Milk formula with specific beta-lactoglobulin peptides

Abstract

FIELD: immunology.SUBSTANCE: compositions for use in the induction of oral immune tolerance, and / or for use in prevention, and / or for reducing the risk of developing oral immune intolerance, and / or for improving or enhancing oral tolerance to cow's milk protein. The inventions are based on the use of Bifidobacterium and cow's milk protein hydrolysate containing the amino acid sequence SEQ ID NO: 1 or 4 and at least one peptide having the amino acid sequence SEQ ID NO: 2–4, for obtaining a composition for the prophylaxis, prevention or reduction of the risk of developing oral immune intolerance to milk protein.EFFECT: obtaining a composition for improving the induction of oral immune tolerance to cow's milk protein.22 cl, 2 dwg, 10 tbl, 8 ex

Description

[0001] FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The invention relates to the field of milk formulas containing hydrolyzed cow's milk protein for infants who suffer from or are at risk of developing an allergy to cow's milk protein.

[0003] STATE OF THE ART

[0004] Food proteins are exposed to the immune system in the gastrointestinal tract. As a default immune response, oral tolerance to these harmless food proteins develops. The development of oral immune tolerance is largely evident in infants, who are exposed to numerous harmless proteins at an early age through exposure to food proteins. If this natural response to harmless proteins is disrupted, food allergies will develop. It is generally recognized that the prevalence of food allergies has been increasing in recent decades, especially in Western countries. In infancy, cow's milk allergy is the most common food allergy: currently, 2-5% of infants are allergic to cow's milk, and a higher percentage is at risk of developing allergies.

[0005] For infants with cow's milk protein allergies, there are infant formulas available that contain highly hydrolyzed proteins (deep protein hydrolysate) or even only free amino acids as a nitrogen source. These formulas contain virtually no allergen-causing proteins or peptides, if any.

[0006] Infants born to parents one or both of whom suffer from an atopic disease, or who have one or more siblings with an atopic disease, have a higher risk of developing food protein allergies. For this group, in addition to the preferred breastfeeding, hypoallergenic infant formulas containing partially hydrolyzed proteins (partially hydrolyzed proteins) are available. These partially hydrolyzed proteins have reduced allergenicity. This approach has been proven effective in preventing sensitization to native proteins present in adapted infant formulas. Typically, the degree of protein hydrolysis is less than that of highly hydrolyzed proteins for infants with pre-existing allergies.Such infant formulas not only reduce the risk of allergic reactions by preventing protein sensitization, but also support the natural development of oral immune tolerance to the intact protein. This allows the native protein to be introduced into the diet later with a reduced risk of allergic reactions.

[0007] Strict allergen avoidance for the prevention and treatment of allergen sensitivity has been recommended for many decades. The emphasis on complete avoidance of food allergens may inadvertently contribute to the rise in food allergies in recent decades. The best way to prevent or treat allergies is to develop strategies that promote oral tolerance induction to allergens, such as oral immunotherapy or presentation of food allergens in a form that promotes the induction of natural tolerance mechanisms (Allen et al, 2009, Pediatr Allerg Immunol 20:415-422).

[0008] EP 2044851 discloses a nutritional composition with partially hydrolyzed milk protein having a degree of hydrolysis of from 15% to 25% and from 50 to 1000 ng TGF-beta per 100 ml, for the primary prevention of allergic reactions to food protein and the prevention of the development of atopic diseases in young mammals.

[0009] EP 0629350 discloses the use of non-allergenic whey protein hydrolysates which are said to be capable of inducing tolerance to cow's milk protein. EP 0827697 discloses the use of whey that has been enzymatically hydrolyzed to produce compositions that induce oral tolerance to cow's milk in susceptible mammals. The whey has an immunological detection level of allergy-causing proteins ≥ 100 times lower than that of non-hydrolyzed whey.

[0011] WO 00 / 42863 discloses a hypoallergenic composition for inducing protein tolerance in infants at risk of developing protein allergy, comprising a non-allergenic base of highly hydrolyzed protein and / or a base of free amino acids, said composition containing as an active substance at least one tolerogenic peptide of an allergy-causing protein.

[0012] WO 2011 / 151059 discloses infant food with partially hydrolyzed proteins and non-digestible oligosaccharides for use in inducing oral tolerance to native food proteins.

[0013] WO 2015 / 090347 relates to identified peptides that are capable of inducing tolerance to cow's milk, especially β-lactoglobulin.

[0014] WO 2017 / 144730 discloses a strategy for achieving desensitization or tolerance induction to milk protein allergens, such as β-lactoglobulin, in humans or animals, comprising preparing and using a composition comprising purified intact expressed milk protein together with one or more purified peptides from said intact milk protein.

[0015] WO 2016 / 0148572 discloses oral immune tolerance to a specific mixture of synthetic beta-lactoglobulin peptides and probiotics.

[0016] However, hypoallergenic compositions from the prior art often achieve their effect by simply preventing the presence of potential allergens—therefore, without solving the underlying problem—and / or by requiring the presence of special components, such as growth promoters or synthetic or purified peptides. Furthermore, the peptides identified in the prior art may be unsuitable for inducing oral immune tolerance in a significant portion of the human population. Therefore, there remains a need for nutrition containing a specific bovine beta-lactoglobulin hydrolysate with specific peptides and components that further enhance oral immune tolerance in subjects at risk of developing or suffering from food allergies, with improved effects on inducing oral immune tolerance to food proteins, in particular cow's milk protein.

[0017] Thus, the technical problem underlying the present invention is to obtain compositions, methods, uses and means for overcoming the above-mentioned disadvantages, in particular for providing improved induction of oral immune tolerance to food proteins in humans, in particular in humans at risk of developing food allergies, in particular in infants, and in particular in infants at risk of developing cow's milk protein allergy.

[0018] SUMMARY OF THE INVENTION

[0019] The inventors promote the assumption in the art that the best way to prevent or treat allergies is to develop strategies that promote the induction of oral tolerance to allergens, such as oral immunotherapy or the presentation of food allergens in a form that promotes the induction of natural tolerance mechanisms (Allen et al, 2009, Pediatr Allerg Immunol 20:415-422)

[0020] The basis of all T cell responses, including the induction of regulatory T cells (Treg), consists of three interrelated processes: T cell receptor activation, costimulation, and cytokine signaling. T cell receptor activation on CD4+ T cells is facilitated by the recognition of specific peptides, called T cell epitopes, presented by MHC class II molecules on antigen-presenting cells. The presence of T cell epitopes in protein hydrolysate is essential for the induction of cow's milk-specific Tregs. Amino acid (AA) #13-48 of mature beta-lactoglobulin (BLG) is of particular interest because synthetic peptides encompassing this region can significantly reduce the acute allergic skin reaction in a mouse model of cow's milk allergy for prophylactic purposes. Overall, this region of BLG appears to be important for the development of whey protein tolerance.

[0021] Using a new method, the researchers found that a specific partially hydrolyzed whey-based infant formula contained specific identical beta-lactoglobulin (BLG) peptides that function as T-cell epitopes to promote the development of oral tolerance to cow's milk protein among a significant portion of the human population, for example, those with different HLA-DRB1 alleles. These specific alleles are particularly relevant because HLA-DR alleles are the predominant MHC class II isotype and play a central role in the selection and activation of CD4+ T cells. HLA-DR is a heterodimeric molecule consisting of the HLA-DRA and HLA-DRB chains. While the HLA-DRA gene is highly conserved in humans, there are multiple HLA-DRB genes. Among them, HLA-DRB1 is the most polymorphic gene and is expressed five times higher than its functional paralogs.

[0022] First, a new liquid chromatography-mass spectrometry (LC-MS) method was developed to identify a limited set of specific BLG-derived peptides naturally present at highest frequencies in whey protein hydrolysate mixtures, with a focus on the AA region #13-48 of mature bovine BLG. The inventors were able to identify a total of thirteen BLG peptides from at least nine amino acids in this region of interest, six of which were identified consistently across different production lots.

[0023] Second, the mixture was subjected to ProImmune ProPresent® antigen presentation assay and MHC class II binding algorithm to identify relevant HLA-DRB1-restricted peptides. These peptides were confirmed to be processed and presented by human dendritic cells. The above peptides were grouped into two groups of unique sequences, i.e., DIQ…DIS (AA #11-30) and AMA…APL (AA #23-39). The BLG fragments of the identified two groups of unique sequences were found to have high binding affinity to the selected HLA-DRB1 alleles. The BLG peptide mixture was found to be the most preferable to ensure coverage of interactions with several HLA-DRB1 alleles commonly present in the human population. In particular, a peptide mixture comprising at least the BLG peptide of SEQ ID NO:5 together with one of the BLG peptides of SEQ ID NO:2, 3 or 4 covers the majority of the HLA-DRB1 alleles examined.Reference is made to examples 2 and 3.

[0024] Third, the sequences of the identified BLG peptides were examined and found to be recognized by cow's milk-specific human T cell lines obtained from various donors suffering from an allergic disease and to induce T cell proliferation / activation. Reference is made to Example 4 and the associated Fig. 2. When tested on these various T cell lines, different patterns of T cell reactivity were observed, and although not every peptide interacted with every cell line, when taken together (specifically, BLG peptide SEQ ID NO: 4 alone or a combination of BLG peptide SEQ ID NO: 1 with one of SEQ ID NO: 2, 3, or 5), the reactivity of each cell line was covered. These results indicate an improved effect of hydrolysates with these identical BLG-derived peptides on the development of oral immune tolerance to whey protein or a reduced risk of developing oral immune tolerance.

[0025] As shown in Example 6, the peptides have a higher number of MHC class II binding domains and therefore an increased probability of being presented to T cells.

[0026] BLG peptides in the AA region No. 13-48 provide a further improvement in the effect on the induction of oral immune tolerance or a further reduction in the risk of developing oral immune tolerance when used together with a lactic acid bacteria strain, together or separately with indigestible oligosaccharides, and therefore, these results also indicate a further improvement in the effect of hydrolyzed protein compositions containing a mixture of such BLG-derived peptides consisting of an amino acid sequence corresponding to the consecutive amino acids of the beta-lactoglobulin protein represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, preferably together with a lactic acid bacteria strain, together or separately with indigestible oligosaccharides (probiotics or synbiotics).

[0027] Furthermore, the attached experimental section demonstrates that the presence of lactic acid bacteria increases the expression of HLA-DR molecules on the surface of dendritic cells (Example 6). Increased HLA-DR expression also indicates improved peptide presentation to T cells, and therefore, the induction of oral immune tolerance.

[0028] LIST OF PREFERRED IMPLEMENTATION OPTIONS

[0029] 1. A nutritional composition comprising a protein hydrolysate from mammalian milk, preferably milk from species of the genus Bos, Bison, Bubalus or Capra, more preferably from the genus Bos, preferably from cow's milk, for use in

[0030] induction of oral immune tolerance to milk protein; and / or

[0031] prevention or treatment of oral immune intolerance to milk protein; and / or

[0032] reducing the risk of developing oral immune intolerance to milk protein,

[0033] in a human, wherein the protein hydrolysate comprises at least two peptides having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5,

[0034] wherein said composition preferably further comprises at least one strain of lactic acid bacteria.

[0035] 2. The use according to claim 1, wherein the protein hydrolysate comprises:

[0036] (i) at least a peptide having a sequence according to SEQ ID NO:5, and at least one peptide having a sequence according to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; or

[0037] (ii) at least one peptide having a sequence according to SEQ ID NO:1 or SEQ ID NO:4, and at least one peptide having a sequence according to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:5.

[0038] 3. A nutritional composition for use according to claim 1 or 2, wherein the composition contains less than 6 μg of allergy-causing beta-lactoglobulin, preferably less than 3.5 μg of allergy-causing beta-lactoglobulin per 1 g of total protein.

[0039] 4. A nutritional composition for use according to one of the preceding claims, wherein the composition comprises at least 50% by weight, preferably at least 95% by weight of hydrolyzed whey protein, based on total protein.

[0040] 5. A nutritional composition for use according to one of the preceding claims, containing less than 10% by weight, preferably less than 6% by weight, of peptides or proteins having a size of 5 kDa or more, calculated on the total protein.

[0041] 6. A nutritional composition for use according to one of the preceding claims, wherein at least 1% by weight of the peptides or proteins present in the composition have a size of 1 kDa or more, calculated as total protein, preferably at least 5% by weight, more preferably at least 10% by weight, calculated as total protein.

[0042] 7. A nutritional composition for use according to one of the preceding claims, wherein the composition contains an amount of allergy-causing beta-lactoglobulin greater than 0.8 μg per 1 g of total protein.

[0043] 8. A nutritional composition for use according to one of the preceding claims, wherein the milk protein is from a species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, preferably cow's milk protein.

[0044] 9. A nutritional composition for use according to one of the preceding claims, wherein the human being is an infant.

[0045] 10. The nutritional composition for use according to one of the preceding claims, wherein a human being is at risk of developing or suffers from an allergy to milk protein from milk of species of the genus Bos, Bison, Bubalus or Capra, more preferably from the genus Bos.

[0046] 11. A nutritional composition for use according to one of the preceding claims, wherein the composition comprises a strain of lactic acid bacteria belonging to the genus Bifidobacterium, preferably to the species Bifidobacterium breve.

[0047] 12. A nutritional composition for use according to one of the preceding claims, comprising one or more indigestible oligosaccharides selected from the group consisting of fructooligosaccharide, indigestible dextrin, galactooligosaccharide, xylooligosaccharide, arabinooligosaccharide, arabinogalactooligosaccharide, glucooligosaccharide, glucomannooligosaccharide, galactomannooligosaccharide, mannanoligosaccharide, chitooligosaccharide, uronic acid oligosaccharide, sialyloligosaccharide and fucooligosaccharide, and mixtures thereof, preferably fructooligosaccharides.

[0048] 13. The nutritional composition for use according to claim 12, wherein the indigestible oligosaccharides comprise at least two indigestible oligosaccharides selected from the group consisting of fructooligosaccharides and galactooligosaccharides, preferably a mixture of long-chain fructooligosaccharides with short-chain fructooligosaccharides or with short-chain galactooligosaccharides.

[0049] 14. A nutritional composition for use according to one of the preceding claims, containing from 10 5 up to 10 11 CFU of the lactic acid bacteria strain per gram of dry weight and, optionally, at least 2% by weight of indigestible oligosaccharides on a dry weight basis.

[0050] 15. A nutritional composition for use according to one of the preceding claims, containing long-chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35% by weight of DHA based on the total amount of fatty acids.

[0051] 16. The nutritional composition for use according to one of the preceding items, which is an infant formula, a second-level formula, or an infant formula.

[0052] 17. Nutritional composition containing:

[0053] a. A strain of lactic acid bacteria belonging to the genus Bifidobacterium;

[0054] b. a milk protein hydrolysate from a species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, wherein the milk protein hydrolysate comprises at least two peptides having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5

[0055] c. less than 6 mcg of allergenic beta-lactoglobulin, preferably less than 3.5 mcg of beta-lactoglobulin, per 1 g of total protein,

[0056] d. less than 10% by weight of peptides or proteins having a size of 5 kDa or more, calculated as total protein, and

[0057] e. at least 50 wt.%, preferably at least 95 wt.% hydrolyzed whey protein based on total protein,

[0058] f. optionally, one or more indigestible oligosaccharides selected from the group consisting of fructooligosaccharide, indigestible dextrin, galactooligosaccharide, xylooligosaccharide, arabinooligosaccharide, arabinogalactooligosaccharide, glucooligosaccharide, glucomannooligosaccharide, galactomannooligosaccharide, mannanoligosaccharide, chitooligosaccharide, uronic acid oligosaccharide, sialyloligosaccharide and fucooligosaccharide, and mixtures thereof, preferably fructooligosaccharides, and

[0059] g. optionally long-chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35% by weight DHA based on total fatty acids.

[0060] 18. The nutritional composition according to claim 17, wherein the protein hydrolysate comprises:

[0061] (i) at least a peptide having a sequence according to SEQ ID NO:5, and at least one peptide having a sequence according to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; or

[0062] (ii) at least one peptide having a sequence according to SEQ ID NO:1 or SEQ ID NO:4, and at least one peptide having a sequence according to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:5.

[0063] 19. The nutritional composition according to claim 17 or 18, wherein the amount of allergy-causing beta-lactoglobulin is more than 0.8 μg per 1 g of protein, and / or wherein the composition contains more than 1 wt.% of peptides or proteins of 1 kDa or more in size based on total protein, more preferably at least 5 wt.%, more preferably at least 10 wt.%.

[0064] 20. The nutritional composition according to one of paragraphs 17-19, wherein the strain of lactic acid bacteria belongs to the species Bifidobacterium breve.

[0065] 21. The nutritional composition according to one of claims 17-20, wherein the indigestible oligosaccharides comprise at least two indigestible oligosaccharides selected from the group consisting of fructooligosaccharides and galactooligosaccharides, preferably a mixture of long-chain fructooligosaccharides with short-chain fructooligosaccharides or with short-chain galactooligosaccharides.

[0066] 22. A nutritional composition according to one of items 17-21, containing from 10 5 up to 10 11 CFU of lactic acid bacteria per g dry weight.

[0067] 23. The nutritional composition according to one of paragraphs 17-22, containing at least 2% by weight of indigestible oligosaccharides, calculated on a dry weight basis.

[0068] 24. The nutritional composition according to one of items 17 to 23, which is an infant formula, a second-level formula, or an infant formula.

[0069] 25. A method for providing nutrition to a human subject at risk of developing or suffering from an allergy, more preferably an allergy to milk protein, comprising administering to the test human subject a nutritional composition according to one of paragraphs 17-24.

[0070] 26. The method according to claim 25, wherein the human is an infant or young child, preferably an infant.

[0071] 27. The method according to claim 25 or 26, wherein the milk protein comes from a species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, preferably cow's milk protein.

[0072] 28. Method:

[0073] induction of oral immune tolerance to milk protein; and / or

[0074] prevention or treatment of oral immune intolerance to milk protein; and / or

[0075] reducing the risk of developing oral immune intolerance to milk protein,

[0076] in a human, comprising administering to the subject a nutritional composition comprising a protein hydrolysate from mammalian milk, preferably milk from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, wherein the protein hydrolysate comprises at least two peptides having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, wherein said composition preferably further comprises at least one strain of lactic acid bacteria.

[0077] 29. Applications of protein hydrolysate to obtain nutritional composition for:

[0078] induction of oral immune tolerance to milk protein; and / or

[0079] prevention or treatment of oral immune intolerance to milk protein; and / or

[0080] reducing the risk of developing oral immune intolerance to milk protein,

[0081] wherein the nutritional composition comprises a protein hydrolysate from mammalian milk, preferably milk from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, wherein the protein hydrolysate comprises at least two peptides having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, wherein said composition preferably further comprises at least one strain of lactic acid bacteria.

[0082] DETAILED DESCRIPTION OF THE INVENTION

[0083] Thus, the present invention relates to a nutritional composition comprising a protein hydrolysate from mammalian milk,

[0084] for use in the induction of oral immune tolerance to milk protein and / or

[0085] for use in the prevention or treatment of oral immune intolerance to milk protein and / or

[0086] to reduce the risk of developing oral immune intolerance to milk protein and / or

[0087] for improving or enhancing oral immune tolerance to milk protein,

[0088] in a human, wherein the protein hydrolysate comprises at least two peptides having an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. In a preferred embodiment, the composition comprises at least a peptide having an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO:2 and / or SEQ ID NO:4. In a preferred embodiment, the composition comprises at least a peptide having an amino acid sequence corresponding to SEQ ID NO:4. In a preferred embodiment, the composition comprises at least a peptide having an amino acid sequence corresponding to SEQ ID NO:5.In a preferred embodiment, the composition comprises at least three peptides selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, more preferably at least four peptides or even all five peptides selected from this group.

[0089] In a preferred embodiment, the protein hydrolysate comprises:

[0090] (i) at least a peptide consisting of an amino acid sequence corresponding to the consecutive amino acids represented by the sequence SEQ ID NO:5, and at least one peptide consisting of an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; or

[0091] (ii) at least a peptide consisting of an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO:4; or at least one peptide consisting of an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO:1, and at least one peptide consisting of an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5.

[0092] These peptides are also referred to herein as "BLG-derived peptides." The composition preferably further comprises a lactic acid bacteria strain, more preferably further comprises one or more non-digestible oligosaccharides (NDO(s)), as defined further below. Mammalian milk is preferably milk from species of the genus Bos, Bison, Bubalus, or Capra, more preferably from the genus Bos, preferably cow's milk.In other words, the invention relates to the use of a protein hydrolysate or BLG-derived peptides in the production of the above-mentioned nutritional composition for inducing oral immune tolerance to milk protein and / or for preventing or treating oral immune intolerance to milk protein and / or for reducing the risk of developing oral immune intolerance to milk protein in humans, preferably milk protein from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, wherein the nutritional composition comprises a protein hydrolysate as defined above, preferably in accordance with (i) or (ii) as defined herein above, said composition preferably further comprising lactic acid bacteria, more preferably comprising one or more NDO(s), as defined further below.The invention also relates to a method for inducing oral immune tolerance to milk protein and / or use for the prevention or treatment of oral immune intolerance to milk protein and / or reducing the risk of developing oral immune intolerance to milk protein in humans, preferably milk protein from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, the method comprising administering to a human the above-mentioned nutritional composition comprising a protein hydrolysate as defined above, preferably in accordance with (i) or (ii) as defined herein above, said composition preferably further comprising a strain of lactic acid bacteria, more preferably comprising one or more NDO(s), as defined further below.

[0093] The present invention also relates to one or more BLG-derived peptides

[0094] for use in the induction of oral immune tolerance to milk protein and / or

[0095] for use in the prevention or treatment of oral immune intolerance to milk protein and / or

[0096] to reduce the risk of developing oral immune intolerance to milk protein and / or

[0097] for improving or enhancing oral immune tolerance to milk protein,

[0098] in humans, preferably to a milk protein from a species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, wherein said one or more BLG-derived peptides are characterized by including at least two peptides having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, preferably according to (i) or (ii) as defined herein above.In other words, the invention relates to the use of one or more BLG-derived peptides in the manufacture of a nutritional composition for the induction of oral immune tolerance to milk protein and / or for use in the prevention or treatment of oral immune intolerance to milk protein and / or for reducing the risk of developing oral immune intolerance to milk protein in humans, preferably milk protein from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, wherein said one or more BLG-derived peptides are characterized by comprising at least two peptides having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, preferably according to (i) (ii) as defined herein above.

[0099] The invention also relates to a (non-therapeutic) method

[0100] for use in the induction of oral immune tolerance to milk protein and / or

[0101] for use in the prevention or treatment of oral immune intolerance to milk protein and / or

[0102] to reduce the risk of developing oral immune intolerance to milk protein and / or

[0103] for improving or enhancing oral immune tolerance to milk protein,

[0104] in a human, preferably to a milk protein from a species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably from cow's milk, comprising administering to the human one or more BLG-derived peptides, wherein the peptides are characterized by including at least two peptides having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, preferably according to (i) or (ii) as defined herein above. In a preferred embodiment of the above use or method, one or more BLG-derived peptides are preferably administered in combination with a lactic acid bacteria strain, more preferably additionally in combination with one or more non-digestible oligosaccharides (NDO(s)), as defined further below.

[0105] In one embodiment

[0106] induction of oral immune tolerance to milk protein and / or

[0107] Prevention or treatment of oral immune intolerance to milk protein and / or

[0108] reducing the risk of developing oral immune intolerance to milk protein and / or

[0109] Improving or enhancing oral immune tolerance to milk protein

[0110] refers to milk protein from a specific mammalian species, and the hydrolyzed milk protein included in the composition is produced by the same mammalian species, preferably both belong to the genus Bos.

[0111] The term "improving or enhancing oral immune tolerance to milk protein" means that oral immune tolerance to milk protein is improved compared to oral immune tolerance to milk protein before the start of administration of the composition of the invention.

[0112] The related invention also relates to a nutritional composition comprising:

[0113] a. A strain of lactic acid bacteria belonging to the genus Bifidobacterium;

[0114] b. a milk protein hydrolysate from a species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, wherein the milk protein hydrolysate comprises at least two peptides having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5

[0115] c. less than 6 mcg of allergenic beta-lactoglobulin, preferably less than 3.5 mcg of beta-lactoglobulin, per 1 g of total protein,

[0116] d. less than 3% by weight of peptides or proteins having a size of 5 kDa or more, calculated as total protein,

[0117] e. at least 50 wt.%, preferably at least 95 wt.% hydrolyzed whey protein based on total protein,

[0118] f. optionally, one or more indigestible oligosaccharides selected from the group consisting of fructooligosaccharide, indigestible dextrin, galactooligosaccharide, xylooligosaccharide, arabinooligosaccharide, arabinogalactooligosaccharide, glucooligosaccharide, glucomannooligosaccharide, galactomannooligosaccharide, mannanoligosaccharide, chitooligosaccharide, uronic acid oligosaccharide, sialyloligosaccharide and fucooligosaccharide, and mixtures thereof, preferably fructooligosaccharides, and

[0119] g. optionally long-chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35% by weight of DHA based on total fatty acids.

[0120] In this regard, the invention also relates to a method for providing nutrition to a test subject who is at risk of developing or suffering from an allergy, more preferably an allergy to milk protein, comprising administering to the subject a nutritional composition as defined above.

[0121] The nutritional composition in the context of the present invention is defined herein below, and all embodiments apply to all aspects of the invention, including the composition, the composition for use, the methods and use according to the invention.

[0122] Peptides / hydrolysate

[0123] The composition of the invention comprises at least two peptides consisting of an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. These peptides are derived from or obtainable from beta-lactoglobulin and, thus, are considered as peptides obtained from beta-lactoglobulin. In the description and claims, the amino acid sequences of the BLG peptides with SEQ ID NOs: 1-5 are identified herein below:

[0124] SEQ ID NO:1: XIVTQTMKGLDIQKVAGTWYSLAMAAS

[0125] SEQ ID NO:2: XIVTQTMKGLDIQKVAGTWYSLAMAASDISLL

[0126] SEQ ID NO:3: TMKGLDIQKVAGTWYSLAMAASDISLL

[0127] SEQ ID NO:4: TMKGLDIQKVAGTWYSLAMAASDISLLDAQ

[0128] SEQ ID NO:5: DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY.

[0129] Here, X is an amino acid selected from leucine (L) or isoleucine (I), preferably X is leucine. SEQ ID NO:1, where X=L is also referred to herein as SEQ ID NO:9. SEQ ID NO:1, where X=I is also referred to herein as SEQ ID NO:37. SEQ ID NO:2, where X=L is also referred to herein as SEQ ID NO:11. SEQ ID NO:2, where X=I is also referred to herein as SEQ ID NO:38. The choice of either leucine or isoleucine as X is a direct consequence of the source of mammalian milk. Leucine is preferred since the milk of mammals from the genus Bos, preferably cow's milk, is most preferred.

[0130] Preferred option (i) is based on the ProImmune ProPresent® antigen presentation assay, where this peptide mixture was found to cover more of the HLA-DRB1 alleles tested; preferred option (ii) is based on experiments targeting T cell recognition of T cells in cow's milk allergic infants, where these peptides were found to cover the reactivity of each cell line.

[0131] The peptides are preferably provided by a protein hydrolysate (i.e., hydrolyzed proteins) obtained from mammalian milk, preferably milk from species of the genera Bos, Bison, Bubalus, or Capra, more preferably the genus Bos, most preferably cow's milk (Bos taurus). The amino acid sequence of BLG is the same in the genera Bos and Bison, and SEQ ID NOs: 9, 11, 3, 4, and 5 can be obtained in hydrolysates from the milk protein of these genera. In the genera Capra and Bubalus, amino acid 1 is different (isoleucine instead of leucine in SEQ ID NOs: 37 and 38), and SEQ ID NOs: 37, 38, 3, 4, and 5 can be obtained in hydrolysates from the milk protein of these genera. In a preferred embodiment, the peptides are obtained from whey protein. The nutritional composition preferably contains at least 50 wt.%, more preferably at least 70 wt.%, even more preferably at least 95 wt.% hydrolyzed whey protein, based on total protein.A suitable source is a mixture of acid whey protein and demineralized sweet whey protein. Acid whey and sweet whey are commercially available. Sweet whey is a by-product of rennet-coagulated cheese and contains caseinoglycomacropeptide (CGMP), while acid whey (also called fermented whey) is a by-product of acid-coagulated cheese and does not contain CGMP. Suitable whey protein sources are demineralized whey (Deminal, Friesland Campina, the Netherlands) and / or whey protein concentrate (WPC80, Friesland Campina, the Netherlands). The whey protein preferably contains acid whey, more preferably at least 50 wt.%, more preferably at least 70 wt.% acid whey, based on total whey protein. Acid whey has an improved amino acid profile compared to sweet whey protein.

[0132] Hydrolysis can be carried out using a mixture of microbial endopeptidases and exopeptidases using the method described in Example 1 of International Application WO 2011 / 151059, incorporated herein by reference. Hydrolysates with BLG peptides according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 can be preferably obtained using the formulation according to Example 1. Preferably, a mixture of endoprotease and exoprotease is used. To determine whether any given composition contains one or more BLG-derived peptides, identification means are described in the experimental parts.

[0133] The composition preferably comprises less than 10 wt.%, preferably less than 6 wt.% of peptides or proteins of 5 kDa or more in size based on total protein. In one embodiment, the composition comprises more than 0.3 wt.%, preferably more than 0.5 wt.%, more preferably more than 1 wt.%, even more preferably more than 1.5 wt.% of these peptides of 5 kDa or more in size based on total protein. Preferably, more than 1 wt.% of the peptides or proteins present in the composition have a size of 1 kDa or more in size based on total protein, more preferably at least 5 wt.%, more preferably at least 10 wt.%, based on total protein. More preferably, more than 1% by weight of the peptides or proteins present in the composition have a size of 3 kDa or more based on total protein, more preferably at least 5% by weight, more preferably at least 10% by weight, based on total protein.

[0134] The size distribution of peptides in a protein hydrolysate can be determined using high-pressure size-exclusion liquid chromatography, which is known in the art. An example of this is described in the article by Saint-Sauveur et al. “Immunomodulating properties of a whey protein isolate, its enzymatic digest and peptide fractions” Int. Dairy Journal (2008) vol. 18(3) pages 260–270. Briefly, the total surface area of ​​the chromatograms is integrated and divided into mass ranges expressed as a percentage of the total surface area. The mass ranges are calibrated using peptides / proteins with known molecular weights. The protein hydrolysate containing said peptides is preferably characterized in that it contains from 64 to 89 wt.% peptides with a molecular weight below 1000 Da, from 10 to 30 wt.% peptides with a molecular weight from 1000 to 5000 Da and from 1 to 6 wt.% peptides or proteins with a molecular weight above 5 kDa, all based on total protein.

[0135] While the peptide mass distribution is informative, it does not provide any information about the sequence and activity of the peptides present in the infant formula. Structural information is crucial for understanding the overall biological activity of hydrolyzed infant formula.

[0136] Several specific peptides derived from the BLG region, which is known to contain T-cell epitopes, were consistently identified in different production lots. Only two studies had previously examined the peptide profile of pHP (Wada et al. Peptides 2015 73:101–105; Catala-Clariana et al., Electrophoresis 2013 Jul; 34 (13): 1886–1894). Although these studies focused on bioactive peptides in general, rather than specifically on T-cell epitopes, none of the peptides with SEQ ID NOs: 1–5 identified in the present application were detected in these previously tested products. In addition to the profiles of these hydrolyzed protein products, the peptide profile of an uncommercially available BLG-based hydrolysate was determined (Pecquet et al. J Allergy Clin Immunol 2000 Mar; 105 (3):514-521).Two peptides (AA#21-40 and AA#25-40) that overlap with the region of interest were also identified in this hydrolysate, but again, not peptides with SEQ ID NOs: 1-5. At least two of the three studies described above analyzed hydrolysate from different manufacturers (Wada and Pecquets), and, as noted above, the main reason for these differences may be differences in manufacturing processes. This is further supported by a recent study in which highly hydrolyzed infant formulas from different manufacturers were characterized using peptidomics. The authors showed that infant formulas from different manufacturers have a different signature based on their peptide profile and may therefore have different effects in clinical trials (Lambers et al. Food Sci Nutr 2015 Jan; 3(1):81-90. Hochwallner et al. al, 2017, Allergy 72:416-425).

[0137] In a preferred embodiment of the present invention, the present composition comprises, per gram of total protein, at least 10 μg (micrograms), more preferably from 10 to 5000 μg, more preferably from 20 to 2000 μg, more suitably from 30 to 500 μg, and particularly preferably from 50 to 250 μg of the sum of beta-lactoglobulin-derived peptides consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, more preferably consisting of the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The BLG-derived peptides are preferably present in therapeutically effective amounts.

[0138] Preferably, the nutritional composition contains less than 6 μg of allergenic beta-lactoglobulin BLG per 1 g of protein, preferably less than 5 μg per 1 g of protein, more preferably less than 3.5 μg per 1 g of protein. In the context of the invention, the expression "allergenic beta-lactoglobulin" refers to intact or immunogenic BLG and does not take into account hydrolyzed BLG. Preferably, the nutritional composition contains an amount of allergenic beta-lactoglobulin greater than 0.8 μg per g of protein. Allergenic BLG is determined by an ELISA method known in the art, and an amount greater than 0.8 μg per g of protein indicates a partial protein hydrolyzate. For comparison, highly hydrolyzed whey protein exhibits less than 0.2 μg of allergenic BLG per g of protein.Small but significant amounts of allergenic BLG together with hydrolyzed BLG or BLG-derived peptides, more preferably also in combination with a lactic acid bacteria strain, even more preferably also in combination with lactic acid bacteria and one or more NDO(s) enhance the tolerogenic activity of the composition, however, the amount of BLG still remains low enough to reduce the allergenicity of the nutritional composition and for the nutritional composition to be hypoallergenic.From this point of view, it is preferable to use a protein hydrolysate containing an insignificant amount of allergy-causing beta-lactoglobulin and containing BLG-derived peptides according to the invention compared to synthetic peptides consisting of the amino acid sequence presented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, more preferably consisting of the amino acid sequence presented by SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 (without allergy-causing BLG).

[0139] Lactic acid bacteria

[0140] The composition of the present invention preferably comprises a strain of lactic acid bacteria that enhances the oral immune tolerogenic activity of the BLG peptides of the present invention. The bacterial strain is preferably a probiotic. Evidence is provided in Examples 5 and 7. The presence of lactic acid bacteria was found to increase the expression of HLA-DR molecules on the surface of dendritic cells. Increased HLA-DR expression also indicates improved peptide presentation to T cells and, consequently, the induction of oral immune tolerance.

[0141] Suitable lactic acid bacteria include strains of the genus Bifidobacteria (e.g., B. breve, B. longum, B. infantis, B. bifidum), Lactobacillus (e.g., L. acidophilus, L. paracasei, L. johnsonii, L. plantarum, L. reuteri, L. rhamnosus, L. casei, L. lactis) and Streptococcus (e.g., S. thermophilus). Bifidobacterium breve and Bifidobacterium longum are particularly suitable lactic acid bacteria.

[0142] The composition preferably comprises a strain of lactic acid bacteria belonging to the genus Bifidobacterium, preferably the species Bifidobacterium breve. B. breve preferably has at least 95% identity to the 16S rRNA sequence compared to the type strain B. breve ATCC 15700, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). Suitable B. breve strains can be isolated from the faeces of healthy breastfed infants. They are typically commercially available from lactic acid bacteria manufacturers, but they can also be directly isolated from the faeces, identified, characterized, and obtained. According to one embodiment, the present composition comprises B. breve selected from the group consisting of B. breveBb-03 (Rhodia / Danisco),B. breveM-16V (Morinaga),B. breveR0070 (Rosell Institute, Lallemand),B. breveBR03 (Probiotic),B.breveBR92 (Cell Biotech), DSM 20091, LMG 11613, YIT4065, FERM BP-6223, and CNCM I-2219. B. breve can be B. breveM-16V and B. breveCNCM I-2219, most preferably B. breveM-16V. B. breveI-2219 was published in international application WO 2004 / 093899 and deposited at the Collection National des Cultures des Microorganisms, Institut Pasteur, Paris, France, on May 31, 1999, by Compagnie Gervais Danone. B. breveM-16V was deposited as BCCM / LMG23729 and is commercially available from Morinaga Milk Industry Co., Ltd.

[0143] The lactic acid bacteria may be present in the composition in any suitable concentration, preferably in a therapeutically effective amount or "amount effective for treatment" in the context of the invention. Preferably, the lactic acid bacteria strain is included in the present composition in an amount of 10 4 -10 13 CFU per g dry weight of the composition, preferably 10 5 -10 11 CFU / g, most preferably 10 6 -10 10 CFU / g.

[0144] Indigestible oligosaccharides

[0145] In a preferred embodiment, the present composition further comprises one or more non-digestible oligosaccharides (NDOs). Like lactic acid bacteria, these NDOs further enhance oral immune tolerance by inducing the properties of BLG protein peptides. Evidence of this enhanced effect is provided in Example 5.

[0146] Advantageously and most preferably, the indigestible oligosaccharide is water-soluble (according to the method disclosed in L. Prosky et al, J. Assoc. Anal. Chem 71:1017-1023, 1988) and is preferably an oligosaccharide with a degree of polymerization (DP) of from 2 to 200. The average DP of the indigestible oligosaccharide is preferably below 200, more preferably below 100, even more preferably below 60, most preferably below 40.

[0147] Non-digestible oligosaccharides are preferably prebiotics. They are not digested in the intestine by digestive enzymes present in the upper gastrointestinal tract (small intestine and stomach). Indigestible oligosaccharides are fermented by the human gut microbiota. For example, glucose, fructose, galactose, sucrose, lactose, maltose, and maltodextrins are considered digestible. Oligosaccharide starting materials may include monosaccharides such as glucose, fructose, fucose, galactose, rhamnose, xylose, glucuronic acid, GalNac, and the like, but they are not part of the oligosaccharides.The indigestible oligosaccharide is preferably selected from the group consisting of fructooligosaccharide, indigestible dextrin, galactooligosaccharide, xylooligosaccharide, arabinooligosaccharide, arabinogalactooligosaccharide, glucooligosaccharide, glucomannooligosaccharide, galactomannooligosaccharide, mannanoligosaccharide, chitooligosaccharide, uronic acid oligosaccharide, sialyloligosaccharide and fucooligosaccharide, and mixtures thereof, preferably fructooligosaccharides. Examples of sialyloligosaccharide are 3-sialyllactose, 6''-sialyllactose, sialyllactose-N-tetraose, disiallyllactose-N-tetraose. Examples of fucooligosaccharides are (un)sulfated fucoidan oligosaccharides, 2'-fucosyllactose, 3'-fucosyllactose, lacto-N-fucopentaose I, II, III, LNDH, lactodifucotetraose, lacto-N-difucohexaose I and II.

[0148] One suitable type of oligosaccharide is a short-chain oligosaccharide having an average degree of polymerization below 10, preferably no higher than 8, preferably in the range of 2-7. The short-chain oligosaccharide preferably includes galactooligosaccharides and / or fructooligosaccharides (i.e., scGOS and / or scFOS). In one embodiment, the composition comprises galactooligosaccharides, preferably beta-galactooligosaccharides, preferably transgalactooligosaccharides. The galactooligosaccharides preferably have an average degree of polymerization in the range of 2 to 8, preferably 3 to 7, i.e., are short-chain oligosaccharides in the context of the invention. (Trans)galactooligosaccharides are commercially available, for example, under the trade name Vivinal®GOS (Friesland Campina Domo Ingredients, The Netherlands), Bimuno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate55 (Yakult).The composition preferably comprises short-chain fructooligosaccharides and / or short-chain galactooligosaccharides, preferably at least short-chain fructooligosaccharides. The fructooligosaccharides may be inulin hydrolysate products having an average DP in the above-mentioned (sub)ranges; such FOS products are, for example, commercially available as Raftilose P95 (Orafti) or from Cosucra.

[0149] Another suitable type of oligosaccharides is long-chain fructooligosaccharides (lcFOS), which have an average degree of polymerization above 10, usually in the range of 10-100, preferably 15-50, most preferably above 20. A specific type of long-chain fructooligosaccharides is inulin, such as Raftilin HP.

[0150] The present composition may comprise a mixture of two or more types of non-digestible oligosaccharides, most preferably a mixture of two non-digestible oligosaccharides. If the NDO comprises or consists of a mixture of two different oligosaccharides, one oligosaccharide may be short-chain, as defined above, and one oligosaccharide may be long-chain, as defined above. Most preferably, the short chain oligosaccharides and long chain oligosaccharides are present in a short chain to long chain weight ratio in the range of 1:99 to 99:1, more preferably 1:1 to 99:1, more preferably 4:1 to 97:3, even more preferably 5:1 to 95:5, even more preferably 7:1 to 95:5, even more preferably 8:1 to 10:1, most preferably about 9:1.

[0151] In one embodiment, the composition comprises at least two fructooligosaccharides and / or galactooligosaccharides. Suitable mixtures include mixtures of long-chain fructooligosaccharides with short-chain fructooligosaccharides or with short-chain galactooligosaccharides, most preferably long-chain fructooligosaccharides with short-chain fructooligosaccharides.

[0152] The present composition preferably contains from 0.05 to 20% by weight of said indigestible oligosaccharides, more preferably from 0.5 to 15% by weight, even more preferably from 1 to 10% by weight, most preferably from 2 to 10% by weight, based on the dry weight of the present composition. In liquid form, the present composition preferably contains from 0.01 to 2.5% by weight of indigestible oligosaccharide, more preferably from 0.05 to 1.5% by weight, even more preferably from 0.25 to 1.5% by weight, most preferably from 0.5 to 1.25% by weight per 100 ml.

[0153] In one embodiment, the NDO mixture does not contain detectable amounts of acid oligosaccharides.

[0154] If the indigestible oligosaccharide is a mixture, the average values ​​of the relevant parameters are used to determine the present invention.

[0155] The combination of NDO and lactic acid bacteria, as defined above, is also called a "synbiotic." The presence of therapeutically effective amounts of NDO along with lactic acid bacteria is believed to further improve oral immune tolerance by inducing the properties of a Bifidobacterium strain, preferably B. breve, along with fructooligosaccharides. Reference is made to Example 5.

[0156] Other components

[0157] The composition may further comprise long-chain polyunsaturated fatty acids (LC-PUFA). LC-PUFA are fatty acids in which the acyl chain has a length of 20 to 24 carbon atoms (preferably 20 or 22 carbon atoms), and in which the acyl chain contains at least two unsaturated bonds between said carbon atoms in the acyl chain. More preferably, the present composition comprises at least one LC-PUFA selected from the group consisting of eicosapentaenoic acid (EPA, 20:5 n3), docosahexaenoic acid (DHA, 22:6 n3), arachidonic acid (ARA, 20:4 n6) and docosapentaenoic acid (DPA, 22:5 n3), preferably DHA, EPA and / or ARA. These LC-PUFAs have additional beneficial effects in reducing the risk of allergies.

[0158] The preferred content of LC-PUFA in the present composition does not exceed 15 wt.% of the total fatty acids, preferably does not exceed 10 wt.%, even more preferably does not exceed 5 wt.%. Preferably, the present composition contains at least 0.2 wt.%, preferably at least 0.25 wt.%, more preferably at least 0.35 wt.%, even more preferably at least 0.5 wt.% LC-PUFA of the total fatty acids, more preferably DHA. The present composition preferably contains ARA and DHA, wherein the weight ratio of ARA / DHA is preferably more than 0.25, preferably more than 0.5, more preferably 0.75-2, even more preferably 0.75-1.25. The weight ratio is preferably less than 20, more preferably from 0.5 to 5. The amount of DHA is preferably more than 0.2 wt.%, more preferably more than 0.3 wt.%, more preferably at least 0.35 wt.%, even more preferably 0.35-0.6 wt.% of total fatty acids.

[0159] Nutritional composition

[0160] The composition according to the invention can be used as a nutritional composition, nutritional therapy, nutritional support, as a therapeutic food, as a food product for special medical purposes, or as a food supplement. The present composition is preferably an enteral (oral) composition. The composition is administered orally or is intended for oral administration to a subject in need thereof, in particular to children and infants, including toddlers, preferably children under 6 years of age, preferably infants or young children, usually aged 0-36 months, more preferably infants aged 0-12 months, most preferably 0-6 months.Thus, in some embodiments, the present composition is a first-level infant formula, a second-level infant formula, or an infant formula (also referred to as a third-level infant formula), preferably a first-level infant formula or a second-level infant formula, most preferably a first-level infant formula. The term "first-level infant formula" is clearly defined and is constantly monitored by regulatory authorities at the international level. In particular, CODEX STAN 73-1981 "Standard for Infant Formula and Special Medicinal Products Intended for Infants" is generally recognized. It recommends, in terms of nutritional value and composition, that the prepared milk contain at least 60 kcal (250 kJ) and no more than 70 kcal (295 kJ) of energy per 100 ml. The FDA and other regulatory authorities have established nutrient requirements in accordance with them.

[0161] Preferably, the present enteral, preferably nutritional, composition is intended to provide the daily nutritional needs of humans, in particular for administration, in particular for feeding, to humans, in particular infants, including toddlers, preferably at risk of developing an allergy to milk protein, preferably to milk protein from species of the genus Bos, Bison, Bubalus, or Capra, more preferably of the genus Bos, most preferably an allergy to cow's milk protein. In one embodiment, the allergy to milk protein concerns milk from a specific mammalian species, and the hydrolyzed milk protein contained in the composition belongs to the same mammalian species, preferably both belong to the genus Bos.

[0162] To meet the caloric needs of an infant, the present enteral composition preferably contains 50 to 200 kcal / 100 ml of liquid, more preferably 60 to 90 kcal / 100 ml of liquid, and even more preferably 60 to 75 kcal / 100 ml of liquid. This caloric content ensures an optimal ratio between water and calorie intake. The osmolarity of this composition is preferably 150 to 420 mOsmol / L, more preferably 260 to 320 mOsmol / L. Low osmolarity aims to reduce gastrointestinal stress.

[0163] Preferably, the present enteral composition is in liquid form, preferably with a viscosity of below 35 mPa⋅s, more preferably below 6 mPa⋅s, measured on a Brookfield viscometer at 20°C and a shear rate of 100 s -1Accordingly, the present enteral composition is in the form of a powder, which can preferably be reconstituted with water to form a liquid, or as a liquid concentrate, which must be diluted with water. If the present enteral composition is in liquid form, the preferred volume administered daily is in the range of about 80 to 2500 ml, more preferably about 450 to 1000 ml per day.

[0164] The composition of the invention preferably comprises a lipid component, preferably a lipid component suitable for infant nutrition, as known in the art. The lipid component of the present composition preferably provides from 2.9 to 6.0 g, more preferably from 4 to 6 g per 100 kcal of the composition. In liquid form, the composition preferably contains from 2.1 to 6.5 g lipids per 100 ml, more preferably from 3.0 to 4.0 g per 100 ml. On a dry weight basis, the present first-tier or second-tier infant formula preferably contains from 12.5 to 40% by weight of lipids, more preferably from 19 to 30% by weight.

[0165] The composition of the invention may contain additional protein material in addition to the beta-lactoglobulin-derived peptide(s) of the invention. In the context of the present invention, additional "protein" or "protein material" or "protein equivalents" encompasses proteins, peptides, free amino acids, and partially or highly hydrolyzed proteins.

[0166] Preferably, the additional protein material added to the beta-lactoglobulin-derived peptide(s) does not cause an allergic reaction or is hypoallergenic, such as free amino acids and hydrolyzed protein. As an additional protein component, i.e., in addition to the beta-lactoglobulin-derived peptide(s), the composition of the present invention preferably contains free amino acids, hydrolyzed whey protein, preferably partially hydrolyzed whey proteins.

[0167] The composition of the present invention preferably contains less than 1% by weight of intact mammalian (cow) milk protein. The composition may contain an additional protein component selected from the group consisting of free amino acids, hydrolyzed whey protein, and proteins from other sources such as soy, pea, rice, collagen, or the like, in intact form, in partially hydrolyzed form, and / or in highly hydrolyzed form.

[0168] The present composition preferably contains at least 50% by weight of a protein component derived from milk of non-human origin, more preferably at least 90% by weight based on the dry weight of the total protein.

[0169] The present composition preferably contains from 4 to 25%, more preferably from 5 to 20%, more preferably from 7 to 16%, most preferably from 7 to 12% protein, based on total calories. The present composition, if in liquid form, preferably contains from 0.5 to 6.0 g, more preferably from 0.8 to 3.0 g, even more preferably from 1.0 to 2.5 g protein per 100 ml. The present composition preferably contains at least 7.0 wt.%, more preferably at least 8.0 wt.%, most preferably at least 9 wt.% or at least 10 wt.% protein, based on the dry weight of the total composition. Preferably, the present composition contains no more than 40 wt.%, more preferably no more than 15 wt.%, preferably no more than 20 wt.% protein, based on the dry weight of the total composition.

[0170] The composition may contain digestible carbohydrate(s). Typically, digestible carbohydrates are those known in the art to be suitable for use in nutritional compositions for infants, such as those selected from digestible polysaccharides (e.g., starch, maltodextrin), digestible monosaccharides (e.g., glucose, fructose), and digestible disaccharides (e.g., lactose, sucrose). Particularly suitable are lactose and / or maltodextrin. In one embodiment, the composition does not contain lactose.

[0171] The digestible carbohydrate component preferably contains at least 60% by weight of lactose based on the total amount of digestible carbohydrates, more preferably at least 75% by weight, even more preferably at least 90% by weight of lactose based on the total amount of digestible carbohydrates.

[0172] Test subjects

[0173] The target test subjects or population are preferably infants (0-12 months), preferably infants at risk of developing allergy, preferably allergy to milk protein, preferably milk protein from species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, most preferably allergy to cow's milk protein, preferably infants suffering from allergy to milk protein, preferably to milk protein from species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, most preferably allergy to cow's milk protein. Infants at risk are preferably defined by a family history, preferably at least one parent, or a history of allergy.

[0174] A human being preferentially has a particular HLA-DRB1 allele selected from the groups consisting of HLA-DRB1*01:01, HLA-DRB1*04:01, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1*7:01, and HLA-DRB1*09:01. HLA-DRB1 is the most predominant human MHC class II isotype (>90%), the HLA-DRB1 gene locus is polymorphic, while the HLA-DRA1 gene locus is monomorphic (that is, the HLA-DRB1 genotype determines the entire HLA-DR molecule), and the HLA-DRB1 allele is expressed fivefold higher than its paralogs (HLA-DRB3, -DRB4, or -DRB5) and is present in all humans.

[0175] Induction of oral immune tolerance

[0176] The invention relates to

[0177] inducing oral immune tolerance to milk protein, preferably to milk protein from a species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, most preferably to cow's milk protein in a human or population, or

[0178] prevention or treatment of oral immune intolerance to milk protein, preferably milk protein from a species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, most preferably cow's milk protein in a human or population, or

[0179] reducing the risk of occurrence or development of oral immune intolerance to milk protein, preferably milk protein from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, most preferably cow's milk protein in a human or population, or

[0180] improving or enhancing oral immune tolerance to milk protein, preferably milk protein from a species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, most preferably cow's milk protein in a human or population.

[0181] As described herein above, the test person or population is preferably an infant test person or population.

[0182] In the context of the present invention, the term “oral tolerance” means oral immune tolerance.

[0183] In a preferred embodiment, the invention relates to a nutritional composition as defined herein above for use in the prevention of oral immune intolerance to cow's milk protein in a test infant or infant population at risk of developing an allergy to milk protein, preferably to milk protein from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, most preferably an allergy to cow's milk protein.In a preferred embodiment, the invention relates to a nutritional composition as defined herein above for use in reducing the risk of occurrence or development of oral immune intolerance to milk protein, preferably milk protein from a species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, most preferably cow's milk protein in a test infant or infant population at risk of developing an allergy to milk protein, preferably to milk protein from a species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, most preferably an allergy to cow's milk protein, wherein the partial hydrolysate of mammalian protein comprises at least 1 wt.%, more preferably at least 5 wt.%, even more preferably at least 10 wt.% peptides with a molecular weight above 1 kDa and / or more than 0.8 μg of allergenic BLG per 1 g of total protein.

[0184] LEGALS FOR FIGURES

[0185] Figure 1: Comparison of fragmentation spectra of a representative peptide (SEQ ID NO:3; shown at the top), experimentally detected (B), and its synthetic equivalent (A). The mass difference between the annotated fragment ions of the experimental and synthetic peptide corresponds to the presence of a stable isotope labeled with lysine in the synthetic peptide, highlighted in italics in the sequence (+8 Da). The * symbol indicates the loss of water from the fragments of the corresponding b-ions.

[0186] Fig. 2: T cell responses after stimulation with the identified peptides. Cow's milk-specific T cell lines (TCLs) from three different donors were stimulated with synthetic equivalents of the identified peptides (LIV…AAS=SEQ ID NO:9; LIV…SLL=SEQ ID NO:11; TMK…SLL=SEQ ID NO:3; TMK…DAQ=SEQ ID NO:4; DIQ…RVY=SEQ ID NO:5). Cow's milk protein (CMP) was used as a control. A stimulation index ≥2 was considered significant.

[0187] EXAMPLE 1. HYDROLYZED WHEY PROTEIN AND A NEW METHOD FOR IDENTIFICATION OF BETA-LACTOGLOBULIN PEPTIDES

[0188] A mixture of acid whey protein and demineralized sweet whey protein (protein weight ratio of 77:23) was dissolved in water (purified by reverse osmosis) and then hydrolyzed under specific conditions. Acid whey and sweet whey are commercially available. Sweet whey is a by-product of rennet-coagulated cheese and contains caseinoglycomacropeptide (CGMP), while acid whey (also called fermented whey) is a by-product of acid-coagulated cheese and does not contain CGMP. Suitable sources of acid whey protein include demineralized whey (Deminal, Friesland Campina, the Netherlands) and whey protein concentrate (WPC80, Friesland Campina, the Netherlands). A mixture of microbial endopeptidases and exopeptidases was used to hydrolyze these two protein sources using the method described in Example 1 of international application WO 2011 / 151059. The hydrolyzed protein solution was then spray dried.The peptide size distribution in this protein hydrolysate was determined using high-pressure liquid chromatography (HPLC), a technique known in the art. Briefly, the total surface area of ​​the chromatograms is integrated and divided into mass ranges expressed as a percentage of the total surface area. The mass ranges are calibrated using peptides / proteins of known molecular weight. Whey protein hydrolysate can be classified as either a partial or moderate protein hydrolysate.

[0189] The resulting hydrolysate powder was used as the sole protein source in infant formula. Ten different batches of infant formula were prepared in a similar manner. The beta-lactoglobulin content, determined by an ELISA method known in the art, ranged from approximately 0.8 to 3.5 μg / g total protein.

[0190] For determining the presence of specific sequences in biological samples, mass spectrometry (MS) is the preferred method, compared to more traditional methods currently used to characterize protein hydrolysates. A recent development in the field of MS, termed peptidomics, allows for the characterization of peptide sequences with high sensitivity and specificity (Dallas et al. J Nutr 2015; 145:425-433). This method has the potential to bridge the gap between understanding the impact of sequence specificity on the biological activity that a protein hydrolysate may have.

[0191] Samples were prepared essentially as described by Butre et al., with the addition of a reduction and alkylation step (Butre et al. Anal Bioanal Chem 2014; 406:5827–5841). All chemicals were obtained from Sigma Aldrich. Briefly, batches of protein partial hydrolysate (pHP) were diluted to 0.5% (v / v) using 50 mM ammonium bicarbonate, followed by peptide reduction with 4 mM DTT and alkylation with 8 mM iodoacetamide. The mixture was clarified by centrifugation at 20,000×g for 10 min and diluted to 0.1% (v / v) using 0.1 M acetic acid.

[0192] All samples were analyzed by nanoflow liquid chromatography using an Agilent 1200 HPLC system (Agilent Technologies) connected online to an LTQ Velos mass spectrometer (Thermo Fisher Scientific). The liquid chromatography portion of the system was set up essentially as described previously (14). Peptides were trapped at 5 μL / min in 100% solvent A (0.1 M acetic acid in water) on a 2-cm trap column (100 μm i.d., packed in-house with Aqua C18, 5 μm resin (Phenomenex)) and eluted from a 20-cm IntegraFrit column (50 μm i.d., ReproSil-Pur C18-AQ 3 μm, New Objective) at ~100 nL / min in a 90-min gradient from 10% to 40% solvent B (0.1 M acetic acid in 8:2 (v / v) acetonitrile / water). The eluent was nebulized through standard coated emitter tips (New Objective) coupled end-to-end with the analytical column.The mass spectrometer operated in data-driven mode, automatically switching between MS and MS / MS. Full-scan mass spectra (from 300 to 1200 m / z) were acquired by increasing the scan rate after accumulation to a target value of 3000. The five most intense ions with a threshold above 500 were selected for collision-induced spectra at a normalized collision energy of 35% after accumulation to a target value of 10000.

[0193] All MS data were processed using Proteome Discoverer (version 2.1, Thermo Scientific). Peak lists were generated using a standard workflow. Peptide identification was performed by searching individual peak lists of CID fragmentation spectra against a database containing selected bovine whey and casein proteins using Mascot (version 2.4.1, Matrix Science). The enzyme was not defined, and missed cleavages were not allowed. The precursor ion mass tolerance was set to 0.2 Da, and the product ion mass tolerance was set to 0.5 Da. Carbamidomethylation (C) was set as a fixed modification.

[0194] Of the 314 peptides identified by this method, 101 could be assigned to beta-lactoglobulin (BLG), resulting in 90% sequence coverage of BLG. Most of the remaining peptides were derived from other common serum proteins, such as α-lactalbumin and serum albumin. A total of 13 beta-lactoglobulin peptides were identified in the region of interest (AAs 13–48) from a minimum of 9 AAs (see Table 1 and Fig. 1). Six of these were consistently identified in all 10 batches. Unambiguous peptide identification was obtained by comparing the characteristics (retention time, peptide mass, and fragmentation spectrum) of the experimental peptide with its stable isotope-labeled synthetic equivalent. An example is shown in Fig. 1, where the fragmentation spectra of the experimental and synthetic peptides show excellent correlation. Thus, the identity of six peptides found in all 10 batches was confirmed.

[0195] Table 1. Beta-lactoglobulin peptide sequences identified in 10 different lots of partially hydrolyzed whey-based infant formula Amino acid number in the BLG sequence Subsequence Number of batches in which the peptide was identified SEQ ID NO AA 1-22 LIVTQTMKGLDIQKVAGTWYSL 7 6 AA 1-25 LIVTQTMKGLDIQKVAGTWYSLAMA 9 7 AA 1-26 LIVTQTMKGLDIQKVAGTWYSLAMAA 10 8 AA 1-27 LIVTQTMKGLDIQKVAGTWYSLAMAAS 10 9 AA 1-30 LIVTQTMKGLDIQKVAGTWYSLAMAASDIS 2 10 AA 1-32 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLL 10 11 AA 1-35 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQ 5 12 AA 1-39 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPL 2 13 AA 1-42 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 2 14 AA 5-35 QTMKGLDIQKVAGTWYSLAMAASDISLLDAQ 2 15 AA 6-32 TMKGLDIQKVAGTWYSLAMAASDISLL 10 3 AA 6-35 TMKGLDIQKVAGTWYSLAMAASDISLLDAQ 10 4 AA 11-42 DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 10 5

[0196] A similar analysis was conducted with batches of Nutrilon pepti, an infant formula containing highly hydrolyzed whey protein, marketed for the dietary management of cow's milk allergy. The BLG peptides listed in Table 1 were not present in Nutrilon pepti.

[0197] EXAMPLE 2. IN VITRO IDENTIFICATION OF HLA-DR-RESTRICTED PEPTIDES

[0198] Identification of BLG-derived peptides presented by human DCs was performed using ProImmune as described (Lamberth et al, Sci Transl Med 2017 11; 9(372):10.1126 / scitranslmed.aag1286.). Briefly, peripheral blood mononuclear cell samples were obtained from 12 healthy adult donors with the HLA-DR type. Donors were selected based on the 11 common HLA-DRB1 alleles (Table 2). In this study, HLA-DRB1 was prioritized because the HLA-DR molecule is the most predominant human MHC class II isotype (<90%) (Sturniolo et al. Nat Biotechnol 1999; 17:555–561) and that the HLA-DRB1 gene locus is polymorphic, whereas the HLA-DRA1 gene locus is monomorphic (i.e., the HLA-DRB1 genotype determines the entire HLA-DR molecule) (Marsh et al., 2010. Tissue Antigens 75:291–455). Furthermore, the HLA-DRB1 allele is expressed fivefold higher than its paralogs (HLA-DRB3, -DRB4, or -DRB5) and is present in all individuals (O'Leary et al.Nucleic Acids Res 2016 4; 44(D1):D733 -45).

[0199] Table 2. Donors with HLA-DRB1 typing information Donor ID DRB1_1 DRB1_2 P1 *03:01 *03:01 P2 *01:01 *04:01 P3 *01:01 *07:01 P4 *01:01 *04:05 P5 *04:01 *07:01 P6 *13:02 *14:01 P7 *03:01 *09:01 P8 *11:01 *15:01 P9 *03:01 *11:01 P10 *07:01 *15:01 P11 *03:01 *15:01 P12 *01:01 *04:04

[0200] Immature monocytic DCs were generated in vitro and matured in the presence of the test hydrolyzed protein (HP). DCs were collected and lysed to obtain HLA-DR complexes using a specific immunoaffinity method. Peptides were eluted from the HLA-DR complexes and then analyzed by high-resolution sequencing LC-MS / MS. The presence of six endogenous relevant proteins (i.e., ITGAM, ApoB, CLIP, TFRC, FcER2 / FcGR2, and LAMP-1 / 3) was assessed as a control for this analysis. Each donor sample had to express at least three relevant proteins to be qualified for subsequent analysis. The obtained HLA-DR-restricted peptide data were compiled and evaluated using sequence analysis software with reference to the Swiss-Prot Human Proteome Database with the sequences of the tested elements included.The probability of actually detecting peptides is described by their expected value ≤0.05. The false discovery rate was defined as <1%.

[0201] Using the ProPresent® antigen presentation assay, focusing on BLG-derived sequences, 15 relevant peptides with an expected value of ≤0.05 were identified in 5 donor samples (Table 3). These peptides could be further grouped into 2 groups of unique sequences, i.e., DIQ…DIS (AA #11-30) and AMA…APL (AA #23-39) (Table 4). Importantly, both sequence groups overlap the region of interest (i.e., AA #13-48 of mature BLG). This finding demonstrated that the studied BLG-derived peptides could be presented by HLA-DR molecules on human DCs when incubated with specific HP.

[0202] Table 3. Significant fragments identified from HLA-DR peptide complexes with an expected value of <0.05 Donor ID DRB1_1 DRB1_2 Fragment sequence (mature BLG) [SEQ ID NO] Amino acid start / end Expected value P2 *01:01 *04:01 AMAASDISLLDAQSAPL -

[16] - MAASDISLLDAQSAPLR

[17] 23-39 24-40 0,043 0,0013 P3 *01:01 *07:01 - MAASDISLLDAQSAPL -

[18] 24-39 0,009 P4 *01:01 *04:05 DIQKVAGTWYSLAMAASDI -

[19] - - - - VAGTWYSLAMAASDIS

[20] - - - - - - GTWYSLAMAASDIS

[21] 11-29 15-30 17-30 0,000052 0,00021 0,00037 P7 *03:01 *09:01 DIQKVAGTWYSLAMAASDI - -

[19] DIQKVAGTWYSLAMAASDIS -

[22] - - - - VAGTWYSLAMAASDI - -

[23] - - - - VAGTWYSLAMAASDIS -

[20] - - - - - AGTWYSLAMAASDIS -

[24] - - - - - - GTWYSLAMAASDI - -

[25] - - - - - - GTWYSLAMAASDIS -

[21] 11-29 11-30 15-29 15-30 16-30 17-29 17-30 0,00019 0,00053 0,002 0,000019 0,0014 0,00023 0,000015 P12 *01:01 *04:04 AMAASDISLLDAQSAPL -

[16] - MAASDISLLDAQSAPL -

[18] 23-39 24-39 0,028 0,042

[0203] Table 4. The association of HLA-DR alleles in the unique region of mature BLG with significant fragments was determined by the expected value <0.05 Donor ID DRB1_1 DRB1_2 Unique region (mature BLG) [SEQ ID NO] Amino acid beginning - end Number of significant fragments and individual fragment P2 *01:01 *04:01 AMAASDISLLDAQSAPLR

[39] 23-40 2 (23-39 and 24-40) P3 *01:01 *07:01 MAASDISLLDAQSAPL

[18] 24-39 1 (24-39) P4 *01:01 *04:05 DIQKVAGTWYSLAMAASDIS

[22] VAGTWYSLAMAASDIS

[20] GTWYSLAMAASDIS

[21] 11-30 3 (11:29, 15:30 and 17:30) P7 *03:01 *09:01 DIQKVAGTWYSLAMAASDIS

[22] 11-30 7 (11-29, 11-30, 15-29, 15-30, 16-30, 17-29, 17-30) P12 *01:01 *04:04 AMAASDISLLDAQSAPL

[16] 23-39 2 (23-39, 24-39)

[0204] Not all donor antigen-presenting cells bind peptides. This was the case for HLA-DRB1*03:01 in the homozygous donor P1. Similar arguments apply to donors P6 and P11.

[0205] EXAMPLE 3. MHC CLASS II BINDING IN SILICO

[0206] Limitation of the ProPresent Antigen Presentation Assay ® The use of a general anti-HLA antibody, rather than a specific antibody to a specific HLA-DRB1 allele (e.g., anti-DRB1*01:01 antibodies), is essential for isolating the HLA-DRB1 peptide complex under study. Therefore, for a donor with heterozygous HLA-DRB1 genotypes, such as *01:01 and *04:01, it is unclear which allele will present the identified peptide.

[0207] Thus, the identified HLA-DR-restricted peptides were calculated in the IEDB MHC class II binding prediction software (http: / / tools.iedb.org / mhcii / ) to evaluate the binding of the detected peptides to the selected HLA-DRB1 alleles as described (Wang P, et al. PLoS Comput Biol 2008 4; 4(4):e1000048; Wang et al, BMC Bioinformatics 2010, 11:568-2105-11-568). The recommended IEDB prediction method was selected by default. For each peptide sequence (15-mer length), a percentile rank was obtained by comparing the peptide score with the scores of five million random 15-mer peptides selected from the Swiss-Prot database. A lower percentile rank indicates a higher binding affinity of the peptide to a particular MHC class II allele, where the IEDB recommends making a choice based on the consensus percentile rank of the top 10%.To ensure greater rigor when using in silico scoring, a choice was made based on the consensus percentile rank of the top 3%.

[0208] MHC class II prediction software was used to assess whether fragments of the identified two groups of unique BLG sequences would have high binding affinity to selected HLA-DRB1 alleles of characterized donors from the ProPresent® assay, i.e., HLA-DRB1 *01:01, *03:01, *04:01, *04:04, *04:05, *07:01, and *09:01. As shown in Table 5, at an arbitrary percentile rank cutoff set at <3% (i.e., the top 3% of binders), DIQ…DIS fragments (AA#11-30) were predicted to have high binding affinity to 5 HLA-DRB1 alleles, i.e., DRB1*01:01, *04:01, *04:04, *04:05, and *09:01. In contrast, only one AMA…APL fragment (AA#23-39) was predicted to bind to HLA-DRB1*07:01 with high affinity. Taken together, these results suggest that AA#11-30 fragments had a higher probability of being presented as T-cell epitopes than AA#23-39 fragments.Furthermore, these data suggest that multiple common HLA-DRB1 alleles may present fragments derived from the identified two groups of BLG-derived unique sequences.

[0209] These groups of unique BLG sequences (AA#11-30 and #23-39) overlap and can be combined into a single sequence (i.e., AA#11-39). This combined sequence was highly correlated with one consistently identified peptide in the studied HP (AA#11-42; DIQ…RVY). Therefore, the AA#11-42 sequence was tested by MHC class II prediction software. Importantly, the predicted AA#11-42 complexes confirmed the predicted results of the two in vitro identified unique sequences (Table 6), suggesting that both unique BLG sequences can be derived from the AA#11-42 peptide, which is present in the studied infant formula. Furthermore, AA regions #11-42 were predicted to have high affinity for the same groups of identified HLA-DRB1 alleles, i.e., DRB1 *01:01, *04:01, *04:04, *04:05, *07:01, and *09:01.In conclusion, in silico evaluation confirms in vitro results that BLG-derived peptides can bind to common HLA-DRB1 alleles.

[0210] Table 5. In silico evaluation of potential HLA-DRB1-peptide complexes of two identified BLG-derived peptides with a percentile rank <3% HLA-DRB1 allele Predicted 15-mer fragment DIQKVAGTWYSLAMAASDIS (SEQ ID NO:25) or AMAASDISLLDAQSAPL (SEQ ID NO:19) [SEQ ID NO] Percentile rank HLA-DRB1*09:01 AGTWYSLAMAASDIS

[24] 0,86 HLA-DRB1*04:05 AGTWYSLAMAASDIS

[24] 1,38 HLA-DRB1*04:05 VAGTWYSLAMAASDI

[23] 1,40 HLA-DRB1*09:01 VAGTWYSLAMAASDI

[23] 1,56 HLA-DRB1*04:05 KVAGTWYSLAMAASD

[26] 2,26 HLA-DRB1*01:01 AGTWYSLAMAASDIS

[24] 2,37 HLA-DRB1*04:04 AGTWYSLAMAASDIS

[24] 2,44 HLA-DRB1*09:01 KVAGTWYSLAMAASD

[26] 2,53 HLA-DRB1*04:01 AGTWYSLAMAASDIS

[24] 2,84 HLA-DRB1*07:01 AASDISLLDAQSAPL

[27] 2,39

[0211] Table 6. In silico evaluation of potential HLA-DRB1-peptide complexes of long BLG-derived peptide with percentile rank <3% (top 3% of binders) HLA-DRB1 allele Predicted 15-mer fragment DIQKVAGTWYSLAMAASDIS (SEQ ID NO:25) or AMAASDISLLDAQSAPL (SEQ ID NO:19) [SEQ ID NO] Percentile rank HLA-DRB1*09:01 GTWYSLAMAASDISL

[28] 0,65 HLA-DRB1*09:01 AGTWYSLAMAASDIS

[24] 0,86 HLA-DRB1*04:05 GTWYSLAMAASDISL

[28] 1,30 HLA-DRB1*09:01 TWYSLAMAASDISLL

[29] 1,32 HLA-DRB1*04:05 AGTWYSLAMAASDIS

[24] 1,38 HLA-DRB1*04:05 VAGTWYSLAMAASDI

[23] 1,40 HLA-DRB1*04:05 TWYSLAMAASDISLL

[29] 1,40 HLA-DRB1*09:01 VAGTWYSLAMAASDI

[23] 1,56 HLA-DRB1*09:01 WYSLAMAASDISLLD

[30] 2,12 HLA-DRB1*09:01 YSLAMAASDISLLDA

[31] 2,14 HLA-DRB1*01:01 GTWYSLAMAASDISL

[28] 2,18 HLA-DRB1*04:05 KVAGTWYSLAMAASD

[26] 2,26 HLA-DRB1*01:01 AGTWYSLAMAASDIS

[24] 2,37 HLA-DRB1*07:01 AASDISLLDAQSAPL

[27] 2,39 HLA-DRB1*07:01 SDISLLDAQSAPLRV

[32] 2,44 HLA-DRB1*04:04 AGTWYSLAMAASDIS

[24] 2,44 HLA-DRB1*04:04 GTWYSLAMAASDISL

[28] 2,44 HLA-DRB1*04:04 TWYSLAMAASDISLL

[29] 2,44 HLA-DRB1*01:01 TWYSLAMAASDISLL

[29] 2,46 HLA-DRB1*09:01 KVAGTWYSLAMAASD

[26] 2,53 HLA-DRB1*04:04 YSLAMAASDISLLDA

[31] 2,57 HLA-DRB1*04:01 GTWYSLAMAASDISL

[28] 2,83 HLA-DRB1*04:01 AGTWYSLAMAASDIS

[24] 2,84 HLA-DRB1*04:04 WYSLAMAASDISLLD

[30] 2,91

[0212] Some in silico assessments confirmed the in vitro ProPresent® results for HLA-DR peptide complexes. However, we noted that some in silico predicted complexes did not correlate with those detected in the ProPresent® assay, for example, DRB1 *04:04 with AGT…DIS. This discrepancy could be explained by the small size of the study cohort in the ProPresent assay. ® (n=12), or the fact that each donor had a different combination of HLA-DRB1 allele types (i.e., interindividual variability) and / or the tendency of in silico assessment to be overly predictive (which requires strict limitation). Therefore, it is advisable to combine both in vitro and in silico approaches to identify peptide-MHC class II complexes.

[0213] EXAMPLE 4. T-CELL PROLIFERATION ASSAY

[0214] Synthetic peptides were produced using JPT technology. Only peptides containing at least 9 AAs were considered, as this is the minimum size for binding to MHC class II molecules and subsequent recognition by T cells (Holland et al, Front Immunol 2013 Jul 1; 4:172). Synthetic peptides (JPT technology), identical to the peptides identified in all HP lots from Example 1, were dissolved in dimethyl sulfoxide (Sigma Aldrich) at a concentration of 5.3 mM. The peptides were tested on cow's milk-specific T cell lines (TCLs) from three different donors. Peptides were tested on cow's milk protein-specific T cell lines (TCLs) from three human infant donors diagnosed with cow's milk protein allergy (CMPA) at ages <1 year, 7.5 months, and 6 years. These TCLs had been previously generated and were shown to recognize epitopes in the region of interest (AA #13-48 of mature BLG).Proliferation was determined as described previously (Ruiter et al, Clin Exp Allergy 2006, 36(3):303–310). Stimulation indices (SIs, the ratio between the proliferation of allergen / peptide-stimulated and unstimulated T cells) were calculated, and SI ≥ 2 was considered positive.

[0215] To confirm that the peptides identified in HP were recognized by T cells, synthetic peptides identical to these identified peptides were tested on cow's milk-specific human TCLs. Since there was overlap between the identified peptides, five peptides (Table 7) that were identified in all lots and differed from each other by more than 9 A were tested. All tested peptides were able to induce proliferation (Fig. 2). However, each donor demonstrated its own pattern recognition. This was also observed with in silico prediction data (data not shown). All five peptides induced proliferation of TCL B, suggesting that this TCL recognizes either the overlapping portion of the peptides (AA #11-27) or multiple T cell epitopes in this region. TCL A also showed a proliferative response after stimulation with several peptides.Since the LIV…AAS (AA#1-27) peptide was not able to induce proliferation in TCL A, while the LIV…SLL (AA#1-32) peptide could, the region containing AA#28-32 (DISLL) is essential for the TCL A response. Surprisingly, TCL C recognized the LIV…AAS (AA#1-27) peptide, while a longer peptide containing the same sequence with 5 additional AA, LIV…SLL (AA#1-32), was not able to induce a proliferative response, indicating that longer peptides are not always better presented.

[0216] Table 7. Beta-lactoglobulin peptide sequences identified in 10 different batches of partially hydrolyzed whey-based infant formula and tested for T-cell proliferation activity Amino acid number in the sequence Subsequence SEQ ID NO AA 1-27 LIVTQTMKGLDIQKVAGTWYSLAMAAS 9 AA 1-32 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLL 11 AA 6-32 TMKGLDIQKVAGTWYSLAMAASDISLL 3 AA 6-35 TMKGLDIQKVAGTWYSLAMAASDISLLDAQ 4 AA 11-42 DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 5

[0217] Not all peptides were recognized by each donor, indicating interdonor differences. Each donor expressed a different panel of MHC class II molecules. The same algorithm described above was used to determine whether and which HLA-DRB1 allele was expressed by the donors of the presented peptides. In silico prediction confirmed that the HLA-DRB1 alleles (*11:01 and *04:04) expressed by donor B are capable of presenting all peptides. Based on the algorithm, TCL A should recognize only the DIQ...RVY peptide (AA #11-42), but this TCL showed a proliferative response after stimulation with four of the five peptides. A possible explanation may be that the other three peptides were presented not by HLA-DRB1, but by other MHC class II molecules.

[0218] In conclusion, examples 1-4 demonstrate that the specific whey protein hydrolysate contains functional T-cell epitopes. Specific beta-lactoglobulin peptides, which are HLA-DRB1-restricted peptides, were identified. They are presented and recognized by T cells from various donors, including healthy donors and infants with cow's milk protein allergy. This interaction was confirmed by in silico analysis. Therefore, HP may stimulate the development of oral immunotolerance to the protein.

[0219] EXAMPLE 5. A SYNBIOTICS MIXTURE DEMONSTRATES AN EVEN IMPROVED TOLERANCE-INDUCING EFFECT COMPARED TO ALONE PROBIOTICS OR PREBIOTICS

[0220] Several published studies support the idea that food antigens (including peptides) in the intestinal lumen can be absorbed and then taken up and presented by intestinal antigen-presenting cells, i.e., antigen selection mechanisms, without the need to disrupt the intestinal barrier. There are at least two distinct mechanisms that continuously operate to select food antigens in the healthy state: microfold / M cell-mediated transcytosis and goblet cell-mediated antigen passage. Both pathways deliver food antigens to CD103+ DCs in the intestinal lamina propria, which, in turn, can implicate intestinal homing molecules on T and B cells, can promote the differentiation of IgA-producing intestinal plasma cells, and can also generate intestinal Tregs, a key element in the state of hyporesponsiveness to the absorbed antigen known as oral tolerance.This suggests that peptides in the tested HPs can be absorbed orally, followed by uptake and presentation by intestinal CD103+DCs, which then leads to the formation of functional Tregs.

[0221] For oral tolerance to develop, T-cell epitopes in the HP test cells must be presented under appropriate conditions. In addition to TCR activation, costimulation and cytokine signaling play an important role in the generation of Tregs. Probiotics and prebiotics play a crucial role in creating the appropriate environment for this predisposition to Tregs. The preventive effect of synthetic BLG peptides was enhanced when combined with a probiotic or prebiotic diet.

[0222] To demonstrate the effect of pro-, pre- and synbiotics, an experiment similar to that described in Example 4 of international application WO 2016 / 148572 was conducted using the same protocol, the same concentration and a mixture of synthetic peptides (“Pepmix”), Pepmix is ​​identified in Table 8.

[0223] Table 8. Pepmix according to international application WO 2016 / 148572 Peptide Subsequence 1 (18AA) SEQ ID NO: 33 QKVAGTWYSLAMAASDIS 2 (18AA) SEQ ID NO: 34 WYSLAMAASDISLLDAQS 3 (18AA) SEQ ID NO: 35 AASDISLLDAQSAPLRVY 4 (18AA) SEQ ID NO: 36 LLDAQSAPLRVYVEELKP

[0224] A number of diets were compared:

[0225] (a) synbiotic diet from example 1 of international application WO 2016 / 14472 (1 wt.% scFOS / lcFOS in a weight ratio of 9:1+2 wt.% 2×10 9 CFU / gBifidobacterium breveM-16V,

[0226] (b) the probiotic diet from example 4 of international application WO 2016 / 148572 using 2 wt.% 2×10 9 CFU / gBifidobacterium breveM-16V, and

[0227] (c) Prebiotic diet containing 1 wt% scFOS / lcFOS with a weight ratio of 9 / 1.

[0228] Short-chain (sc-) and long-chain (lc-) fructooligosaccharides (FOS) were marketed as Raftilose P95 (Orafti) and Raftiline HP, respectively.

[0229] All groups were pre-treated with Pepmix and one of the above diets, sensitized with serum+cholera toxin, and challenged with serum.

[0230] After serum challenge, the ear swelling delta of mice consuming (c) scFOS / lcFOS diet was about 174 μm, that of mice consuming (b) probiotic diet was about 158 ​​μm, and that of mice consuming (a) synbiotic diet was about 112 μm. The ear swelling in the synbiotic group (a) was statistically significantly lower than that in the corresponding scFOS / lcFOS group (c), and there was a tendency for a lower response compared with the probiotic group (b) (p=0.08). These results indicate further improvement of the effect on oral immunotolerance induction:

[0231] when using lactic acid bacteria, in particular the Bifidobacterium species B. breve, and

[0232] especially when using lactic acid bacteria in combination with NDOs.

[0233] EXAMPLE 6. MHC-BINDING DOMAINS IN NATURAL, IDENTIFIED HYDROLYSATE PEPTIDES COMPARED TO SYNTHETIC BETA-LACTOGLOBULIN PEPTIDES

[0234] Method: Beta-lactoglobulin peptide sequences identified in hydrolyzed infant formula (ID_PEP) and synthetic beta-lactoglobulin peptide sequences published in international application WO2016 / 148572 (SYN_PEP, Table 9) were tested in silico using the IEDB MHC class II binding prediction software (http: / / tools.iedb.org / mhcii / ) to determine the number of MHC class II binding domains in each peptide. The software selected the IEDB-recommended prediction method and a complete set of HLA references. This set of HLA references provides population coverage of <99%. The software calculates a percentile rank by comparing the binding affinity of the predicted binding domain to a large group of peptides. The higher the affinity, the lower the percentile rank. In this experiment, the top 2% of binders were selected.

[0235] Results

[0236] The number of MHC class II binding domains in the identified peptides is higher than in the synthetic peptides (see Table 9). The software predicted at least one binding domain for all identified peptides and a total of six different domains. For the synthetic peptides, four different domains were predicted. However, all of these domains were derived from a single sequence, namely SYN_PEP_1. No MHC class II binding domains were predicted for the other two synthetic peptides.

[0237] Table 9. Number of MHC class II binding domains per beta-lactoglobulin peptide predicted by IEBD Peptide Subsequence Number of MHC class II binding domains ID_PEP_1 LIVTQTMKGLDIQKVAGTWYSLAMAAS 1 ID_PEP_2 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLL 6 ID_PEP_3 TMKGLDIQKVAGTWYSLAMAASDISLL 6 ID_PEP_4 TMKGLDIQKVAGTWYSLAMAASDISLLDAQ 6 ID_PEP_5 DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 6 SYN_PEP_1 QKVAGTWYSLAMAASDIS 4 SYN_PEP_2 WYSLAMAASDISLLDAQS 0 SYN_PEP_3 AASDISLLDAQSAPLRVY 0

[0238] The number of MHC class II binding domains is higher in the identified peptides than in the synthetic peptides. In other words, more T-cell epitopes can be obtained from the identified peptides than from the synthetic peptides. Consequently, the likelihood that the identified peptides will be presented to T cells is higher. Such presentation to T cells is a prerequisite for the induction of oral immune tolerance.

[0239] EXAMPLE 7: EXPRESSION OF HLA-DR ON DENDRITIC CELLS AFTER MATURATION USING LACTIC ACID BACTERIA

[0240] Method: Monocytes (CD14+ cells) were cultured for 7 days with GM-CSF and IL-4 to obtain immature dendritic cells (DCs). After 7 days, immature DCs were washed and incubated with either medium, LPS (100 ng / mL), or Bifidobacterium breve M-16V (Morinaga) at a bacteria:DC ratio of 10:1. After 48 hours, mature DCs were collected and stained with APC-Cyanine7-labeled human HLA-DR antibodies to determine cell surface HLA-DR expression. Samples were analyzed on a BD FACS Canto flow cytometer with FACS DIVA software. HLA-DR expression is given as mean fluorescence intensity (MFI).

[0241] Results: Maturation with Bifidobacterium breve M-16V (M16v-DC) increased HLA-DR expression to a level similar to LPS (LPS-DC, see Table 10).

[0242] Table 10. Number of MHC class II binding domains per beta-lactoglobulin peptide predicted by IEBD DCs MFI HLA-DR expression (SD) Immature DCs 11338 (1919,1) LPS-DCs 24676 (9694,4) M16v-DCs 21714 (1516)

[0243] DC=dendritic cells, MFI=mean fluorescence intensity, SD=standard deviation

[0244] Bifidobacterium breveM-16V increases the expression of HLA-DR molecules on the surface of dendritic cells. Increased HLA-DR expression indicates enhanced peptide presentation to T cells.

[0245] EXAMPLE 8. INFANT FORMULA

[0246] Powdered infant formula, packaged with instructions to reconstitute with water at 40°C and add 3 scoops of powder (13.74 g) to 90 ml of water to obtain a final volume of 100 ml. The product is suitable from birth to 6 months.

[0247] 100 ml of infant formula milk contains 66 kcal, 1.5 g of protein (hydrolyzed whey protein from Example 1), 3.3 g of fat (vegetable oils, single-cell oil and fish oil containing 0.4 wt% DHA and 0.35 wt% ARA based on total fatty acids), 7.2 g of digestible carbohydrates (mainly lactose), 0.8 g of indigestible oligosaccharides (mixture of scGOS and lcFOS 9 / 1 wt / wt), Bifidobacterium breveM-16V in an amount of about 3×10 7 CFU / g, as well as vitamins, minerals, trace elements and other biologically active substances in accordance with international guidelines for infant formula.

[0248] --->

[0249] SEQUENCE LIST

[0250] <110> NV Nutricia

[0251] <120> MILK FORMULA WITH SPECIFIC BETA-LACTOGLOBULIN PEPTIDES

[0252] <130> P6075124PCT1

[0253] <150> PCT / NL2018 / 050281

[0254] <151> 2018-04-30

[0255] <160> 39

[0256] <210> 1

[0257] <211> 27

[0258] <212> protein

[0259] <213> Artificial sequence

[0260] <220>

[0261] <223> beta-lactoglobulin peptide fragment

[0262] <220>

[0263] <221> misc_feature

[0264] <222> (1)..(1)

[0265] <223> Haa can be Ile or Leu

[0266] <400> 1

[0267] Xaa Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0268] 1 5 10 15

[0269] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser

[0270] 20 25

[0271] <210> 2

[0272] <211> 32

[0273] <212> protein

[0274] <213> Artificial sequence

[0275] <220>

[0276] <223> beta-lactoglobulin peptide fragment

[0277] <220>

[0278] <221> misc_feature

[0279] <222> (1)..(1)

[0280] <223> Haa can be Ile or Leu

[0281] <400> 2

[0282] Xaa Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0283] 1 5 10 15

[0284] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0285] 20 25 30

[0286] <210> 3

[0287] <211> 27

[0288] <212> protein

[0289] <213> Artificial sequence

[0290] <220>

[0291] <223> beta-lactoglobulin peptide fragment

[0292] <400> 3

[0293] Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala Gly Thr Trp Tyr Ser

[0294] 1 5 10 15

[0295] Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0296] 20 25

[0297] <210> 4

[0298] <211> 30

[0299] <212> protein

[0300] <213> Artificial sequence

[0301] <220>

[0302] <223> beta-lactoglobulin peptide fragment

[0303] <400> 4

[0304] Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala Gly Thr Trp Tyr Ser

[0305] 1 5 10 15

[0306] Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln

[0307] 20 25 30

[0308] <210> 5

[0309] <211> 32

[0310] <212> protein

[0311] <213> Artificial sequence

[0312] <220>

[0313] <223> beta-lactoglobulin peptide fragment

[0314] <400> 5

[0315] Asp Ile Gln Lys Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala

[0316] 1 5 10 15

[0317] Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg Val Tyr

[0318] 20 25 30

[0319] <210> 6

[0320] <211> 22

[0321] <212> protein

[0322] <213> Artificial sequence

[0323] <220>

[0324] <223> beta-lactoglobulin peptide fragment

[0325] <400> 6

[0326] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0327] 1 5 10 15

[0328] Gly Thr Trp Tyr Ser Leu

[0329] 20

[0330] <210> 7

[0331] <211> 25

[0332] <212> protein

[0333] <213> Artificial sequence

[0334] <220>

[0335] <223> beta-lactoglobulin peptide fragment

[0336] <400> 7

[0337] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0338] 1 5 10 15

[0339] Gly Thr Trp Tyr Ser Leu Ala Met Ala

[0340] 20 25

[0341] <210> 8

[0342] <211> 26

[0343] <212> protein

[0344] <213> Artificial sequence

[0345] <220>

[0346] <223> beta-lactoglobulin peptide fragment

[0347] <400> 8

[0348] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0349] 1 5 10 15

[0350] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala

[0351] 20 25

[0352] <210> 9

[0353] <211> 27

[0354] <212> protein

[0355] <213> Artificial sequence

[0356] <220>

[0357] <223> beta-lactoglobulin peptide fragment

[0358] <400> 9

[0359] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0360] 1 5 10 15

[0361] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser

[0362] 20 25

[0363] <210> 10

[0364] <211> 30

[0365] <212> protein

[0366] <213> Artificial sequence

[0367] <220>

[0368] <223> beta-lactoglobulin peptide fragment

[0369] <400> 10

[0370] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0371] 1 5 10 15

[0372] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

[0373] 20 25 30

[0374] <210> 11

[0375] <211> 32

[0376] <212> protein

[0377] <213> Artificial sequence

[0378] <220>

[0379] <223> beta-lactoglobulin peptide fragment

[0380] <400> 11

[0381] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0382] 1 5 10 15

[0383] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0384] 20 25 30

[0385] <210> 12

[0386] <211> 35

[0387] <212> protein

[0388] <213> Artificial sequence

[0389] <220>

[0390] <223> beta-lactoglobulin peptide fragment

[0391] <400> 12

[0392] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0393] 1 5 10 15

[0394] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0395] 20 25 30

[0396] Asp Ala Gln

[0397] 35

[0398] <210> 13

[0399] <211> 39

[0400] <212> protein

[0401] <213> Artificial sequence

[0402] <220>

[0403] <223> beta-lactoglobulin peptide fragment

[0404] <400> 13

[0405] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0406] 1 5 10 15

[0407] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0408] 20 25 30

[0409] Asp Ala Gln Ser Ala Pro Leu

[0410] 35

[0411] <210> 14

[0412] <211> 42

[0413] <212> protein

[0414] <213> Artificial sequence

[0415] <220>

[0416] <223> beta-lactoglobulin peptide fragment

[0417] <400> 14

[0418] Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0419] 1 5 10 15

[0420] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0421] 20 25 30

[0422] Asp Ala Gln Ser Ala Pro Leu Arg Val Tyr

[0423] 35 40

[0424] <210> 15

[0425] <211> 31

[0426] <212> protein

[0427] <213> Artificial sequence

[0428] <220>

[0429] <223> beta-lactoglobulin peptide fragment

[0430] <400> 15

[0431] Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala Gly Thr Trp Tyr

[0432] 1 5 10 15

[0433] Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln

[0434] 20 25 30

[0435] <210> 16

[0436] <211> 17

[0437] <212> protein

[0438] <213> Artificial sequence

[0439] <220>

[0440] <223> beta-lactoglobulin peptide fragment

[0441] <400> 16

[0442] Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro

[0443] 1 5 10 15

[0444] Leu

[0445] <210> 17

[0446] <211> 17

[0447] <212> protein

[0448] <213> Artificial sequence

[0449] <220>

[0450] <223> beta-lactoglobulin peptide fragment

[0451] <400> 17

[0452] Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu

[0453] 1 5 10 15

[0454] Arg

[0455] <210> 18

[0456] <211> 16

[0457] <212> protein

[0458] <213> Artificial sequence

[0459] <220>

[0460] <223> beta-lactoglobulin peptide fragment

[0461] <400> 18

[0462] Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu

[0463] 1 5 10 15

[0464] <210> 19

[0465] <211> 19

[0466] <212> protein

[0467] <213> Artificial sequence

[0468] <220>

[0469] <223> beta-lactoglobulin peptide fragment

[0470] <400> 19

[0471] Asp Ile Gln Lys Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala

[0472] 1 5 10 15

[0473] Ser Asp Ile

[0474] <210> 20

[0475] <211> 16

[0476] <212> protein

[0477] <213> Artificial sequence

[0478] <220>

[0479] <223> beta-lactoglobulin peptide fragment

[0480] <400> 20

[0481] Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

[0482] 1 5 10 15

[0483] <210> 21

[0484] <211> 14

[0485] <212> protein

[0486] <213> Artificial sequence

[0487] <220>

[0488] <223> beta-lactoglobulin peptide fragment

[0489] <400> 21

[0490] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

[0491] 1 5 10

[0492] <210> 22

[0493] <211> 20

[0494] <212> protein

[0495] <213> Artificial sequence

[0496] <220>

[0497] <223> beta-lactoglobulin peptide fragment

[0498] <400> 22

[0499] Asp Ile Gln Lys Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala

[0500] 1 5 10 15

[0501] Ser Asp Ile Ser

[0502] 20

[0503] <210> 23

[0504] <211> 15

[0505] <212> protein

[0506] <213> Artificial sequence

[0507] <220>

[0508] <223> beta-lactoglobulin peptide fragment

[0509] <400> 23

[0510] Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile

[0511] 1 5 10 15

[0512] <210> 24

[0513] <211> 15

[0514] <212> protein

[0515] <213> Artificial sequence

[0516] <220>

[0517] <223> beta-lactoglobulin peptide fragment

[0518] <400> 24

[0519] Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

[0520] 1 5 10 15

[0521] <210> 25

[0522] <211> 13

[0523] <212> protein

[0524] <213> Artificial sequence

[0525] <220>

[0526] <223> beta-lactoglobulin peptide fragment

[0527] <400> 25

[0528] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile

[0529] 1 5 10

[0530] <210> 26

[0531] <211> 15

[0532] <212> protein

[0533] <213> Artificial sequence

[0534] <220>

[0535] <223> beta-lactoglobulin peptide fragment

[0536] <400> 26

[0537] Lys Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp

[0538] 1 5 10 15

[0539] <210> 27

[0540] <211> 15

[0541] <212> protein

[0542] <213> Artificial sequence

[0543] <220>

[0544] <223> beta-lactoglobulin peptide fragment

[0545] <400> 27

[0546] Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu

[0547] 1 5 10 15

[0548] <210> 28

[0549] <211> 15

[0550] <212> protein

[0551] <213> Artificial sequence

[0552] <220>

[0553] <223> beta-lactoglobulin peptide fragment

[0554] <400> 28

[0555] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu

[0556] 1 5 10 15

[0557] <210> 29

[0558] <211> 15

[0559] <212> protein

[0560] <213> Artificial sequence

[0561] <220>

[0562] <223> beta-lactoglobulin peptide fragment

[0563] <400> 29

[0564] Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0565] 1 5 10 15

[0566] <210> 30

[0567] <211> 15

[0568] <212> protein

[0569] <213> Artificial sequence

[0570] <220>

[0571] <223> beta-lactoglobulin peptide fragment

[0572] <400> 30

[0573] Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp

[0574] 1 5 10 15

[0575] <210> 31

[0576] <211> 15

[0577] <212> protein

[0578] <213> Artificial sequence

[0579] <220>

[0580] <223> beta-lactoglobulin peptide fragment

[0581] <400> 31

[0582] Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala

[0583] 1 5 10 15

[0584] <210> 32

[0585] <211> 15

[0586] <212> protein

[0587] <213> Artificial sequence

[0588] <220>

[0589] <223> beta-lactoglobulin peptide fragment

[0590] <400> 32

[0591] Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg Val

[0592] 1 5 10 15

[0593] <210> 33

[0594] <211> 18

[0595] <212> protein

[0596] <213> Artificial sequence

[0597] <220>

[0598] <223> Pepmix peptide 1

[0599] <400> 33

[0600] Gln Lys Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp

[0601] 1 5 10 15

[0602] Ile Ser

[0603] <210> 34

[0604] <211> 18

[0605] <212> protein

[0606] <213> Artificial sequence

[0607] <220>

[0608] <223> Pepmix peptide 2

[0609] <400> 34

[0610] Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala

[0611] 1 5 10 15

[0612] Gln Ser

[0613] <210> 35

[0614] <211> 18

[0615] <212> protein

[0616] <213> Artificial sequence

[0617] <220>

[0618] <223> Pepmix peptide 3

[0619] <400> 35

[0620] Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg

[0621] 1 5 10 15

[0622] Val Tyr

[0623] <210> 36

[0624] <211> 18

[0625] <212> protein

[0626] <213> Artificial sequence

[0627] <220>

[0628] <223> Pepmix peptide 4

[0629] <400> 36

[0630] Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg Val Tyr Val Glu Glu Leu

[0631] 1 5 10 15

[0632] Lys Pro

[0633] <210> 37

[0634] <211> 27

[0635] <212> protein

[0636] <213> Artificial sequence

[0637] <220>

[0638] <223> beta-lactoglobulin peptide fragment

[0639] <400> 37

[0640] Ile Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0641] 1 5 10 15

[0642] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser

[0643] 20 25

[0644] <210> 38

[0645] <211> 32

[0646] <212> protein

[0647] <213> Artificial sequence

[0648] <220>

[0649] <223> beta-lactoglobulin peptide fragment

[0650] <400> 38

[0651] Ile Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

[0652] 1 5 10 15

[0653] Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

[0654] 20 25 30

[0655] <210> 39

[0656] <211> 18

[0657] <212> protein

[0658] <213> Artificial sequence

[0659] <220>

[0660] <223> beta-lactoglobulin peptide fragment

[0661] <400> 39

[0662] Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro

[0663] 1 5 10 15

[0664] Leu Arg

[0665] <---

Claims

1. Use of a nutritional composition to obtain a product for: - induction of oral immune tolerance to milk protein; and / or - prevention of oral immune intolerance to milk protein; and / or - reducing the risk of developing oral immune intolerance to milk protein, and / or - improvement or strengthening of oral immune tolerance to milk protein, in humans, wherein the nutritional composition comprises a protein hydrolysate from mammalian milk, preferably milk from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, wherein the protein hydrolysate comprises at least a peptide having a sequence according to SEQ ID NO: 1 or SEQ ID NO: 4, and at least one peptide having a sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5, wherein said composition preferably further comprises one strain of lactic acid bacteria belonging to the genus Bifidobacterium.

2. The use according to claim 1, wherein the composition contains less than 6 μg of allergy-causing beta-lactoglobulin, preferably less than 3.5 μg of allergy-causing beta-lactoglobulin per 1 g of total protein.

3. The use according to any one of claims 1 or 2, wherein the composition comprises at least 50% by weight, preferably at least 95% by weight of hydrolyzed whey protein, based on total protein.

4. The use according to any one of claims 1 to 3, comprising less than 10% by weight, preferably less than 6% by weight, of peptides or proteins having a size of 5 kDa or more, calculated on the total protein.

5. The use according to any one of claims 1 to 4, wherein at least 1% by weight of the peptides or proteins present in the composition have a size of 1 kDa or more, calculated on the total protein, preferably at least 5% by weight, more preferably at least 10% by weight, calculated on the total protein.

6. Use according to any one of claims 1-5, wherein the composition contains an amount of allergy-causing beta-lactoglobulin greater than 0.8 μg per 1 g of total protein.

7. The use according to any one of claims 1 to 6, wherein the milk protein comes from a species of the genus Bos, Bison, Bubalus or Capra, more preferably the genus Bos, preferably cow's milk protein.

8. Use according to any of paragraphs 1-7, where the person is an infant.

9. The use according to any one of claims 1 to 8, wherein the human is at risk of developing or suffers from an allergy to milk protein from milk of species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, and wherein the human is preferably an infant.

10. The use according to any one of claims 1 to 9, wherein the composition comprises a strain of lactic acid bacteria belonging to the species Bifidobacterium breve.

11. The use according to any one of claims 1 to 10, comprising one or more indigestible oligosaccharides selected from the group consisting of fructooligosaccharide, indigestible dextrin, galactooligosaccharide, xylooligosaccharide, arabinooligosaccharide, arabinogalactooligosaccharide, glucooligosaccharide, glucomannooligosaccharide, galactomannooligosaccharide, mannanoligosaccharide, chitooligosaccharide, uronic acid oligosaccharide, sialyloligosaccharide and fucooligosaccharide, and mixtures thereof, preferably fructooligosaccharides.

12. The use according to claim 11, wherein the indigestible oligosaccharides comprise at least two indigestible oligosaccharides selected from the group consisting of fructooligosaccharides and galactooligosaccharides, preferably a mixture of long-chain fructooligosaccharides with short-chain fructooligosaccharides or with short-chain galactooligosaccharides.

13. The use according to any one of claims 1 to 12, comprising long-chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35% by weight DHA based on total fatty acids.

14. The use according to any one of paragraphs 1-13, which is an infant formula, a second-level formula, or an infant formula.

15. A nutritional composition comprising: a) a strain of lactic acid bacteria belonging to the species Bifidobacterium breve; b) a milk protein hydrolysate from a species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, wherein the milk protein hydrolysate comprises at least a peptide having a sequence according to SEQ ID NO: 1 or SEQ ID NO: 4, and at least one peptide having a sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5; c) less than 6 mcg of allergenic beta-lactoglobulin, preferably less than 3.5 mcg of beta-lactoglobulin, per 1 g of total protein; d) less than 10% by weight of peptides or proteins having a size of 5 kDa or greater, calculated as total protein; and e) at least 50% by weight, preferably at least 95% by weight of hydrolyzed whey protein, based on total protein; f) optionally, one or more indigestible oligosaccharides selected from the group consisting of fructooligosaccharide, indigestible dextrin, galactooligosaccharide, xylooligosaccharide, arabinooligosaccharide, arabinogalactooligosaccharide, glucooligosaccharide, glucomannooligosaccharide, galactomannooligosaccharide, mannanoligosaccharide, chitooligosaccharide, uronic acid oligosaccharide, sialyloligosaccharide and fucooligosaccharide, and mixtures thereof, preferably fructooligosaccharides; and g) optionally long-chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35% by weight DHA, based on total fatty acids, wherein the nutritional composition is an infant formula, a second-level formula, or an infant formula.

16. The nutritional composition of claim 15, wherein the amount of allergy-causing beta-lactoglobulin is more than 0.8 μg per 1 g of protein and / or wherein the composition contains more than 1 wt.% of peptides or proteins of 1 kDa or more in size based on total protein, more preferably at least 5 wt.%, more preferably at least 10 wt.%.

17. The nutritional composition according to claim 15 or 16, wherein the indigestible oligosaccharides comprise at least two indigestible oligosaccharides selected from the group consisting of fructooligosaccharides and galactooligosaccharides, preferably a mixture of long-chain fructooligosaccharides with short-chain fructooligosaccharides or with short-chain galactooligosaccharides.

18. A nutritional composition according to any one of paragraphs 15-17, containing from 10 5 up to 10 11 CFU of lactic acid bacteria per 1 g of dry weight.

19. A nutritional composition according to any one of claims 15-18, comprising at least 2% by weight of indigestible oligosaccharides on a dry weight basis.

20. A method of providing nutrition to a human being at risk of developing or suffering from an allergy, more preferably an allergy to milk protein, comprising administering to the human being a nutritional composition according to any one of claims 15-19.

21. The method of claim 20, wherein the human is an infant or young child, preferably an infant.

22. Non-therapeutic method: - induction of oral immune tolerance to milk protein; and / or - prevention of oral immune intolerance to milk protein; and / or - reducing the risk of developing oral immune intolerance to milk protein, and / or - improvement or strengthening of oral immune tolerance to milk protein, in humans, comprising administering to the subject a nutritional composition comprising a protein hydrolysate from mammalian milk, preferably milk from species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, preferably cow's milk, wherein the protein hydrolysate comprises at least a peptide having a sequence according to SEQ ID NO: 1 or SEQ ID NO: 4, and at least one peptide having a sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5, wherein said composition preferably further comprises one strain of lactic acid bacteria belonging to the genus Bifidobacterium.

Images