Characterisation Of Polyphenol Oxidase(PPO) From Chaenomeles Sinensis(Thouin) Koehne,Browning Control And Quality Of Its Juice

Chaenomeles sinensis(Thouin)Koehne fruit,belonging to Pomoideae of Rosaceae,is reported to be rich in bioactive constituents such as polyphenols,polysaccharide,flavonoids and oleanolic acid.It possesses biological activities such as antiviral,hepatoprotective effect,antibacterial properties,anti-inflammatory action,antioxidant capacity,anti-tumor activity,and immunomodu-latory effect.Enzymatic browning often occurs when the physiological injury happened resulting from essential processing operations,including drying,crushing and squeezing process.Browning reactions change sensory properties,reduce nutritional quality and decrease consumer acceptance of the final products.Polyphenol oxidase(PPO)is the main enzyme involved in the browning process.In this paper,purification and characterization of polyphenol oxidase from Chaenomeles sinensis(Thouin)Koehne fruit,effect of microwave and various inhibitors combination on inhibition of enzymatic browning in Chaenomeles Sinensis(Thouin)Koehne juice,effect of microwave and thermal treatments on PPO activity and quality of Chaenomeles Sinensis(Thouin)Koehne juice,and the antioxidant capacity in vitro of the juice were studied,the main results are as follows:1.Purification and characterization of polyphenol oxidase from Chaenomeles sinensis(Thouin)Koehne fruit:Polyphenol oxidase(PPO)from Chaenomeles Sinensis(Thouin)Koehne was extracted with cold acetone and its enzymatic characteristic were studied using catechol as the substrate.Stable and highly active PPO extracts were obtained using 1%(w/v)PVP and 1%(w/v)TritonX-100 which is 2.9 higher than that of using acetone powder without adding PVP and TritonX-100.Crude PPO extracts was purified through ammonium sulfate precipitation,dialysis and gel chromatography by Sephadex G-75.The specific activity of purified PPO is 3790U/mg and the molecular weight is 44KDa.The purification factor for PPO was 8.3%yield.The optimum pH for PPO was found to be 6.5.The enzyme had a temperature optimum of 20℃.The heat-stability of PPO followed first-order kinetics.Z value and the activation energy(Ea)were calculated to be 16.72℃ and 142.04 kJ/mol,respectively.The kinetic parameters Km and Vmax were 0.015mol/L and 250 U/min,respectively.Heating treatment for 5 min at 95℃ and microwave treatment for 10s at 700W could completely inactivate the enzyme activity.The inhibitory effects of eight inhibitors used in the study were placed in relative order of inhibition:Vc>L-cysteine>GSH>NaHSO3>citric acid>DL-malic acid>EDTA-2Na>NaCl.PPO activity was inhibited by both 1 mmol/L and 10 mmol/L of different metal ions,the inhibitory effects order were:Ca2+>Mg2+>Cu2+>Zn2+>Mn2+.2.The optimum condition of microwave and various inhibitors combination on inhibition of enzymatic browning in Chaenomeles Sinensis(Thouin)Koehne juice:compared with thermal treatment,microwave treatment before pressing increased the content of polyphenolics,and also presented lower Vc destruction.Vc,L-cys as well as microwave treatment were found to be the most significant anti-browning agents,while the additive content of citric acid did not show significant effect on inhibition.The optimum conditions for the inhibition were:Microwave treatment 100g fruit sized in 1cm×1cm.After 14s,adding 0.11%Vc,0.12%L-cys,0.34%citric acid and 20%water.After that,fruit was mashed,filtered at 4000 r/min.Under these conditions,90.57%inhibition was obtained.3.Microwave and thermal inhibition of enzyme of Chaenomeles Sinensis(Thouin)Koehne juice were comparatively evaluated and their impact on quality parameters of PPO activity,color,total polyphenols,vitamin C,amino nitrogen and antioxidant capacity were examined.The results showed that Microwave treatment could kill PPO in a shorter time.PPO was totally inactivated when treated with 231W for 60s,385W for 30s,539W for 20s andb700W for 10s,respectively.Complete inactivation of PPO was in 15min at 85℃ and 5min at 95℃.With the extension of processing time,thermal treatment caused remarkable increase of absorbance of juice at 410nm.Other quality parameters such as total polyphenols,VC and amino nitrogen content were reduced during the processing of these two treatments.Moreover,antioxidant capacity that evaluated by DPPH free radical scavenging capacity appeared a slight decrease and then increased slowly.4.The antioxidant capacity in vitro of Chaenomeles Sinensis(Thouin)Koehne juice:Chaenomeles Sinensis(Thouin)Koehne juice possessed strong scavenging activity of ABTS radical,DPPH radical,hydroxyl radical and superoxide anion radical.Their IC50 values were 0.0043,0.021,0.39 and 0.22 mL,respectively.The juice showed strong reducing ability and significant inhibitory effects on peroxidation of yolk lipoprotein induced by Fe2+.The correlation analysis showed that total phenolics content correlated with antioxidant capacity on DPPH,ABTS and peroxidation of yolk lipoprotein were more greatly than with Vc.