Glycation of the Major Milk Allergen β‐Lactoglobulin Changes Its Allergenicity by Alterations in Cellular Uptake and Degradation

1 Scope

During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β‐lactoglobulin (BLG), in their interactions with cells crucially involved in allergy.

2 Methods and results

BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T‐cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco‐2 monolayer. Uptake of glycated BLG by bone marrow–derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor‐mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4⁺ T‐cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG‐specific IgE sensitized basophil cells.

3 Conclusions

This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy.     

Effects of orally administered immunodominant T-cell epitope peptides on cow's milk protein allergy in a mouse model

Immunodominant T-cell epitopes have been reported to induce oral tolerance against egg and pollen allergy. However, whether immunodominant T-cell epitopes have effects on cow's milk allergy is unknown. In this study, two T-cell dominant epitopes of β-lactoglobulin (BLG) were used in pre-sensitized Balb/c mice. Female mice of 3 weeks old were randomly assigned to four experimental groups (n = 18/group). Mice were intraperitoneally (i.p.) sensitized with 3 weekly doses of BLG as well as alum for 3 weeks starting at 4 weeks of age. One week after the final sensitization, mice were gavaged with T-cell dominant peptides for 4 weeks (1 mg/mouse/d and 3d/week). After mice were orally challenged with BLG, clinical and immunological parameters were examined. Compared to untreated control mice, both peptide treated groups had lower hypersensitivity scores with unchanged rectal temperatures, but had similar BLG-specific serum immunoglobulin levels and Treg cell populations. The two T-cell dominant peptides used in our therapeutic study failed to induce CD25⁺ Foxp3⁺ regulatory T cells and reduce allergen-specific systemic markers in BLG-sensitized mice. However, clinical signs for allergic reactions were alleviated in peptide treated mice. Further studies that focus not only on Treg-mediated tolerance but also on IgG, B cells and other T-cell assisted tolerance development mechanisms are required to elucidate the allergy improving effect of these peptides.     

Foaming characteristics of chemical and enzymatic hydrolysates of bovine β‐lactoglobulin

The foaming properties of bovine β‐lactoglobulin (BLG( BNPS‐skatole (2‐(2′‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine) (BNPS( trypsin (T) and pepsin in 25, 30 and 40% ethanol (P25, P30, P40) hydrolysates were investigated in the 0.2 to 1 mg/ml range. Foaming capacity and foam stability were assessed in terms of drained liquid volume and foam volume. Foam texture was analyzed from video images obtained during foam decay. The foaming capacity of BNPS, P30 and P40 was similar to that of BLG and greater than that of T or P25. All hydrolysates except BNPS were less stable than BLG at all concentrations tested. This result was insured by texture analysis. Principal component analysis confirmed the distribution of the samples into three groups based on their increasing stability: (i): P25, (ii): P30 and P40, and (iii): BLG and BNPS. Tryptic hydrolysate had the poorest foaming properties. The results are considered in relation to the molecular characteristics of the peptides, particularly their size and hydrophobicity.     

Emulsification of chemical and enzymatic hydrolysates of β‐lactoglobulin: characterization of the peptides adsorbed at the interface

Bovine β‐Lactoglobulin (BLG) was cleaved by BNPS‐skatole (2‐(2′‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine( trypsin, or pepsin in 40% ethanol before emulsification with hexadecane in order to characterize the peptides active at the interfaces. The total digests and the different phases obtained after emulsification were analyzed by RP‐HPLC to separate the peptides according to their gradual order on a hydrophilicity‐to‐hydrophobicity scale. In each case, hydrophobic peptides were recovered in the creamed phase and characterized by mass spectrometry and sequencing. After tryptic hydrolysis, short peptides were identified at the interfacial layer as fragments S₂₁–L₃₂, V₄₁–L₅₇, V₄₁–K₆₀, and W₆₁–K₇₀linked to L₁₄₉–I₁₆₂ by a C₆₆–C₁₆₀bond. It indicates that the hydrophilic/hydrophobic distribution of the amino acids in the sequence of the fragments is more relevant to adsorption than the length of the peptide. BNPS‐skatole and peptic hydrolysis produced larger hydrophobic peptides which were also recovered in the creamed phase of the emulsion and characterized.     

Allergenicity of acidic peptides from bovine β-lactoglobulin is reduced by hydrolysis with Bifidobacterium lactis NCC362 enzymes

Cow milk proteins, particularly β-lactoglobulin (BLG) are a leading cause of food allergy in children. In vivo, gastrointestinal hydrolysis of milk proteins decreases their allergenicity, but the intestinal microbiota also contributes to their breakdown. This study investigated the effect of hydrolysis of BLG-derived tryptic-chymotryptic (TC) peptides by Bifidobacterium lactis NCC362 enzymes, on their allergenicity. B. lactis enzymes have been shown to hydrolyze both acidic and basic peptide fractions isolated from tryptic-chymotryptic hydrolysate, but not whole proteins. The IgE binding capacity of acidic TC peptides was reduced by B. lactis peptidases-driven hydrolysis. This is partly explained by the breakdown of known allergenic peptides. Moreover, the resulting peptide fragments significantly up-regulated IFN-γ and IL-10 production and down-regulated IL-4 secretion by murine splenocytes. These results indicate that B. lactis NCC362 could be a potential probiotic for preventing cow's milk allergy through hydrolysis of the allergenic portion of BLG and the release of peptides which down-regulate the allergic immune response, but further studies using living bacteria are needed to confirm these data.     

Allergenic and immunogenic potential of cow's milk β-lactoglobulin and caseins evidenced without adjuvant in germ-free mice

Scope: In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ‐free (GF) mice exhibit Th2‐polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant. Methods and results: β‐lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward κ‐ and αS1‐caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG‐reactivation, IL‐4, IL‐5, IL‐13 and IFN‐γ productions by splenocytes were higher in GF mice than in CV mice. Heat‐treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat‐treatment increased protein immunogenicity and led to the production of anti‐BLG and anti‐κ‐casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL‐4, IL‐5 and IL‐13 in BLG‐reactivated splenocytes from CV mice. Conclusion: Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins.     

The (193–209) 17‐residues peptide of bovine β‐casein is transported through Caco‐2 monolayer

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β‐casein (β‐CN, 193–209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β‐CN (193–209) peptide across Caco‐2 cell monolayer. In addition, we evaluated the possible routes of the β‐CN (193–209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β‐CN (193–209) peptide resisted the action of brush‐border membrane peptidases, and that it was transported through the Caco‐2 cell monolayer. The main route involved in transepithelial transport of the β‐CN (193–209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight‐junctions could not be excluded. Our results demonstrated the transport of an intact long‐chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.     

Citrus Tachibana Leaf Extract Mitigates Symptoms of Food Allergy by Inhibiting Th2‐Associated Responses

Although the incidence of food allergy continues to rise, there have been no effective therapeutic strategies. Citrus fruits contain a number of bioactive flavonoids with immune‐regulatory functions. The objective of this study was to determine whether Citrus tachibana (fruit body with peel, leaves, and branch) can protect against the development of food allergy and the mechanism behind it, and to identify the active compound(s) responsible. We found that C. tachibana leaf extract (CLE) mitigated ovalbumin (OVA)‐induced food allergy symptoms including increased rectal temperature, diarrhea, and anaphylaxis. This mitigation was likely due to CLE‐mediated decreases in cytokine release from T‐helper 2 cells (Th2 cells) in mesenteric lymph nodes. Moreover, higher levels of CLE attenuated systemic Th2 cell–mediated responses in mouse splenocytes sensitized with OVA+Alum. This was evidenced by CLE‐mediated reductions in Th2 cytokine release, including interleukin (IL)‐4, IL‐5, and IL‐13, but not the Th1 cytokines IL‐12 and interferon (IFN)‐γ, which was attributable to decreased gene expression levels. We also identified kaempferol as the most potent compound for reducing Th2‐associated responses in splenocytes. The findings of this study suggest that CLE suppresses Th2‐cell–mediated immune responses, contributing to alleviation of food allergy symptoms, and that kaempferol is a flavonoid with potential antiallergenic activity that targets Th2 cell–induced responses.     

Hydrolysate of β-lactoglobulin by Lactobacillus delbrueckii subsp. bulgaricus CRL 656 suppresses the immunoreactivity of β-lactoglobulin as revealed by in vivo assays

Cows' milk allergy, common in young infants, is often solved by the age of five; however, it can persist in some adults. The in vivo immunomodulatory potential of β-lactoglobulin (BLG) hydrolysates obtained with Lactobacillus delbrueckii subsp. bulgaricus CRL656 (H656) was studied. Sera from mice gavaged with H656 showed lower specific BLG-IgE values than those of allergic mice. Secretion of IL-10, INF-γ and IL-6 was increased and that of IL-4 decreased when allergic spleen cells were stimulated with H656. IL-4 secretion was significantly reduced and concentrations of IL-10, IL-17A and IL-6 were increased when H656 was orally administrated. Highest expression of IL-4 and IL-12 was observed for allergic mice and H656 gavaged mice, respectively. H656 immunisation had a positive effect on intestinal mucosa. Overall, H656 displayed an immunomodulating effect, balancing the allergic Th2 response by stimulating Th1 type cytokine secretion. The hydrolysate might promote oral tolerance towards BLG in infant foods.     

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

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

Maillard glycation of β‐lactoglobulin induces conformation changes

Glycation by the Maillard reaction is an ubiquitous reaction of condensation of a reducing sugar with amino groups of proteins, which products could improve the functional and/or biological properties for food and non‐food uses. It can induce structural modifications in proteins, modifying their properties. The aim of this work was to investigate the association behavior and the conformational changes of β‐lactoglobulin (BLG) after its glycation by the Maillard reaction with several alimentary sugars (arabinose, galactose, glucose, lactose, rhamnose and ribose). Protein samples were heated in the presence or in the absence (heated control) of different sugars during 3 days at 60°C. Glycation induced oligomerization of BLG monomers. Depending on the reactivity of the sugar, the population of produced oligomers showed smaller or greater heterogeneity in molecular masses. Analysis of modified BLG by circular dichroism and by its susceptibility to pepsinolysis showed that the conditions of heating used did not significantly alter the conformation of BLG. Heating of BLG in presence of sugars induced only minor structural modification, when using the less reactive sugars such as lactose and rhamnose. It was, however, at the origin of major three‐dimensional destructuring in the case of the more reactive sugars such as arabinose and ribose. Pepsinolysis of glycated BLG did not affect about 62 and 35% of the protein molecules modified with lactose or rhamnose, and arabinose or ribose, respectively. The increase of susceptibility of glycated BLG to pepsinolysis could be related to the alteration of the conformation of the protein when glycation was performed with highly reactive sugars, as observed by circular dichroism and calorimetry analysis.     

The effect of pH and calcium ion on rheological behaviour of β‐lactoglobulin‐basil seed gum mixed gels

Effect of pH (4.5–7.5) and Ca²⁺ (0.01–0.5 m ) on gelation of single and mixed systems of 10% β‐lactoglobulin (BLG) and 1% basil seed gum (BSG) was investigated. The gelling point of BLG and BSG gels was strongly pH‐dependent, and stiffer gels formed at higher pH. The BLG gels were formed upon heating to 90 °C and reinforced on cooling to 20 °C; however, the gelation of BSG occurred at temperatures below 70 °C. By increasing Ca²⁺ concentration, storage modulus of BLG and BSG gels were increased, although pH had a greater effect than Ca²⁺. In contrast, mixed systems showed two distinct types of behaviour: BLG gel formation and BSG network, suggesting that phase‐separated gels were formed. In addition, higher strength was obtained for BLG‐BSG mixture at higher Ca²⁺ concentration.     

Effect of prior dietary exposure to cows' milk protein on antigen-specific and nonspecific cellular proliferation in mice

The impact of dietary components on the immune system is gaining increased attention in the effort to develop safe food products, some even with health-promoting potential, as well as to improve the basic understanding of the immunomodulatory potential of common food components. In such studies, which are mainly based on experiments in vitro, it is important to be able to differentiate nonspecific activation of immune cells induced by dietary components from ex vivo restimulation of antigen-specific cells that might be present in cell cultures owing to prior dietary exposure to the antigens in cell donors. Focusing on the immunostimulatory potential of cows' milk proteins and peptides, we studied the impact of prior dietary exposure to cows' milk on proliferation of murine immune cells upon ex vivo stimulation with bovine milk proteins. Nonspecific proliferation induced by beta-casein peptides was further assessed on cells from mice bred on a cows'-milk-free diet. Regarding the dietary effect, we found that prior oral intake of cows' milk proteins affected cell proliferation induced by culturing with cows' milk proteins in vitro, as spleen cells from mice fed a milk-containing diet showed a significantly greater proliferative response than did cells from mice bred on a cows'-milk-free diet. Studies of immune enhancing potentials of beta-casein peptides showed that some peptides stimulate proliferation of immune cells nonspecifically. In conclusion, these findings stress the importance of employing immune cells from mice unexposed to cows' milk for studies of the immunomodulating capacity of cows' milk proteins and peptides, in order to rule out the interference caused by antigen-specific immune responses. By using such cells, we here show that some beta-casein peptides possess the potential to induce proliferation in immune cells in a nonspecific manner.     

A nondigestible saccharide,fructooligosaccharide, increases the promotive effect of a flavonoid, α‐glucosyl‐isoquercitrin,on glucagon‐like peptide 1 (GLP‐1) secretion in rat intestine and enteroendocrine cells

This study conducted in vivo and in situ experiments with rats to investigate the glucagon‐like peptide‐1 (GLP‐1) secretion in response to oral or ileal administration of α‐glucosyl‐isoquercitrin (20–40 mmol in 2 mL; Q3G), fructooligosaccharides (200 mmol in 2 mL; FOS) and Q3G + FOS. Direct effects on GLP‐1‐producing l ‐cells were also examined by an in vitro study using a murine enteroendocrine cell line, GLUTag. To evaluate the plasma GLP‐1 level, blood samples from jugular cannula for in vivo and portal cannula for in situ experiments were obtained before and after administration of Q3G, FOS, or Q3G + FOS. We found tendencies for increases but transient stimulation of GLP‐1 secretion by Q3G in in vivo and in situ experiments. Although FOS alone did not have any effects, Q3G + FOS enhanced and prolonged high plasma GLP‐1 level in both experiments. In addition, application of Q3G on GLUTag cells stimulated GLP‐1 secretion while FOS enhanced the effect of Q3G. Our results suggest that Q3G + FOS possess the potential for the management or prevention of Type 2 diabetes mellitus (T2DM) by enhancing and prolonging the GLP‐1 secretion via direct stimulation of GLP‐1 producing l ‐cell.     

Two-dimensional gel electrophoresis of proteins and peptides in bovine milk

Proteins and peptides in bovine milk and colostrum were separated by two-dimensional gel electrophoresis (2-DE). The proteins were separated in the first dimension by isoelectric focusing in the pH ranges 4–7 and 6–11, and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis on 12.5% T homogeneous gels. The peptides were separated in the pH range 3–10 in the first dimension, while 15% T homogeneous gels were used in the second dimension. 2-DE evaluation of samples with various somatic cell counts showed an increased number of peptides with increased cell count. 2-DE analysis of samples heated at 65 and 85 °C for 30 min showed a decrease in the number of proteins and peptides with increased temperature. Colostrum exhibited a peptide pattern in which the intensity and number of spots decreased with time postpartum. These results show that 2-DE is a useful tool to detect variations of proteins and peptides in milk.     

Immunomodulating peptides for food allergy prevention and treatment

Among the most promising strategies currently assayed against IgE-mediated allergic diseases stands the possibility of using immunomodulating peptides to induce oral tolerance toward offending food allergens or even to prevent allergic sensitization. This review focuses on the beneficial effects of food derived immunomodulating peptides on food allergy, which can be directly exerted in the intestinal tract or once being absorbed through the intestinal epithelial barrier to interact with immune cells. Food peptides influence intestinal homeostasis by maintaining and reinforcing barrier function or affecting intestinal cell-signalling to nearby immune cells and mucus secretion. In addition, they can stimulate cells of the innate and adaptive immune system while supressing inflammatory responses. Peptides represent an attractive alternative to whole allergens to enhance the safety and efficacy of immunotherapy treatments. The conclusions drawn from curative and preventive experiments in murine models are promising, although there is a need for more pre-clinical studies to further explore the immunomodulating strategy and its mechanisms and for a deeper knowledge of the peptide sequence and structural requirements that determine the immunoregulatory function.     

Removal of N-terminal peptides from β-lactoglobulin by proteolytic contaminants in a commercial phenol oxidase preparation

The use of enzymes may improve the functional properties of various food ingredients. The aim of this study was to examine the effects of proteolytic contaminants in phenol oxidases on β-lactoglobulin (BLG). In the presence of Trametes versicolor laccase and Agaricus bisporus tyrosinase, both variants of BLG (A and B) underwent removal of a peptide from the N-terminus. The truncated forms were more susceptible to digestion by pepsin. The truncation of BLG resulted from contaminating proteases and not due to the action of phenol oxidases. The removal of N-terminal peptides proceeded quickly, while the rest of the globular protein remained resistant to proteolysis for up to 3 h. In the case of the application of enzymes in food bioprocessing, it may be important to carefully monitor the effects of contaminating proteases in enzyme preparations used.     

Relationship between composition and stability of bovine milk lysozyme.

The amino acid analysis, peptide mapping, and heat stability of bovine milk lysozyme are presented. The bovine milk lysozyme molecule contains approximately 154 amino acids and is strikingly different in amino acid content from human milk lysozyme and egg white lysozyme. Tryptic hydrolysis yielded 26 peptides, all of which are unique from tryptic peptides of human milk lysozyme and egg white lysozyme. In addition, bovine milk lysozyme was more heat stable than human milk lysozyme at pH 4.0 but more labile at pH 7.0 and 9.0. Possible explanations for the differences in heat stability are discussed.     

Preparation,Bioavailability, and Mechanism of Emerging Activities of Ile‐Pro‐Pro and Val‐Pro‐Pro

Ile‐Pro‐Pro and Val‐Pro‐Pro are two most well‐known food‐derived bioactive peptides, initially identified as inhibitors of angiotensin I‐converting enzyme (ACE) from a sample of sour milk. These two peptides were identified in fermented and enzymatic hydrolyzed cow and non‐cow (that is, goat, sheep, buffalo, yak, camel, mare, and donkey) milk, as well as sourdough prepared from wheat, rye, and malt. Similar to other bioactive peptides, bioavailability of these peptides is low (about 0.1%), reaching picomolar concentration in human plasma; they showed blood pressure lowering activity in animals and in human, via improved endothelial function, activation of ACE2, and anti‐inflammatory property. Emerging bioactivities of these two peptides toward against metabolic syndrome and bone‐protection received limited attention, but may open up new applications of these peptides as functional food ingredients. Further studies are warranted to determine the best source as well as to identify novel enzymes (particularly from traditional fermented milk products) to improve the efficiency of production, to characterize possible peptide receptors using a combination of omics technology with molecular methods to understand if these two peptides act as signal‐like molecules, to improve their bioavailability, and to explore new applications based on emerging bioactivities.