Conventional Enzymatic Hydrolysis Research Articles

Production, identification and characterization of antioxidant peptides from potato protein by energy-divergent and gathered ultrasound assisted enzymatic hydrolysis

Effects of energy divergent ultrasound (EDU, 30, 40 and 50 W/L) and energy gathered ultrasound (EGU)-assisted enzymatic hydrolysis on production, identification and characterization of antioxidant peptides from potato protein hydrolysates (PPH) were investigated. EDU and EGU significantly increased degree of hydrolysis and recovery of PPH compared with conventional (C) enzymatic hydrolysis. PPH by EGU40 showed the highest Fe2+-chelating activity (55.33 µg EDTA/mL), ·OH scavenging activity (230.05 µg Vc/mL) and oxygen radical absorbance capacity (82.24 µg TE/mL). Peptides with molecular weight <1 and 1–2 kDa of PPH by EGU40 displayed higher antioxidant activities than those by C and EDU40, which might be contributed to their more hydrophobic, polar charged and antioxidant amino acids at terminals and/or inside the sequences identified by LC-MS/MS. Additionally, structure–activity relationship was revealed by peptide conformation and position of some specific amino acids. Therefore, EGU has great potential in production of functional peptides with enhanced antioxidant activity.

Sunlight active TiO2-Bi2WO6 photocatalyst pretreatment of biomass for simultaneous hydrolysis and saccharification in bioethanol production

In this work we examine photocatalysis as an alternative to the conventional enzymatic hydrolysis and liquefaction in the upstream processing of biomass for bioethanol production. A highly efficient visible light active TiO2-Bi2WO6photocatalyst having a band gap of 2.7 eV was used to deconstruct potato mash and casuarina sawdust. Thebiomass was pretreated with thephotocatalyst under sunlight for 5 h and thesawdust was subjected toprolonged treatmentsunder a 65 W fluorescent light source. Photocatalytically deconstructed biomass was subjected to fermentation by yeast. Control biomass was subjected to standard liquefaction byα amylasefollowed by yeast fermentation. Analysis of photocatalytic deconstructed products by ESI-MS reveals increasing glucose accumulation up to two hours followed by decrease upon prolonged pretreatment, suggesting possible cleavageof evolving glucose moieties. Increased concentration of lower carbonfragments in the products further corroborates this finding. HPLC analysis of fermentation products show ethanol yield on samples subjected to photocatalysis as upstream processing is comparable to that of standard enzymatic fermentation. It is thus demonstrated that visible light active photocatalysis of biomass upstream of the fermentation process can be used in place of enzymatic or chemical hydrolysis inethanol production which also constitute an environmentally benign approach.

Hydrolysis of Anhydrosugars over a Solid Acid Catalyst for Saccharification of Cellulose via Pyrolysis

Glucose is a key compound for future biomass-based energy and chemical industry. An availability of glucose from abundant lignocellulosic biomass is limited because of a slow reaction rate and costly feed materials in a conventional enzymatic hydrolysis of cellulose. The present work investigated the production of glucose with hydrolysis of anhydrosugars produced by cellulose pyrolysis that is a fast reaction with no requirement for other chemicals to feed. A commercially available solid acid was employed as the hydrolysis catalyst for enabling a direct use of glucose aqueous solution without posttreatment such as separation. The experiments using a model anhydrosugar, levoglucosan (LGA), as feedstock revealed a selective activity of the catalyst to produce glucose even at the high concentration of 2.7 M and the catalytic stability in 15 h run of the reaction using a continuous flow reactor. The catalyst worked for the reaction with a cellulose-derived bio-oil as the feedstock to selectively produce glucose mainly from LGA. However, the activity gradually decreased due to deposition of carbonaceous materials from compounds other than LGA over the catalyst, indicating the necessity for eliminating those compounds before the hydrolysis.

Production of Demineralized Antibacterial, Antifungal and Antioxidant Peptides from Bovine Hemoglobin Using an Optimized Multiple-Step System: Electrodialysis with Bipolar Membrane.

Numerous studies have shown that bovine hemoglobin, a protein from slaughterhouse waste, has important biological potential after conventional enzymatic hydrolysis. However, the active peptides could not be considered pure since they contained mineral salts. Therefore, an optimized multi-step process of electrodialysis with bipolar membranes (EDBM) was carried out to produce discolored and demineralized peptides without the addition of chemical agents. The aim of this study was to test the antibacterial, antifungal and antioxidant activities of discolored and demineralized bovine hemoglobin hydrolysates recovered by EDBM and to compare them with raw and discolored hydrolysates derived from conventional hydrolysis. The results demonstrate that discolored–demineralized hydrolysates recovered from EDBM had significant antimicrobial activity against many bacterial (gram-positive and gram-negative) and fungal (molds and yeast) strains. Concerning antibacterial activity, lower MIC values for hydrolysates were registered against Staphylococcus aureus, Kocuria rhizophila and Listeria monocytogenes. For antifungal activity, lower MIC values for hydrolysates were registered against Paecilomyces spp., Rhodotorula mucilaginosa and Mucor racemosus. Hemoglobin hydrolysates showed fungicidal mechanisms towards these fungal strains since the MFC/MIC ratio was ≤4. The hydrolysates also showed a potent antioxidant effect in four different antioxidant tests. Consequently, they can be considered promising natural, low-salt food preservatives. To the best of our knowledge, no previous studies have identified the biological properties of discolored and demineralized bovine hemoglobin hydrolysates.

Hybridising plasma functionalized water and ultrasound pretreatment for enzymatic protein hydrolysis of Larimichthys polyactis: Parametric screening and optimization

Hybridising plasma functionalized water and ultrasound pretreatment for the enzymatic hydrolysis (HPUEH) of Larimichthys polyactis was evaluated by adopting Plackett-Burman design for parametric screening of six key variables, and Box-Behnken design for optimizing three most significant variables including plasma generating voltage (PV), ultrasound treatment time (UT), and enzyme concentration (EC). The models developed for predicting the degree of hydrolysis (DoH), protein recovery (PVY), and soluble protein content (SPC) were sufficiently fitted to the experimental data (R2 ≥ 0.966) with non-significant lack of fit and used for determining the optimum conditions as PV of 70 V, UT of 15 min, and EC of 1.787%, with predictive values of 27.74%, 85.62%, and 3.28 mg/mL for DoH, PVY, and SPC, respectively. HPUEH presented hydrolysates with smaller peptide sizes and molecular weights, enhanced DoH, PVY, SPC, amino acids and antioxidant activity, but reduced emulsifying and foaming properties when compared with conventional enzymatic hydrolysis.

AF4-UV-ICP-MS for detection and quantification of silver nanoparticles in seafood after enzymatic hydrolysis

A method based on asymmetric flow field-flow fractionation (AF4) coupled to ultraviolet–visible (UV–vis) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) has been developed for silver nanoparticles (Ag NPs) detection and quantification in bivalve molluscs. Samples were pre-treated using a conventional enzymatic (pancreatin and lipase) hydrolysis procedure (37 °C, 12 h). AF4 was performed using a regenerated cellulose (RC) membrane (10 kDa, 350 μm spacer) and aqueous 5 mM Tris-HCl pH = 7.4 as carrier. AF4 separation was achieved with a program that included a focusing step with tip and focus flows of 0.20 and 3.0 mL min−1, respectively, and an injection time of 4.0 min. Elution of different size fractions was performed using a cross flow of 3.0 mL min−1 for 15 min, followed by linear cross flow decrease for 7.5 min, and a washing step for 9.4 min with no cross flow. Several bivalve molluscs (clams, oysters and variegated scallops) were analysed for total Ag content (ICP-MS after microwave assisted acid digestion), and for Ag NPs by the method presented here. Results show that Ag NPs are detected at the same elution time than proteins (UV monitoring at 280 and 405 nm), which suggests a certain interaction occurred between Ag NPs with proteins in the enzymatic extracts. AF4-UV-ICP-MS fractograms also suggest different Ag NPs size distributions for selected samples. Membrane recoveries, determined by peak area comparison of fractograms with and without application of cross flow, were within the 49–121% range. Confirmation of the presence Ag NPs in the investigated enzymatic extracts was demonstrated by SEM after an oxidative pre-treatment based on hydrogen peroxide and microwave irradiation.

Bovine Hemoglobin Enzymatic Hydrolysis by a New Ecoefficient Process-Part I: Feasibility of Electrodialysis with Bipolar Membrane and Production of Neokyotorphin (α137-141).

Neokyotorphin (α137-141) is recognized as an antimicrobial peptide and a natural meat preservative. It is produced by conventional enzymatic hydrolysis of bovine hemoglobin, a major component of cruor, a by-product of slaughterhouses. However, during conventional hydrolysis, chemical agents are necessary to adjust and regulate the pH of the protein solution and the mineral salt content of the final hydrolysate is consequently high. To produce this peptide of interest without chemical agents and with a low salt concentration, electrodialysis with bipolar membrane (EDBM), an electromembrane process recognized as a green process, with two different membrane configurations (cationic (MCP) and anionic (AEM) membranes) was investigated. Hydrolysis in EDBM showed the same enzymatic mechanism, “Zipper”, and allowed the generation of α137-141 in the same concentration as observed in conventional hydrolysis (control). EDBM-MCP allowed the production of hydrolysates containing a low concentration of mineral salts but with fouling formation on MCP, while EDBM-AEM allowed the production of hydrolysates without fouling but with a similar salt concentration than the control. To the best of our knowledge, this was the first time that EDBM was demonstrated as a feasible and innovative technology to produce peptide hydrolysates from enzymatic hydrolysis.

Intensification of delignification of sawdust and subsequent enzymatic hydrolysis using ultrasound.

The current work investigates intensification of delignification of sawdust and subsequent enzymatic hydrolysis to produce reducing sugars with the use of ultrasound. Alkaline hydrolysis of sawdust was initially performed to remove lignin which hampers the rate of enzymatic hydrolysis. Effect of different parameters in the case of ultrasound (US) assisted and conventional processes such as alkali concentration (0.5-2.5 N), substrate loading (0.2-1.0% w/v) and temperature (40-80 °C) have been investigated. Optimized parameters obtained for US assisted process showed better trends as compared to conventional process with about 1.25 times higher yields and significant reduction in time by about 4 h. The process parameters for US assisted and conventional enzymatic hydrolysis to produce reducing sugars were also optimized by varying substrate loading (0.5-10% w/v), reaction temperature (30-70 °C) along with variation in US power (10-80 W) and US duty cycle (30-90%). US assisted enzymatic hydrolysis performed at 4% w/v substrate loading along with 50 W US power and 50% duty cycle at 50 °C resulted in 7.46 mg/mL of reducing sugars yield within 1 h while conventional stirring with 6% w/v substrate loading and 50 °C resulted in approximately same yield of reducing sugars within 3 h. The requirement of lower time for similar yields or in other words higher yield in same time clearly highlights the process intensification benefits due to the use of ultrasound. Overall it can be concluded from the study that US assisted processes resulted in efficient delignification along with higher yield of reducing sugars in lower treatment time as compared to conventional process.

EFFECTS OF ULTRASOUND TREATMENT ON STRUCTURAL, CHEMICAL AND FUNCTIONAL PROPERTIES OF PROTEIN HYDROLYSATE OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) BY-PRODUCTS

In this study, the effects of ultrasound treatment on biochemical, physical, structural and functional properties of fish protein hydrolysate of rainbow trout (Oncorhynchus mykiss) by-products were investigated. Enzymatic hydrolysis was conducted by Alcalase 2.4 L, pH 8, 1 h at 60°C, and enzyme/substrate ratio at 0.5%. A probe-type ultrasound was used for ultrasound assisted hydrolysis (UH) process. Higher protein recovery was obtained in UH than in the conventional enzymatic hydrolysis (CH). The highest foaming capacities of CH and UH were measured as 137.5% and 152.5%, respectively (p<0.05). Overall, our data suggest that ultrasound treatment helps to improve the functional properties such as foaming capacity and stability.

Convenient preparation of 2″-O-rhamnosyl icariside II, a rare secondary flavonol glycoside, by recyclable and integrated biphase enzymatic hydrolysis

Background: 2″-O-rhamnosyl icariside II, a rare secondary flavonol glycoside in Epimedii Folium (EF), has much better in vivo bioactivities than its original glycoside epimedin C. Its preparation methods, such as acidic hydrolysis, are of low efficiency, with by-products generated. Objective: The objective of this study was to establish a novel catalysis system for convenient preparation of this compound based on the recyclable and integrated biphase enzymatic hydrolysis. Materials and Methods: β-dextranase was selected from five commercial enzymes due to the best catalysis performance. After optimization of conditions, the biphase system was constructed with propyl acetate and HAc-NaAc buffer (pH 4.5) (3:2, v/v) containing β-dextranase/epimedin C (1:2, w/w), and the hydrolysis was performed at 60°C for 40 min. Results: Epimedin C was completely hydrolyzed to 2″-O-rhamnosyl icariside II, and 93.38% of the product has been transferred into organic phase; moreover, a high conversion rate had been achieved at 91.69% even after the enzyme solution was used for four cycles. In addition, the procedure was much simplified compared with conventional enzymatic hydrolysis. Conclusion: The newly proposed strategy is an efficient and promising approach for the preparation of 2″-O-rhamnosyl icariside II in industrial application. Abbreviations used: EF: Epimedii Folium; ACN: Acetonitrile; MeOH: Methanol; PTFE: Polytetrafluoroethylene; SD: Standard deviation; ILO-ICSC: International Labor Organization-The International Chemical Safety Cards; HMDB: Human Metabolome Database; HSDB: Hazardous Substances Data Bank.

Convenient preparation of sagittatoside B, a rare bioactive secondary flavonol glycoside, by recyclable and integrated biphase enzymatic hydrolysis

Sagittatoside B, a rare secondary flavonol glycoside in Epimedii Folium, has much better in vivo bioactivities than its original glycoside epimedin B. Its preparation methods, such as acidic hydrolysis, are of low efficiency, and byproducts are generated. The objective of this study was to establish a novel catalysis system for convenient preparation of this compound based on recyclable and integrated biphase enzymatic hydrolysis. β-glucanase was selected from five commercial enzymes based on the best catalysis performance. After optimization of the conditions, the biphase system was constructed with propyl acetate and HAc-NaAc buffer (pH 4.5) (1:1, v/v) containing β-glucanase/epimedin B (1:2, w/w), and the hydrolysis was performed at 60 °C for 1 h. Consequently, epimedin B was completely hydrolyzed to sagittatoside B, and 95.7% of the product was transferred into the organic phase. Moreover, a high conversion ratio of 94.0% was achieved, even after the enzyme solution was used for six cycles. Additionally, the procedure was much simplified compared with conventional enzymatic hydrolysis. The newly proposed strategy is an efficient and promising approach for the preparation of sagittatoside B in industrial applications.

Efficient preparation of rare Sagittatoside A from epimedin A, by recyclable aqueous organic two-phase enzymatic hydrolysis

The rare secondary flavonol glycoside Sagittatoside A has much better in vivo bioactivities than epimedin A in Epimedii Folium. However, its current preparation methods are of low efficiency, with byproducts generated. The aim of this study was to establish a novel catalysis system for effective and convenient preparation of Sagittatoside A from epimedin A based on recyclable and integrated aqueous organic two-phase enzymatic hydrolysis. The system was consisted of propyl acetate and HAc-NaAc buffer (pH4.5) containing β-dextranase/epimedin A, and the hydrolysis was performed at 60 °C for 1 h. Consequently, epimedin A was completely hydrolyzed to sagittatoside A, and 95.02% of the product was transferred into the organic phase. Moreover, 90% of its initial activity was retained after seven cycles of hydrolysis. Additionally, the procedure was simpler than conventional enzymatic hydrolysis. Collectively, the newly proposed strategy is an efficient and promising approach for the preparation of sagittatoside A in industrial application.

Bacteria as cell factories for producing selenium nanoparticles: their synthesis by the rhizobacterium Azospirillum brasilense and characterisation

Sagittatoside B, a rare secondary flavonol glycoside in Epimedii Folium, has much better in vivo bioactivities than its original glycoside epimedin B. Its preparation methods, such as acidic hydrolysis, are of low efficiency, and byproducts are generated. The objective of this study was to establish a novel catalysis system for convenient preparation of this compound based on recyclable and integrated biphase enzymatic hydrolysis. β-glucanase was selected from five commercial enzymes based on the best catalysis performance. After optimization of the conditions, the biphase system was constructed with propyl acetate and HAc-NaAc buffer (pH 4.5) (1:1, v/v) containing β-glucanase/epimedin B (1:2, w/w), and the hydrolysis was performed at 60 °C for 1 h. Consequently, epimedin B was completely hydrolyzed to sagittatoside B, and 95.7% of the product was transferred into the organic phase. Moreover, a high conversion ratio of 94.0% was achieved, even after the enzyme solution was used for six cycles. Additionally, the procedure was much simplified compared with conventional enzymatic hydrolysis. The newly proposed strategy is an efficient and promising approach for the preparation of sagittatoside B in industrial applications.

Transformation of residual starch from brewer’s spent grain into fermentable sugars using supercritical technology

This work provides the suitability of using near-critical CO2 to assist the enzymatic hydrolysis of starch from brewer’s spent grain (BSG). Total reducing sugars (TRS) were obtained in different conditions of temperature (30 °C–50 °C), pressure (100 bar–250 bar) and moisture (40%–80%) using a central composite rotatable design. The concentration of enzyme in relation to substrate (BSG) was maintained constant at 8.2 wt.%. The highest TRS yield (219.39 g/kg) was reached with enzyme-substrate mixture and CO2 at 40 °C, 175 bar, and 80% moisture for 240 min. In this condition, approximately 26.5 wt.% glucose, 2.2 wt.% cellobiose, 1.7 wt.% xylose and 0.7 wt.% arabinose were quantified in the hydrolyzed medium. Using near-critical CO2 increased the sugars yield by 3.6 times higher than that of conventional enzymatic hydrolysis.

Changes on antioxidant activity of microwave-treated protein hydrolysates after simulated gastrointestinal digestion: Purification and identification

Two samples of trout frame protein hydrolysates were prepared by Microwave Pretreatment followed by Conventional Enzymatic hydrolysis (MPCE) and Non-Pretreated followed by Microwave-assisted Enzymatic hydrolysis (NPME), respectively, were subjected to simulated gastrointestinal digestion. Changes on degree of hydrolysis, antioxidant activity, molecular weight, and amino acid composition between undigested and after gastrointestinal digestion of peptides were investigated. Comparing to undigested peptides, a breakdown of MPCE and NPME into smaller molecules was observed. Degree of hydrolysis, ABTS+ radical scavenging activity and reducing power increased (P < 0.05) for both samples after gastrointestinal digestion. A purified peptide from GI-MPCE had two possible sequences, NGRLGYSEGVM or GNRLGYSWDD (1182.65 Da). Whereas GI-NPME had two peptides IRGPEEHMHR or RVAPEEHMHR (1261.77 Da) and SAGVPRHK or SARPRHK (962.63 Da). These results indicate that digested hydrolysates can be a rich source of antioxidants. Isolated peptides extracted from trout frame by-products could be new food ingredients used as natural antioxidants.

Microwave-assisted enzymatic hydrolysis of DNA for mass spectrometric analysis: A new strategy for accelerated hydrolysis

Study of DNA base composition, DNA adducts and modification in its primary structure is a subject of interest in different fields of scientific research. Various methods like immunochemistry, capillary electrophoretic separation, chromatographic separation coupled with mass spectrometric (LC-MS/MS) detection have been developed for DNA analysis. During the past decade LC-MS/MS has emerged as a more sensitive and selective technique and now frequently used for the analysis of DNA. The workflow for the DNA analysis include DNA extraction, hydrolysis of DNA into mononucleosides and analysis. Though high-throughput methods are available for the analysis DNA hydrolysis oftentimes is a rate limiting step. Conventional enzymatic hydrolysis of DNA using a multienzyme mixture will take a minimum of 6–17 h to complete the hydrolysis. In this work, we developed an accelerated enzymatic hydrolysis of DNA using microwave-technology for the first time. 1.00 μg of calf thymus DNA was used to demonstrate the microwave assisted enzymatic hydrolysis of DNA. The resultant mononucleosides were separated on Atlantis T3 (50 × 2.1 mm i.d.; 3 μ particle size) C18 column and analyzed on ABSCIEX QTRAP 5500 LC/MS system. The sample through put and recovery of microwave-assisted digestion were compared with the conventional enzymatic hydrolysis. Efficient digestion of DNA with a performance similar to that obtained by the conventional overnight digestion procedure was attained in just 30 min with a hydrolysis yield of ≥90%. Furthermore, our method was found to be much more accurate and easier to perform. Thus, this new application of microwave technology to DNA enzymatic digestion will facilitate the application of DNA analysis in biological and clinical research.

Construction of a novel catalysis system for clean and efficient preparation of Baohuoside I from Icariin based on biphase enzymatic hydrolysis

Baohuoside I has better in vivo bioactivities than Icariin. Its preparation methods, such as acid hydrolysis, are of low efficiency, with undesirable byproducts. The objective of this study was to establish a novel catalysis system for its clean and efficient preparation via biphase enzymatic hydrolysis. β-glucosidase was selected from five commercial enzymes due to the best catalysis performance. After optimization of conditions, a biphase system was constructed with EtOAc and HAc-NaAc buffer (pH 4.5) (5:1, v/v) containing Icariin/β-glucosidase (3.2:1, w/w), and hydrolysis was performed at 60 °C for 2 h. Consequently, the conversion rate of Icariin was 99.92 ± 0.11% (n = 3), and the processing capacity in the system was increased from 20 mg/mL to 50 mg/mL, 2.5 times that of conventional enzymatic hydrolysis, meanwhile the procedure was much simplified. Additionally, complete hydrolysis with conversion rate at 99.14% had been achieved after the enzyme solution was used twice. The newly proposed strategy is a clean and efficient approach for the preparation of Baohuoside I and also a promising technology for producing secondary glycosides or aglycones.

Sustainability assessment of glucose production technologies from highly recalcitrant softwood including scavengers

AbstractThe utilization of abandoned lignocellulosic residues for chemical production has a strong potential to partially substitute chemicals, which are traditionally produced from non‐renewable resources. Softwood especially, with its high availability, presents a sustainable resource for the conversion to higher value‐added products such as biofuels and bioplastics. In this study, we investigate mature and innovative technologies for the conversion of softwood to the platform chemical sugar from an economic and environmental perspective. We show that the conventional enzymatic hydrolysis has high economic as well as environmental burdens and that the increase of enzyme availability via a carbocation scavenger process is the key solution to overcome them. Furthermore, we present a process design based on concentrated acid hydrolysis, which is both environmentally and economically competitive compared to conventional production from sugarbeet. The low energy and raw material requirements combined with heat integration and moderate capital costs makes this technology attractive for utilization of softwood residues. This proves that lignocellulosic residues have the potential to become an important raw material in the future bioeconomy. © 2017 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Effect of Microwave Treatments on Antioxidant Activity and Antigenicity of Fish Frame Protein Hydrolysates

Protein solubility, protein recovery, antioxidant activity, and antigenicity of microwave pretreatment and/or microwave-assisted hydrolysis of trout frame protein hydrolysates was investigated. Treatments consisted of (1) microwave pretreatment at low temperature (55 °C) followed by conventional enzymatic hydrolysis or (2) high temperature (90 °C) followed by conventional enzymatic hydrolysis; (3) microwave pretreatment at high temperature (90 °C) followed by microwave-assisted enzymatic hydrolysis, and (4) no microwave pretreatment followed by microwave-assisted enzymatic hydrolysis. Compared to controls, microwave treatments significantly improved (P < 0.05) protein solubility, protein recovery, degree of hydrolysis, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) free radical scavenging radical scavenging activity. Decrease of antigenicity (55–93 %) was obtained in all microwave-treated samples. Based on the results of this study, the use of microwave pretreatment for 5 min at 90 °C, followed by conventional enzymatic hydrolysis with alcalase for 2–10 min was the best treatment condition to produce fish peptides with antioxidant activity and the lowest immunoreactivity. These peptides have potential to be applied as hypoallergenic ingredients in food formulations and nutraceuticals.

Ultrasound Pretreatment as an Useful Tool to Enhance Egg White Protein Hydrolysis: Kinetics, Reaction Model, and Thermodinamics.

The impact of ultrasound waves generated by probe-type sonicator and ultrasound cleaning bath on egg white protein susceptibility to hydrolysis by alcalase compared to both thermal pretreatment and conventional enzymatic hydrolysis was quantitatively investigated. A series of hydrolytic reactions was carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations, and temperatures using untreated, and pretreated egg white proteins (EWPs). The kinetic model based on substrate inhibition and second-order enzyme deactivation successfully predicts the experimental behavior providing an effective tool for comparison and optimization. The ultrasound pretreatments appear to greatly improve the enzymatic hydrolysis of EWPs under different conditions when compare to other methods. The apparent reaction rate constants for proteolysis (k2 ) are 0.009, 0.011, 0.053, and 0.045 min-1 for untreated EWPs, and those pretreated with heat, probe-type sonicator, and ultrasound cleaning bath technologies, respectively. The ultrasound pretreatment also decreases hydrolysis activation (Ea ) and enzyme deactivation (Ed ) energy, enthalpy (ΔH), and entropy (ΔS) of activation and for the probe-type sonication this decrease is 61.7%, 61.6%, 63.6%, and 32.2%, respectively, but ultrasound has little change in Gibbs free energy value in the temperature range of 318 to 338 K. The content of sulfhydryl groups and ζ potential show a significant increase (P < 0.05) for both applied ultrasound pretreatments and the reduction of particle size distribution are achieved, providing some evidence that the ultrasound causes EWP structural changes affecting the proteolysis rate.