The Inactivation Mechanism Of Organic Acid Combined With Physcial Methods On Polyphenoloxidase And Its Application

Polyphenoloxidase(PPO)is a group of copper-containing enzymes,which are widely distributed in animals,plants,fungi and bacteria.PPO is responsible for the browning of damaged fruits and vegetables by catalyzing two different reactions: the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to oquinones,which polymerize to brown pigments subsequently.Besides the color changes,enzymatic browning deteriorates the nutritional quality of fruits and vegetables and cause a reduction of market value.Therefore,the inactivation of PPO is necessary for controlling browning.The current studies mainly focus on the characterasation of enzyme and inhibition of activity.Little information is available about the inactivation mechanisms and the effect of organic acid comined with physical methods on the inhibition of PPO and browning of fruits and vegetables.Therefore,this thesis would mainly focus on the following three subjects.Firstly,investigate the inactivation mechanisms of organic acid and thermal treatment on PPO.Secondly,analyze the refolding of PPO at different acid conditions and its effect on the thermal,high pressure and ultrasound inactivation of PPO.At last,apply the combined processing method on products such as pear puree and fresh cut yam and investigate the changes in enzyme activity and related qualities.The main contents of this thesis are summarized as follows:(1)Cinnamic acid,citric acid and malic acid inhibited PPO in different modes.The cinnamic acid reversibly inhibited PPO in a mixed-type manner,while the decrease of pH was the main cause for citric acid and malic acid.Cinnamic acid inserted into the hydrophobic cavity of PPO by forming π–π stacking with His residual and induced the rearrangement of secondary structure.During the heating process,the inactivation of PPO can be achieved by high temperature-short time or mild temperature-long time treatment.Heating process induced the rearrangement of secondary structure and the disruption of tertiary structure.Red shifts of fluorescence spectra showed positive correlations with the inactivation rate of PPO.There were significant differences in the conformation and molecular microstructure among PPO samples with the same relative activity,which were obtained by treating PPO at 45,55 and 65 °C for different times.PPO molecules were deformed at mild temperature,while higher temperature induced the formation of large aggregates.These results indicated that PPO with the same relative activity might exist in different forms.(2)The acid p H can enhance the inactivation of PPO by physical treatment.PPO gradually unfolded at acid pH condition.At pH ≥ 4.0,the activity of PPO decreasd and PPO was mildly unfoled.After adjusting to pH 6.8,the activity recoveried and conformation refolded.At pH ≤ 4.0,PPO was strongly inactived and denatued,and the changes in activtiy and conformational are irreversible.Acid pH improved the sensitivity of PPO to thermal inactivation and the inactivation followed a biphasic model.Combination of acid and heat enhanced the conformational changes of PPO.Neither medium concentrations(10,20,30 mM)malic acid nor individual ultrasound treatment could remarkably inactivate PPO.However,the inactivation during their combination was more significant than the sum of ultrasound and malic acid inactivation.Ultrasound broke large molecular groups of PPO into small ones and combined treatment resulted in greater changes in conformation and microstructure.(3)Organic acid combined with high pressure or thermal processing can effectively inhibit the browning of fruits and vegetables and maintain the quality of products.Citric acid inhibited peroxidase and PPO and preserved the color of pear puree.The addition of citric acid(2%,w/w)increased total phenolic content(TPC)and oxygen radical absorbance capacity of pear puree by ~50%.Citric acid enhanced thermal and high pressure thermal inactivation.Thermal processing resulted in a slightly better color retention of acidified samples than high pressure thermal processing under conditions of equivalent enzyme inactivation.For the fresh cut yam,thermal processing at 80 °C and combined 50 °C with cinnamic acid can effectively inhibit browning of yam.Compared to heat processing with 80 °C,the combined method improved the nutritional quality,reflecting in the higher TPC and antioxidant capacity.Besides,combined method maintained the texture quality of yam.