Whey Protein Isolate Hydrolysates Research Articles

Effect of enzymatic hydrolysis and ultrasound pretreatment on allergenicity, functional properties and bioactivity of whey protein isolates

Whey protein isolate (WPI) is an important food ingredient and a major allergen in food. Enzymatic hydrolysis is an important method for WPI modification, but there is limited information on the effect of ultrasound pretreatment on WPI hydrolysates (WPH). The aim of this study was to analyze the effect of enzymatic hydrolysis and ultrasound pretreatment on the allergenicity, functional properties and bioactivity of WPH by multispectroscopy and peptidomics combined with bioinformatics. The results showed that ultrasound pretreatment contributed to the production of WPH with low allergenicity (IgE binding capacity is 55.59%) and good functional properties (emulsifying activity index is 27.02%, foaming characteristic is 50.00%) and bioactivity (total antioxidant capacity is 65.94%). This was attributed to the synergistic hydrolysis of WPI by endopeptidases and exopeptidases, and ultrasound pretreatment that altered the accessibility of WPI to proteases and the peptide profiles and main proteins of WPH, affecting the changes in their spatial and primary structure. This research will advance the application of WPI in hypoallergenic foods.

Effects of ultrasound pretreatment on the structure, IgE binding capacity, functional properties and bioactivity of whey protein hydrolysates via multispectroscopy and peptidomics revealed

Whey protein is an important food ingredient, but it is also considered a major food allergen. The aim of this study was to investigate the effect of ultrasound pretreatment on the structure, IgE binding capacity, functional properties and biological activity of whey protein isolate (WPI) hydrolysates (WPH), including WPI hydrolyzed by a combination of enzymes from Bromelain and ProteAXH (BA-WPI) and WPI hydrolyzed by a combination of enzymes from Papain W-40 and ProteAXH (PA-WPI). The IgE binding capacity of BA-WPI and PA-WPI was reduced to 40.28% and 30.17%, respectively, due to disruption/exposure/shielding of conformational and linear epitopes. The IgE binding capacity of sonicated WPI was increased, but ultrasound pretreatment further reduced the IgE binding capacity of the hydrolysates to 32.89% and 28.04%. This is due to the fact that ultrasound pretreatment leads to conformational changes including increased α-helix and β-sheet structure, exposure of aromatic amino acids, surface hydrophobicity, and increased sulfhydryl content, which increases the accessibility of allergenic epitopes to WPI by the enzyme. Multispectral and LC-MS/MS results further indicated that ultrasound pretreatment altered the conformational and primary structural changes of the hydrolysates. The thermograms showed that ultrasound pretreatment mainly altered the epitope spectra of β-lactoglobulin hydrolysates, while it had less effect on the epitope spectra of α-lactalbumin hydrolysates. Additionally, ultrasound pretreatment significantly improved the foaming properties, antioxidant activity, and α-glucosidase inhibition of the hydrolysates without impairing the solubility and emulsification properties of the hydrolysates. Therefore, ultrasound pretreatment is a feasible method to reduce the allergenicity of WPH and to improve their functional properties and bioactivity. Notably, ultrasonic pretreatment improved the effectiveness and efficiency of WPI hydrolysis, which is a feasible method to produce high-quality protein feedstock in a green, efficient, and economical way.

Stability of whey protein bioactive peptide‐stabilised nanoemulsions: effect of pH, ions, heating and freeze–thawing

SummaryWhey protein hydrolysates are important food emulsifiers and bioactive ingredients. This study investigated the stability of whey protein isolate (WPI) bioactive peptide fraction nanoemulsions under representative food processing and storage conditions: pH (3–9), ion concentration (Na+, 0–200 mm and Ca2+, 0–15 mm), thermal treatment (30–90 °C) and freeze–thawing. Bioactive peptide fractions, UC–10 and UP–10, were obtained by ultrafiltration of chymotrypsin or pepsin WPI hydrolysates, respectively. The nanoemulsions produced with these fractions had droplet diameters of 177 ± 3.5 nm (UC–10) and 154 ± 1.6 nm (UP–10). Nanoemulsions destabilised at pH 3–5, around the isoelectric point of WPI proteins but were stable at higher pH values, 6–9. Nanoemulsion instability escalated above critical Na+ (25 mm) and Ca2+ (2.5 mm) concentrations, but Ca2+ accelerated droplet aggregation more strongly than Na+. Furthermore, nanoemulsions were moderately stable to heating and freeze–thawing. Overall, both WPI bioactive peptide‐stabilised nanoemulsions showed consistent stability to the processing conditions. This study expands on designing, producing and utilising nanoemulsions based on WPI bioactive peptides.

PSVIII-B-10 Feeding Senior Labrador Retrievers Hydrolyzed Whey Protein Isolate to Prevent Sarcopenia

Abstract Conditions which affect humans are often closely followed by our companion animals, especially in aging populations. Canines often present with muscle wasting diseases such as sarcopenia and cachexia as a result of aging or chronic disease. In this 26wk study, our goal was to evaluate the effects of feeding 1.5x AAFCO CP from whey protein isolate and a pea isolate compared to feeding senior dogs a standard AAFCO CP diet (45g CP/1000 kcal DM) on body composition in exercised senior Labrador Retrievers. Thirty-six (36) Labrador Retrievers (18m/18f; 8-12yrs) were sorted into three equal groups and fed basal AAFCO diet plus hydrolyzed whey isolate, pea isolate, or added fat and sugar (control). All dogs ran 1.6km run twice weekly throughout the trial. Body composition was determined by dual-energy x-ray absorptiometry (DXA) at Weeks 0, 12, 20, and 26. All dogs were weighed weekly and feed intake measured daily. Body weights and feed intake were similar between groups. Whey group gained 1.69% fat and 0.6kg fat mass compared to control group gaining 7.97% fat and 3.05kg fat mass (p=0.045; p=0.005). From Wk0 to Wk26, whey group lost only 0.38kg lean/fat ratio compared to control group’s -1.75kg loss (p=0.021). From Wk12 to Wk26 and Wk18 to Wk26, whey group maintained a higher lean:fat ratio compared to pea and control group (p=0.101; p=0.049). Based on these results, senior Labrador Retrievers fed 1.5x AAFCO CP from basal plus hydrolyzed whey protein isolate during an exercise regimen maintained higher lean:fat ratio compared to seniors fed basal plus pea protein and seniors fed only basal. Senior dogs fed hydrolyzed whey protein isolate also had increased fat loss vs seniors consuming only the basal diet.

Limited hydrolysis as a strategy to improve the non-covalent interaction of epigallocatechin-3-gallate (EGCG) with whey protein isolate near the isoelectric point

This study was aimed to investigate the interaction mechanisms and structural changes of whey protein isolate (WPI) and whey protein isolate hydrolysates (WPIHs) with epigallocatechin-3-gallate (EGCG) near the isoelectric point through multiple spectroscopic techniques and field emission scanning electron microscopy. Fluorescence spectra results indicated that limited hydrolysis endowed WPIHs with higher affinity for the EGCG but the increased degree of hydrolysis led to an opposite result. Thermodynamic analysis revealed that EGCG bound WPI primarily through hydrogen bonds and van der waals forces, while the hydrophobic force was the main driving force in the interaction of EGCG with WPIHs. Synchronous fluorescence and three-dimensional spectra confirmed that EGCG induced conformational alterations of WPI and WPIHs, which was further supported by Ultraviolet–Visible spectra. Raman spectra indicated that binding to EGCG resulted in changes in the microenvironment of tryptophan residues, CH bending vibration and the secondary structure arrangements of WPI and WPIHs. Furthermore, compared with a sheet-like structure of WPI-EGCG complexes, the morphology of WPIHs with limited hydrolysis presented an uneven blocky structure after complexing with EGCG. Our findings might be helpful to better understand the interactions of milk protein hydrolysates-EGCG and suggest the potential application of the formed complexes as bioactive ingredients in food industry.

Novel xanthine oxidase inhibitory peptides derived from whey protein: identification, in vitro inhibition mechanism and in vivo activity validation

As the development of hyperuricemia (HUA) and gout continues to accelerate worldwide, there is increasing interest in the use of xanthine oxidase (XO) inhibitors as therapeutic agents for the management of HUA and gout. In the present study, XO inhibitory peptides were identified from whey protein isolate (WPI) hydrolysates, and the underlying inhibitory mechanism and in vivo activities was investigated. WPI hydrolysates were isolated and purified, and two peptides (ALPM and LWM) with lower binding energy were screened by molecular docking. The result showed that these two peptides interacted with residues around the active site of XO through hydrogen bond and hydrophobic interaction. The IC50 values of ALPM and LWM were 7.23 ± 0.22 and 5.01 ± 0.31 mM, respectively. According to the Lineweaver-Burk curve, the inhibition types of ALPM and LWM were non-competitive inhibition. Circular dichroism (CD) spectra indicated ALPM and LWM could change the secondary structure of XO. Molecular dynamics simulations revealed that XO-peptide complexes were more stable and compact than XO. Moreover, animal studies have shown that ALPM and LWM have anti-hyperuricemia effects in vivo. This study suggested that ALPM and LWM can be considered as natural XO inhibitors for the treatment of HUA.

The Influence of Whey Protein Heating Parameters on Their Susceptibility to Digestive Enzymes and the Antidiabetic Activity of Hydrolysates.

The inhibition of dipeptidyl peptidase-IV (DPP-IV) and the release of glucagon-like peptide-1 (GLP-1) could normalize blood glucose levels in diabetic patients. This study evaluated the susceptibility of whey proteins to enzyme hydrolysis and the antidiabetic properties of protein hydrolysates from β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) solutions compared with whey protein isolate (WPI) solution treated at different heating temperatures (65, 75, and 85 °C). α-LA hydrolysate provided the lowest degree of hydrolysis (DH). Those heating temperatures did not significantly affect the DH of all protein hydrolysates. α-LA hydrolysate significantly increased GLP-1 levels and DPP-IV inhibitory activity more than β-LG hydrolysate. WPI hydrolysate inhibited DPP-IV activity less than an α-LA hydrolysate, but they were no significant differences for GLP-1 release activity. Heat treatment could affect the antidiabetic properties of all protein hydrolysates. Heating at 75 °C resulted in greater inhibition of the activity of DPP-IV than at 65 and 85 °C. The highest increase in GLP-1 release was also observed by heating at 75 °C. The recently obtained information is useful for the utilization of α-LA, heated at 75 °C for 30 min, in the preparation of antidiabetic food supplements.

PH-responsive delivery of rebaudioside a sweetener via mucoadhesive whey protein isolate core-shell nanocapsules

Rebaudioside (Reb A) is a natural high-intensity sweetener available for commercial use. Its application has been hampered, however, by its unnatural sweetness profile and a bitter aftertaste at higher concentration thresholds. The goal of this research was to mitigate these shortcomings by increasing the sweetening power, thus lowering the amount needed to achieve the desired sweetness, specifically in acidic liquid media. We achieved the stated goal via the fabrication of a mucoadhesive delivery vehicle that can be retained on the tongue mucosa and allowed for a more targeted and prolonged release of the sweetener near the taste bud. We have chosen hydrolyzed whey protein isolate and pectin as shell materials and crosslinked them via an ultrasonic process. In the pursuit of our goal, we found that WPI-Pectin was the most suitable of all the protein-polysaccharide combinations tested, yielding nanocapsules with average particle sizes of 393 nm and polydispersity index (PDI) of 0.43. These capsules could be resuspended in water with ζ-potential of −20.4 mV. The WPI-pectin system was also shown to be twice as effective at depositing Reb A to the surface of the tongue as regular sweetener solution. Finally, we have demonstrated that WPI and pectin capsules can achieve a desirable pH-responsive profile for application in beverages with minimal release in acidic conditions (7.1 %) and higher release in neutral conditions (19.5 %).

Characterization and application of a crude bacterial protease to produce antioxidant hydrolysates from whey protein

Bacillus sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca2+, Mg2+, and Mn2+ (5 mM) enhanced activity (12–26%), whereas Co2+, Cu2+, Fe2+, and Zn2+ inhibited it (50–94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21–27%), β-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca2+, protease was optimally active at 65 °C and completely stable after 20 min at 40–55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca2+) for 0–180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50–52% (60–180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; ∼34%, 60–180 min) and Fe2+-chelating ability (56%, 0 min; ∼74%, 45–180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.

Effects of sequential enzymatic hydrolysis and transglutaminase crosslinking on functional, rheological, and structural properties of whey protein isolate

This work investigated the effect of transglutaminase (TGase) crosslinking condition on the functional, rheological, and structural properties of hydrolyzed whey protein isolate (WPI) at three hydrolysis degrees (DHs) (7.61%, 9.22%, 18.55%). Between three DHs crosslinking, it was found that low DH coexistent with higher fluorescence intensity, sulfhydryl content and emulsifying activity, and moderate DH had higher thermal stability and viscosity. In crosslinking process, increasing the temperature had significant effect on solubility, emulsification, and viscosity of TGase-induced hydrolysate with moderate DH (p < 0.05), while the sulfhydryl contents decreased to various degrees. And the best reaction temperatures of hydrolysate with moderate DH were identified for thermal stability (50 °C/30 °C), solubility (30 °C), emulsifying activity/emulsifying stability (EAI/ESI) (50 °C/40 °C) and viscosity (40 °C), respectively. With increasing TGase concentrations, hydrolysate with moderate DH had best thermal stability at 30 U/g, while the highest EAI and the lowest ESI was at 40 U/g. The process conditions adopted enzymatic hydrolysis coupled with TGase-induced crosslinking could significantly influence the protein conformation, consequently determining their specific functional properties. The understanding of hydrolysis and crosslinking effects on properties could assist in selecting the appropriate method to optimize the utilization of WPI in the food industry.

Production of biologically active peptides by hydrolysis of whey protein isolates using hydrodynamic cavitation

Whey protein isolate (WPI) hydrolysates have higher solubility in aqueous phase and enhanced biological properties. Hydrolysis of WPI was optimized using operating pressure (ΔP, bar), number of passes (N), and WPI concentration (C, %) as deciding parameters in hydrodynamic cavitation treatment. The optimum conditions for generation of WPI hydrolysate with full factorial design were 8 bar, 28 passes, and 4.5% WPI concentration yielding 32.69 ± 1.22 mg/mL soluble proteins. WPI hydrolysate showed alterations in binding capacity over WPI. SDS-PAGE and particle size analysis confirmed the hydrolysis of WPI. Spectroscopic, thermal and crystallinity analyses showed typical properties of proteins with slight variations after hydrodynamic cavitation treatment. ABTS, DPPH and FRAP assays of WPI hydrolysate showed 7–66, 9–149, and 0.038–0.272 µmol/mL GAE at 1–10, 0.25–4, and 3–30 mg/mL concentration, respectively. Further, a considerable enhancement in fresh weight, chlorophyll, carotenoids, reducing sugars, total soluble sugars, soluble proteins content and total phenolics content was noticed during in vitro growth of sugarcane in WPI hydrolysate supplemented medium at 50–200 mg/L concentration over the control. The process cost (INR/kg) to hydrolyze WPI was also calculated.

Functional and Technological Potential of Whey Protein Isolate in Production of Milk Beverages Fermented by New Strains of Lactobacillus helveticus

With their desired functional and technological properties, whey protein preparations are used in the food industry. In turn, lactic acid fermentation may contribute to release of a wide range of biologically active peptides (BAPs) (known also as bioactive peptides or biopeptides) from whey and milk proteins, which are perceived as potential therapeutic tools and important constituents of personalized food suitable for prevention of many civilization and diet-related diseases. Therefore, the objective of this study was to determine the suitability of new Lb. helveticus strains for production of fermented milk beverages (drinking type) supplemented with whey protein isolate (WPI). Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was employed to assess if WPI (water solution) might be a suitable precursor for BAPs produced by selected strains of Lb. helveticus. In order to identify the bioactivities of the peptides generated in WPI hydrolysates, the procedures indicated in databases were used. The fermented products differed from each other in some texture parameters, the content of protein, total nitrogen, and non-protein nitrogen, and the proteolysis index, which was dependent on the strain. Strain B734 was found to exhibit technological potential for development of new health-oriented fermented milk beverages with characteristics of functional food. Additionally, it proved to be able to release a wide range of BAPs from WPI with antioxidative, antibacterial, and immuno- and cyto-modulatory effects, as well as ACE (angiotensin-converting enzyme) inhibitory and antihypertensive activities.

Effect of pH-dependent fibrillar structure on enzymatic hydrolysis and bioactivity of nanofibrillated whey protein

The functional properties of fibrillar protein make them an excellent novel food ingredient. However, for industrial applications, it is necessary to have an insight into their digestibility and bioactivity. Therefore, the objectives of this work were to study the enzymatic hydrolysis and bioactivity of nanofibrillated whey protein isolate (WPI), prepared at 90 °C under acidic condition, followed by pH adjustment to 2, 4, and 6. Fourier transform infrared spectroscopy (FTIR) showed that the secondary structure of protein changed and the intermolecular β-sheets structure was formed over fibrillization. The results showed that by increasing the rate of fibril formation at lower pH level monitored by Thioflavin T (ThT) fluorescence spectrophotometry, the degree of pepsin and trypsin hydrolysis in simulated gastrointestinal environments increased significantly. As the extent of fibrillar structure decreased at higher pH, Diphenyl-picryl hydrazinyl (DPPH) radical scavenging activity and Ferric reducing ⁄ antioxidant power (FRAP) of nanofibrillated WPI hydrolysates were reduced. Likewise, in all samples at different hydrolysis times, the antioxidant activities of pepsin-derived hydrolysates were higher than those of tryptic hydrolyzed samples, which probably was resulted from the increased hydrolyzed peptides with higher antioxidant activity. Overall, these findings may reinforce the applications of nanofibrillated proteins as functional food ingredients.

Proteins and Amino Acids Treated with Atmospheric Plasma Show Significantly Increased Bioavailability in Humans.

Background:Muscle mass is an important determinant of metabolic health and physical function. It has previously been demonstrated that the postprandial rise in circulating essential amino acids acts as the main stimulus for muscle protein synthesis (MPS). The current study investigated the postprandial plasma essential amino acid (EAA) and branched-chain amino acid (BCAA) responses of (1) Hydrolyzed whey protein isolate (HWPI) compared to plasma treated non-hydrolyzed whey protein isolate (PT-NHWPI), (2) standard branch-chain amino acids (S-BCAA) compared to plasma treated branch-chained amino acids (PT-BCAA), (3) standard pea protein (S-PP), compared to plasma treated pea protein (PT-PP), and (4) HWPI compared to PT-PP.Methods:Ten subjects (24.6 ± 5.3 years; 178.8 ± 8.1 cm; 78.6 ± 10.1 kg) participated in a double-blind, randomized, crossover trial comparing four separate protein conditions (HWPI, PT-NHWPI, S-PP, PT-PP). A separate cohort of ten subjects (26.4 ± 7.4 years; 178.8 ± 5.9 cm; 85 ± 12.3 kg) participated in a double-blind randomized, crossover trial comparing two branch-chain amino acid conditions: S-BCAA and PT-BCAA. All conditions were administered following a 7-day washout. Plasma EAA and BCAA concentrations were assessed from blood donated by subjects at pre-consumption, 30-, 60-, 90-, 120-, and 180 minutes post-consumption.Results:Blood plasma levels of total EAA and BCAA concentration were significantly greater in all treated conditions at 30-, 60-, 90-, and 120 minutes post consumption (P < .05). There were no differences between PT-PP and HWPI.Discussion:All proteins significantly elevated EAAs, and BCAAs from basal levels. However, we conclude that the consumption of the treated proteins significantly raises blood levels of EAAs, and BCAAs to a greater extent across multiple dairy, vegan, and isolated BCAA conditions. Moreover, atmospheric plasma treatment of a vegan protein source makes its amino acid response similar to whey. Thus, protein supplementation with that has undergone Ingredient Optimized® atmospheric plasma treatment technology may be highly beneficial for improving the blood plasma amino acid response.

Influence of whey protein hydrolysis in combination with dextran glycation on immunoglobulin E binding capacity with blood sera obtained from patients with a cow milk protein allergy

Food protein allergies are a major global concern. Hydrolysis of food proteins reduces their allergenicity, but another novel approach is the covalent attachment of polysaccharides to proteins via the Maillard reaction (i.e., glycation), which blocks some IgE binding epitopes on the protein allergen. We wanted to examine whether enzymatic hydrolysis, combined with glycation, could further reduce IgE binding for people with a cow milk protein allergy. Whey protein isolate (WPI) was hydrolyzed by immobilized trypsin and chymotrypsin to degree of hydrolysis (DH) values of 17 to 27%. Immobilized enzymes were used to avoid heat-treating the hydrolysate (to inactivate the enzymes, because heating could also affect the IgE binding ability of the protein). The resultant whey protein isolate hydrolysates (WPIH) were then glycated with 10-kDa dextran (DX) in aqueous solutions held at 62°C for 24 h. We analyzed the molar mass (MW) of WPIH samples and their corresponding glycates (WPIH-DX) using size-exclusion chromatography with multi-angle laser light scattering. We obtained blood sera from 8 patients who had been diagnosed with a cow milk protein allergy, and we used a composite serum for IgE binding analysis. The average MW values of samples WPIH-1 to WPIH-3 decreased from 11.15, 9.46, and 7.57 kDa with increasing DH values of 18.7, 22.5, and 27.1%. Glycation significantly reduced the high bitterness of the WPIH samples, as assessed by a trained sensory panel. The WPIH-DX glycates had significantly reduced WPI-specific IgE binding capacity compared to WPI or unglycated WPIH; we found an almost 99% reduction in IgE binding for the WPIH-DX glycate made from WPIH with a DH value of 27.1%. Hydrolysis of WPI followed by glycation with DX via the Maillard reaction significantly decreased the allergenicity of whey proteins.

Covalent conjugation of whey protein isolate hydrolysates and galactose through Maillard reaction to improve the functional properties and antioxidant activity

Galactose was grafted onto whey protein isolate (WPI) and whey protein hydrolysates (WPHs) via the Maillard reaction (95 °C, 0–4 h). The pH, browning intensity, structure, functional properties and antioxidant activity of conjugates were evaluated. The pH, browning and degree of conjugation were at maximum after heating for 4 h. The Maillard reaction changed the WPI and WPHs to a looser secondary structure. Conjugates of WPI and WPHs with galactose had higher water holding capacity, foaming capacity and stability, and antioxidant activity. Compared with the WPI-Galactose conjugates, WPH-Galactose conjugates showed better functional properties and higher antioxidant activity, and its highest reducing power was 0.923; ABTS+ radical scavenging ability was 69.81%; hydroxyl radical scavenging ability was 89.05%. These findings indicate that the covalent conjugation of WPH-Galactose conjugates can be used as a potential ingredient and a novel wall material to encapsulate bioactive compounds.

Estimation of the antioxidant properties of milk protein preparations hydrolyzed by Lactobacillus helveticus T80, T105 and B734

The investigation determined the influence of Lactobacillus helveticus strains (T80, T105, B734 and DSMZ 20075) and Lactobacillus acidophilus La-05 (probiotic strain) on the antioxidant activity of whey and milk protein preparations. In order to indicate possible mechanism of antioxidant activity of the obtained hydrolysates, the reducing power (RP) assay, ability to neutralize free radicals and Fe2+ chelating activity analysis were employed. The results suggest that antioxidant activity of tested hydrolysates depends on the type of protein preparation (substrate) and the applied bacterial strain. The strongest antioxidant activity was exhibited by whey protein isolate hydrolysate obtained by using L. helveticus T80. A majority of fermented products obtained with the use of the probiotic strain were characterized by higher antioxidant properties than those obtained by application of L. helveticus. The solution of caseinoglicomacropeptide fermented by strain T105 exhibited the highest RP values, while α-lactalbumin hydrolysed by probiotic strain (La-5) was characterized by the strongest Fe2+ chelating activity. The analysed protein preparations and their hydrolysates obtained with using tested bacteria might potentially be applied in food products in order to inhibit oxidation processes.

Separation and Enrichment of Antioxidant Peptides from Whey Protein Isolate Hydrolysate by Aqueous Two-Phase Extraction and Aqueous Two-Phase Flotation.

At present, peptides are separated by molecular exclusion chromatography and liquid chromatography. A separation method is needed in any case, which can be scaled up for industrial scale. In this study, aqueous two-phase extraction (ATPE) and aqueous two-phase flotation (ATPF) were applied to separate and enrich antioxidant peptides from trypsin hydrolysates of whey protein isolates (WPI). The best experimental conditions were investigated, and the results were evaluated using the 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radical scavenging activity of the peptides-per-unit concentration and the recovery rate (Y) of peptides in the top phase of both ATPE and ATPF. Under optimal conditions, the Y and ABTS free radical scavenging activity per unit concentration in top phase of ATPE could reach 38.75% and 12.94%, respectively, and in ATPF could reach 11.71% and 29.18%, respectively. The purified peptides were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and reversed-phase high-performance liquid chromatography (RP-HPLC). PeptideCutter and PeptideMass were applied to analyze and calculate the peptide sequencing. KILDKVGINYWLAHK, VGINYWLAHKALCSEK, and TPEVDDEALEKFDKALK sequences having antioxidant activity were detected in the top phase of ATPE, and VGINYWLAHKALCSEK, KILLDKVGINYWLAHK, ILLDKVGINYWLAHK, IIAEKTKIPAVFK, KIIAEKTKIPAVFK, and VYVEELKPTPEGDLEILLQK sequences having antioxidant activity were detected in the top phase of ATPF. In conclusion, antioxidant peptides were successfully separated from the WPI hydrolysate by ATPE and ATPF; compared with ATPE, ATPF has superior specificity in separating antioxidant peptides.

Separation of Antioxidant Peptides from Pepsin Hydrolysate of Whey Protein Isolate by ATPS of EOPO Co-polymer (UCON)/Phosphate

An aqueous two-phase system (ATPS) consisting of poly(ethylene glycol-ran-propylene glycol) monobutyl ether (UCON)/phosphate was developed for the separation of the antioxidant peptides from pepsin hydrolysate of Whey Protein Isolate (WPI). The efficiency of the separation was evaluated based on the DPPH radical scavenging activity, ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP) of the separated peptides. The effects of some parameters on the partition of antioxidant peptides were investigated. An efficient separation of antioxidant peptides was achieved using ATPS with pH of 4.0, 4 mL of UCON solution (40%, w/w), 4 mL of KH2PO4 solution (15.5%, w/w), 2 mL of WPI hydrolysate and 0.40 g/10 mL of NaCl. Reversed-phase high-performance liquid chromatography (RP-HPLC), amino acid analyzer and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to characterize the purified peptides separated by the ATPS. The peptides in top phase were less polar than those in bottom phase. More antioxidative and hydrophobic amino acids were extracted to the top phase of ATPS, and the peptides with the amino acid sequences with antioxidant activities moved to the top phase as well. In conclusion, antioxidant peptides were successfully separated from the WPI hydrolysate by UCON/phosphate ATPS.

Whey protein isolate hydrolysates obtained with free and immobilized Alcalase: Characterization and detection of residual allergens

Protein antigenicity can be reduced by enzymatic hydrolysis, which can be performed either by free or immobilized enzyme. The immobilized enzyme is removed from the reaction medium and reused, while the free enzyme must be inactivated to stop the reaction, generally by heating. Here we have shown that hydrolysates produced with free or immobilized Alcalase on glyoxyl-agarose bead presented different physicochemical properties (hydrophilicity profile, molecular mass distribution, surface hydrophobicity) and different levels of residual milk allergens (α-lactalbumin and β-lactoglobulin). Although, under the studied conditions, the hydrolysis with immobilized enzyme did not reduce the residual allergen levels as efficiently as the free enzyme, the results suggest potential applications of immobilized Alcalase for production of hypoallergenic hydrolysates.