Multivariate strategy in screening of enzymes to be used for whey protein hydrolysis in an enzymatic membrane reactor

Proteinase activities for Alcalase^® 2.4L (EC 3.4.21.62), Flavourzyme^® (EC 3.4.11.1), Protease A (EC 3.4.24.39) and Protease N (IUB 3.4.24.28) were determined using 2% whey protein isolate (WPI) and 2% casein. The optimum substrate and enzyme concentrations and temperature were determined by the pH-stat method. Residual enzyme activity, hydrolysate molecular weight and free amino acid (FAA) content were determined. Protease N and Alcalase^® 2.4L had the highest proteinase activities on casein and WPI, respectively. Alcalase^® 2.4L was more stable in the presence of WPI while Protease N was inhibited by hydrolysates, and like Protease A which released high FAAs, they produced shorter peptides. Flavourzyme^® hydrolysed WPI poorly and released the highest FAAs. Short peptides were removed by 5% trichloroacetic acid (TCA) and 3.5% 5-sulphosalicylic acid before FAA analysis by reversed phase high-performance liquid chromatography (RP-HPLC) of Flavourzyme^® and Protease A hydrolysates, but were detected in Alcalase^® 2.4L and Protease N hydrolysates. The enzyme activities for WPI hydrolysis in an enzymatic membrane reactor were Flavourzyme^®<Protease A<Alcalase^® 2.4L⩽Protease N.     

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 24 h) 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.     

Production of whey protein isolate hydrolysate fractions with enriched ACE-inhibitory activity

A study on the enrichment of angiotensin-converting enzyme (ACE) inhibitory activity in whey protein isolate (WPI) hydrolysate fractions is presented. A previously identified low molecular mass fraction (1 kDa permeate) of an enzymatically hydrolysed heat-treated WPI with elevated ACE-inhibition (IC₅₀ = 0.23 g L⁻¹) was subjected to cascade membrane ultrafiltration (UF) and diafiltration steps at lab-scale. Assaying for ACE-inhibition revealed that the 1 kDa retentate demonstrated the highest ACE-inhibitory activity (IC₅₀ = 0.17 g L⁻¹). Isoelectric focussing (IEF) of the hydrolysate fraction further increased ACE-inhibition in fractions collected within the pH range 6.1–6.6. Overall, both UF and IEF enriched the ACE inhibitory activity in the original fraction by ∼52%, demonstrating the potential for enrichment of bio-functional activities in enzymatic hydrolysates of whey proteins.     

Emulsifying Property of Whey Peptide Fractions as a Function of pH and ionic Strength

Solubility and emulsifying properties of whey protein concentrate (WPC), heat-treated WPC (90°C, pH 2.5, 10 min), and their tryptic or chymotryptic peptide fractions obtained by ultrafiltration were measured from pH 3 to 9 at two ionic strengths (uμ=0 and 0.6). There was no correlation between solubility and emulsifying capacity (EC). Heat treatment induced some conformational changes in WPC, resulting in better EC. Some peptide fractions had better EC than proteins at pH 7 and 9. Treatments resulting in more hydrophobic peptides or a higheT content of larger peptides produced fractions with better emulsifying properties. pH and ionic strength affected conformation and emulsifying properties of proteins and peptides.     

Release of multifunctional peptides by gastrointestinal digestion of sea cucumber (Isostichopus badionotus)

Sea cucumber is a benthic marine organism distributed worldwide and used as food in several Asian countries. The species Isostichopus badionotus is captured intensively off the Yucatan Peninsula, Mexico. Boiled I. badionotus was subjected to in vitro simulated gastrointestinal digestion using pepsin and a pepsin–Corolase PP^® mixture. ACE-inhibitory and radical scavenging activities, iron reducing capacity and cytotoxic effects against colorectal cancer cells were evaluated in the hydrolysates and their ultrafiltered fractions. ACE-inhibitory activity was potent in fractions containing peptides <3000 Da, an effect augmented with combined action of gastric (pepsin) and intestinal (Corolase PP^®) enzymes (IC₅₀ = 0.038 ± 0.004 mg/mL). Antioxidant activity was exerted by peptides with low and high molecular weights, depending on hydrolysis method. This is the first report of cytotoxic capacity against colorectal HT-29 cells in peptides from sea cucumber. Sea cucumber hydrolysates and ultrafiltered fractions are potential ingredients for development of functional foods.     

Peptic hydrolysis of bovine beta‐lactoglobulin under microwave treatment reduces its allergenicity in an ex vivo murine allergy model

In this study, the in vivo allergenicity of bovine beta‐lactoglobulin (BLG) in peptic whey protein hydrolysates generated during microwave and conventional heating treatments was assessed. The allergenicity of the hydrolysates was explored by studying the reaction of the murine jejunum from previously immunised Balb/c mice to treated BLG in an Ussing chamber. Intestinal anaphylactic reactions after stimulation of the gut‐associated immune system are a good indicator of potential in vivo allergenicity of whey hydrolysates. Fifty‐two per cent of BLG was hydrolysed by pepsin after only 3 min of microwave irradiation at 200 watts (W), yet it remained intact under conventional heating. Far‐ and near‐UV circular dichroism spectra indicated significant changes in BLG secondary and tertiary structures with microwave irradiation at 200 W. Pepsin whey protein hydrolysates obtained with microwave irradiation at 200 W for 3 min did not stimulate secretion of chloride in the Ussing chamber, as shown by the intensity of the short current values recorded (27.86 μA cm^?2), compared to the conventional pepsin hydrolysates (68.21 μA cm^?2). This demonstrates the low allergenicity of whey protein hydrolysates generated in this manner. These results confirm that microwave treatment combined with peptic hydrolysis could be applied to produce low allergenicity milk peptides.     

Peptide–peptide and protein–peptide interactions in mixtures of whey protein isolate and whey protein isolate hydrolysates

The extent of aggregation in whey protein isolate (WPI) hydrolysates induced by Bacillus licheniformis protease was quantified as a function of degree of hydrolysis (DH), temperature and ionic strength. The capacity of the hydrolysates to aggregate added intact protein was also studied. The amount of aggregated material and the size of the aggregated peptides were measured by nitrogen content and size exclusion chromatography, respectively. Aggregation increased with DH up to the practical end point of hydrolysis (DH 6.8%). The aggregates formed under the various conditions studied consisted of peptides with masses ranging from 1.4 to 7.5 kDa. The hydrolysates were also able to aggregate added WPI. The additional amount of aggregated material increased with increasing DH. Peptides involved in peptide–peptide interactions were also involved in protein–peptide interactions. It is hypothesized that hydrophobic interactions dominated peptide–peptide interactions, while protein–peptide interactions depended on the balance between hydrophobic attraction and electrostatic repulsion.     

Reducing and radical-scavenging activities of whey protein hydrolysates prepared with Alcalase

Antioxidant activities of whey protein isolate (WPI) hydrolysates prepared by Alcalase treatment at different concentrations and times were investigated. The antioxidant activity of WPI hydrolysates, indicated by peroxide value and thiobarbituric acid-reactive substance values in a liposome-oxidizing system, increased with increasing hydrolysis time up to 5 h (P < 0.05). The WPI hydrolysates also showed greater radical-scavenging ability, greater Cu²⁺-chelating ability and improved reducing power when compared with non-hydrolysed WPI (P < 0.05). An increase in protein concentration was shown to significantly enhance antioxidant activities (P < 0.05). Although non-hydrolysed WPI displayed an antioxidative effect, it was far less potent than the hydrolysed WPI. This study shows that enzyme-hydrolysed WPI can act as a hydrogen donor, a metal ion chelator, and a radical stabiliser to inhibit lipid oxidation. The WPI hydrolysates produced by Alcalase could be employed in the food industry as an antioxidant to replace synthetic antioxidants.     

Isolation and identification of antimicrobial peptides derived by peptic cleavage of whey protein isolate

The antimicrobial potential of whey protein isolate hydrolyzed by gastrointestinal enzymes was determined by attempting to identify and characterize the antimicrobial peptides responsible. While tryptic and chymotryptic hydrolysates did not show antibacterial activity, whey proteins hydrolyzed for 45–90 min by pepsin exhibited significant activity. Fractionation of 60-min hydrolysate by reversed-phase high performance liquid chromatography yielded 5 fractions that were antibacterial, with minimum inhibitory concentrations comprised between 20 and 35 μg/mL. These fractions contained short peptides not previously identified as antimicrobial. Fragment 14–18 (KVAGT) of β-lactoglobulin is very close to a sequence previously identified as antibacterial and is found in antimicrobial sequences of diverse origin. Five other peptides derived from β-lactoglobulin, and one fragment from α-lactalbumin (f117–121, KVGIN), were also identified as antibacterial. The identified peptides do not match pepsin action exactly, indicating modified proteolysis of unknown origin. Protein by-products of the dairy industry offer potential for large-scale production of antimicrobial peptides.     

Effect of ultrasound on the structure and functional properties of transglutaminase-crosslinked whey protein isolate exposed to prior heat treatment

Effects of ultrasound treatment (20 kHz, 41–45 W cm⁻², 0, 20, 40 or 60 min) on the physicochemical, functional properties and elements of the secondary structure of transglutaminase (TGase)-crosslinked whey protein isolate (WPI) exposed to prior thermal treatment (75 °C, 15 min) were investigated. The largest molecular size of proteins in the TGase-crosslinked WPI was observed after the ultrasound and thermal pre-treatment (HU-WPI-TGase). HU-WPI-TGase had the maximum intrinsic fluorescence intensity, with highest loss of free amino groups. Ultrasound-treated WPI (U-WPI) showed more (13%) emulsifying activity and more (63%) foaming ability than untreated WPI, but HU-WPI-TGase had higher foam stability and lower emulsifying activity than U-WPI. FTIR analysis indicated that ultrasound, heat treatment and TGase cross-linking had effects on the β-sheet, β-turn and random coil of WPI. The outcomes from this study show a potential application in providing novel functional ingredients for the dairy industry.     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

Inhibition of the in vitro activities of α-amylase, α-glucosidase and pancreatic lipase by yellow field pea (Pisum sativum L.) protein hydrolysates

The aim of this work was to produce yellow field pea protein-derived peptides as inhibitors of α-amylase, α-glucosidase and pancreatic lipase activities. A pea protein concentrate was hydrolysed with alcalase, chymotrypsin, pepsin or trypsin and the hydrolysates separated into different fractions (<1, 1–3, 3–5, 5–10 kDa) by membrane ultrafiltration. Peptide sequence analysis showed that the alcalase hydrolysate had higher levels of di- and tripeptides when compared with the chymotrypsin, pepsin and trypsin hydrolysates. The peptide fractions inhibited α-amylase and α-glucosidase activities at levels that were similar to the unfractionated hydrolysates. The peptides were more active against α-amylase (inhibition at μg level) than α-glucosidase (mg level). In contrast, the fractionated peptides had reduced ability (IC₅₀ >4.2 mg mL⁻¹) when compared with the unfractionated hydrolysate (IC₅₀ <4.2 mg mL⁻¹) to inhibit lipase activity. Enzyme kinetic studies revealed that the peptides reduced α-amylase activity through competitive inhibition. However, inhibition of α-glucosidase activity was non-competitive.     

乳清多肽对猪肉糜氧化和凝胶特性的影响

目的：研究乳清多肽对猪肉糜氧化和凝胶特性的影响作用。方法：实验分为6 组,第1组为空白对照组,第2组加入20%（质量分数,下同）的乳清分离蛋白未水解物,第3～5组中分别加入10%、15%、20%的乳清分离蛋白水解物冻干粉,第6组中加入0.02%的丁基羟基茴香醚（butylated hydroxyanisole,BHA）。在肉糜冷藏7 d过程中测定过氧化值、羰基含量、巯基含量及凝胶的质构、白度、保水性、流变学特性的变化。结果：在贮藏初期,处理组和对照组之间无显著差异（P＞0.05）。贮藏7 d后,添加乳清分离蛋白水解物组在抑制氧化和保护凝胶品质方面都要比对照组和未水解组好（P＜0.05）。其中添加20%乳清分离蛋白水解物冻干粉的储能模量的极大值最高,而在抑制脂肪、蛋白氧化及保护凝胶质构、保水性方面,添加15%乳清分离蛋白水解物冻干粉效果更好（P＜0.05）,接近甚至达到了BHA组水平。结论：乳清多肽具有抑制猪肉糜氧化和保护肉糜凝胶品质的作用。     

Identification of potent antioxidant bioactive peptides from goat milk proteins

Goat milk proteins have gained increasing attention especially the bioactive peptides released from the parent proteins by digestive enzymes. Specifically, the interest in bioactives of goat milk is intensifying due to its reduced allergenicity compared to bovine milk. In this study, proteins of goat milk were fractionated into caseins (GCP) and whey proteins (GWP), hydrolyzed by pepsin and the generated peptides were examined for radical scavenging activities. The hydrolysates of whey (P-GWP) and casein (P-GCP) proteins exhibited potent superoxide anion (O₂^･−) scavenging activity in a dose-dependent manner, as investigated using the natural xanthine/xanthine oxidase (X/XOD) system. The P-GWP and P-GCP dramatically quenched the O₂^･− flux but had negligible effect on the catalytic function of the enzyme, indicating specificity to scavenge O₂^･− but not oxidase inhibition. Further, both P-GWP and P-GCP were able to remarkably quench the chemical DPPH radical. Fractionation of hydrolysates by size-exclusion chromatography produced four fractions (F1-F4) from both hydrolysates, with variable O₂^･− scavenging activities. However, the slow eluting fractions (F4) of both hydrolysates and fast eluting fraction (F2) of P-GCP contained peptides with the highest scavenging activities. Peptides in the active fractions of P-GWP and P-GCP, isolated by reversed phase-HPLC, exhibited significantly strong O₂^･− scavenging activities. MALDI-TOF-MS allowed the identification of several antioxidant peptides derived from both caseins and whey proteins, with β-casein and β-lactoglobulin being the major contributors, respectively. The results demonstrate that digestion with pepsin generates multiple soluble peptides from goat milk protein fractions with remarkable ability to scavenge superoxide radicals and thus providing a fascinating opportunity for their potential candidacy as antioxidant bioactive peptides.     

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 °C and hydrolyzed using the enzymes Alcalase, α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 °C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate.     

Antibody binding and functional properties of whey protein hydrolysates obtained under high pressure

This paper examines the potential of high hydrostatic pressure to produce whey protein hydrolysates that combine low immunoglobulin (Ig)G- and IgE-binding with acceptable functional properties, with the aim to produce milk-based ingredients with reduced potential allergenicity that could be used in hypoallergenic foods. Treatment with pepsin and chymotrypsin under high pressure produced, in minutes, hydrolysates in which α-lactalbumin and β-lactoglobulin were totally proteolysed, giving rise to large and hydrophobic peptides. Such hydrolysates presented reduced antigenicity and human IgE-binding properties. The hydrolysates obtained with pepsin at 400 MPa showed improved heat stability, particularly at a pH, close to the isoelectric point of the whey proteins, and their emulsion activity indexes at pH 7.0 were superior to those of the untreated whey proteins. These results suggest that the peptides present retained low antigenicity together with sufficient capacity to form emulsions.     

Angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein fractions

Casein and whey protein fractions from goat milk were hydrolysed by subtilisin and trypsin, individually and in combination, to release angiotensin converting enzyme (ACE)-inhibitory peptides. Selected hydrolysates were fractionated by size exclusion chromatography (SEC) and further characterised. The highest ACE-inhibitory activity was obtained from the casein fraction hydrolysed by the combination of enzymes. SEC presented 4 fractions with fraction F2 (<2.3 kDa) containing the highest concentration of peptides and the highest activity. F2 contained a number of peptides not previously identified from caprine caseins but with structural similarity to other ACE-inhibitory peptides. The most active fraction in relation to protein content was F4 with IC₅₀ between 9.3 and 5.1 μg mL⁻¹. This fraction contained a compound tentatively identified as WY, an active dipeptide not previously reported from caseins. The high inhibitory capacity of these fractions points towards the advantage of implementing a membrane process to concentrate the most active peptides.     

Use of macroporous adsorption resin for simultaneous desalting and debittering of whey protein hydrolysates

Whey protein isolate (WPI) was hydrolysed to whey protein hydrolysates (WPH) of degree of hydrolysis equal to 15% using Protease N ‘Amano’ G (IUB 3.4.24.28) in a batch reactor at 55 °C and pH 7.0 according to the pH‐stat procedure. Ash was removed by adsorbing WPH onto macroporous adsorption resins (MAR). Following rinsing with deionised water, desorption was achieved by washing with 20%, 40% and 75% alcohol (v v^?1) to obtain the three fractions HS20, HS40 and HS75. Ash reduced from 15.71% (WPH) to 4.38% (HS20), 2.02% (HS40) and 2.38% (HS75). Similarly, the protein content was enriched from a low of 64.89% (WPH) to 94.74% (HS20), 95.32% (HS40) and 92.00% (HS75). The fractions were analysed for surface hydrophobicity (SH_o), angiotensin‐I converting enzyme (ACE) inhibition, emulsifying activity index, total amino acids composition and molecular weight distribution. Fraction HS75 was objectionably bitter, showed superior ACE inhibition (lowest IC₅₀), had the highest content of hydrophobic and essential amino acids and contained about 71% of <600 Da with no fractions exceeding 4142 Da. Desorption with alcohol weakened the hydrophobic interaction forces between the peptides and resins and hence eluted the peptides, with the bitter HS75 being extracted.     

Inhibition of dipeptidyl peptidase IV by enzymatic hydrolysates derived from primary and secondary whey of fresh and Oaxaca cheeses

Currently, there is an interest in studying natural inhibitors of dipeptidyl peptidase IV (DPP‐IV) for the treatment of type II diabetes. To look for novel inhibitors, the primary and secondary whey of fresh and Oaxaca cheeses was obtained, and the electrophoretic patterns were determined. Hydrolysis with pepsin and trypsin of primary and secondary whey from fresh and Oaxaca cheeses, respectively, showed DPP‐IV inhibition with values of ~51 and 55%, and also an IC50 of 92.86 μg/mL for Oaxaca cheese‐derived peptides. Data show the potential of whey enzymatic hydrolysates from fresh and Oaxaca cheeses as antidiabetic agents.     

Inhibition of frozen storage‐induced oxidation and structural changes in myofibril of common carp (Cyprinus carpio) surimi by cryoprotectant and hydrolysed whey protein addition

The objective of the present study was to evaluate the efficacy of combined cryoprotectants (sucrose + sorbitol) and whey protein isolate (WPI) hydrolysates to inhibit protein oxidation and quality loss in common carp (Cyprinus carpio) surimi during frozen storage at ?25 °C. With increasing storage time, the carbonyl content of myofibrillar proteins increased from 4.02 nmolmg^‐1 protein (0 day) to 7.25, 6.31, 5.26 and 4.83 nmol mg^?1 protein (180 days; P < 0.05) for the control and samples with cryoprotectants, with cryoprotectants + WPI hydrolysates and with cryoprotectants + propyl gallate, respectively; protein surface hydrophobicity and turbidity increased in a similar trend, while sulfhydryl content, Ca‐ATPase activity, protein solubility and protein thermal stability decreased (P < 0.05). These results suggest that treatments with combined cryoprotectants and antioxidative WPI hydrolysates offer an effective approach to reducing the extent of protein oxidation in common carp surimi, thereby limiting protein structural changes known to impair texture of surimi products.