract, separate, purify and select internalbrowning substrates of pineapple fruit flesh by PPO of pineapple fruit tissues, and finallythe composition and structure of these endogenous substrates were identified. Theenzymatic reaction characteristics and kinetics parameters of pineapple PPO werecomparatively analyzed by using endogenous substrate and exogenous substrate to confirmthe optimal endogenous substrate, and experiments were carried to study the influence ofexternal factors on endogenous substrates properties and changes of substrate content inpineapple fruit during storage at ambient temperature. Through the in-depth study of PPOand the substrate, It can provide a theoretical basis for the research of enzymatic browningmechanism and the control of the black heart in postharvest pineapple. The results showsas follow:1. PPO and phenolic compounds were extracted from the fruit pulp of pineapple andthe phenolic extracts were isolated by polyamide column chromatography, silica gelcolumn chromatography and sephadex LH-20chromatography. Four browning substrateswere selected by a combination of1%FeCl3and PPO. The four kinds of substrates werecharacterized as catechin, epicatechin, chlorogenic acid and catechol by ultravioletspectrum, HPLC and electrospray ionization mass spectrometry (ESI-MS).2. By comparative analysis of kinetics parameters of PPO enzymatic reaction withfour substrates, catechins was identified as the optimal endogenous substrates of pineapplePPO. Studying the enzymatic reaction properties of catechins and common exogenous substrates catechol with pineapple PPO, it was found that when the substrate is different,the characteristics of pineapple PPO reaction have some differences. When catechin is usedfor the substrate, the optimum pH and the optimum temperature of pineapple PPO activityis4.8and55℃separately, and Fe2+, Fe3+, Cu2+and Ca2+can promote the enzymaticreaction but K+has a strong inhibiting effect, and ascorbic acid is the best inhibitors of theenzymatic reaction. When catechol is used for substrate, the optimum pH and the optimumtemperature of pineapple PPO activity is4.8and50℃, and Fe2+, Fe3+, Cu2+and Ca2+canpromote the enzymatic reaction but K+can effectively inhibited the pineapple PPO activity,and the best inhibitor of reaction catalyzed by pineapple PPO is sodium sulfite.3. The experiments were carried to study the effect of external factors on pineapplePPO substrates. Pineapple PPO substrates were unstable in structure and susceptible to bebrown in presence of sunlight, heat (greater than40℃), alkalinescence (pH>9) and metalion (Ca2+, Cu2+, Fe2+, Pb2+and Zn2+). O2and H2O2increased the enzymatic reaction ofthese substrates, while CO2, N2, and reducing agent inhibited the oxidation of thesubstrates by pineapple PPO.4. Through the quantitative analysis by high performance liquid chromatography(HPLC), the PPO substrates content in pineapple fruit is extremely low just after harvest,and the substrates content gradually increased with storage time extended, reached thepeak at2~3weeks, and then decreased until storage duration was over.