Effects of Enzymatic Hydrolysis Assisted by High Hydrostatic Pressure Processing on the Hydrolysis and Allergenicity of Proteins from Ginkgo Seeds

The purpose of this work was to study the combined effect of high hydrostatic pressure (HHP) and enzymatic hydrolysis treatment on the hydrolysis and allergenicity of ginkgo seed proteins (GSPs). Four food-grade proteases (papain, alcalase, pepsin, and neutrase) were used, and HHP (200, 300, and 400 MPa separately) was applied prior to hydrolysis. The extent of hydrolysis was measured with the o-phthaldialdehyde method, SDS-PAGE, and MALDI-TOF-MS, and the allergenicity was assessed with a Western blot and enzyme-linked immunosorbent assay (ELISA). The results showed that HHP could significantly improve the extent of proteolysis by papain, alcalase, or pepsin and reduce the antigenicity of GSP, whereas neutrase showed poor effects at any pressure. Papain and alcalase showed the highest proteolysis at 300 MPa, followed by pepsin at 400 MPa, and all of the obtained hydrolysates showed molecular weights lower than 10 kDa; furthermore, papain or alcalase at 300 MPa as well as pepsin at 400 MPa reduced antigenicity by more than 95 %, and all of the immunoreactive bands disappeared in the obtained hydrolysates. These results suggest that HHP can enhance the hydrolysis of GSP by certain enzymes and reduce the residual antigenicity of the hydrolysates. The obtained hypoallergenic hydrolysates could be used as a source of peptides for food ingredients.     

Proteolytic pattern, antigenicity, and serum immunoglobulin E binding of beta-lactoglobulin hydrolysates obtained by pepsin and high-pressure treatments

This study evaluates the use of high pressure to enhance pepsin hydrolysis of β-lactoglobulin (β-LG). The protein was subjected to high pressure before and during the proteolytic process. Analysis of remnant β-LG, identification of the peptides produced, and evaluation of antigenicity (binding to commercial antibodies) and binding to IgE of allergic patients’ sera were conducted in the hydrolysates. The results showed that the application of high pressure before the enzyme treatment slightly improved the proteolytic process but did not reduce the antigenicity or IgE binding of the hydrolysates. The application of high pressure during the enzymatic treatment enhanced the production of large intermediate fragments that were further proteolysed to smaller fragments as proteolysis proceeded for longer periods. At 400 MPa, all the intact protein was removed in minutes, simultaneously decreasing its antigenicity and serum IgE binding properties. However, for considerable reduction of the antigenicity and IgE binding of β-LG, extending the incubation time with the enzyme was needed to reduce the amount of potentially allergenic intermediate peptides. Changes of β-LG under pressure at acidic pH are discussed.     

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.     

Identification and characterization of novel α-amylase and α-glucosidase inhibitory peptides from camel whey proteins

This study explores the inhibitory properties of camel whey protein hydrolysates (CWPH) toward α-amylase (AAM) and α-glucosidase (AG). A general full factorial design (3 × 3) was applied to study the effect of temperature (30, 37, and 45°C), time (120, 240, and 360 min), and enzyme (pepsin) concentration (E%; 0.5, 1, and 2%). The results showed that maximum degree of hydrolysis was obtained when hydrolysis was carried out at higher temperature (45°C; P < 0.05), compared with lower temperatures of 30 and 37°C. Electrophoretic pattern displays degradation of all protein bands upon hydrolysis by pepsin at various hydrolysis conditions applied. All the 27 CWPH generated showed significant AAM and AG inhibitory potential as indicated by their lower IC₅₀ values (mg/mL) compared with intact whey proteins. In total 196 peptides were identified from selected hydrolysates and 15 potential peptides (PepSite score > 0.8; http://pepsite2.russelllab.org/) were explored via in silico approach. Novel peptides PAGNFLMNGLMHR, PAVACCLPPLPCHM, MLPLMLPFTMGY, and PAGNFLPPVAAAPVM were identified as potential inhibitors for both AAM and AG due to their high number of binding sites and highest binding probability toward the target enzymes. CCGM and MFE, as well as FCCLGPVPP were identified as AG and AAM inhibitory peptides, respectively. This is the first study that reports novel AG and AAM inhibitory peptides from camel whey proteins. The future direction for this research involves synthesis of these potential AG and AAM inhibitory peptides in a pure form and investigate their antidiabetic properties in the in vitro, as well as in vivo models. Thus, CWPH can be considered for potential applications in glycaemic regulation.     

Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100°C) WPC (2% protein solution) were incubated at 50°C for 30, 60, 90, and 120 min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120 min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The α-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60 min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30 min completely removed the BSA and α-lactalbumin. The β-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The β-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of β-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey.     

Whey Protein Antigenicity Reduction by Fungal Proteinases and a Pepsin/Pancreatin Combination

Whey protein components were hydrolyzed with Corolase 7092? (peptidases from Aspergillus strains), pepsin and Corolase PP? (a mixture of pancreatic enzymes), either individually or in combination, in trials to eliminate protein allergenicity. The hydrolysates were characterized by physico-chemical and by immunological techniques using sera from patients allergic to milk proteins. Enzyme specificity rather than degree of hydrolysis or molecular mass distribution of hydrolysates determined the residual antigenicity of the whey proteins. Ultrafiltration was a prerequisite for obtaining hypoallergenic whey protein hydrolysates.     

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.     

高静压协同酶法处理对白果蛋白抗原性的影响

本文研究了高静压结合酶解处理对白果蛋白抗原性的影响,分别采用4种蛋白酶水解白果蛋白,水解前分别采用不同压力的高静压对白果蛋白进行预处理,酶解产物水解率和分子量采用OPA法和SDS-PAGE测定,致敏性采用western-blotting和ELISA法测定。结果表明,木瓜蛋白酶,碱性蛋白酶或胃蛋白酶为水解酶时,高静压能显著提高白果蛋白的水解率和降低其致敏性;而中性蛋白酶为水解酶时,白果蛋白的水解和脱敏效果很差,即使高压处理也未见明显提高。木瓜蛋白酶或碱性蛋白酶在处理压力为300 MPa时,而胃蛋白酶在400 MPa时,其水解和脱敏效果最好,在此条件下白果蛋白能被水解为分子量小于15 ku的多肽,95%以上的白果蛋白致敏性能被消除,酶解产物中致敏蛋白条带全部消失。因此,高静压处理能明显提高蛋白酶对白果蛋白的水解效率和脱敏效果,但是取决于选择的蛋白酶种类和处理压力的大小。     

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.     

High hydrostatic pressure enhances whey protein digestibility to generate whey peptides that improve glutathione status in CFTR-deficient lung epithelial cells

Whey protein isolates (WPI) may provide anti-inflammatory benefits to cystic fibrosis (CF), which could be mediated via peptides, as proteolytic digests of WPI enhance intracellular glutathione (GSH) concentrations. The objectives of this study were to investigate whether high hydrostatic pressure can (i) improve the in vitro digestibility of WPI; and (ii) generate low molecular weight (< 1 kDa) peptides from WPI hydrolysates that exert GSH-enhancing and anti-inflammatory properties in wild type and mutant CF transmembrane conductance regulator (CFTR) tracheal epithelial cells. Hydrostatic pressure processing enhanced the in vitro digestibility of WPI to proteolytic enzymes resulting in altered peptide profiles as assessed by CZE and GC-MS. The exposure of mutant CFTR cells to low molecular weight (< 1 kDa) peptides isolated from WPI hydrolysates exposed to pressure processing (pressurized WPI hydrolysates, pWPH), showed increased intracellular levels of reduced GSH and total GSH relative to treatment with peptides obtained from native WPI hydrolysates (nWPH). A tendency for decreased interleukin-8 secretion was associated with the pWPH and nWPH treatments in mutant CFTR cells, which was not observed in wild type cells. Hydrostatic pressure processing of whey proteins appears to enhance their impact on cellular GSH status in cells with the mutant CFTR condition.     

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.     

Dual function peptides from pepsin hydrolysates of whey protein isolate

The aim of this study was to investigate the effect of pepsin hydrolysates of whey protein isolate (WPI) on vascular relaxation and emulsifying capacity. WPI was subjected to pepsin hydrolysis for 5 h. The chromatographic profiles of the samples showed the formation of a wide variety of peptides. Addition of WPI hydrolysates in phenylephrine-contracted rat aortic rings induced a similar concentration-dependent relaxation in both endothelium-intact and endothelium-denuded preparations. In endothelium-denuded vessels the maximum relaxation induced by WPI fractions increased along the time, reaching over 70% after 3 h-hydrolysis on. In addition, the vascular relaxation was not associated with an inhibition of the angiotensin-converting enzyme or activation of K⁺ channels. Hydrolysed fractions were further evaluated for the emulsifying capacity (EC) and all tested fractions were able to keep an EC over 60%. These results reinforce the potential of WPI pepsin-hydrolysates as an option in the search for dual function peptides from whey proteins.     

Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins

The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100–300 MPa for 15 min at 37 °C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The β-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed α-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae.     

Hydrolysis under high hydrostatic pressure as a means to reduce the binding of beta-lactoglobulin to immunoglobulin E from human sera

Cows' milk allergy is the most frequent food allergy in children, and beta-lactoglobulin (beta-Lg) is a major allergen. Milk-based hypoallergenic ingredients are manufactured by enzymatic hydrolysis, a process that could be improved by the application of high-pressure treatments. This study showed that the treatment of beta-Lg dissolved in buffer with chymotrypsin and trypsin under high pressure for relatively short times accelerated proteolysis by leading to a rapid removal of the intact protein. The rapid proteolysis of the beta-Lg substrate under pressure led to the production, in 20 min, of hydrolysates with lower immunoglobulin (Ig) G binding than those produced in 8 h (chymotrypsin) or 48 h (trypsin) at atmospheric pressure. However, those hydrolysates retained some residual IgE-binding properties that could be traced to the preferential release, during the initial stages of proteolysis, of peptides containing IgE epitopes, such as (Val-41-Lys-60), (Leu-149-Ile-162), and (Ser-21-Arg-40). The formation of these fragments was favored when proteolysis was conducted under high pressure due to the preferential hydrolysis of Arg-40 and Arg-148 by trypsin, and Tyr-42 and Leu-149 by chymotrypsin, all located at the dimer interface of beta-Lg or very close to it. Although our results do not support that trypsin and chymotrypsin under high pressure selectively address the allergenic regions of beta-Lg, it is possible to select the conditions that quickly produce hydrolysates with reduced potential allergenicity that could be used in hypoallergenic foods.     

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.     

Use of selected sourdough lactic acid bacteria to hydrolyze wheat and rye proteins responsible for cereal allergy

This work was aimed at showing the capacity of selected sourdough lactic acid bacteria to hydrolyze wheat and rye allergens. Hydrolysis was investigated after wheat sourdough fermentation and after treatment of wheat and rye sourdough breads with pepsin, trypsin and pancreatin, which mimicked the digestive process. As shown by immunoblotting with sera from allergic patients, wheat sourdough fermentation caused the disappearance of some IgE-binding proteins (albumins/globulins and gliadins mainly) with respect to the chemically acidified dough used as the control. The IgE-binding protein profile of wheat and rye sourdough breads differed from those of baker's yeast breads. The signals of the IgE-binding proteins contained in the sourdough breads disappeared after in vitro digestion with pepsin, trypsin and pancreatin. The same effect by digestive enzymes was not found for baker's yeast breads which showed persistent IgE-binding proteins. As shown by ELISA inhibition assays, the presence of IgE-binding low molecular weight proteins/peptides in sourdough breads significantly decreased with respect to baker's yeast breads. Proteolytic activity by selected sourdough lactic acid bacteria may have an importance during food processing to produce pre-digested wheat and rye dough which contains IgE-binding proteins degradable by digestive enzymes.     

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).     

Pepsin treatment of whey proteins under high pressure produces hypoallergenic hydrolysates

BALB/c mice were used to assess the ability of a whey protein hydrolysate obtained by pepsin treatment under high pressure (400 MPa, 37 °C, 30 min, HWP), to induce anaphylaxis, antibody production and cytokine responses in comparison with the whey protein isolate (WP) from which it is derived. HWP did not contain intact allergens and 50% of its peptides ranged between 10 and 3 kDa. Challenge with HWP did not induce clinical signs, body temperature changes or release of mast cell proteinase-1 in mice sensitized to WP. Immunization of mice with HWP did not produce WP-specific antibodies or allergic reactions upon HWP or WP challenge and thus, it can be considered hypoallergenic. However, HWP stimulated Th2 responses in splenocytes from sensitized mice. These characteristics make HWP a good candidate to be used in the management of milk allergy in diagnosed patients or to induce tolerance to whey proteins.Industrial relevanceHydrolysis with pepsin under high pressure produces in minutes a whey protein hydrolysate that complies with the health claims of the European guidelines on infant and follow-on formulas related to the reduction of risk to allergy to milk proteins. This process constitutes an alternative to the exhaustive enzymatic hydrolysis treatments used in the processing of hypoallergenic formulas that release short peptides and free amino acids to adversely affect organoleptic properties and technological value.     

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.     

Opioid peptides derived from in-vitro proteolysis of bovine whey proteins

The formation of opioid peptides by in-vitro proteolysis of whey proteins was investigated. Bovine β-lactoglobulin (β-LG) or -lactalbumin (-LA) were predigested with pepsin and subsequently treated with either trypsin (Try) or trypsin+chymotrypsin (Try+Chy). For separation and identification of the peptides. HPLC chromatography, protein sequencing and amino acid analysis were used. Identified peptides were synthesized by Peninsula Laboratories Europe Ltd. UK. Binding to rat brain homogenates was tested against ³H-naloxone. The effects of the peptides on smooth muscle were tested in coaxially stimulated guinea pig ileum in vitro. Digestion of β-LG with pepsin plus Try, or Try+Chy yielded Tyr-Leu-Leu-Phe (β-lactorphin). Proteolysis of -LA with pepsin alone produced Tyr-Gly-Leu-Phe (-lactorphin) although a higher degree of hydrolysis was achieved by addition of Try. Among hydrolysates of whey proteins at least -lactorphin exerted a weak but continuous opioid property both in terms of receptor binding and smooth muscle effects.