| Chinese chestnut fruit has unique flavor and is a kind of favorite food. However, mechanical damage during the processing will make the chestnut fruit turning browning, which influences the sensory quality of chestnut product To study the mechanism of chestnut enzymatic browning, the present paper investigated the changes of interior and surface browning, polyphenol oxidase (PPO) and peroxidase (POD) activities, and polyphenol content of sliced chestnut during 6-day cold storage period. Then the influence of several chemical compounds (salicylic acid, ascorbic acid, phytic acid,4-hexylresorcinol, EDTA-2Na and neutral protease) on both chestnut browning and the activities of PPO and POD were detected. The kinetics of PPO and POD with the attendance of salicylic acid, ascorbic acid, phytic acid and 4-hexylresorcinol were measured to investigate their inhibit modes on PPO and POD. After that AutoDock4.2 was applied to determine the binding modes of salicylic acid, ascorbic acid, phytic acid and 4-hexylresorcinol with PPO and POD. Finally, response surface experiment was carried out to optimize the ratio of browning inhibitor in making chestnut paste. The experimental results are as follows:(1) The browning degree of chestnut surface was low but fast, while that of chestnut interior was higher with slower changing speed. During the 6-day storage, chestnut total phenol content decreased by 34% in the first two days then it didn’t change significantly. The PPO activity increased by 42% during the first day, and plummeted to the bottom in the next 2 days. After 4-day storage it deceased to 13% of fresh chestnut POD activity of chestnut had been declining during the whole storing period, it decreased by 13% after 1-day storage, and decreased by 50% in the second day.(2) After measuring the effect of salicylic acid, ascorbic acid, phytic acid,4-hexylresorcinol, EDTA-2Na and neutral protease on the chestnut browning and the activities of chestnut PPO and POD, it was observed that 0.3g/L of salicylic acid had the best effect on the inhibition of chestnut enzymatic browning, and salicylic acid could also inhibit PPO activity. When the ascorbic acid concentration was lower than 0.4g/L, it would strengthen chestnut browning. But ascorbic acid would almost completely suppress the activity of chestnut POD when its concentration was higher than 0.1g/L while it rarely had influence on the PPO of Chinese chestnut When the concentration of phytic acid was between 0.01% and 0.05%, it could control both surface and internal chestnut browning.4-hexylresorcinol could inhibit chestnut internal browning only if the concentration was O.lg/L or less. EDTA-2Na of 0.3g/L could simultaneously inhibit both surface and internal browning of chestnut, but it would affect chestnut color when the concentration reached 0.5g/L. Neutral protease could effectively suppress the surface and internal browning when the treatment time was less than 30min. After treated at 40℃ for 120min by neutral protease, PPO and POD activity decreased by 96% and 68%, respectively.(3) The kinetics experiments showed that salicylic acid, phytic acid and 4-hexylresorcinol were competitive inhibitors of PPO and ascorbic acid was anti-competitive inhibitor. As for POD, ascorbic acid and phytic acid were competitive inhibit agents,4-hexylresorcinol was an anti-competitive inhibitor, while salicylic acid had no obvious effect on it.(4) The docking modes of PPO with salicylic acid, phytic acid and 4-hexylresorcinol were more than hydrogen bond monitor, which was the reason why they are all competitive inhibitors. The docking between POD and phytic acid was π-cation monitor in addition of hydrogen bond monitor, which also made phytic acid a competitive inhibitor to POD. However, ascorbic acid, another competitive inhibit agent of POD, had no other binding with POD except hydrogen bond. The hydrogen bond with serine of POD was the only difference of ascorbic acid from other agents, which made serine an important amino acid in the activity center of POD.(5) The optimal browning inhibitors for preparing chestnut paste were obtained by response surface experiment:0.44g/L of salicylic acid,0.6g/L of ascorbic acid and 0.03% of phytic acid. Chestnut paste could maintain fresh color under above treatment and meet the none-browning desire in industrial production. In addition, the chestnut paste treated with both anti-browning agents and antioxidants during processing showed lower browning degree after high-temperature sterilization. The browning degree of that only treated with anti-browning agents was also obviously lower (p<0.01) than that of control. It indicated that the browning inhibit agents were effective and could be considered to use in industrial production. |
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