Peptide Characterization and Functional Stability of a Partially Hydrolyzed Whey-Based Formula over Time.

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = -0.95; -0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level (p < 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers.

Proteomics Analysis Reveals Altered Nutrients in the Whey Proteins of Dairy Cow Milk with Different Thermal Treatments.

Thermal treatments of milk induce changes in the properties of milk whey proteins. The aim of this study was to investigate the specific changes related to nutrients in the whey proteins of dairy cow milk after pasteurization at 85 °C for 15 s or ultra-high temperature (UHT) at 135 °C for 15 s. A total of 223 whey proteins were confidently identified and quantified by TMT-based global discovery proteomics in this study. We found that UHT thermal treatment resulted in an increased abundance of 17 proteins, which appeared to show heat insensitivity. In contrast, 15 heat-sensitive proteins were decreased in abundance after UHT thermal treatment. Some of the heat-sensitive proteins were connected with the biological immune functionality, suggesting that UHT thermal treatment results in a partial loss of immune function in the whey proteins of dairy cow milk. The information reported here will considerably expand our knowledge about the degree of heat sensitivity in the whey proteins of dairy cow milk in response to different thermal treatments and offer a knowledge-based reference to aid in choosing dairy products. It is worth noting that the whey proteins (lactoperoxidase and lactoperoxidase) in milk that were significantly decreased by high heat treatment in a previous study (142 °C) showed no significant difference in the present study (135 °C). These results may imply that an appropriately reduced heating intensity of UHT retains the immunoactive proteins to the maximum extent possible.

Selenium Modulates the Allergic Response to Whey Protein in a Mouse Model for Cow's Milk Allergy.

Cow's milk allergy is a common food allergy in infants, and is associated with an increased risk of developing other allergic diseases. Dietary selenium (Se), one of the essential micronutrients for humans and animals, is an important bioelement which can influence both innate and adaptive immune responses. However, the effects of Se on food allergy are still largely unknown. In the current study it was investigated whether dietary Se supplementation can inhibit whey-induced food allergy in an animal research model. Three-week-old female C3H/HeOuJ mice were intragastrically sensitized with whey protein and cholera toxin and randomly assigned to receive a control, low, medium or high Se diet. Acute allergic symptoms, allergen specific immunoglobulin (Ig) E levels and mast cell degranulation were determined upon whey challenge. Body temperature was significantly higher in mice that received the medium Se diet 60 min after the oral challenge with whey compared to the positive control group, which is indicative of impaired anaphylaxis. This was accompanied by reductions in antigen-specific immunoglobulins and reduced levels of mouse mast cell protease-1 (mMCP-1). This study demonstrates that oral Se supplementation may modulate allergic responses to whey by decreasing specific antibody responses and mMCP-1 release.

Comparison of allergenicity among cow, goat, and horse milks using a murine model of atopy.

Due to the prevalence and severity of cow milk (CM) allergy (CMA), an ideal substitute is urgently needed to develop hypoallergenic infant formula for infants who experience anaphylaxis to typical whey-based CM formula. Goat milk (GM) and horse milk (HM) are considered appropriate substitutes; however, whether GM and HM are less allergenic than CM is unclear. In the present study, the difference in allergenicity among CM, GM, and HM was investigated using the Balb/c mouse model. The number of mice with severe respiratory symptoms was significantly lower in the GM- and HM-sensitised groups than in the CM-sensitised group. Furthermore, histologic examination of intestinal and lung tissues revealed a thinner lamina propria of the small intestine and obvious inflammation and congestion in lungs in the CM-sensitised group than in the GM- and HM-sensitised groups. CM-specific immunoglobulin (Ig) E, serum IgG1, and plasma histamine levels were also higher in CM-sensitised mice than in GM- or HM-sensitised mice. In addition, higher interleukin (IL) 4 and IL-17A levels and lower interferon-γ (IFN-γ) and IL-10 levels were observed in CM-sensitised mice compared with GM- and HM-sensitised mice, according to qPCR, indicating Th1/Th2 and Treg/Th17 imbalances. The CM-sensitised group had a higher proportion of IL-4- and IL-17A-producing CD3+ T cells but a lower proportion of IFN-γ- and IL-10-producing CD3+ T cells compared with the GM- and HM-sensitised groups, confirming the Th1/Th2 and Treg/Th17 imbalances. In conclusion, GM and HM were less allergenic than CM in mice as a result of a shift in the Th1/Th2 and Treg/Th17 imbalances; however, HM was less allergenic than GM and can be used as an alternative milk to develop infant formulas for children with CMA.

The effect of atmospheric cold plasma treatment on the antigenic properties of bovine milk casein and whey proteins.

Casein, ß-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, ß-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the ß-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of ß-sheet and ß-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of ß-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors.

Denaturation of selected bioactive whey proteins during pasteurization and their ability to modulate milk immunogenicity.

This research communication relates to the hypothesis that the consumption of raw or unprocessed cow's milk contributes to lowered prevalence of allergies. Thermal pasteurization of bovine milk can result in denaturation of minor whey proteins and loss of their bioactivity. Denaturation of bovine serum albumin (BSA), immunoglobulin G (IgG) and lactoferrin (LF) in skim milk was studied under different temperature (72, 75 or 78°C) and time (0-300 s) combinations. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that denaturation of all 3 proteins occurred at 72°C and progressed with increase in temperature and holding time. About 59% of LF and 12% of IgG denatured under high-temperature short-time (72°C/ 15 s) pasteurization, while BSA was least impacted. To assess modulation of milk immunogenicity, secretion of selected T helper (Th)-type cytokines by human peripheral blood mononuclear cells (PBMCs) was studied in vitro in response to different concentrations of BSA (0.4-1.0 mg/ml) and IgG (0.8-1.6 mg/ml) in unheated skim milk. Addition of IgG at 1.6 mg/ml induced a prominent Th1-skewed cytokine profile that may not trigger a Th2-skewed allergic reaction. BSA did not appear to modulate milk immunogenicity to any significant extent.

Effect of Low-Immunogenic Yogurt Drinks and Probiotic Bacteria on Immunoreactivity of Cow's Milk Proteins and Tolerance Induction-In Vitro and In Vivo Studies.

There is no effective therapy for milk allergy. The role of lactic acid bacteria (LAB) and probiotics in protection against allergy-related outcomes is still under investigation. The aim of the study was to evaluate the immunomodulative and therapeutic potential of yogurt drinks in cow's milk allergy (CMA) management. We compared immunoreactivity of α-casein (α-CN), ß-casein (ß-CN), κ-casein (κ-CN), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) in 27 yogurt drinks fermented with different basic yogurt cultures, or yogurt cultures enriched with Lactobacillus plantarum and/or Bifidobacterium lactis strains, by competitive ELISA assay. Drinks with the lowest antigenic potential were used as allergoids for CMA therapy. BALB/c mice were sensitized via intraperitoneal injection of α-CN + ß-LG mixture with aluminum adjuvant, and gavaged with increasing doses of selected low-immunogenic drinks (YM-basic, or YM-LB-enriched with L. plantarum and B. lactis) to induce tolerance. Milk- or phosphate-buffered saline (PBS)-dosed mice served as controls. Compared to milk, the immunoreactivity of proteins in drinks increased or decreased, depending on the bacterial sets applied for fermentation. Only a few sets acted synergistically in reducing immunoreactivity. The selected low-immunogenic drinks stimulated allergic mice for profiling Th2 to Th1 response and acquire tolerance, and the effect was greater with YM-LB drink, which during long-lasting interventional feeding strongly increased the secretion of regulatory cytokines, i.e., IL-10 and TGF-ß, and IgA and decreased IL-4, IgE, and anti-(α-CN + ß-LG) IgG1. The studies revealed variations in the potency of yogurt bacteria to change allergenicity of milk proteins and the need for their strict selection to obtain a safe product for allergy sufferers. The YM-LB drink with reduced antigenic potential may be a source of allergoids used in the immunotherapy of IgE mediated CMA, but further clinical or volunteer studies are required.

A multi-center assessment to compare residual allergenicity of partial hydrolyzed whey proteins in a murine model for cow's milk allergy - Comparison to the single parameter guinea pig model.

INTRODUCTION: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas. AIM: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers. METHODS: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study. RESULTS: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers. CONCLUSIONS: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account.

Thermally-Induced Lactosylation of Whey Proteins: Identification and Synthesis of Lactosylated ß-lactoglobulin Epitope.

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, ß-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a ß-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Whey milk proteomics from Schistosoma mansoni-infected mice reveals proteins involved in immunomodulation of the offspring.

Milk from schistosomotic mothers can modulate the immune response of their offspring. However, its characterization and potential of modulating immunity has not yet been fully elucidated. Thus, the aim of this study was to evaluate whey proteins from the milk of Schistosoma mansoni-infected mice in order to identify the fractions which can act as potential immunomodulatory tools. For this, we did a mass spectrometry (nanoUPLC-MSE) analysis to characterize the proteomic profile of milk from infected (MIM) and non-infected mice (MNIM). It was possible to identify 29 differentially expressed proteins: 15 were only found in MIM, 10 only found in MNIM, and 4 were downregulated in MIM group. Gene Ontology (GO), pathway enrichment analysis, and protein-protein interaction (PPI) analyses indicated differentially expressed proteins linked to biological processes and pathways in MIM group such as the following: fructose 1,6-biphosphate metabolic and glycolytic processes, glucose metabolism, and neutrophil degranulation pathways. The downregulated and unique proteins identified in MNIM group were involved in the positive regulation of B cell activation and receptor signaling pathway, in the innate immune response, complement activation, and phagocytosis. The present findings revealed a protein profile that may be involved in the activation and deactivation of the offspring's immune system in the long term, conferring a protective character due to the previous contact with milk from infected mothers.

Cow's milk allergy prevention and treatment by heat-treated whey-A study in Brown Norway rats.

BACKGROUND: Food processing, including heat-treatment, can affect protein structure and stability, and consequently affect protein immunogenicity and allergenicity. A few studies have shown that structural changes induced by heat-treatment impact the intestinal protein uptake and suggest this as a contributing factor for altered allergenicity. OBJECTIVE: To investigate the impact of heat-treatment of a whey-based protein product on allergenicity and tolerogenicity as well as on intestinal uptake in various animal models. METHODS: Immunogenicity and sensitizing capacity of the heat-treated whey product were compared to that of the unmodified product by intraperitoneal and oral exposure studies, while tolerogenic properties were assessed by oral primary prevention and desensitization studies in high-IgE responder Brown Norway rats. RESULTS: Heat-treatment of whey induced partial protein denaturation and aggregation, which reduced the intraperitoneal sensitizing capacity but not immunogenicity. In contrast, heat-treatment did not influence the oral sensitizing capacity, but the heat-treated whey showed a significantly reduced eliciting capacity compared to unmodified whey upon oral challenge. Heat-treatment did not reduce the tolerogenic properties of whey, as both products were equally good at preventing sensitization in naïve rats as well as desensitizing already sensitized rats. Results from inhibitory ELISA and immunoblots with sera from sensitized rats demonstrated that heat-treatment caused an altered protein and epitope reactivity. Protein uptake studies showed that heat-treatment changed the route of uptake with less whey being absorbed through the epithelium but more into the Peyer's patches. CONCLUSION AND CLINICAL RELEVANCE: These results support the notion that the physicochemical features of proteins affect their route of uptake and that the route of uptake may affect the protein allergenicity. Furthermore, the study highlights the potential for heat-treatment in the production of efficient and safe cow's milk protein-based products for prevention and treatment of cow's milk allergy.

A free amino acid-based diet partially prevents symptoms of cow's milk allergy in mice after oral sensitization with whey.

BACKGROUND: Amino acid-based formulas (AAFs) are used for the dietary management of cow's milk allergy (CMA). Whether AAFs have the potential to prevent the development and/or symptoms of CMA is not known. OBJECTIVE: The present study evaluated the preventive effects of an amino acid (AA)-based diet on allergic sensitization and symptoms of CMA in mice and aimed to provide insight into the underlying mechanism. METHODS: C3H/HeOuJ mice were sensitized with whey protein or with phosphate-buffered saline as sham-sensitized control. Starting 2 weeks before sensitization, mice were fed with either a protein-based diet or an AA-based diet with an AA composition based on that of the AAF Neocate, a commercially available AAF prescribed for the dietary management of CMA. Upon challenge, allergic symptoms, mast cell degranulation, whey-specific immunoglobulin levels, and FoxP3+ cell counts in jejunum sections were assessed. RESULTS: Compared to mice fed with the protein-based diet, AA-fed mice had significantly lower acute allergic skin responses. Moreover, the AA-based diet prevented the whey-induced symptoms of anaphylaxis and drop in body temperature. Whereas the AA-based diet had no effect on the levels of serum IgE and mucosal mast cell protease-1 (mMCP-1), AA-fed mice had significantly lower serum IgG2a levels and tended to have lower IgG1 levels (P = .076). In addition, the AA-based diet prevented the whey-induced decrease in FoxP3+ cells. In sham-sensitized mice, no differences between the two diets were observed in any of the tested parameters. CONCLUSION: This study demonstrates that an AA-based diet can at least partially prevent allergic symptoms of CMA in mice. Differences in FoxP3+ cell counts and serum levels of IgG2a and IgG1 may suggest enhanced anti-inflammatory and tolerizing capacities in AA-fed mice. This, combined with the absence of effects in sham-sensitized mice indicates that AAFs for the prevention of food allergies may be an interesting concept that warrants further research.

Epicutaneous immunogenicity of partially hydrolyzed whey protein evaluated using tape-stripped mouse model.

BACKGROUND: Hydrolyzed cow's milk protein formulas are widely used for infants with a history or risk of cow's milk allergy. Based on the current theory that food allergen sensitization occurs via the skin, we investigated the epicutaneous immunogenicity of partially hydrolyzed whey proteins, which are ingredients in infant formulas. METHODS: BALB/c mice were exposed epicutaneously to whey protein concentrate (WPC) or partial whey protein hydrolysates (PWH1 or PWH2) on tape-stripped skin. Sensitization was assessed by evaluating serum ß-lactoglobulin (ß-LG)-specific antibodies, basophil activation, and cytokine production from ß-LG-stimulated lymphoid cells. The anaphylaxis reaction was evaluated by measuring the rectal temperature and plasma level of mouse mast cell protease-1 after oral ß-LG challenge. Immune cell accumulation in the skin was also analyzed. RESULTS: Substantive sensitization and ß-LG-induced anaphylaxis reaction were observed in WPC-exposed mice, whereas no significant changes were observed in PWH1- or PWH2-exposed mice. The basophil and eosinophil counts increased in WPC-exposed murine skin, not but in PWH1- or PWH2-exposed mice. CONCLUSION: The epicutaneous immunogenicity of PWH1 and PWH2 is markedly decreased, which may reduce the risk of allergen sensitization. Further studies are required to investigate the clinical value of these partial hydrolysates for high-risk infants.

Effect of ultrasonic-ionic liquid pretreatment on the hydrolysis degree and antigenicity of enzymatic hydrolysates from whey protein.

With the aim to reduce the antigenicity of whey protein hydrolysate in milk, the pretreatment method of coupling ultrasonic and ionic liquid (US-IL) and further enzymatic treatments were studied. Papain and alcalase were found to be suitable for ultrasonic-ionic liquid pretreatment. After ultrasound-ionic liquid treatment, the antigenic decline rates of ALA and BLG upon alcalase hydrolysis were 82.82% and 88.01%, and that of the papain hydrolysis was 81.87% and 88.46%, respectively. Upon ultrasonic-ionic liquid pretreatment, the molecular weight of whey protein did not change significantly, but the small molecular weight proportion of components in the enzymatic hydrolysate obviously increased. The findings showed that combining with US-IL pretreatment for further protease hydrolysis of whey proteins, the hydrolysate can be used in order to produce hypoallergenic bovine whey proteins.

Tolerogenic Effect Elicited by Protein Fraction Derived From Different Formulas for Dietary Treatment of Cow's Milk Allergy in Human Cells.

Several formulas are available for the dietary treatment of cow's milk allergy (CMA). Clinical data suggest potentially different effect on immune tolerance elicited by these formulas. We aimed to comparatively evaluate the tolerogenic effect elicited by the protein fraction of different formulas available for the dietary treatment of CMA. Five formulas were compared: extensively hydrolyzed whey formula (EHWF), extensively hydrolyzed casein formula (EHCF), hydrolyzed rice formula (HRF), soy formula (SF), and amino acid-based formula (AAF). The formulas were reconstituted in water according to the manufacturer's instructions and subjected to an in vitro infant gut simulated digestion using a sequential gastric and duodenal static model. Protein fraction was then purified and used for the experiments on non-immune and immune components of tolerance network in human enterocytes and in peripheral mononuclear blood cells (PBMCs). We assessed epithelial layer permeability and tight junction proteins (occludin and zonula occludens-1, ZO-1), mucin 5AC, IL-33, and thymic stromal lymphopoietin (TSLP) in human enterocytes. In addition, Th1/Th2 cytokine response and Tregs activation were investigated in PBMCs from IgE-mediated CMA infants. EHCF-derived protein fraction positively modulated the expression of gut barrier components (mucin 5AC, occludin and ZO-1) in human enterocytes, while SF was able to stimulate the expression of occludin only. EHWF and HRF protein fractions elicited a significant increase in TSLP production, while IL-33 release was significantly increased by HRF and SF protein fractions in human enterocytes. Only EHCF-derived protein fraction elicited an increase of the tolerogenic cytokines production (IL-10, IFN-γ) and of activated CD4+FoxP3+ Treg number, through NFAT, AP1, and Nf-Kb1 pathway. The effect paralleled with an up-regulation of FoxP3 demethylation rate. Protein fraction from all the study formulas was unable to induce Th2 cytokines production. The results suggest a different regulatory action on tolerogenic mechanisms elicited by protein fraction from different formulas commonly used for CMA management. EHCF-derived protein fraction was able to elicit tolerogenic effect through at least in part an epigenetic modulation of FoxP3 gene. These results could explain the different clinical effects observed on immune tolerance acquisition in CMA patients and on allergy prevention in children at risk for atopy observed using EHCF.

Reduced IgE and IgG antigenic response to milk proteins hydrolysates obtained with the use of non-commercial serine protease from Yarrowia lipolytica.

The aim of the study was to investigate the use of serine protease from Yarrowia lipolytica yeast for reduction of milk proteins allergenicity. Whey protein concentrate (WPC-80), αs-casein and their hydrolysates were analyzed for the capacity to bind IgE and IgG antibodies present in sera from patients with cow milk protein allergy using a competitive ELISA. The hydrolysis of αs-casein and whey protein concentrate contributed to a significant reduction of their immunoreactive epitopes. In case of IgE antibodies, the lowest binding capacity was detected in the 24 h hydrolysates of both proteins in which the inhibition of the reaction was ≤20 and ≤68% for αs-casein and whey protein concentrate respectively. One hour hydrolysis of WPC-80 reduced the protein antigenicity, while the longer time (5 h) might lead to the exposure of new IgE - reactive epitopes.

Influence of whey protein hydrolysis in combination with dextran glycation on immunoglobulin E binding capacity with blood sera obtained from patients with a cow milk protein allergy.

Food protein allergies are a major global concern. Hydrolysis of food proteins reduces their allergenicity, but another novel approach is the covalent attachment of polysaccharides to proteins via the Maillard reaction (i.e., glycation), which blocks some IgE binding epitopes on the protein allergen. We wanted to examine whether enzymatic hydrolysis, combined with glycation, could further reduce IgE binding for people with a cow milk protein allergy. Whey protein isolate (WPI) was hydrolyzed by immobilized trypsin and chymotrypsin to degree of hydrolysis (DH) values of 17 to 27%. Immobilized enzymes were used to avoid heat-treating the hydrolysate (to inactivate the enzymes, because heating could also affect the IgE binding ability of the protein). The resultant whey protein isolate hydrolysates (WPIH) were then glycated with 10-kDa dextran (DX) in aqueous solutions held at 62°C for 24 h. We analyzed the molar mass (MW) of WPIH samples and their corresponding glycates (WPIH-DX) using size-exclusion chromatography with multi-angle laser light scattering. We obtained blood sera from 8 patients who had been diagnosed with a cow milk protein allergy, and we used a composite serum for IgE binding analysis. The average MW values of samples WPIH-1 to WPIH-3 decreased from 11.15, 9.46, and 7.57 kDa with increasing DH values of 18.7, 22.5, and 27.1%. Glycation significantly reduced the high bitterness of the WPIH samples, as assessed by a trained sensory panel. The WPIH-DX glycates had significantly reduced WPI-specific IgE binding capacity compared to WPI or unglycated WPIH; we found an almost 99% reduction in IgE binding for the WPIH-DX glycate made from WPIH with a DH value of 27.1%. Hydrolysis of WPI followed by glycation with DX via the Maillard reaction significantly decreased the allergenicity of whey proteins.

Reducing antigenicity of bovine whey proteins by Kluyveromyces marxianus fermentation combined with ultrasound treatment.

This work investigated the reduction of bovine whey proteins antigenicity by ultrasonic pretreatment and microbial fermentation. Firstly, bovine whey proteins was pretreated by ultrasonic techniques, and its secondary structure was detected by circular dichroism. The pretreated whey proteins was used as the fermentation substrate by Kluyveromyces marxianus for microbial transformation. The single factor design and Box-Behnken Design (BBD) were carried out with the aim to optimize culture temperature, initial pH, inoculum volume and rotation speed. After optimization process, culture temperature, initial pH, inoculum volume and rotation speed were determined. Under culture temperature 35 °C, pH 7.25, inoculum level 10% and shaking speed 150 rpm, the α-LA and ß-LG antigenicity in bovine whey proteins were reduced by 29% and 53%, respectively. The findings showed that combined with microbial fermentation for hydrolysis of whey proteins, ultrasonic pretreatment can be used in order to produce hypoallergenic bovine whey proteins.

Effect of chlorogenic acid covalent conjugation on the allergenicity, digestibility and functional properties of whey protein.

We investigated the allergenicity, digestibility and functional properties of whey protein isolate (WPI) after covalent conjugation with chlorogenic acid (CHA). The covalent conjugation of CHA may cause an unfolded protein structure. The WPI-CHA conjugate showed lower IgE binding capacity but higher intestinal digestibility than unmodified WPI. Furthermore, after digestion, the IgE binding capacity of ß-lactoglobulin and α-lactoalbumin was lower in the digested WPI-CHA conjugate than digested WPI. Moreover, the solubility, emulsifying activity, foaming properties and antioxidant capacity of WPI were enhanced by covalent conjugation of CHA. Covalent conjugation with CHA might reduce the allergenicity in vitro of WPI by improving the functional properties of the protein.

Confirmed Hypoallergenicity of a Novel Whey-Based Extensively Hydrolyzed Infant Formula Containing Two Human Milk Oligosaccharides.

BACKGROUND: We sought to determine whether an extensively hydrolyzed formula (EHF) supplemented with two human milk oligosaccharides (HMO) was tolerated by infants with cow's milk protein allergy (CMPA). METHODS: A whey-based EHF (Test formula) containing 2'fucosyl-lactose (2'FL) and lacto-N-neotetraose (LNnT) was assessed for clinical hypoallergenicity and safety. The Control formula was a currently marketed EHF without HMO. Children with CMPA, aged 2 months to 4 years, were assessed by double-blind, placebo-controlled food challenges (DBPCFC) to both formulas, in randomized order. If both DBPCFC were negative, subjects participated in a one-week, open food challenge (OFC) with the Test formula. Symptoms and adverse events were recorded. Hypoallergenicity was accepted if at least 90% (with 95% confidence intervals) of subjects tolerated the Test formula. RESULTS: Of the 82 children with CMPA that were screened, 67 (intention-to-treat [ITT] cohort-mean age 24.5 ± 13.6 months; range 2-57; 45 [67.2%] male) were randomized to receive either the Test or the Control formula during the first DBPCFC. Of these, 64 children completed at least one DBPCFC (modified intention-to-treat [mITT] cohort). Three children were excluded due to protocol deviations (per protocol [PP] cohort; n = 61). There was one allergic reaction to the Test, and one to the Control formula. On the mITT analysis, 63 out of 64 (98.4%; 95% CI lower bound 92.8%), and on the PP analysis 60 out of 61 (98.4%; 95% CI lower bound 92.5%) participants tolerated the Test formula, confirming hypoallergenicity. CONCLUSION: The whey-based EHF supplemented with 2'FL and LNnT met the clinical hypoallergenicity criteria and can be recommended for the management of CMPA in infants and young children.