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This study focuses on the influence of operating conditions on Alcalase-catalyzed egg white protein hydrolysis performed in a continuously stirred tank reactor coupled with ultrafiltration module (10 kDa). The permeate flow rate did not significantly affect the degree of hydrolysis (DH), but a significant increase in process productivity was apparent above flow rate of 1.9 cm min. By contrast, an increase in enzyme/substrate ( E/ S) ratio provided an increase in DH, but a negative correlation was observed between E/ S ratio and productivity. The relationship between operating conditions and antioxidant properties of the hydrolysates, measured by three methods, was studied using Box-Behnken experimental design and response surface methodology. The statistical analysis showed that each variable (impeller speed, E/ S ratio, and permeate flow rate) had a significant effect on the antioxidant capacity of all tested systems. Nevertheless, obtained response functions revealed that antioxidative activity measured by DPPH, ABTS and FRAP methods were affected differently by the same operating conditions. High impeller speeds and low permeate flow rates favor ABTS while high impeller speeds and high permeate flow rates had a positive effect on the DPPH scavenging activity. On the other hand, the best results obtained with FRAP method were achieved under moderate operating conditions. The integration of the reaction and ultrafiltration membrane separation in a continuous manner appears to be a right approach to improve and intensify the enzymatic process, enabling the production of peptides with desired antioxidant activity. [ABSTRACT FROM AUTHOR]

The objective of this research was to investigate the impact of high-intensity ultrasound (HIU) generated by a probe-type sonicator (frequency 20 ± 0.2 kHz and an amplitude of 40%) for 2-20 min on the selected functional and structural properties of egg white proteins (EWPs) and their susceptibility to hydrolysis by alcalase. The protein solubility, foaming, and emulsifying properties were studied as a function of ultrasonication time and related to protein particle and structural properties. The length of ultrasonication exhibited important effect on EWP particle size, uniformity, and charge, affecting also the protein conformation and susceptibility to alcalase hydrolysis and determining functional properties. There was a linear correlation between the particle size decrease and the solubility while a two-step linear correlation between the foam capacity (FC)/foam stability (FS) and particle size was apparent. Specifically, FC and FS sharply increased with decreasing particle size for range from ∼370 to ∼260 nm, and below this range from 260.6 to 68.4 nm, the changes were not that substantial. Besides, the solubility, FC, and FS were directly and linearly related with the absolute value of the particle zeta potential. The overall emulsifying properties were also improved with an increase of sonication time, through both the decrease of the mean particle diameter and the increase of zeta potential, but there was no direct correlation between the emulsion activity/stability index and protein particle size and/or charge. Analysis of EWP structure by Raman spectroscopy revealed that the HIU leads to changes in the secondary structure, while heat and ultrasound generated by the ultrasound bath were not sufficient to exhibit this effect. [ABSTRACT FROM AUTHOR]

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer-Emmett-Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of HO) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi-Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effects of different ultrasound pretreatment on enzymatic hydrolysis of egg white proteins (EWPs) by Alcalase as well as evaluating some functional and antioxidant properties of hydrolysates obtained by various proteases treatment and ultrasound technology. The effects of chosen ultrasound pretreatment parameters including frequency of ultrasonic waves (35 and 40 kHz), temperature (25 and 55 °C), time of pretreatment (15-60 min) and pH of egg white solution (7.00-10.00) were examined. It appeared that controlled ultrasound treatment can improved the hydrolysis process compared with untreated samples, but optimization of the power and length of sonication was important. The optimal ultrasound pretreatment at calorimetric power of 21.3 W and frequency of 40 kHz for 15 min at 25 °C and with naturally basic egg white (pH 9.25) resulted in increased initial rate and equilibrium degree of Alcalase hydrolysis by about 139.8 and 13.86 % compared with the control, respectively. EWP hydrolysates with ≈27.0 % degree of hydrolysis obtained with heat pretreatment and ultrasound pretreatments under optimal conditions were further separated by sequential ultrafiltration into 4 hydrolysate fractions (<1, 1-10, 10-30 and >30 kDa) which were investigated for protein content, peptide yield and antioxidant activity. The hydrolysis after heat pretreatment generated more peptides <1 kDa (19.04 ± 1.02 %) than ultrasound pretreatment did (11.90 ± 0.53 %), whereas the proportion of peptides <10 kDa were higher in the second case (28.80 ± 0.07 vs. 20.46 ± 0.39 %). The fraction obtained by the ultrasound pretreatment containing peptides with a molecular weight between 1 and 10 kDa demonstrated the strongest ABTS radical scavenging efficacy among the fractions (97.54 ± 0.30) with IC value of 4.31 mg/mL. Compared with single-enzyme processes, the two-stage enzymatic processes did not significantly improve both antioxidant and functional hydrolysates' properties. [ABSTRACT FROM AUTHOR]