The Effect Of Dynamic High-Pressure Microfluidzation On HRP And Browning Enzymes Of Fuji Apple

Polyphenoloxidases and peroxidas are the main enzymes which is responsible for the ezymatic browning in fruits and vegetables, especially in apple. This paper studied the characterization of PPO and POD extracted from fuji apple, and using the dynamic high-pressure microfluidization to inactivated the these enzymes at the same time. Effects of DHPM on the activity and structures of horseradish peoxidase (HRP) in buffered solution were studied, and explored the synergistic effect on the HRP activity for DHPM combining with heat.Crude polyphenoloxidases (PPO) solusion extracted from fuji apple was characterized using catechol as a substrate, the resultes showed:PPO had a Km of 21.17mmol/L and a Vmax of 115.60U/min, the optimum pH and temperature for PPO activity were 5.5 and 15℃respectively. In addition, the fuji apple solution was treated by dynamic high-pressure microfluidization, and it has an inactivated effect on PPO. The PPO activity decreased with treatment pressure and teatment pass. After one pass of 15000 psi and 25000 psi at room temperature, the reduction of PPO relative activity were 20.17% and 27.07%, respectively. After three passes of 25000 psi, the reduction of PPO relative activity was 34.24%.Apple juice was pressed from Fuji apple and the crude peroxidase (POD) solution was extracted from the juice. Then enzymatic property of crude POD was characterized using guaiacol as substrate. The results showed:POD had a Vmax of 476.19 U/min and a Km of 129.09 mmol/L, the optimum temperature for the Fuji apple POD was 40℃and the optimum pH was 5.0. The activity of POD was inactivated by high-pressure microfluidization treatment. The activity of POD decreased with the increase of treatment pressure, passes and temperature. The activity of POD was decrease by 23.62% and 42.14% after one pass and three passes treatment under 25000 psi at room temperature (20℃), respectively; the activity of POD reduced by 37.25% after one pass of 25000 psi at 50℃.Effects of DHPM and DHPM combined with Heat on the activity of horseradish peoxidase (HRP) in buffered solution were studied while the corresponding changes to their secondary and structures was demonstrated by far-UV Circular dichroism (CD) and UV-VIS absorption spectra. HRP is a fairly stable protein against pressure denaturation. The relative residual activity of HRP decreased with the increase in pressure, however the activity of HRP decreased slowly. The greatest reduction of the activity was achieved for HRP at 180MPa with reductions of 9.6%. Increasing pass is not significant on HRP activity. It has a synergistic effect on the HRP activity for DHPM combining with heat. HRP has a good stability after heat treatment, however it loss the stability after DHPM treatment and after DHPM combined with heat treatment. Hydrophobic groups of HRP exposured, the prosthetic group heme may split out from the soret band and the a-helix decreased after DHPM, heat and DHPM combined with heat treatment, the change of HRP structure was in close agreement with the strength of all treatments. After a 5-day storage at 4℃, hydrophobic groups exposured further, the loss of the a-helix content and decrease of intensity of soret band changed by days.