Hydrolysis Of Whey Protein Isolate Research Articles

Milk Protein Hydrolysis by Actinidin-Kinetic and Thermodynamic Characterisation and Comparison to Bromelain and Papain.

Plant proteases, including actinidin, papain and bromelain, have been widely used in the food industry but with limited application in dairy systems. This research aimed to establish and compare operational parameters (kinetics, temperature, enzyme type, time and thermodynamics) relevant to the applications of these enzymes in the hydrolysis of whey protein isolates (WPI), whey protein concentrates (WPC) or milk protein concentrates (MPC). The degree of hydrolysis (DH) increased with the rise in temperature, and the maximum DH was achieved at 60 °C for all three dairy systems. The addition of papain resulted in a greater %DH of whey proteins in comparison to bromelain. The cleavage of proteins was clearly time-dependent (p < 0.05), while the pH did not change significantly (p > 0.05) during this time. PAGE analysis revealed that all three enzymes mainly acted on α-lactalbumin and αs-casein in WPI and MPC, respectively. Kinetic parameters from the Lineweaver-Burk plot at 60 °C using WPC and MPC as a substrate varied widely, establishing that WPC hydrolysis was characterised by a lower KM, higher kcat, kcat/KM and Vmax compared to MPC in the case of all three enzymes. The difference in kcat/KM values amongst all enzymes (actinidin > papain > bromelain) indicated the difference in the strength of substrate binding sites. The thermodynamic parameters of these enzymes with MPC and WPC were also determined at a temperature range of 15-60 °C, and the results indicate the potential application of papain and actinidin in the dairy industry.

Hydrogels assembled from hybrid of whey protein amyloid fibrils and gliadin nanoparticles for curcumin loading: Microstructure, tunable viscoelasticity, and stability.

Food grade hydrogel has become an ideal delivery system for bioactive substances and attracted wide attention. Hybrids of whey protein isolate amyloid fibrils (WPF) and gliadin nanoparticles (GNP) were able to assemble into WPF-GNP hydrogel at a low protein concentration of 2 wt%, among which WPF and GNP were fabricated from the hydrolysis of whey protein isolate under 85°C water bath (pH 2.0) and antisolvent precipitation, respectively. Atomic force microscope (AFM) images indicated that the ordered nanofibrillar network of WPF was formed at pH 2.0 with a thickness of about 10 nm. Cryo-SEM suggested that WPF-GNP hydrogel could arrest GNP within the fibrous reticular structure of the partially deformed WPF, while the hybrids of native whey protein isolate (WPI) and GNP (WPI-GNP hybrids) only led to protein aggregates. WPF-GNP hydrogel formed at pH 4.0 (85°C, 3 h, WPF:GNP = 4:1) possessed the largest elastic modulus (G’ = 419 Pa), which far exceeded the elastic modulus of the WPI-GNP hybrids (G’ = 16.3 Pa). The presence of NaCl could enhance the strength of WPF-GNP hydrogel and the largest value was achieved at 100 mM NaCl (∼105 mPa) in the range of 0∼500 mM due to electrostatic screening. Moreover, WPF-GNP hydrogel showed a high encapsulation efficiency for curcumin, 89.76, 89.26, 89.02, 85.87, and 79.24% for pH 2.0, 3.0, 4.0, 5.0, and 6.0, respectively, which suggested that the formed hydrogel possess good potential as a delivery system. WPF-GNP hydrogel also exhibited a good protection effect on the photodegradation stability of the loaded curcumin with the retention of up to 75.18% after hydrogel was exposed to ultraviolet radiation for 7 days. These results suggested that the viscoelasticity of WPF-GNP hydrogel was tunable via pH-, ion-, or composition-adjustment and the hydrogel showed excellent protection on the thermal and photodegradation stability of curcumin.

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.

In-flow SAXS investigation of whey protein isolate hydrolyzed by bromelain

In-flow small angle X-ray scattering (SAXS) was used to probe in real-time (typically every second) the hydrolysis of whey protein isolate (WPI) by bromelain. The WPI concentration was 2.5%, the enzyme to substrate ratio was 1:10, and the enzymatic reaction was followed for 90 min at 50 °C and pH 7. SAXS showed that the average size of WPI molecules was about 20 Å and that even at the completion of the enzymatic reaction some intact molecules of similar size remained; these are likely bromelain molecules and trace amount of BSA. SAXS allowed us to monitor the hydrolysis course through the calculation of the power-law exponent (P) and the Guinier scale factor (G) using a theoretical unified model fitting. The fitting exercise also indicated that bromelain hydrolysis transforms the globular WPI molecules into Gaussian polypeptides. The hydrolysis of WPI, which was completed within 40 min of hydrolysis, was confirmed by the degree of hydrolysis and turbidity measurements and by SDS-PAGE, which showed that bromelain has a broad specificity. This study demonstrates that SAXS is a powerful method to monitor in situ and real-time protein hydrolysis and can offer insights into protein structural changes that occur.

The Influence of Enzymatic Hydrolysis of Whey Proteins on the Properties of Gelatin-Whey Composite Hydrogels.

Amino-acids, peptides, and protein hydrolysates, together with their coordinating compounds, have various applications as fertilizers, nutritional supplements, additives, fillers, or active principles to produce hydrogels with therapeutic properties. Hydrogel-based patches can be adapted for drug, protein, or peptide delivery, and tissue healing and regeneration. These materials have the advantage of copying the contour of the wound surface, ensuring oxygenation, hydration, and at the same time protecting the surface from bacterial invasion. The aim of this paper is to describe the production of a new type of hydrogel based on whey protein isolates (WPI), whey protein hydrolysates (WPH), and gelatin. The hydrogels were obtained by utilizing a microwave-assisted method using gelatin, glycerol, WPI or WPH, copper sulfate, and water. WPH was obtained by enzymatic hydrolysis of whey protein isolates in the presence of bromelain. The hydrogel films obtained have been characterized by FT-IR and UV-VIS spectroscopy. The swelling degree and swelling kinetics have also been determined.

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.

Changes of structural and techno-functional properties of high hydrostatic pressure (HHP) treated whey protein isolate over refrigerated storage

This work assessed the effect of storage time on the structure and functionality of HHP-treated whey protein isolate (WPI). Different pressures (100–600 MPa) and treatment times (15–30 min) were applied to aqueous WPI dispersions (5% w/v). The induced degree of unfolding was evaluated to select optimal HHP treatment conditions of WPI before storage at 4 °C. Conformational and techno-functional properties of untreated and optimally HHP-treated WPI samples were determined by UV–Vis and IR spectroscopy, foaming capacity, and interfacial tension measurements, respectively. Further tests of HHP-assisted hydrolysis of WPI were performed by α-chymotrypsin, bromelain, or their mixture (1:1 w/w), with the degree of hydrolysis (DH%) and electrophoretic patterns analyzed.The maximum unfolding degree was detected after a treatment of 400 MPa and 15 min and, at these processing conditions, no aggregation occurred. However, the structural changes achieved upon HHP were gradually lost during storage through a first-order refolding process (kREF. = 0.031 h−1), with restoring of native functionality.Hydrolysis performances of selected enzymes towards WPI were significantly promoted by high-pressure. Interestingly, a clear synergistic effect of α-chymotrypsin and bromelain combination on the WPI hydrolysis yield was detected, which resulted in the highest protein rupture (DH = 17%).

Low purity enzymes and ultrasound pretreatment applied to partially hydrolyze whey protein

The hydrolysis of whey protein isolate with low purity enzymes was studied with application of ultrasound pre-treatment. The aim was to increase the degree of hydrolysis and produce new biologically active peptides. The experiments were carried out with hydrolysis times up to 24 h. The influence of temperature, pH, enzymes (pepsin and papain), substrate concentration, and use or not of the ultrasonic probe was investigated. The peptide's structure and profile were examined after exposure to ultrasound. The highest degree of hydrolysis occurred with the use of pepsin for 10 h, 37 °C, and pH 2.5. The ultrasound pre-treatment allowed a reduction of 6 h in the process. After partial enzymatic hydrolysis and ultrasound pretreatment a higher proportion of low molar mass peptides were observed at 1000–2000 g.mol-1. In vitro simulated digestion showed slight differences in peptide profile indicating that active compounds were preserved intact with a slight impact on bioactivity.

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.

Exploring the Use of a Modified High-Temperature, Short-Time Continuous Heat Exchanger with Extended Holding Time (HTST-EHT) for Thermal Inactivation of Trypsin Following Selective Enzymatic Hydrolysis of the β-Lactoglobulin Fraction in Whey Protein Isolate

Tryptic hydrolysis of whey protein isolate under specific incubation conditions including a relatively high enzyme:substrate (E:S) ratio of 1:10 is known to preferentially hydrolyse β-lactoglobulin (β-LG), while retaining the other major whey protein fraction, i.e., α-lactalbumin (α-LA) mainly intact. An objective of the present work was to explore the effects of reducing E:S (1:10, 1:30, 1:50, 1:100) on the selective hydrolysis of β-LG by trypsin at pH 8.5 and 25 °C in a 5% (w/v) WPI solution during incubation periods ranging from 1 to 7 h. In addition, the use of a pilot-scale continuous high-temperature, short-time (HTST) heat exchanger with an extended holding time (EHT) of 5 min as a means of inactivating trypsin to terminate hydrolysis was compared with laboratory-based acidification to <pH 3 by the addition of HCl, and batch sample heating in a water bath at 85 °C. An E:S of 1:10 resulted in 100% and 30% of β-LG and α-LA hydrolysis, respectively, after 3 h, while an E:S reduction to 1:30 and 1:50 led >90% β-LG hydrolysis after respective incubation periods of 4 and 6 h, with <5% hydrolysis of α-LA in the case of 1:50. Continuous HTST-EHT treatment was shown to be an effective inactivation process allowing for the maintenance of substrate selectivity. However, HTST-EHT heating resulted in protein aggregation, which negatively impacts the downstream recovery of intact α-LA. An optimum E:S was determined to be 1:50, with an incubation time ranging from 3 h to 7 h leading to 90% β-LG hydrolysis and minimal degradation of α-LA. Alternative batch heating by means of a water bath to inactivate trypsin caused considerable digestion of α-LA, while acidification to <pH 3.0 restricted subsequent functional applications of the protein.

Formation of whey protein isolate nanofibrils by endoproteinase GluC and their emulsifying properties

Nanofibrils have favorable functional properties that facilitate their applications in the food industry. Whey protein isolate (WPI) is widely used to prepare nanofibrils via acid hydrolysis. In this research, the effect of hydrolysis by endoproteinase GluC (GluC) on WPI nanofibrils (WPNFs) was evaluated based on their structural and emulsifying properties. Following the hydrolysis of WPI by GluC at 37 °C and pH 8.0, the peptides self-assembled into WPNFs at 37 °C and pH 2.0. WPNFs with several micrometric length and nanometric (1–10 nm) diameter exhibited high aspect ratio (length/diameter). Based on microscopic images, circular dichroism spectroscopy, and infrared spectroscopy, we concluded that β-sheets played an important role in the formation and stability of WPNFs. The main factor affecting WPNF formation was found to be hydrolysis by GluC for 10 h followed by aggregation at pH 2.0. Whey protein hydrolysates with glutamic acid or aspartic acid residues, obtained from WPI hydrolysis by GluC for 10 h, might be positively charged in an acidic environment, thereby favoring the formation of ordered aggregates instead of random ones. WPNFs prepared under such conditions have a strong ability of improving the oxidative stability, activity and stability of emulsions. This new strategy for producing WPNFs will potentially expand their application in the food industry.

Comparison of antioxidant activities of bovine whey proteins before and after simulated gastrointestinal digestion

Oxidative stress caused by free radicals has been implicated in several human disorders. Dietary antioxidants can help the body to counteract those reactive species and reduce oxidative stress. Antioxidant activity is one of the multiple health-promoting attributes assigned to bovine whey products. The present study investigated whether this activity was retained during upper gut transit using a static simulated in vitro gastrointestinal digestion (SGID) model. The capacity to scavenge free radicals and reduce ferric ion of whey protein isolate (WPI), individual whey proteins, and hydrolysates pre- and post-SGID were measured and compared using various antioxidant assays. In addition, the free AA released from individual protein fractions in physiological gut conditions were characterized. Our results indicated that the antioxidant activity of WPI after exposure to the harsh conditions of the upper gut significantly increased compared with intact WPI. From an antioxidant bioactivity viewpoint, this exposure negates the need for prior hydrolysis of WPI. The whey protein α-lactalbumin showed the highest antioxidant properties post-SGID (oxygen radical absorbance capacity = 1,825.94 ± 50.21 μmol of Trolox equivalents/g of powder) of the 4 major whey proteins tested with the release of the highest amount of the antioxidant AA tryptophan, 6.955 μmol of tryptophan/g of protein. Therefore, α-lactalbumin should be the preferred whey protein in food formulations to boost antioxidant defenses.

Comparative Study of in Situ and ex Situ Enzymatic Hydrolysis of Milk Protein and Separation of Bioactive Peptides in an Electromembrane Reactor

Tryptic hydrolysis of whey protein isolate was performed simultaneously during (in situ) and before (ex situ) fractionation by electrodialysis with ultrafiltration membrane (EDUF) to obtain bioactive peptides. Peptide migration to anionic (ARC–) and cationic (CRC+) peptide recovery compartments was strongly dependent on the digestion strategy used. Indeed, peptide migration to the ARC– was observed to be higher with in situ digestion while peptide migration to the CRC+ was higher in an ex situ digestion: a final peptide concentration of 103.10 ± 2.76 μg/mL was found in the CRC+ (ex situ) while it was 49.65 ± 6.13 μg/mL in the ARC– (in situ). HPLC-MS studies showed 23 major peaks that were generated by tryptic digestion of whey protein isolate. Seven of these peptides migrated to the ARC– while nine and eight peptides migrated to the CRC+ for ex situ and in situ digestions, respectively. Among them, different antihypertensive, antimicrobial, and hypocholesterolemic peptides were recovered depending on the ...

Demasking kinetics of peptide bond cleavage for whey protein isolate hydrolysed by Bacillus licheniformis protease

During enzymatic hydrolysis of proteins, some cleavage sites will be accessible in the intact protein. Others may only become accessible after demasking of the cleavage site, i.e. after cleavage of other bonds in the protein. Studies on demasking so far have used data of the release of few peptides in a hydrolysate. To obtain a more complete understanding of the hydrolysis kinetics and to validate the demasking model, this study used the absolute quantification of all peptides formed during hydrolysis of whey protein isolate by Bacillus licheniformis protease. This allowed the determination of the rate constants of hydrolysis of each individual cleavage site in the substrate protein. For five cleavage sites, no cleavages were found. For nine cleavage sites, the hydrolysis was best described with first order kinetics addition. The other 13 cleavage sites were better described with the two-step demasking model, which consists of two consecutive first order reaction equations. In this way, the demasking concept was validated, showing that some peptide bonds are only cleaved when present in intermediate peptides.

PH-stat vs. free-fall pH techniques in the enzymatic hydrolysis of whey proteins

Enzymatic hydrolysis of a commercial whey protein isolate (WPI) using either trypsin or Protamex® was compared using controlled (pH-stat) and uncontrolled (free-fall) pH conditions. pH-stat control at the enzyme’s optimum value led to a more rapid rate of WPI hydrolysis by trypsin, while the opposite was the case when Protamex® was used. Furthermore, the choice of alkaline solution used to maintain constant pH during pH-stat experiments appeared to affect the reaction rate, being higher when KOH is added to the reaction mixture instead of NaOH. It would appear that potassium may play a role as co-factor or activator for the activity of this particular protease preparation. Although pH-stat techniques are usually considered to yield better hydrolysis kinetics, these findings suggest that the response of proteolytic enzyme preparations to static or free-fall pH control should be checked in advance, particularly when undertaking large scale production of WPI hydrolysates.

Spontaneous, non-enzymatic breakdown of peptides during enzymatic protein hydrolysis

It is expected that during the hydrolysis of proteins with specific enzymes only peptides are formed that result from hydrolysis of the specific cleavage sites (i.e. specific peptides). It is, however, quite common to find a-specific peptides (i.e. resulting from a-specific cleavage), which are often ignored, or explained by impurities in the enzyme preparation. In recent work in a whey protein isolate (WPI) hydrolysate obtained with the specific Bacillus licheniformis protease (BLP), 13 peptides of 77 identified were found to be the result of a-specific cleavage. These were formed after degradation of 6 specific peptides, after 5 different types of amino acids. The fact that other peptides were not hydrolyzed after these 5 amino acids suggests that the cleavages were not the result of a contamination with a different enzyme. In other systems, certain peptide sequences have been described to degrade chemically, under relatively mild conditions. This process is referred to as spontaneous cleavage. To test if the a-specific peptides observed in the WPI hydrolysis are the results of spontaneous cleavages, the parental peptides were synthesized. Surprisingly, 4 of the 5 synthesized peptides were indeed spontaneously cleaved under the mild conditions used in this study (i.e. 40°C and pH8) showing that peptides are less stable than typically considered. The rate of cleavage on the a-specific bonds was found to be enhanced in the presence of BLP. This suggests that the formation of a-specific peptides is not due to side activity but rather an enhancement of intrinsic instability of the peptides.

Iron-binding peptides from whey protein hydrolysates: Evaluation, isolation and sequencing by LC–MS/MS

Iron–peptide complexes have been considered a promising source of more bioavailable iron, with reduced side effects as compared to iron salts. Whey protein isolate (WPI) hydrolyzed by alcalase, pancreatin or flavourzyme was ultrafiltered (cut off 5kDa) and their fractions – retentates and filtrates – were evaluated for iron-binding capacity. The Fe–hydrolysate complexation reaction resulted in a dramatic increase in iron solubility at pH7.0, from 0% to almost 100%. This result was obtained regardless of the molecular mass profile or the enzyme used to obtain the samples. Fractions from hydrolysate obtained with pancreatin (HP) were chosen to continue the study. The complexes formed with both fractions from HP were stable under simulated gastric digestion (50.8–89.4%). To identify the peptides with iron-binding capacity, the HP fractions were isolated by IMAC-Fe3+, and the retentate showed higher relative concentrations of iron-binding peptides than the filtrate. Iron-binding peptide sequencing, accomplished by LC–MS/MS, showed Glu and/or Asp in all the sequences, and their carboxylic groups were amongst the main iron-binding sites. WPI hydrolysis with pancreatin yields peptides that can form iron complexes with the potential to increase iron bioavailability and reduce its pro-oxidant effect.

Screening of whey protein isolate hydrolysates for their dual functionality: Influence of heat pre-treatment and enzyme specificity

Heat pre-treated and non heat pre-treated whey protein isolate (WPI) were hydrolysed using α-chymotrypsin (chymotrypsin), pepsin and trypsin. The in vitro antioxidant activity, ACE-inhibition activity and surface hydrophobicities of the hydrolysates were measured in order to determine if peptides with dual functionalities were present. Dual functional peptides have both biological (e.g. antioxidant, ACE-inhibition, opioid activities) and technological (e.g. nanoemulsification abilities) functions in food systems. Heat pre-treatment marginally enhanced the hydrolysis of WPI by pepsin and trypsin but had no effect on WPI hydrolysis with chymotrypsin. With the exception of the hydrolysis by trypsin, heat pre-treatment did not affect the peptide profile of the hydrolysates as analysed using size exclusion chromatography, or the antioxidant activity (P>0.05). Heat pre-treatment significantly affected the ACE-inhibition activities and the surface hydrophobicities of the hydrolysates (P<0.05), which was a function of the specificity of the hydrolysing enzyme. Extended hydrolysis (up to 24h) had no significant effect on the DH and the molecular weight profiles (P>0.05) but in some instances caused a reduction in the antioxidant activity of WPI hydrolysates. The chymotrypsin hydrolysate showed a broad MW size range, and was followed by pepsin and then trypsin. The bioactivities of the hydrolysates generally decreased in the order; chymotrypsin>trypsin>pepsin. This study showed that by manipulating protein conformation with pre-hydrolysis heat treatment, combined with careful enzyme selection, peptides with dual functionalities can be produced from WPI for use as functional ingredients in the manufacture of functional foods.

Chymotrypsin selectively digests β-lactoglobulin in whey protein isolate away from enzyme optimal conditions: Potential for native α-lactalbumin purification

The present study examines the resistance of the α-lactalbumin to α-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7.0, 7.8 and 8.5; temperature 25, 37 and 50 °C; enzyme-to-substrate ratio, E/S, of 0.1%, 0.5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman-Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8.5 selective hydrolysis of β-lactoglobulin was improved because of a dimer-to-monomer transition while α-la remained relatively resistant. The best conditions for the recovery of native and pure α-la were at 25 °C, pH 8.5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman-Birk inhibitor with around 81% of original α-la in WPI was recovered with no more β-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of β-lg with production of whey protein concentrates containing low or no β-lg and pure native α-la. This method also offers the possibility for production of β-lg-depleted milk products for sensitive populations.

Effect of Additives on Subcritical Water Hydrolysis of Whey Protein Isolate

The objective was to examine the effect of the additives acetic acid, lactic acid, sodium bicarbonate, sodium chloride, and sodium hydroxide on the hydrolysis of whey protein isolate with subcritical water. A screening experimental design was used to study the effect of temperature, time, and additives. The most influential additive, sodium bicarbonate, along with temperature and time was used in a second experimental design to predict the treatment conditions to maximize the degree of hydrolysis and production of free amino acids. The maximum degree of hydrolysis achieved was 50% at a concentration of 1.24 M sodium bicarbonate, 291 °C, and 28 min. The highest concentration of total amino acids was 83.0 mg/g of whey protein isolate with 0.83 M sodium bicarbonate at 264 °C for 29 min. Compared to water alone, sodium bicarbonate increased the degree of hydrolysis 4-fold and the production of amino acids by 44% and decreased peptides' molecular weight.