The effect of different frequencies of ultrasound on the activity of horseradish peroxidase

Abstract

Please cite this article as: Tsikrika, K., Chu, B.-S., Bremner, D.H., Lemos, M.Adí., The effect of different frequencies of ultrasound on the activity of horseradish peroxidase, Abstract 6Ultrasound technology has been studied by food researchers as an alternative 7 method for thermal processing. The use of ultrasound as a way to inactivate and/or 8 activate enzymes has been widely studied at low frequencies (20-40 kHz), however, 9 little research on the effect of high frequencies has been reported. Thus, the effect 10 of high and low frequency ultrasound on commercial horseradish peroxidase with a 11 concentration of 0.005 mg mL -1 is described. Experiments were performed for 60 12 minutes using 20, 378, 583, 862, 995, 1144 and 1175 kHz ultrasound at power levels 13 (acoustic energy) between 2.1 to 64 W. Residual activity was monitored using a 14 spectrophotometric method and data analysis was performed using ANOVA. A 15 significant enhancement of enzyme inactivation (p<0.05) was observed at each 16 frequency with an increase of sonication time and power. Inactivation of peroxidase 17 by ultrasound followed first order kinetics and an increase of the rate constant with 18 the power applied was observed for all the frequencies studied. Overall, low 19 frequency (20 kHz) and low power are not effective on the enzyme inactivation and 20 the level of residual activity remained high. The use of 378 and 583 kHz (48 W) is 21 particularly effective for complete enzyme inactivation. 22 ACCEPTED MANUSCRIPT 2

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“…Free radical-mediated deactivation mechanisms and cleavage of the functional groups from enzymes could occur during US treatment and is the likely reason for the modifications in proteins and enzymes. For example, the inactivation of peroxidase as a result of haem group dissociation and the loss of iron was facilitated by hydroxyl radicals from US cavitation [ 69 ].…”

“…The position of the disulfide motif in the center of the macromolecular framework (e.g. Hydrocarbon) enabled the efficient mechanochemical scission and the release of this monoterpene molecule [ 69 ]. A US mechanophore breakage (mechanochemically breakage of the polymer reactive units like cyclic rings) can occur in extracts solutions via the shear stress caused by the collapse of US-induced cavitation bubbles [ [73] , [74] ].…”

“…As mentioned earlier, CPT extraction from the Camptotheca acuminata herb by US transformed the disulfide bonds to thiol bonds of these molecules, which resulted in the disulfide motif to be in the center of the macromolecular framework (e.g. Hydrocarbon) and enabled efficient mechanochemical scission and the release of this monoterpene molecule [ 69 ]. High-intensity US using a titanium horn at frequency 19.5 kHz and intensity 140 W increased the extraction efficiency of carnosic (13%) and rosmarinic (6.8% of dry extract) acids from rosemary leaves [ [60] , [61] ].…”

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“…e inactivation of horseradish peroxidase was investigated by Tsikrika et al [98] who determined that sonication for 60 min using 20, 378, 583, 862, 995, 1144, and 1175 Hz ultrasound at power levels (acoustic energy) between 2.1 and 64 W was very effective at inactivating the enzymes with little effect observed at the 20 kHz lower frequency. e fact that the greatest levels of inactivation were observed at 378 and 583 nm suggests that some radical effect may be the cause.…”

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“…Moreover, it needs to be considered that during acoustic cavitation the extremely high local temperature, pressure, and shearing forces – attributable to the collapse of cavitation bubbles and the generation of high-velocity microjets – can also affect both the enzyme molecule and the solid substrate, modifying their chemical and physical properties. Previous studies have shown that intensive ultrasonication can change the protein molecules, resulting in a decrease in the activity of enzymes [12] , [13] , [14] .…”