Characterization Of Polyphenol Oxidase And Regulation Methods Of Browning From Toona Sinensis

Toona sinensis is an unique and traditional woody vegetable in China,which contains unique flavour and various nutritional components.However,enzymatic browning is prone to take place in the processing and storage of Toona sinensis after the harvest.These would destroy the appearance,flavor and nutritional value of Toona sinensis products.Generally speaking,enzymatic browning found in fruits and vegetables are caused by polyphenol oxidase(PPO).Nevertheless,the researches of Toona sinensis PPO have not been reported.In order to control enzymatic browning and protect the sensory quality or nutritional value of Toona sinensis,the fresh tender buds were used as raw material.PPO taked from Toona sinensis was extracted and purified with the modern isolation and purification technology,and its enzymatic properties were probed.The antioxidant capacity and isozyme of Toona sinensis PPO during different growth periods were analyzed.A preliminary study about the preservation of Toona sinensis was carried out in order to control the enzymatic browning and preservation of Toona sinensis products.The main conclusions are as follows:After fractionated by 25-70%(NH4)2SO4 and purified by Sephacryl S-100 column,the specific activity of PPO was 900.619U/mg.Using SDS-PAGE gel electrophoresis purified enzyme protein molecular weight of about 84.55 kDa.Optimal pH and temperature were 6.2,40℃ and 8.5,80℃ for catechol and pyrogallol,respectively.PPO showed high and stable activity at pH ranging from 5.0 to 7.2 for catechol and pH 7.4 to 9.5 for pyrogallol.Activation energy(ΔEa#)values were 263.79 KJ/mol for catechol and 103.91 KJ/mol for pyrogallol.At temperature ranging from 65℃ to 80℃,the time required for 50%reduction in activity(t1/2)and the D-values of Toona sinensis PPO were 51.10-0.84min and 169.82-2.81min for catechol,25.17-5.57min and 83.65-18.10min for pyrogallol,respectively.From heat-inactivation studies,PPO had a stronger thermostability using pyrogallol as a substrate.PPO was stable below 75℃,but its thermal stability decreased significantly when the temperature was above 75℃.Based on the results of substrate specificity,the Vm/Km ratio were 0.0151,0.0543 and 0.0053 for catechol,pyrogallol and gallic acid,respectively.Pyrogallol was the most suitable substrate,and PPO exhibited no affinity with methyl gallate and epicatechin.The most effective chemical inhibitors were sulfite,followed by ascorbic acid,L-cysteine and citric acid.The inhibitory effect of sodium benzoate,SDS and EDTA-2Na were very weak.The results of the effect of metal ions on the activity of PPO showed that Na+,Ca2+and Al3+ had no significant inhibitory effect,Cu2+and Fe3+even increase enzymic activity.Purple onion peel extract and pomegranate rind extract were effective natural inhibitors,chestnut shell extract showed no inhibitory effect.The half maximal inhibitory concentration(IC50)on PPO was 5.19 mg/mL and 3.69 mg/mL for pomegranate rind extract,3.3 mg/mL and 2.78 mg/mL for purple onion peel extract using catechol and pyrogallol as substrates,respectively.The third growth period of Toona sinensis had the highest antioxidant activity,followed by the second and the first growth period.The antioxidant activity and phenolic constituents showed a dose-response relationship.The order of PPO activity of Toona sinensis during different growth periods was the first growth period,the third and the second growth period.The contents of total phenols showed a gradually increasing tendency during growth period.Toona sinensis of the first growth period had the lowest contents of total flavonoid,and the difference was not significant between the second and the third growth period.The contents of nitrite showed a gradually decreasing tendency during growth period.A total of 5 kinds of isozymes(A1-A5)were separated,and A3 and A4 were the the particular enzyme bands of Toona sinensis PPO.A1,A2 and A5 showed the different enzymatic activity during three growth periods,which leaded to the changes of PPO activity.The most influential band for PPO activity was A5,followed by A1 and A2.When the blanching condition were 90℃ and 90s without inhibitors,the activity of Toona sinensis PPO had been reduced to 24.95%.However,when Toona sinensis were blanched with the three inhibitors(L-cysteine,ascorbic acid and sulfite)for 60s at 90℃respectively,the activity of Toona sinensis PPO had fell below 22%and the browning degree decreased obviously.Sulfite was the most effective inhibitors,followed by ascorbic acid and L-cysteine.Compared with the above results,the blanching time was decreased obviouly after the inhibitors were increased.The best composition of combined inhibitors was 0.05g/100g Vc and 0.10g/100g sodium sulfite,which had better effect on color protection of Toona sinensis.