Evaluation of the residual antigenicity of dairy whey hydrolysates obtained by combination of enzymatic hydrolysis and high-pressure treatment

Dairy whey was hydrolyzed for 15 min with five food-grade enzymes (Alcalase, Neutrase, Corolase 7089, Corolase PN-L, and Papain) at atmospheric pressure (0.1 MPa) and in combination with high pressure (HP) at 100, 200, and 300 MPa, applied prior to or during enzymatic digestion. The peptide profile of the hydrolysates obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their residual antigenicity was assessed by immuno-blotting with anti-beta-lactoglobulin monoclonal antibodies and the sera from pediatric patients allergic to cow's milk proteins. Moreover, to evaluate the presence of residual trace amounts of casein in bovine whey hydrolysates, immunoblotting with anti-cow's milk protein polyclonal antibodies was performed. SDS-PAGE analysis showed that HP treatment increased hydrolysis by the proteases assayed, especially when it was applied during the enzymatic digestion. Positive reactions at the band corresponding to beta-lactoglobulin were detected for Corolase PN-L and Corolase 7089 hydrolysates, except for those obtained under 300 MPa by the last protease. However, the immunochemical reaction was lower in the hydrolysis products obtained under HP than in those obtained at atmospheric pressure and after the HP treatment. On the contrary, no residual immunochemical reactivity associated with beta-lactoglobulin was observed in the hydrolysates obtained by Alcalase and Neutrase under HP, and none was observed in any of the hydrolysis products obtained by Papain. The presence of traces of casein was not significant. These results suggest that HP combined with selected food-grade proteases is a treatment that can remove the antigenicity of whey protein hydrolysates for their use as ingredients of hypoallergenic infant formulae.     

Zymogen Activation in Pancreatic Endoproteolytic Preparations and Influence on Some Whey Protein Hydrolysate Characteristics

A proteolytic preparation from porcine pancreas was isolated. Trypsin, chymotrypsin and elastase were characterized and their time-dependent stability at 37°C was studied. The supematant of a 30% (w/v) saturated ammonium sulfate precipitation of a pancreatic extract (30S) was developed to pilot-scale level. The influence of zymogen activation time on molecular characteristics of whey protein hydrolysates produced by 30S and the commercial pancreatic preparation Corolase PP were compared. Amino acid analysis and gel permeation chromatography were used to characterize lactalbumin hydrolysates produced. Physicochemical characteristics of whey protein hydrolysates could be altered by manipulation of zymogen activation conditions in pancreatic proteinase preparations to be used during subsequent protein hydrolysis.     

Enzymatic proteolysis,under high pressure of soybean whey: Analysis of peptides and the allergen Gly m 1 in the hydrolysates

Soybean (Glycine max) whey was hydrolyzed with Alcalase, Neutrase, Corolase 7089 and Corolase PNL during high pressure (HP) treatment at 100, 200 and 300 MPa and at atmospheric pressure for 15 min. The protein content and the degree of hydrolysis were determined. Furthermore, the allergen Gly m 1 in the treated soybean whey and the hydrolysates was detected. The results showed that HP treatments increased the hydrolysis by the four proteases used. Pressure at 200 and 300 MPa proved to be better pressures to enhance the proteolysis. The immunochemical response of soybean whey to anti-Gly m 1 monoclonal antibodies decreased after the HP treatments and this decrease was greater after the combined treatment of high pressure and enzymatic hydrolysis. Soybean whey proteins hydrolysed at high pressure could be used as sources of peptides with low antigenicity when incorporated as food ingredients.     

High pressure can reduce the antigenicity of bovine whey protein hydrolysates

The combined effect of high pressure (HP) and enzymatic treatments on the proteolysis and antigenicity of hydrolysates from bovine whey proteins (WP) was studied. Four food grade protease preparations (Alcalase, Neutrase, Corolase 7089 and Corolase PN-L) were used. Hydrolysis was performed at 40 °C for Corolase PN-L and 50 °C for the other enzymes, for 15 min, after or during HP treatment. The degree of hydrolysis and RP-HPLC peptide profile were evaluated. An indirect ELISA test using polyclonal rat anti β-lactoglobulin antibodies was used to determine the residual antigenicity. The results showed that HP treatment enhanced the hydrolysis of bovine WP. For most enzymes, the best results were obtained at a pressure of 300 MPa. For two enzymes, Corolase PN-L and Neutrase, differences in peptide profiles were obtained due to the pressure applied during the enzymatic hydrolysis. Based on these differences, the residual antigenicity of these preparations were determined. An important reduction was found in the antigenicity of the hydrolysates obtained with Corolase PN-L and Neutrase in combination with HP treatment (300 MPa), prior to or during enzymatic hydrolysis, respectively. These results suggest that HP can enhance the WP hydrolysis, and, depending on the choice of enzymes, reduce the residual antigenicity of the hydrolysates. The reduced antigenicity of hydrolysates obtained by the combined treatments could have a relevant application in the development of hypoallergenic infant formulae.     

Assessment of the residual immunoreactivity of soybean whey hydrolysates obtained by combined enzymatic proteolysis and high pressure

Soybean (Glycine max) whey was hydrolyzed for 15 min using three food-grade proteases (Alcalase, Neutrase, Corolase PN-L) at atmospheric pressure (0.1 MPa) and under high pressure (HP) at 100, 200, and 300 MPa. All hydrolysates were analyzed by SDS-PAGE and their residual immunoreactivity was assessed by immunoblotting using the sera from children allergic to soybean. As shown in SDS-PAGE, Alcalase, Neutrase, and Corolase PN-L produced different patterns of hydrolysis. Each enzyme showed a similar proteolytic activity at atmospheric pressure and at 100 MPa, while an increased degree of hydrolysis was observed at 200 and 300 MPa. No residual antigenicity was observed in the hydrolysates obtained by Alcalase and Corolase PN-L in all considered conditions of hydrolysis. A positive reaction associated with a band having molecular weight of about 70 kDa was observed in the immunoblotting of the hydrolysates obtained with Neutrase at 0.1, 100, and 200 MPa, while no antigenicity was detected for those samples obtained under high pressure, at 300 MPa. These results suggest that high pressure combined with suitable enzymatic activity could be a useful tool for obtaining hydrolysates with low immunoreactivity to be used in special foods (hypoallergenic foods).     

Rheological and structural characterization of gels from whey protein hydrolysates/locust bean gum mixed systems

The gelling ability of whey proteins can be changed by limited hydrolysis and by the addition of other components such as polysaccharides. In this work the effect of the concentration of locust bean gum (LBG) on the heat-set gelation of aqueous whey protein hydrolysates (10% w/w) from pepsin and trypsin was assessed at pH 7.0. Whey protein concentrate (WPC) mild hydrolysis (up to 2.5% in the case of pepsin and 1.0% in the case of trypsin) ameliorates the gelling ability. The WPC synergism with LBG is affected by the protein hydrolysis. For a WPC concentration of 10% (w/w), no maximum value was found in the G′ dependence on LBG content in the case of the hydrolysates, unlike the intact WPC. However, for higher protein concentrations, the behaviour of gels from whey proteins or whey protein hydrolysates towards the presence of LBG becomes very similar. In this case, a small amount of LBG in the presence of salt leads to a big enhancement in the gel strength. Further increases in the LBG concentration led to a decrease in the gel strength.     

In vitro generation and stability of the lactokinin beta-lactoglobulin fragment (142-148)

The objectives of this study were to investigate the generation of beta-lactoglobulin fragment (142-148) (beta-LG f(142-148) during the hydrolysis of whey proteins, and the in vitro stability of this fragment upon incubation with gastrointestinal and serum proteinases and peptidases. An enzyme immunoassay (EIA) protocol was developed for the quantification of beta-LG f(142-148) in whey protein hydrolysates and in human blood serum. The minimum detection limit was 3 ng/mL. The level of the peptide in whey protein hydrolysates was influenced by the degree of hydrolysis (DH). As expected, highest levels of this peptide were found in hydrolysates generated with trypsin. Sequential incubation of hydrolysates at different DH values with pepsin and Corolase PP, to simulate gastrointestinal digestion, generally resulted in the degradation of beta-LG f(142-148) as determined by EIA. Reversed-phase HPLC and angiotensin-I-converting enzyme (ACE) activity assays demonstrated that synthetic beta-LG f(142-148) was rapidly degraded upon incubation with human serum. Furthermore, beta-LG f(142-148) could not be detected by EIA in the sera of 2 human volunteers following its oral ingestion or in sera from these volunteers subsequently spiked with beta-LG f(142-148). These in vitro results indicate that beta-LG f(142-148) is probably not sufficiently stable to gastrointestinal and serum proteinases and peptidases to act as an hypotensive agent in humans following oral ingestion. The in vitro methodology described herein has general application in evaluating the hypotensive potential of food protein-derived ACE inhibitory peptides.     

Effects of various whey proteins on the physicochemical and textural properties of set type nonfat yoghurt

In this study, the changes during storage in the physicochemical, textural and sensory properties of nonfat yoghurts fortified with whey proteins, namely whey protein concentrates (WPC), whey protein isolates and whey protein hydrolysates, were investigated. Enrichment of nonfat yoghurt with the whey protein additives (1% w/v) had a noticeable effect on pH, titratable acidity, syneresis, water‐holding capacity, protein contents and colour values on the 14th day of storage (P < 0.01). The addition of whey proteins to the yoghurt milk led to increases in the hardness, cohesiveness and elasticity values, resulting in improved textural properties. The addition of WPC improved the texture of set‐type nonfat yoghurt with greater sizes in the gel network as well as lower syneresis and higher water holding capacity. This study suggests that the addition of whey protein additives used for fortification of yoghurt gave the best textural and sensory properties that were maintained constant during the shelf life.     

Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates

Inhibition of dipeptidyl peptidase-IV (DPP-IV) activity is a promising strategy for treatment of type 2 diabetes. In the current study, DPP-IV inhibitory peptides were identi?ed from mare whey protein hydrolysates obtained by papain. The results showed that all the mare whey protein hydrolysates obtained at various hydrolysis durations possessed more potent DPP-IV inhibitory activity compared with intact whey protein. The 4-h hydrolysates showed the greatest DPP-IV inhibitory activity with half-maximal inhibitory concentration of 0.18 mg/mL. The 2 novel peptides from 4-h hydrolysate fractions separated by successive chromatographic steps were characterized by liquid chromatography–electrospray ionization tandem mass spectrometry. The novel peptides Asn-Leu-Glu-Ile-Ile-Leu-Arg and Thr-Gln-Met-Val-Asp-Glu-Glu-Ile-Met-Glu-Lys-Phe-Arg, which corresponded to β-lactoglobulin 1 f(71–77) and β-lactoglobulin 1 f(143–155), demonstrated DPP-IV inhibitory activity with half-maximal inhibitory concentrations of 86.34 and 69.84 μM, respectively. The DPP-IV inhibitory activity of the 2 peptides was retained or even improved after simulated gastrointestinal digestion in vitro. Our findings indicate that mare whey protein-derived peptides may possess potential as functional food ingredients in the management of type 2 diabetes.     

Two‐stage processing of salmon backbones to obtain high‐quality oil and proteins

Traditional processing technologies for fish by‐products containing significant amounts of oils usually either give high amounts of oil or maximised solubilisation of proteins. Due to lower yields and insufficient quality, the proteins or the oil is considered as secondary products. The proposed concept combines a gentle thermal separation of oil followed by enzymatic hydrolysis of the remaining protein‐rich fraction. The first stage, thermal treatment (40 °C) of fresh salmon backbones, separated up to 85% of the oil from the raw material and gave high‐quality oil (PV = 0.2 ± 0.0 meq kg^?1, 0.16 ± 0.05% free fatty acids). Separation of a significant part of the oil gave reduced mass flow into the enzymatic stage, which then requires less enzymes and reduced energy consumption. Among the tested enzymes: Trypsin, Corolase PP and Mixture of Papain and Bromelain gave the highest yield of fish protein hydrolysates (FPH), while use of Protamex and Corolase PP resulted in FPH with the best sensory properties leading to the lowest bitterness.     

Enzymatic Hydrolysis of Milk Proteins Under Alkaline and Acidic Conditions

The pH-stat and osmometric methods were adopted for control of hydrolysis of casein and whey proteins by chymotrypsin, trypsin and pepsin. Experiments in alkaline medium showed that the mean pK values determined for amino groups in milk protein hydrolysates at 52°C were 7.11 for casein hydrolysates and 7.18 for whey protein hydrolysates. During hydrolysis in acidic medium the osmotic coefficients determined for casein and whey proteins enabled calculation of the calibration factor for the osmometer (1.05) which could be assumed as constant. Results will enable monitoring of DH during hydrolysis of milk proteins in such systems.     

Bitterness in Bacillus proteinase hydrolysates of whey proteins

Whey protein concentrate (WPC) hydrolysates were generated with three commercially available Bacillus proteinase preparations (pH 7.0, 50 °C, 20% (w/v) WPC). Alcalase 2.4L hydrolysates were more bitter than Prolyve 1000 and Corolase 7089 hydrolysates when the proteinase activities were included at equivalent high and low addition levels. A glutamyl endopeptidase (GE) activity present in Alcalase was not detected in the Prolyve and Corolase preparations. Hydrolysate bitterness significantly increased when GE activity was included during Prolyve hydrolysis of WPC, indicating that inclusion of the GE activity was linked with the higher bitterness in Alcalase hydrolysates. A peptide present at higher levels in Prolyve compared to Alcalase hydrolysates was identified by mass spectrometry as β-lactoglobulin f(43–57). Hydrolysis of this peptide by GE was shown to release fragments with increased average hydrophobicity (Q-value). This may, in part, explain the higher level of bitterness associated with Alcalase compared to Prolyve hydrolysates of WPC.     

PARTIAL CHARACTERIZATION OF A WHEY PROTEIN CONCENTRATE AND ITS ENZYME HYDROLYSATES

A whey protein concentrate (WPC) was produced from fresh whey by ultrafiltration (MW cut off 10 kDa) and lyophilization. Enzymatic hydrolysis was performed with three enzyme systems: pancreatin (PA), protamex (PR) or alcalase 0.6L (AL) to produce hydrolysates with 20% degree of hydrolysis (DH). The peptide profiles of the hydroly sates were determined by high performance capillary electrophoresis (HPCE). The relationship between enzyme system and preferential protein substrate could be established. The alcalase hydrolysate (ALH) differed from the other two hydrolysates, and the enzyme showed the lowest specificity for β‐lactaglobulin. Considering the protein content from WPC the pancreatin hydrolytic system was the most efficient leaving only 4.69% unhydrolyzed protein in the final hydrolysate (PAH). For 20% degree of hydrolysis alcalase left 7.98% unhydrolyzed protein, while protamex left 9.81% unhydrolyzed protein in the final hydrolysate.     

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.     

Cow and camel milk-derived whey and casein protein hydrolysates demonstrated effective antifungal properties against selected Candida species

Bioactive peptides derived from milk proteins are widely known to possess antibacterial activities. Even though the antibacterial effects of milk-derived peptides are widely characterized, not much focus is given to their antifungal characterization. Therefore, in this study, we investigated the antifungal properties of camel and cow whey and casein hydrolysates against various species of pathogenic Candida. The hydrolysates were produced using 2 enzymes (alcalase and protease) at differing hydrolysis durations (2, 4, and 6 h) and tested for their antifungal properties. The results showed that intact cow whey and casein proteins did not display any anti-Candida albicans properties, whereas the alcalase-derived 2 h camel casein hydrolysate (CA-C-A2) displayed a higher percentage of inhibition against Candida albicans (93.69 ± 0.26%) followed by the cow casein hydrolysate generated by protease-6 h (Co-C-P6; 81.66 ± 0.99%), which were significantly higher than that of fluconazole, a conventional antifungal agent (76.92 ± 4.72%). Interestingly, when tested again Candida krusei, camel casein alcalase 2 and 4 h (CA-C-A2 and CA-C-A4), and cow whey alcalase-6 h (CO-W-A6) hydrolysates showed higher antifungal potency than fluconazole. However, for Candida parapsilosis only camel casein alcalase-4 h (Ca-C-A4) and cow casein protease-6 h (Co-C-P6) hydrolysates were able to inhibit the growth of C. parapsilosis by 19.31 ± 0.84% and 23.82 ± 4.14%, respectively, which was lower than that shown by fluconazole (29.86 ± 1.11%). Overall, hydrolysis of milk proteins from both cow and camel enhanced their antifungal properties. Camel milk protein hydrolysates were more potent in inhibiting pathogenic Candida species as compared with cow milk protein hydrolysates. This is the first study that highlights the antifungal properties of camel milk protein hydrolysates.     

乳清蛋白及其水解物对马铃薯淀粉体外消化性和理化性质的影响

本文通过构建乳清蛋白及乳清蛋白水解物与马铃薯淀粉的共糊化体系来探究乳清蛋白及其水解物对马铃薯淀粉体外消化性和理化性质的影响。结果表明,经过胃蛋白酶和胰酶水解处理的乳清蛋白水解物对淀粉的消化率抑制效果最为明显。其中,天然马铃薯淀粉中快消化淀粉（RDS）含量最高（94.54%）,抗性淀粉（RS）含量最低（3.10%）。而经过胃蛋白酶处理后经胰酶处理120 min的样品中的RDS含量最低（67.51%）,RS含量最高（12.69%）。乳清蛋白水解物对马铃薯淀粉的溶胀和糊化的抑制作用均强于乳清蛋白。这说明乳清蛋白水解物的分子量对马铃薯淀粉的理化特性和消化性均有较大影响。此外,乳清蛋白及其水解物增强了体系中的氢键作用并提高了淀粉结构的有序程度,表明乳清蛋白及其水解物与马铃薯淀粉之间的相互作用会降低淀粉的消化性。     

Preparation of whey protein hydrolysates with ACE-inhibitory activity using cysteine peptidases from Bromelia hieronymi Mez. (Bromeliaceae)

A proteolytic extract from fruits of Bromelia hieronymi was used to hydrolyse bovine whey proteins. The peptide profile obtained exhibited a gradual fading of the main whey proteins and a decrease in the content of hydrophobic peptides. The 180-min hydrolysate showed a hydrolysis degree of 15.2% and inhibited the angiotensin-converting enzyme (ACE), showing an IC₅₀ of 0.17 mg/mL. By bioinformatics analysis, several theoretical sequences of possible ACE-inhibitory peptides were deduced. These results showed that proteolytic extracts from B. hieronymi can be used to prepare whey hydrolysates that would serve as a potential bioactive ingredient of functional foods.     

FUNCTIONAL PROPERTIES OF HIGH HYDROSTATIC PRESSURE-TREATED WHEY PROTEIN

Surface hydrophobicity, solubility, gelation and emulsifying properties of high hydrostatic pressure (HHP)‐treated whey protein were evaluated. HHP treatment of whey protein buffer or salt solutions were performed at 690 MPa and initial ambient temperature for 5, 10, 20 or 30 min. Untreated whey protein was used as a control. The surface hydrophobicity of whey protein in 0.1 M phosphate buffers treated at pH 7.0 increased with an increase in HHP treatment time from 10 to 30 min. HHP treatments of whey protein in salt solutions at pH 7.0 for 5, 10, 20 or 30 min decreased the solubility of whey proteins. A significant correlation was observed between the surface hydrophobicity and solubility of untreated and HHP‐treated whey protein with r = ?0.946. Hardness of HHP‐induced 20, 25 or 30% whey protein gels increased with an increase in HHP treatment time from 5 to 30 min. An increase in the hardness of whey protein gels was observed as whey protein concentration increased. Whey proteins treated in phosphate buffer at pH 5.8 and 690 MPa for 5 min exhibited increased emulsifying activity. Whey proteins treated in phosphate buffer at pH 7.0 and 690 MPa for 10, 20 or 30 min exhibited decreased emulsifying activity. HHP‐treated whey proteins in phosphate buffer at pH 5.8 or 7.0 contributed to an increase in emulsion stability of model oil‐in‐water emulsions. This study demonstrates that HHP treatment of whey protein in phosphate buffer or salt solutions leads to whey protein unfolding observed as increased surface hydrophobicity. Whey proteins treated in phosphate buffers at pH 5.8 and 690 MPa for 5 min may potentially be used to enhance emulsion stability in foods such as salad dressings, sausage and processed cheese.     

Functional and Biological Properties of Peptides Obtained by Enzymatic Hydrolysis of Whey Proteins

The study of peptides released by enzymatic hydrolysis of whey proteins has been initially focusing on improving their functional properties in food model systems. Our first study showed that peptides 41 to 60 and 21 to 40 from β-lactoglobulin (β-LG) were responsible for improved emulsifying properties of a tryptic hydrolysate of whey protein concentrate (WPC). Further work showed that adding negatively charged peptides from tryptic hydrolysates of WPC could prevent phase separation of dairy-based concentrated liquid infant formula, as a replacement for carrageenan. Hydrolysis of whey proteins using a bacterial enzyme was also successful in improving heat stability of whey proteins in an acidic beverage. Some tryptic peptides demonstrated improvement in the heat stability and in modifying thermal aggregation of whey proteins. Recent research has shown that whey peptides could trigger some physiological functions. Within the scope of this research our work has led to the development of a whey protein enzymatic hydrolysate that has demonstrated antihypertensive properties when orally administered to spontaneously hypertensive rats and human subjects. Our work then focused on the fractionation of hydrolysates by nanofiltration to prepare specific peptidic fractions; however, peptide/peptide and peptide/protein interactions impaired membrane selectivity. The study of those interactions has lead to the demonstration of the occurrence of interactions between β-LG and its hydrophobic fragment 102–105 (opioid peptide), which probably binds in the central cavity of the protein. This latest result suggests that β-LG could be used as a carrier for the protection of bioactive peptides from gastric digestion. Our work therefore has shown that the enzymatic hydrolysis of whey proteins is not only improving their functional properties, but it is also providing powerful technology in the exploitation of their biological properties for functional foods and nutraceutical applications.     

Alteration in propagating colonic contractions by dairy proteins in isolated rat large intestine

Gastrointestinal conditions in which the transit of contents is altered may benefit from nutritional approaches to influencing health outcomes. Milk proteins modulate the transit of contents along different regions, suggesting that they have varying effects on neuromuscular function to alter gastrointestinal motility. We tested the hypothesis that bovine whey and casein milk protein hydrolysates could have direct modulatory effects on colonic motility patterns in isolated rat large intestine. Casein protein hydrolysate (CPH), whey protein concentrate (WPC), whey protein hydrolysate (WPH), and a milk protein hydrolysate (MPH; a hydrolyzed blend of 60% whey to 40% casein) were compared for their effects on spontaneous contractile waves. These contractions propagate along the length of the isolated intact large intestine (22 cm) between the proximal colon and rectum and were detected by measuring activity at 4 locations. Milk proteins were perfused through the tissue bath, and differences in contraction amplitude and frequency were quantified relative to pretreatment controls. Propagation frequency was decreased by CPH, increased by MPH, and unaffected by intact whey proteins. The reduced motility with CPH and increased motility with MPH indicate a direct action of these milk proteins on colon tissue and provide evidence for differential modulation by hydrolysate type. These findings mirror actions on lower gastrointestinal transit reported in vivo, with the exception of WPH, suggesting that other factors are required.