Egg White Protein Hydrolysis Research Articles

Efficiency of an enzyme preparation obtained from a new mutant Bacillus subtilis-96 strain in the hydrolysis of whey and egg white proteins

Whey and hen egg white proteins are characterized by high nutritional value, but possess antigenic properties, which limit their use in the production of dietary products. Enzymatic hydrolysis decreases significantly the allergenicity of proteins. The efficiency of hydrolysis depends on the specificity of the proteases used. The aim of this work was to determine the effectiveness of EP-96 enzyme preparation obtained from Bacillus subtilis-96 culture liquid in the hydrolysis of whey and egg white proteins in comparison with commercial bacterial proteases preparations - Alcalase, Neutrase, and Protosubtilin. Material and methods. Whey and egg white protein concentrates were used as substrates. Commercial enzyme preparations Alcalase, Neutrase, and Protosubtilin, and an experimental sample of EP-96 preparation obtained from Bacillus subtilis-96 culture liquid were used for hydrolysis. Hydrolysis was carried out at a substrate concentration of 100 g/L for 3 h at 55 °C or for 24 h at 50 °C. After hydrolysis, the reaction mixture was incubated at 90 °C for 15 min to inactivate the enzymes. The content of peptides with a molecular weight of less than 10 kDa was determined in the obtained hydrolysates. The hydrolysis of the main allergenic proteins was assessed by the disappearance of the corresponding protein bands on the hydrolysate supernatants electrophoregrams. Results and discussion. All the studied preparations showed high efficiency in the hydrolysis of whey proteins and provided the yield of low molecular weight peptides at the level of 18.8-22.8% after 3 h of hydrolysis and 39.4-41.6% after 24 h. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a residual amount of protein with a molecular weight of about 14 kDa, corresponding to α-lactoalbumin, after 3 h of hydrolysis when using Neutrase. The preparations containing serine protease, including EP-96, provided more intensive hydrolysis of whey proteins. In the hydrolysis of egg white protein, Neutrase showed the greatest efficiency. The efficiency of EP-96 was comparable to Neutrase both in the yield of low molecular weight peptides and in the intensity of cleavage of the main allergenic proteins. The effectiveness of preparations with predominant content of serine proteases - Alcalase and Protosubtilin was significantly lower. Conclusion. The optimal ratio of neutral and serine proteases in the EP-96, obtained on the basis of the B. subtilis-96 strain, provided the high efficiency and its versatility in the hydrolysis of the main allergenic proteins of whey and egg white. The parameters of the hydrolysis technology using EP-96 are recommended, which provide intensive conversion of the main immunogenic proteins of whey and egg white to soluble and low molecular weight fractions (duration 3 h at a temperature of 55 °C and the proteolytic activity of the preparation is not less than 2 units per g of substrate) and an increase of subsequent ultrafiltration efficiency in the production of protein hydrolysates for foods for special dietary uses.

Production of egg white protein hydrolysates with improved antioxidant capacity in a continuous enzymatic membrane reactor: optimization of operating parameters by statistical design.

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 (10kDa). 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 1.9cm3 min-1. 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.

Design and characterization of alcalase-chitosan conjugates as potential biocatalysts.

In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated chitosan microbeads were examined. The biocatalysts highest activity was obtained by covalent immobilization of alcalase onto a solid support. The alcalase covalent immobilization onto different types of chitosan beads obtained by inverse emulsion technique and electrostatic extrusion was studied. Parameters examined under different conditions were beads diameter, enzyme loading, enzyme capacity yield, and biocatalyst activity. The highest activity and enzyme loading of 23.6IU/mg protein and 340.2mg/g, respectively, were achieved by the enzyme immobilized onto chitosan microbeads obtained by the electrostatic extrusion technique. FT-IR analysis was used to confirm formation of alcalase-chitosan conjugates. The activity of optimally produced alcalase-chitosan microbeads was then verified in the industrially feasible reaction systems of egg white and soy protein hydrolysis. The high degree of hydrolysis of 29.85±0.967% after 180min and five successive reuses obtained under real conditions (50°C, pH 8) verified the covalently bound alcalase to chitosan beads a promising candidate for use in industrial egg white protein hydrolysis process.

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.

Biochemical characterization of a novel antioxidant and angiotensin I-converting enzyme inhibitory peptide from Struthio camelus egg white protein hydrolysis

A peptide from ostrich (Struthio camelus) egg white protein hydrolysate (OEWPH) was purified, characterized, and its antioxidant and enzyme inhibitory properties were evaluated. The OEWPH was prepared using pepsin and pancreatin, and then fractionated using reversed-phase high performance liquid chromatography. The antioxidant activity of the WG-9 peptide was investigated based on its scavenging capacity for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), superoxide (O2•−), hydroxyl (OH•−), and lipid peroxidation inhibition. The angiotensin-converting enzyme (ACE) inhibitory activity and kinetic parameters of the peptide were determined using N-[3-(2-Furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) as a substrate. Tandem mass spectrometry analysis of the purified peptide revealed a sequence of WESLSRLLG (MW: 1060 Da; WG-9). This peptide inhibited linoleic acid oxidation and acted as a DPPH (IC50 = 15 ± 0.4 μg/mL), ABTS (IC50 = 130 ± 4.5 μg/mL), superoxide (IC50 = 160 ± 6.4 μg/mL), and hydroxyl (IC50 = 150 ± 6.7 μg/mL) radical scavenger. The ACE-inhibitory activity and kinetic parameters of the WG-9 peptide were determined, showing an ACE inhibitory activity with IC50 of 46.7 ± 1.4 μg/mL. The parameters of peptide/ACE interactions were investigated by molecule docking. Furthermore, viability assays showed that the identified peptide had no cytotoxicity against an HFLF-PI-5 cell line. In conclusion, the WG-9 peptide showed potent antioxidant and ACE-inhibitory activity.

Impact of ultrasound on egg white proteins as a pretreatment for functional hydrolysates production

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 ( 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 IC50 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.

A response surface approach on optimization of hydrolysis parameters for the production of egg white protein hydrolysates with antioxidant activities

Abstract Bioactive peptides can be defined as specific protein fractions with amino acid sequence that promotes a positive impact on several biologic activities. The hydrolysis of egg white protein might result in releasing or enhancing their biological activity. The aim of this study was to evaluate the effects of the enzymatic hydrolysis of egg white proteins on their antioxidant properties. The hydrolysates were prepared with protease from Aspergillus oryzae LBA 01 (AO) produced under solid-state fermentation and two commercial proteases: protease from A. oryzae (CAO), and Bacillus licheniformis (CBL). The enzymatic hydrolysis with microbial proteases increased the antioxidant properties of egg white proteins. The egg white protein hydrolysates prepared with commercial protease CAO, presented higher antioxidant activity than the hydrolysates obtained with the other proteases. According CCRD analysis, the hydrolysates prepared with 3.0% of egg white proteins and 20.0 U of protease per mL of reaction mixture, presented high radical-scavenging effect, with an ORAC value of 1193.12±84.62 and DPPH value of 19.05±0.81 Trolox EQ µmol g−1. In ORAC assay, the maximum antioxidant activities were observed in the hydrolysates presenting DH 50.0% obtained after 120 min of hydrolysis. In contrast, for DPPH assay the highest values of antioxidant activity were detected in the hydrolysates obtained in the first 30 min, where the degree of hydrolysis was greater than 60.0%.

Effect of high pressure treatment on egg white protein digestibility and peptide products

Abstract The effect of high pressure treatment on whole egg white was examined using in vitro pepsin digestion and proteomic methods. Pepsin incubations conducted with an enzyme to protein ratio of 3:1 following high pressure treatment (400–800 MPa and 9 °C) resulted in increased hydrolysis of egg white proteins. Pressure treatment of egg white at 800 MPa resulted in greater susceptibility to pepsin hydrolysis than did thermal treatment at 95 °C. The effect of 800 MPa pressure treatment on egg white proteins was additionally examined by incubation with pepsin at an enzyme to protein ratio of 1:20 followed by 2-D electrophoresis. Results of these experiments showed extensive hydrolysis of most egg white proteins. Subsequent LC-MS/MS investigation of the low M r fraction ( Industrial Relevance Short time high pressure treatment of whole egg white at relatively low temperature (9 °C) resulted in increased pepsin digestibility equivalent to or better than heat treatment at 95 °C. Examination of the peptides resulting from high pressure treatment and pepsin digestion revealed sequences with known biological activities. Thus high pressure treatment represents a promising technology for enhancing egg white's healthiness and contributing to its role as a functional food.

Immunoreactivity of hen egg allergens: Influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components.

Effects of sonication and high-pressure carbon dioxide processing on enzymatic hydrolysis of egg white proteins

The objectives of this study were to examine the effect of sonication and high-pressure carbon dioxide processing on proteolytic hydrolysis of egg white proteins and antioxidant activity of the obtained hydrolysates. It appeared that the ultrasound pretreatment resulted in an increase in the degree of hydrolysis of the enzymatic reaction while the high-pressure carbon dioxide processing showed an inhibition effect on the enzymatic hydrolysis of egg white proteins to some extent. The antioxidant activity of the obtained hydrolysates was improved by ultrasound pretreatment of egg white proteins at the pH 8.3. Thus, the combination of ultrasound pretreatment at the pH 8.3 and subsequent enzymatic hydrolysis with alcalase at 50?C and pH 8.0 could offer a new approach to the improvement of the functional properties of egg white proteins and their biological activity.