Hydrolysed ovalbumin offers more effective preventive and therapeutic protection against egg allergy than the intact protein.

BACKGROUND: The application of specific immunotherapy to stimulate oral tolerance towards food allergens is hampered by the high frequency of adverse side-effects and the excessive duration of the treatments. OBJECTIVE: In this work, a hydrolysate of ovalbumin with pepsin (OP), selected for its low IgE reactivity and Th2-stimulating capacity, was assayed for its ability to prevent and treat allergy to egg white (EW). METHODS: As a first step, the safety of OP, in terms of the absence of sensitizing and eliciting potential, was evaluated in BALB/c mice. Then, its suitability for prophylactic and therapeutic applications was compared with that of the intact allergen, paying attention to the molecular and cellular mechanisms involved in the control of the allergic process. To this aim, IgE, IgG1, IgG2a and IgA levels, allergic reactions, expression of genes related to Th1, Th2, Th17 and Treg responses, dendritic and T cell populations were assessed in intestinal tissues and spleens of EW-allergic mice, either untreated or treated with intact ovalbumin (OVA) or OP. RESULTS: The hydrolysate of OVA with pepsin was hypoallergenic, lacked sensitizing potential and offered preventive and therapeutic protection against allergy to EW through the induction of Treg cells and the up-regulation of TGF-ß, IL-10, IL-17, Foxp3 and RORγt in intestinal tissues. This restrained the expression of GATA3 and the differentiation of Th2 cells, leading to low cytokine responses following ex vivo spleen cell stimulation. As compared with intact OVA, OP was more effective against sensitization. In addition, in the therapeutic setting, OP provided quicker desensitization that lasted for at least 3 weeks after discontinuation of the therapy. CONCLUSIONS AND CLINICAL RELEVANCE: This study provides evidence for the superior role of hydrolysed, as compared to intact allergens, in the prevention of allergy development and in the promotion of long-term desensitization, as well as of intermolecular tolerance.

Pepsin Egg White Hydrolysate Ameliorates Obesity-Related Oxidative Stress, Inflammation and Steatosis in Zucker Fatty Rats.

The aim of this work was to evaluate the effect of the administration of egg white hydrolysates on obesity-related disorders, with a focus on lipid metabolism, inflammation and oxidative stress, in Zucker fatty rats. Obese Zucker rats received water, pepsin egg white hydrolysate (750 mg/kg/day) or Rhizopus aminopeptidase egg white hydrolysate (750 mg/kg/day) for 12 weeks. Lean Zucker rats received water. Body weight, solid and liquid intakes were weekly measured. At the end of the study, urine, faeces, different organs and blood samples were collected. The consumption of egg white hydrolysed with pepsin significantly decreased the epididymal adipose tissue, improved hepatic steatosis, and lowered plasmatic concentration of free fatty acids in the obese animals. It also decreased plasma levels of tumor necrosis factor-alpha and reduced oxidative stress. Pepsin egg white hydrolysate could be used as a tool to improve obesity-related complications.

Egg proteins as allergens and the effects of the food matrix and processing.

Hen eggs are an important and inexpensive source of high-quality proteins in the human diet. Egg, either as a whole or its constituents (egg yolk and white), is a key ingredient in many food products by virtue of its nutritional value and unique functional properties, such as emulsifying, foaming, and gelling. Nevertheless, egg is also known because of its allergenic potential and, in fact, it is the second most frequent source of allergic reactions, particularly in children. This review deals with the structural or functional properties of egg proteins that make them strong allergens. Their ability to sensitize and/or elicit allergic reactions is linked to their resistance to gastroduodenal digestion, which ultimately allows them to interact with the intestinal mucosa where absorption occurs. The factors that affect protein digestibility, whether increasing it, decreasing it, or inducing a different proteolysis pattern, and their influence on their capacity to induce or trigger an allergic reaction are discussed. Special attention is paid to the effect of the food matrix and the processing practices on the capacity of egg proteins to modulate the immune response.

Effect of the high-pressure-release phase on the protein composition of the soluble milk fraction.

This paper describes a study on the influence of the high-pressure-release rate on the protein distribution between the soluble and colloidal fractions of milk. Skim milk, without or with sulfhydryl-blocking agents, was pressure treated at 250 and 350 MPa for 5 to 15 min at 25 °C, applying different pressure-release rates (pressure-release times between 0.07 to 10 min). The liberation of caseins to the soluble phase and the denaturation of whey proteins were assessed. A significantly higher increase in the content of soluble casein took place during the pressure-release phase as compared with a pressure-holding phase. Denaturation of ß-lactoglobulin mainly took place by -SH-S-S exchange reactions during the holding phase. The present results, thus, show a negligible influence of whey proteins on the increase in the content of nonsedimentable casein in pressure-treated milk and provide evidence for the importance of the pressure-release rate in this process, so that the slower the pressure release rate, the higher the level of soluble casein.

In vivo methods for testing allergenicity show that high hydrostatic pressure hydrolysates of ß-lactoglobulin are immunologically inert.

The major milk allergen ß-lactoglobulin (ß-LG) exhibits an enhanced susceptibility to proteolysis under high hydrostatic pressure and this may be an efficient method to produce hypoallergenic hydrolysates. The aim of this work was to evaluate the in vivo allergenicity of 3 ß-LG hydrolysates produced under atmospheric pressure or high-pressure conditions. Hydrolysates were chosen based on previous experiments that showed that they provide a complete removal of intact ß-LG but differed in vitro IgE-binding properties that could be traced to the peptide pattern. The ability to trigger systemic anaphylaxis was assessed using C3H/HeJ mice orally sensitized to ß-LG. Outcome measures included symptom score, body temperature, serum mouse mast cell protease 1 (mMCP-1), and quantification of circulating basophils. Mast cell degranulation in vivo was assessed by passive cutaneous anaphylaxis. The 3 tested hydrolysates showed an abrogated allergenicity as revealed by the absence of anaphylactic symptoms and a decrease in body temperature. We demonstrated that the peptides present in the hydrolysates had lost their ability to cross-link 2 human IgE antibodies to induce mast cell degranulation, thus indicating that most of the peptides formed retain just one relevant IgE-binding epitope. The orally sensitized mouse model is a useful tool to address the in vivo allergenicity of novel milk formulas and demonstrates the safety of hydrolysates produced under high-pressure conditions.

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.

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 beta-lactoglobulin (beta-LG). The protein was subjected to high pressure before and during the proteolytic process. Analysis of remnant beta-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 beta-LG, extending the incubation time with the enzyme was needed to reduce the amount of potentially allergenic intermediate peptides. Changes of beta-LG under pressure at acidic pH are discussed.

Application of capillary zone electrophoresis to the characterisation of the human milk protein profile and its evolution throughout lactation.

This work describes the use of capillary zone electrophoresis for the characterisation of human milk proteins. The major proteins were identified following different strategies, such as the treatment with enzymes for selective protein modification. Using this method we studied the proteins in human milk from different donors throughout lactation. Qualitative and quantitative differences in the composition of the individual proteins were observed. The different beta-casein phosphoforms were separated and quantified. The average proportion of the 0P:1P:2P:3P:4P:5P was, approximately, 3:6:9:4:10:2. The evolution of the ratio of the different beta-casein phosphoforms during lactation is reported.

Effect of simulated gastrointestinal digestion on the antihypertensive properties of ACE-inhibitory peptides derived from ovalbumin.

Food-derived bioactive peptides with ACE-inhibitory properties are receiving special attention due to their beneficial effects in the treatment of hypertension. In this work we evaluate the impact of a simulated gastrointestinal digestion on the stability and activity of two bioactive peptides that derive from ovalbumin by enzymatic hydrolysis, YAEERYPIL and RADHPFL. These peptides possess in vitro ACE-inhibitory activity and antihypertensive activity in spontaneously hypertensive rats (SHR). The results showed that YAEERYPIL and RADHPFL were susceptible to proteolytic degradation after incubation with pepsin and a pancreatic extract. In addition, their ACE-inhibitory activity in vitro decreased after the simulated digestion. The antihypertensive activity on SHR of the end products of the gastrointestinal hydrolysis, YAEER, YPI, and RADHP, was evaluated. The fragments YPI and RADHP significantly decreased blood pressure, 2 h after administration, at doses of 2 mg/kg, but they probably did not exert their antihypertensive effect through an ACE-inhibitory mechanism. It is likely that RADHP is also the active end product of the gastrointestinal digestion of the antihypertensive peptides FRADHPFL (ovokinin) and RADHPF (ovokinin 2-7).

Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis.

The hydrolysis of crude egg white with pepsin, trypsin, and chymotrypsin produced peptides with angiotensin-converting enzyme (ACE) inhibitory properties. These peptides were mainly derived from the proteolysis of ovalbumin. The most active hydrolysates were obtained after treatment with pepsin (50% inhibitory concentration [IC50], 55.3 microg/ml), with the fraction having a molecular mass lower than 3,000 Da giving the highest ACE inhibitory activity (IC50, 34.5 microg/ml). Nine subfractions were collected from the fraction with a molecular mass lower than 3,000 Da using semipreparative reversed-phase high-performance liquid chromatography. Considerable ACE inhibitory activity (IC50 < 40 microg/ml) was found in three of them. These subfractions were analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry, and 14 peptides were identified. These sequences were synthesized, and their ACE inhibitory activities were measured. Among the identified peptides, two novel sequences with potent ACE inhibitory activity were found. The amino acid sequences of these inhibitors were identified as Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and showed IC50 values of 6.2 and 4.7 microM, respectively.

Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis.

This work reports the antioxidant activity of peptides produced by enzymatic hydrolysis of crude egg white with pepsin. Four peptides included in the protein sequence of ovalbumin possessed radical scavenging activity higher than that of Trolox. The hydrolysate of egg white with pepsin for 3 h was previously found to exhibit a strong angiotensin I-converting enzyme (ACE) inhibitory activity in vitro. The combined antioxidant and ACE inhibition properties make it a very useful multifunctional preparation for the control of cardiovascular diseases, particularly hypertension. No correlation was found between antioxidant and ACE inhibitory activities. However, the peptide Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu, which was a strong ACE inhibitor (50% inhibitory concentration, 4.7 microM) also exhibited a high radical scavenging activity (oxygen radical absorbance capacity-fluorescein value, 3.8 micromol of Trolox equivalent per micromol of peptide) and delayed the low-density lipoprotein lipid oxidation induced by Cu2+ at a concentration of approximately 0.16 mg/mg of low-density lipoprotein. Present results support that antioxidant peptides and amino acids not only act individually, but also cooperatively and synergistically.

Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine kappa-casein macropeptides and their tryptic hydrolysates.

This work evaluated the angiotensin-converting enzyme (ACE)-inhibitory activities of bovine, ovine, and caprine kappa-casein macropeptides (CMPs) and their tryptic hydrolysates. The results obtained indicate that bovine, ovine, and caprine CMPs exhibited moderate in vitro ACE-inhibitory activities that increased considerably after digestion under simulated gastrointestinal conditions. Active peptides could also be produced from CMPs via proteolysis with trypsin, with tryptic hydrolysates exhibiting a more extensive ACE-inhibitory activity than intact CMPs during simulated gastrointestinal digestion. Two active fractions were chromatographically separated from the tryptic hydrolysate of the bovine CMP, but their complexity hampered the assignment of the ACE-inhibitory activity to specific peptide sequences. Evidence for the release of the strong ACE-inhibitory tripeptide IPP was found upon simulation of the gastrointestinal digestion of peptides released by trypsin from the CMP sequence. These findings might help to promote further exploitation of cheese whey in the preparation of nutraceuticals for inclusion in the composition of functional food products with high added values.

Plasmin activity in pressurized milk.

The effects of pressure (up to 400 MPa), applied at room temperature, on native proteinase activity of milk were investigated by means of plasmin activity, plasmin-derived activity after plasminogen activation and their distribution in different milk fractions, micelle microstructure, beta-LG denaturation, and casein susceptibility to proteolytic attack. The pressure conditions assayed did not lead to plasmin inactivation and only decreased around 20 to 30% total plasmin activity after plasminogen activation. However, pressure caused severe disruption of the micellar structure, releasing high levels of caseins, plasmin, and plasminogen to the soluble fraction of milk. High levels of soluble denatured beta-LG were also found in the ultracentrifugation supernatants of pressurized milks, particularly in those treated at 400 MPa. Probably as a result of micellar disintegration, caseins became more susceptible to proteolysis by exogenous plasmin. However, no enhanced proteolytic degradation was observed when we compared the evolution of pressurized and unpressurized milks during refrigerated storage. Serum-liberated plasmin may become more vulnerable to the action of proteinase inhibitors leading to a reduced proteolysis on refrigerated storage, despite the increased susceptibility of caseins to proteinase action.

Platelet aggregation inhibitory activity of bovine, ovine, and caprine kappa-casein macropeptides and their tryptic hydrolysates.

Kappa-casein macropeptide (CMP) is one of the components of whey and is obtained as a by-product in cheesemaking. There has been increasing interest in research to find new uses of cheese industry by-products in order to improve their value and promote their use. Human platelet aggregation inhibitory activities of bovine, ovine, and caprine CMPs and their tryptic hydrolysates were studied. CMPs from the three species exhibited in vitro antithrombotic properties similar to the activity of the gamma-fibrinogen 400-411 peptide. Inhibitory activities increased following hydrolysis with trypsin. Active sequences were identified among the tryptic peptides by reversed-phase high-performance liquid chromatography with on-line mass spectrometry.

Heterogeneity of caprine kappa-casein macropeptide.

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine kappa-casein due to the silent substitution Ile/Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8-14% and 86-92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

Changes in phosphoglyceride composition during storage of ultrahigh-temperature milk, as assessed by 31P-nuclear magnetic resonance: possible involvement of thermoresistant microbial enzymes.

Soluble phosphoglycerides were studied in ultrahigh-temperature (UHT) milk by 31P-nuclear magnetic resonance. It was shown that, during storage of UHT milk, manufactured from raw milk with poor microbial quality, glycerophosphocholine and glycerophosphoethanolamine disappeared in parallel with an increase in alpha-glycerophosphate (GP). Storage at 10, 20, and 30 degrees C showed a faster transformation as the temperature increased. UHT milk samples manufactured from raw milks with better microbial quality and submitted to severe heat processes did not display changes in phosphoglycerides during storage. Screening of commercial UHT milks showed variations regarding the presence of GP, while in pasteurized milk samples, the appearance of GP occurred when the commercial life had been exceeded. Inoculation of sterile milk with Pseudomonas fluorescens NCIB9046 and incubation at 10 degrees C supported that changes in phosphoglycerides could be the consequence of a phosphodiesterase activity of bacterial origin, able to survive UHT processing. A similar behavior was observed between this activity and proteolytic activity. The potential application of the detection of these compounds as spoilage predictor indices is discussed.

Capillary electrophoresis for the analysis of food proteins of animal origin.

In recent years, capillary electrophoresis (CE) has become an analytical technique with many applications in the study of food proteins and peptides. This review describes the existing CE methods of analysis of milk, egg, meat and fish proteins and peptides. The major developments in the application of CE to solve different problems in food technology, such as the assessment of technological processes, quality, and authenticity control of animal foods, are considered. A section dealing with future directions on the analysis of food proteins by CE is also included.

Does processing of a powder or in-bottle-sterilized liquid infant formula affect calcium bioavailability?

Processing of infant formulas can induce Maillard reaction or lactose isomerization, among other changes. These reactions were evaluated with furosine and lactulose, respectively. Protein alteration was assessed with sodium dodecylsulfate-polyacrylamide gel electrophoresis. Repercussions on calcium bioavailability in powder and in-bottle-sterilized liquid infant formulas were studied. Lactulose, advanced Maillard-reaction products, and denatured proteins were higher in liquid infant formula. After in vitro digestion, soluble non-dialyzed calcium was significantly higher in liquid than in powder infant formula, but there were no differences in dialyzed insoluble calcium. Two-week-old rat pups drank the powder or liquid infant formula for 7 d. Food intake and final body weight were significantly lower in those fed liquid formula. Accordingly, the intake, apparent absorption, and retention of calcium were measured; the percentages of retention versus absorption and retention versus intake were significantly lower, although calcium digestibility (percentage of absorption versus intake) was higher. These results show that, although calcium in the sterilized infant formula was available in vitro and was absorbed more efficiently in vivo, it was poorly used by suckling rats. The low acceptability of this formula and the interaction of calcium with lactulose and advanced but absorbable Maillard-reaction products might explain the results. Thus, for calcium bioavailability, we recommend the powder instead of the conventional sterilized infant formula.

Micellar changes induced by high pressure. influence in the proteolytic activity and organoleptic properties of milk.

We investigated the effects of high-pressure treat-of 25 to 60 degrees C, on micelle structure, proteolytic activity, and sensory properties of milk. Pressure treatments at 25 degrees C considerably reduced micelle size, while pressurization at higher temperatures progressively increased micelle dimensions. Pressure-induced denaturation of beta-lactoglobulin (beta-LG) amounted 76% at 25 degrees C and was almost 100% in milks treated at 40 to 60 degrees C. alpha -Lactalbumin (alpha-LA) was resistant to pressure at temperatures up to 40 degrees C, but its denaturation reached 56% at 60 degrees C. Plasmin resisted pressurization at room temperature; however, pressure treatments at higher temperatures increased plasmin inactivation, which reached 86.5% at 60 degrees C. Pressurization at temperatures from 40 to 60 degrees C reduced the proteolytic activity and improved the organoleptical properties of milk, compared with the same treatments at 25 degrees C, which suggested that these combined treatments could be used to produce milk of good sensory properties with an increased shelf life. These results are discussed in the light of the changes found in micellar structure.

Chromatographic characterization of ovine kappa-casein macropeptide.

Ovine casein macropeptide (CMP) was characterized by anion-exchange FPLC and reversed-phase (RP) HPLC. To study heterogeneity (the degree of glycosylation and phosphorylation), CMP was desialylated with neuraminidase and dephosphorylated with acid phosphatase. Following RP-HPLC, the main CMP components were identified using either on-line or off-line mass spectrometry. The most abundant ovine CMP component was a diphosphorylated carbohydrate-free form, followed by one or two monophosphorylated and a non-phosphorylated asialo-aglyco species. Aglyco non-phosphorylated, monophosphorylated and diphosphorylated forms were in the ratio 3:20:77. Only approximately 30% of ovine CMP was glycosylated. Assuming that the monosaccharide fraction of ovine CMP is composed of N-acetylgalactosamine, galactose and N-glycolylneuraminic acid, molecular masses consistent with the presence of CMP containing tetra-, tri-, di- and monosaccharide were identified.