# Influence of in situ 20 ± 2/28 ± 2 kHz dual-frequency ultrasonication on enzymolysis kinetics, thermodynamics and antioxidant activity of housefly (Musca Domestica) larvae protein hydrolysate

## TL;DR

Using dual-frequency ultrasound improves the breakdown of housefly larvae protein and enhances its antioxidant properties.

## Contribution

In situ dual-frequency ultrasound is shown to significantly enhance enzymatic hydrolysis and antioxidant activity of housefly larvae protein.

## Key findings

- Ultrasound treatment increased the reaction rate constant by 82.67% and reduced the Michaelis constant by 13.54%.
- Thermodynamic parameters like activation energy and enthalpy decreased significantly, while Gibbs free energy remained unchanged.
- Hydrolysate from ultrasound-treated protein showed improved in vitro and in vivo antioxidant activity.

## Abstract

•Employ in-situ dual-frequency ultrasound to accelerate enzymolysis of Housefly larvae protein.•The reaction rate constant (k) increased by 82.67 %, Michaelis constant (Km) reduced by 13.54 %.•Ea, ΔH and ΔS decreased but ΔG didn’t vary, ascribing to Enthalpy-Entropy Compensation Effect.•IC50 of HLPUH on DPPH, •OH and O2•− decreased from 3.02, 4.11 and 5.25 mg/mL to 2.64, 3.47 and 4.68 mg/mL.•HLPUH exhibited better in vivo antioxidant protective effects on HepG2 cells.

Employ in-situ dual-frequency ultrasound to accelerate enzymolysis of Housefly larvae protein.

The reaction rate constant (k) increased by 82.67 %, Michaelis constant (Km) reduced by 13.54 %.

Ea, ΔH and ΔS decreased but ΔG didn’t vary, ascribing to Enthalpy-Entropy Compensation Effect.

IC50 of HLPUH on DPPH, •OH and O2•− decreased from 3.02, 4.11 and 5.25 mg/mL to 2.64, 3.47 and 4.68 mg/mL.

HLPUH exhibited better in vivo antioxidant protective effects on HepG2 cells.

In this study, the influence of in situ 20 ± 2/28 ± 2 kHz dual-frequency ultrasonic irradiation on enzymolysis reaction kinetics, thermodynamics and antioxidant activity of housefly larvae protein (HLP) hydrolysate was investigated to clarify the ultrasonication’s promoting mechanism. The use of in situ dual-frequency ultrasound (DU) treatment significantly increased the reaction rate constant (k) of HLP enzymolysis by 82.67 %, decreased the Michaelis constant (Km) by 13.54 %, and increased the maximum reaction rate (vmax) by 6.12 % compared with conventional HLP hydrolysis. The thermodynamic parameters — activation energy (Ea), enthalpy (ΔH), and entropy (ΔS)—decreased significantly by 24.59 %, 23.95 %, and 18.86 %, respectively. However, no significant change in Gibbs free energy (ΔG) was observed, which may be attributed to the enthalpy–entropy compensation effect (EECE). The IC50 values of HLP hydrolysate obtained with in situ DU treatment (HLPUH) for DPPH, hydroxyl, and superoxide anion radicals decreased from 3.02, 4.11, and 5.25 mg/mL to 2.64, 3.47, and 4.68 mg/mL, respectively, indicating that the in vitro antioxidant activity of HLP hydrolysate (HLPH) was enhanced by ultrasound treatment. According to the results of in vitro HepG2 cell oxidative stress model experiments, HLPUH exhibited better protective effects on HepG2 cells, inhibited intracellular reactive oxygen species (ROS) expression, and enhanced the activities of intracellular oxidases—catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)—compared with the control. HLPUH also promoted HepG2 cell proliferation and reduced the apoptosis rate.