| Alpha-amylase is a commonly utilized enzyme in food processing.The residue after catalysis has a certain influence on the stability of the final product.So,it needs to be inactivated.Thermal enzyme deactivation technology is currently the most commonly used method in the industry.Whereas,the physical or chemical properties of food treated by strong heat will change,resulting in the deterioration of its color,aroma,taste,organizational structure,and loss of nutritional value.As a new food processing technology,pulsed electric field can effectively inactivate enzymes and preserve the original quality of food.However,the inhibition effect and mechanism of pulsed electric field on ?-amylase have not been determined,and the change of the spatial structure of ?-amylase in the process of external electric field treatment is still unclear.In this paper,the effects of different pulsed electric field parameters on the activity and structure of ?-amylase were studied.From the perspective of the unfolding of molecular space structure,the structure-activity relationship between the inactivating enzyme effect and the molecular space structure of the enzyme was constructed.By adding ?-carrageenan,the process of high-voltage pulsed electric field inducing ?-amylase unfolding was regulated.In addition,the changes of enzyme molecular structure caused by the thermal effect of the pulsed electric field were investigated.The multi-field coupling effect during the electric field treatment was evaluated by the simulated ohmic heat treatment.The main research contents and results were as follows:(1)Pulsed electric field has a significant passivation effect on ?-amylase(0.2 mg/m L),and it could reach the best under these conditions(field strength?20 k V/cm,pulse width?40?s,frequency?1.06 k Hz,and 5 cycles).The remaining enzyme activity of ?-amylase in the temperature-controlled(?40°C)electric field treatment was 90%,while it was 50% at the non-temperature-controlled(?60°C)electric field treatment.During the passivation process,the fluorescence intensity of ?-amylase decreased by 90%.The tertiary structure was fully expanded,and the hydrophobic region inside the molecule was exposed,resulting in a significant increase in surface hydrophobicity.The results of CD showed that the content of?-helix and ?-turn structure in ?-amylase molecules decreased,and the content of ?-sheets increased after electric field treatment.It was speculated that the pulsed electric field could inactivate the enzyme activity by destroying the secondary and tertiary structure of ?-amylase molecules.Moreover,through the passivation enzyme experiment,it was found that when the electric field intensity was 15 k V/cm,there was a denaturation intermediate in the denaturation process of ?-amylase,that was,the molten spherical state.(2)The addition of ?-carrageenan could significantly affect the inactivating effect of ?-amylase treated by pulsed electric field.When ?-amylase/?-carrageenan was 100:1,a small amount of ?-carrageenan had little effect on the inactivating.By increasing the ratio to 1:1,excess ?-carrageenan and ?-amylase could form a complex through electrostatic interaction,and protect the structure of ?-amylase during the electric field treatment,and enzyme activity was basically unchanged.The addition ratio of 10:1 could significantly enhance the treatment effect of the pulsed electric field.Through the structural characterization of ?-amylase,it was found that the degree of change in the secondary structure after adding ?-carrageenan was obviously greater than that of the original enzyme alone.(3)The thermal effect of the pulsed electric field could not be ignored in the process of inactivating the enzyme.Through simulating thermal effect,it was found that the thermal passivation effect was significantly better than the pulsed electric field at the same heat treatment temperature for 15 min.Adding ?-carrageenan could obviously inhibit the aggregation of ?-amylase during the heat treatment.At two ratios of ?-amylase/?-carrageenan(10:1 and 1:1),there was no obvious aggregation during the whole heating process,and the particle size maintained at around 300 nm.It was speculated that the complex formed by the electrostatic interaction between ?-amylase and ?-carrageenan changed the thermal denaturation temperature of ?-amylase. |
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