Formulations containing specific beta-lactoglobulin peptides

Abstract

A formulation comprising whey protein hydrolysate containing specific β-lactoglobulin-derived peptides and probiotics, the formulation being used to induce oral immune tolerance to milk proteins, prevent or treat oral immune intolerance to milk proteins, and / or reduce the risk of developing oral immune intolerance to milk proteins.

Description

[0001] This application is a divisional application. The original application was filed on April 18, 2019, with application number 201980035592.1 (international application number PCT / NL2008 / 050281) and titled "Botulinum toxin preparations for oral administration". Technical Field

[0002] This invention belongs to the field of formulations containing hydrolyzed bovine milk proteins for use in infants with bovine milk protein allergy or at risk of developing bovine milk protein allergy. Background Technology

[0003] Dietary proteins are exposed to the immune system in the gastrointestinal tract. As the immune system's default response, oral tolerance to these harmless food proteins develops. Oral immune tolerance is highly prevalent in infants, who are exposed to many harmless proteins early in life through dietary protein intake. When this natural response to harmless proteins goes awry, food allergies occur. It is generally believed that the prevalence of food allergies has been rising in recent decades, particularly in Western countries. In early life, cow's milk allergy is the most common food allergy, currently affecting 2-5% of infants, with an even higher proportion at risk of developing other allergies.

[0004] For infants with bovine protein allergy, commercially available infant formulas contain extensively hydrolyzed proteins (extensive protein hydrolysates), or even just free amino acids as a nitrogen source. These formulas contain little to no allergenic proteins or peptides.

[0005] Infants born to parents with one or both of whom have atopic dermatitis, or infants with one or more siblings who have atopic dermatitis, have a higher risk of developing dietary protein allergies. For this group, in addition to preferred breastfeeding, hypoallergenic formulas containing partially hydrolyzed proteins are commercially available. These partially hydrolyzed proteins have reduced allergenicity. This approach has been shown to effectively prevent sensitization to naturally occurring proteins in adapted formulas. Typically, the degree of protein hydrolysis is lower than that of extensively hydrolyzed proteins used for infants already suffering from allergies. The advantage of these formulas lies not only in reducing the risk of allergic reactions by preventing sensitization to proteins, but also in supporting the natural development of oral immune tolerance to intact proteins. This allows for the subsequent introduction of natural proteins into the diet with a reduced risk of allergic reactions.

[0006] For decades, strict avoidance of allergens has been recommended to prevent and manage allergen sensitivity. In recent decades, the focus on complete avoidance of food allergens may have inadvertently contributed to an increase in food allergies. A better approach to preventing or treating allergies is to develop strategies that promote oral tolerance to allergens, such as oral immunotherapy, or to present food allergens in a form that favors inducing natural tolerance mechanisms (Allen et al, 2009, Pediatr AllergImmunol 20:415-422).

[0007] EP 2 044 851 discloses a nutritional composition containing partially hydrolyzed milk protein with a degree of hydrolysis of 15% to 25% and 50 to 1000 ng of TGF-β / 100 ml for primary prevention of allergic reactions to dietary protein and prevention of atopic diseases in young mammals.

[0008] EP 0 629 350 discloses the use of a non-allergenic whey protein hydrolysate that is said to induce tolerance to bovine milk protein.

[0009] EP 0 827 697 discloses the use of enzymatically hydrolyzed whey in the preparation of compositions for inducing oral tolerance to bovine milk in susceptible mammals. The whey exhibits immunologically detectable levels of allergenic proteins that are >= 100 times lower than those found in unhydrolyzed whey.

[0010] WO 00 / 42863 discloses a hypoallergenic composition for inducing protein tolerance in infants at risk of protein allergy, comprising a deeply hydrolyzed nonallergenic protein basis and / or a free amino acid basis, wherein the composition comprises at least one tolerogenic peptide of an allergenic protein as an active ingredient.

[0011] WO 2011 / 151059 discloses an infant nutrition containing partially hydrolyzed protein and non-digestible oligosaccharides for inducing oral tolerance to natural dietary protein.

[0012] WO 2015 / 090347 relates to peptides that have been identified as capable of inducing tolerance to bovine milk, particularly to β-lactoglobulin.

[0013] WO 2017 / 144730 discloses a strategy for desensitizing or inducing tolerance to a milk protein allergen (such as β-lactoglobulin) in humans or animals, comprising formulating and using a composition comprising a purified, fully expressed milk protein and one or more purified peptides derived from the fully expressed milk protein.

[0014] WO 2016 / 0148572 discloses oral immune tolerance to a specific mixture of synthetic β-lactoglobulin peptide and probiotics.

[0015] However, existing hypoallergenic compositions typically provide their effects only by avoiding the presence of potential allergens (and thus failing to address the underlying problem) and / or by requiring the presence of specific ingredients (such as growth factors or synthetic or purified peptides). Furthermore, peptides identified in the prior art may not be suitable for inducing oral immune tolerance in a broad population. Therefore, there remains a need for nutrients containing specific hydrolysates of bovine β-lactoglobulin along with specific peptides and components that further enhance oral immune tolerance in subjects at risk of developing or suffering from food allergies, and have an improved effect on the induction of oral immune tolerance to food proteins, particularly bovine milk proteins.

[0016] Therefore, the fundamental technical problem of the present invention is to provide compositions, methods, uses and means for overcoming the above-mentioned defects, and in particular for providing improved oral immune tolerance induction to dietary proteins in humans, especially humans at risk of developing food allergies, particularly infants at risk of developing bovine milk protein allergy. Summary of the Invention

[0017] The inventors have contributed to the belief in the art that a better way to prevent or treat allergies is to develop strategies that promote oral tolerance induction to allergens (e.g., oral immunotherapy) or to present food allergens in a form that is conducive to inducing natural tolerance mechanisms (Allen et al, 2009, Pediatr Allerg Immunol 20:415-422).

[0018] All T cell responses (including the induction of regulatory T cells (Tregs)) are based on three interconnected processes: T cell receptor activation, co-stimulation, and cytokine signaling. T cell epitopes, specifically peptides presented by MHC class II molecules on antigen-presenting cells, can promote CD4+ receptor activation. + Activation of T cell receptors on T cells. The presence of T cell epitopes in proteolytic products is crucial for inducing milk-specific Tregs. Amino acid (AA) #13-48 of mature β-lactoglobulin (BLG) is of particular interest because, under prophylactic conditions, synthetic peptides covering this region significantly reduced acute anaphylactic skin reactions in a mouse model of bovine milk allergy. Taken together, this region of BLG appears to be important for the development of whey protein tolerance.

[0019] Researchers used a novel approach to discover that infant formulas based on specific partially hydrolyzed whey contain specific, well-defined β-lactoglobulin (BLG) peptides that function as T-cell epitopes to support oral tolerance to bovine milk proteins in a broader population of human subjects, including those with different HLA-DRB1 alleles. These specific alleles are most relevant because the HLA-DR allele is the major isotype of MHC class II, present in CD4+. + HLA plays a central role in the selection and activation of T cells. HLA-DRB is a heterodimer molecule containing HLA-DRA and HLA-DRB chains. Although the HLA-DRA gene is highly conserved in humans, there are multiple HLA-DRB genes. Among them, HLA-DRB1 is the most polymorphic gene, with its expression level being 5 times higher than that of its functional paralogs.

[0020] First, a novel liquid chromatography-mass spectrometry (LC-MS) method was developed to identify a limited, specific list of BLG-derived peptides that are naturally occurring at the highest rate in formulations containing whey protein hydrolysates, concentrated in the AA#13-48 region of mature bovine BLG. The inventors were able to identify a total of 13 BLG peptides with a minimum of 9 amino acids in this target region, 6 of which were consistently identified across different production batches.

[0021] Secondly, ProImmune was applied to the formulation. Antigen presentation assays and MHC class II binding algorithms were used to identify relevant HLA-DRB1-restricted peptides. These peptides have been confirmed to be processed and presented by human dendritic cells. The aforementioned peptides clustered in two distinct sequence sets, namely DIQ…DIS (AA#11-30) and AMA…APL (AA#23-39). Fragments from the two identified distinct sequence sets of BLG were assessed to have high affinity for binding to selected HLA-DRB1 alleles. It has been found that mixtures of BLG peptides are most preferred to ensure coverage of interactions with several HLA-DRB1 alleles commonly present in the human population. In particular, a peptide mixture containing at least one of BLG peptide SEQ ID NO:5 and BLG peptide SEQ ID NO:2, 3, or 4 covers most of the tested HLA-DRB1 alleles. See Examples 2 and 3.

[0022] Third, the sequences of the identified BLG peptides were tested, and it was found that they were recognized by human bovine milk-specific T cell lines obtained from various allergic donors and induced T cell proliferation / activation. See Example 4 and its associated... Figure 2When tested on these different T cell lines, distinct patterns of T cell responsiveness were observed. While not every peptide responded to every cell line, the combined responses (particularly 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) encompassed the responsiveness of each cell line. These results suggest that hydrolysates containing these specific BLG-derived peptides may improve or reduce the risk of developing oral immune tolerance to whey protein.

[0023] As demonstrated in Example 6, the peptide has more MHC class II binding domains, thus increasing the likelihood of presentation to T cells.

[0024] When consumed with strains of lactic acid bacteria, with or without non-digestible oligosaccharides, the BLG peptides in the AA#13-48 region provide a further improved effect on oral immune tolerance induction or a further reduction in the risk of oral immune intolerance. Therefore, these results also indicate a further improved effect of compositions containing hydrolyzed proteins comprising a mixture of such BLG-derived peptides consisting of an amino acid sequence corresponding to the continuous amino acids of β-lactoglobulin represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, preferably with strains of lactic acid bacteria, with or without non-digestible oligosaccharides (probiotics or synbiotics).

[0025] Furthermore, the experimental section attached demonstrates that the presence of lactic acid-producing bacteria increased 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, thereby improving oral induction of immune tolerance.

[0026] Preferred Terms List

[0027] 1. A nutritional composition comprising protein hydrolysates derived from the milk of mammals, preferably from species of the genera *Bos*, *Bison*, *Bubalus*, or *Capra*, more preferably from species of *Bos*, and most preferably from cow's milk, the nutritional composition being used in human subjects.

[0028] - Inducing oral immune tolerance to milk proteins; and / or

[0029] -Prevention or treatment of oral immuno intolerance to milk proteins; and / or

[0030] -Reduces the risk of developing oral immune intolerance to milk proteins.

[0031] The protein hydrolysate comprises at least two peptides having sequences selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0032] The composition preferably further comprises at least one strain of lactic acid-producing bacteria.

[0033] 2. As per the purpose of Clause 1, the protein hydrolysate comprises:

[0034] (i) at least one peptide having the sequence according to SEQ ID NO:5 and at least one peptide having the sequence according to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; or

[0035] (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 of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:5.

[0036] 3. A nutritional composition for the stated purpose according to Clause 1 or 2, wherein the composition comprises less than 6 μg of allergenic β-lactoglobulin / g of total protein, preferably less than 3.5 μg of allergenic β-lactoglobulin / g of total protein.

[0037] 4. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein the composition contains at least 50% by weight, preferably at least 95% by weight, hydrolyzed whey protein based on total protein content.

[0038] 5. A nutritional composition for the stated purpose according to any of the preceding clauses, based on total protein, comprising less than 10% by weight, preferably less than 6% by weight, of peptides or proteins having a size of 5 kDa or more.

[0039] 6. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein, based on total protein, at least 1% by weight of the peptides or proteins present in the composition have a size of 1 kDa or more, preferably at least 5% by weight, more preferably at least 10% by weight, based on total protein.

[0040] 7. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein the composition comprises more than 0.8 μg of allergenic β-lactoglobulin per gram of total protein.

[0041] 8. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein the milk protein is derived from a species of the genus *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from a species of the genus *Bos*, and preferably bovine milk protein.

[0042] 9. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein the human subject is an infant.

[0043] 10. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein the human subject is at risk of developing or has milk protein allergy, said milk being derived from a species of the genus *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from a species of the genus *Bos*.

[0044] 11. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein the composition comprises a strain of lactic acid-producing bacteria belonging to the genus Bifidobacterium, preferably to the species Bifidobacterium breve.

[0045] 12. A nutritional composition for the stated purpose according to any of the preceding clauses, comprising one or more non-digestible oligosaccharides selected from: fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabino-oligosaccharides, arabinogalacto-oligosaccharides, glucose-glucomannosaccharides, galacto-manno-oligosaccharides, mannose, chitosan oligosaccharides, uronic acid oligosaccharides, sialic acid oligosaccharides, and fucose oligosaccharides, preferably fructooligosaccharides.

[0046] 13. The nutritional composition for the stated purpose according to Clause 12, wherein the non-digestible oligosaccharide comprises at least two non-digestible oligosaccharides selected from fructooligosaccharides and galactooligosaccharides, preferably a mixture of long-chain fructooligosaccharides and short-chain fructooligosaccharides or short-chain galactooligosaccharides.

[0047] 14. A nutritional composition for the stated purpose according to any of the foregoing clauses, comprising 10 5 Up to 10 11 The CFU is the strain of lactic acid-producing bacteria per gram of dry weight, and optionally, at least 2% by weight of non-digestible oligosaccharides based on dry weight.

[0048] 15. A nutritional composition for the stated purpose according to any of the preceding clauses, comprising a long-chain polyunsaturated fatty acid, preferably docosahexaenoic acid (DHA), more preferably at least 0.35% by weight of DHA based on total fatty acids.

[0049] 16. A nutritional composition for the stated purpose according to any of the preceding clauses, wherein it is an infant formula, a follow-up formula, or a young child formula.

[0050] 17. A nutritional composition comprising:

[0051] a. Strains of lactic acid-producing bacteria belonging to the genus Bifidobacterium;

[0052] b. A milk protein hydrolysate derived from a species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from a species of the genus *Bos*, wherein the milk protein hydrolysate comprises at least two peptides having sequences selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0053] c. Less than 6 μg of allergenic β-lactoglobulin / g of total protein, preferably less than 3.5 μg of β-lactoglobulin / g of total protein.

[0054] d. Based on total protein, less than 10% by weight of peptides or proteins with a size of 5 kDa or greater, and

[0055] e. Based on total protein content, at least 50% by weight, preferably at least 95% by weight, of hydrolyzed whey protein.

[0056] f. Optionally, one or more non-digestible oligosaccharides selected from the following: fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinogalactooligosaccharides, arabinogalacto-oligosaccharides, glucose oligosaccharides, glucomannosaccharides, galactomannosaccharides, mannose oligosaccharides, chitosan oligosaccharides, uronic acid oligosaccharides, sialic acid oligosaccharides, and fucose oligosaccharides, and mixtures thereof, preferably fructooligosaccharides, and

[0057] 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.

[0058] 18. The nutritional composition according to Clause 17, wherein the protein hydrolysate comprises:

[0059] (i) at least one peptide having the sequence according to SEQ ID NO:5 and at least one peptide having the sequence according to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; or

[0060] (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.

[0061] 19. A nutritional composition according to Clause 17 or 18, wherein the amount of allergenic β-lactoglobulin is 0.8 μg or more per gram of protein, and / or wherein the composition comprises, based on total protein, more than 1% by weight of a peptide or protein of size 1 kDa or more, more preferably at least 5% by weight, and more preferably at least 10% by weight.

[0062] 20. The nutritional composition according to any one of clauses 17 to 19, wherein the strain of said lactic acid-producing bacteria belongs to the species Bifidobacterium breve.

[0063] 21. A nutritional composition according to any one of clauses 17 to 20, wherein the non-digestible oligosaccharide comprises at least two non-digestible oligosaccharides selected from fructooligosaccharides and galactooligosaccharides, preferably a mixture of long-chain fructooligosaccharides and short-chain fructooligosaccharides or short-chain galactooligosaccharides.

[0064] 22. A nutritional composition according to any one of clauses 17 to 21, comprising 10 5 Up to 10 11 Lactic acid bacteria of CFU / g dry weight.

[0065] 23. A nutritional composition according to any one of clauses 17 to 22, comprising at least 2% by weight of non-digestible oligosaccharides on a dry weight basis.

[0066] 24. A nutritional composition according to any one of clauses 17 to 23, which is an infant formula, a follow-up formula or a toddler formula.

[0067] 25. A method for providing nutrition to a human subject who is at risk of developing or suffers from an allergy, more preferably a milk protein allergy, comprising administering to the human subject a nutritional composition according to any one of clauses 17 to 24.

[0068] 26. The method according to Clause 25, wherein the human subject is an infant or young child, preferably an infant.

[0069] 27. The method according to clause 25 or 26, wherein the milk protein is derived from a species of the genus *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from a species of the genus *Bos*, and preferably bovine milk protein.

[0070] 28. A method for use in human subjects:

[0071] - Inducing oral immune tolerance to milk proteins; and / or

[0072] -Prevention or treatment of oral immuno intolerance to milk proteins; and / or

[0073] -Reduces the risk of developing oral immune intolerance to milk proteins.

[0074] The method includes administering a nutritional composition to the subject, the nutritional composition comprising a protein hydrolysate derived from mammalian milk, preferably from milk of a species of the genus *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from milk of a species of the genus *Bos*, and most preferably from cow's milk, wherein the protein hydrolysate comprises at least two peptides having a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and the composition preferably further comprises at least one strain of lactic acid-producing bacteria.

[0075] 29. Use of protein hydrolysates in the preparation of nutritional compositions, said nutritional compositions being used for:

[0076] - Inducing oral immune tolerance to milk proteins; and / or

[0077] -Prevention or treatment of oral immuno intolerance to milk proteins; and / or

[0078] -Reduces the risk of developing oral immune intolerance to milk proteins.

[0079] The nutritional composition comprises a protein hydrolysate derived from mammalian milk, preferably from milk of species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from milk of species of the genus *Bos*, and most preferably from cow's milk. The protein hydrolysate comprises at least two peptides having sequences selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. The composition preferably further comprises at least one strain of lactic acid-producing bacteria. Detailed Implementation

[0080] Therefore, the present invention relates to a nutritional composition comprising a protein hydrolysate derived from mammalian milk, which has been shown in human subjects to be effective in...

[0081] - Used to induce oral immune tolerance to milk proteins; and / or

[0082] - Used for the prevention or treatment of oral immunodeficiency to milk proteins; and / or

[0083] - To reduce the risk of developing oral immune intolerance to milk proteins; and / or

[0084] - Used to improve or enhance oral immune tolerance to milk proteins.

[0085] The protein hydrolysate comprises at least two amino acid sequences having consecutive amino acid sequences corresponding to those 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 one peptide having an amino acid sequence corresponding to consecutive amino acid sequences represented by SEQ ID NO:2 and / or SEQ ID NO:4. In a preferred embodiment, the composition comprises at least one peptide having an amino acid sequence corresponding to SEQ ID NO:4. In a preferred embodiment, the composition comprises at least one 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 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 or even all five peptides selected from this group.

[0086] In a preferred embodiment, the protein hydrolysate comprises:

[0087] (i) at least one peptide consisting of an amino acid sequence corresponding to the consecutive amino acids represented by 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

[0088] (ii) at least one 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.

[0089] These peptides are also referred to herein as “BLG-derived peptides”. The composition preferably further comprises strains of lactic acid-producing bacteria, more preferably one or more non-digestible oligosaccharides (NDOs) as further defined below. Mammalian milk is preferably milk from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from species of *Bos*, and preferably from cow's milk. In other words, the present invention relates to the use of protein hydrolysates or BLG-derived peptides in the preparation of the above-described nutritional compositions for inducing oral immune tolerance to milk proteins in human subjects, and / or preventing or treating oral immune intolerance to milk proteins, and / or for reducing the risk of developing oral immune intolerance to milk proteins, preferably milk proteins from species of *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably milk proteins from species of *Bos*, and preferably cow's milk, wherein the nutritional composition comprises protein hydrolysates as defined above, preferably according to (i) or (ii) as defined above, and the composition preferably further comprises lactic acid-producing bacteria, more preferably one or more NDOs as further defined below. Additionally, the present invention relates to a method for inducing oral immune tolerance to milk proteins in human subjects and / or for preventing or treating oral immune intolerance to milk proteins and / or for reducing the risk of developing oral immune intolerance to milk proteins, preferably to milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably to milk proteins from species of the genus *Bos*, and preferably to cow's milk. The method comprises administering the above-described nutritional composition to a human subject, the nutritional composition comprising a protein hydrolysate as defined above, preferably a protein hydrolysate according to (i) or (ii) as defined above, the composition preferably further comprising a strain of lactic acid-producing bacteria, more preferably comprising one or more NDOs as further defined below.

[0090] This invention also relates to one or more BLG-derived peptides, which in human subjects

[0091] - Used to induce oral immune tolerance to milk proteins; and / or

[0092] - Used for the prevention or treatment of oral immunodeficiency to milk proteins; and / or

[0093] - To reduce the risk of developing oral immune intolerance to milk proteins; and / or

[0094] - Used to improve or enhance oral immune tolerance to milk proteins.

[0095] Preferably, the protein is a milk protein from a species of the genus *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably a milk protein from a species of the genus *Bos*, and preferably bovine milk, wherein the one or more BLG-derived peptides are characterized by comprising at least two peptides having a sequence selected from 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 above. In other words, the present invention relates to the use of one or more BLG-derived peptides in the preparation of nutritional compositions for inducing oral immune tolerance to milk proteins in human subjects, and / or for preventing or treating oral immune intolerance to milk proteins, and / or for reducing the risk of developing oral immune intolerance to milk proteins, preferably to milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably to milk proteins from species of the genus *Bos*, preferably to bovine milk, wherein the one or more BLG-derived peptides are characterized by comprising at least two peptides having sequences selected from 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) and (ii) as defined above.

[0096] This invention also relates to (non-therapeutic) methods in human subjects.

[0097] - Used to induce oral immune tolerance to milk proteins; and / or

[0098] - Used for the prevention or treatment of oral immunodeficiency to milk proteins; and / or

[0099] - To reduce the risk of developing oral immune intolerance to milk proteins; and / or

[0100] - Used to improve or enhance oral immune tolerance to milk proteins.

[0101] Preferably, the method involves administering one or more BLG-derived peptides to a human subject, wherein the peptides are characterized by comprising at least two peptides having sequences selected from 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 above. In a preferred embodiment of the above use or method, one or more BLG-derived peptides are preferably administered in combination with a strain of lactic acid-producing bacteria, more preferably in combination with one or more non-digestible oligosaccharides (NDOs) as further defined below.

[0102] In one implementation scheme

[0103] - Inducing oral immune tolerance to milk proteins; and / or

[0104] -Prevention or treatment of oral immuno intolerance to milk proteins; and / or

[0105] - Reduce the risk of developing oral immune intolerance to milk proteins; and / or

[0106] - Improve or enhance oral immune tolerance to milk proteins,

[0107] The composition involves milk proteins derived from a specific mammal species, and the hydrolyzed milk proteins contained in the composition are derived from the same mammal species, preferably both from the genus Bovis.

[0108] The term “improved or enhanced oral immune tolerance to milk proteins” should be understood to mean that oral immune tolerance to milk proteins is improved compared to oral immune tolerance to milk proteins prior to the administration of the compositions of the present invention.

[0109] In this regard, the present invention also relates to a nutritional composition comprising:

[0110] a. Strains of lactic acid-producing bacteria belonging to the genus Bifidobacterium;

[0111] b. A milk protein hydrolysate derived from a species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from a species of the genus *Bos*, wherein the milk protein hydrolysate comprises at least two peptides having sequences selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0112] c. Less than 6 μg of allergenic β-lactoglobulin / g of total protein, preferably less than 3.5 μg of β-lactoglobulin / g of total protein.

[0113] d. Based on total protein content, less than 3% by weight of peptides or proteins with a size of 5 kDa or greater.

[0114] e. Based on total protein content, at least 50% by weight, preferably at least 95% by weight, of hydrolyzed whey protein.

[0115] f. Optionally, one or more non-digestible oligosaccharides selected from the following: fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinogalactooligosaccharides, arabinogalacto-oligosaccharides, glucose oligosaccharides, glucomannosaccharides, galactomannosaccharides, mannose oligosaccharides, chitosan oligosaccharides, uronic acid oligosaccharides, sialic acid oligosaccharides, and fucose oligosaccharides, and mixtures thereof, preferably fructooligosaccharides, and

[0116] 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.

[0117] In this regard, the present invention also relates to a method of providing nutrition to human subjects who are at risk of developing or who have allergies, more preferably milk protein allergy, comprising administering to the human subjects a nutritional composition as defined above.

[0118] Nutritional compositions in the context of this invention are defined below, and all embodiments are applicable to all aspects of this invention, including the compositions of this invention, compositions for the stated uses, methods, and uses.

[0119] Peptide / hydrolysate

[0120] The compositions of the present invention comprise at least two peptides consisting of amino acid sequences 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 can be derived from β-lactoglobulin, and are therefore referred to as β-lactoglobulin-derived peptides. Throughout the specification and claims, the amino acid sequences of the BLG peptides having SEQ ID NO:1-5 are identified below:

[0121] SEQ ID NO: 1: XIVTQTMKGLDIQKVAGTWYSLAMAAS

[0122] SEQ ID NO: 2:XIVTQTMKGLDIQKVAGTWYSLAMAASDISLL

[0123] SEQ ID NO: 3: TMKGLDIQKVAGTWYSLAMAASDISLL

[0124] SEQ ID NO: 4: TMKGLDIQKVAGTWYSLAMAASDISLLDAQ

[0125] SEQ ID NO: 5: DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY

[0126] In this document, X is defined as an amino acid selected from leucine (L) or isoleucine (I), preferably 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 selection of X as leucine or isoleucine is a direct result of its mammalian milk source. Leucine is preferred because it is most preferably derived from the milk of bovine mammals, preferably bovine milk.

[0127] The preferred implementation scheme (i) is based on ProImmune. Antigen presentation assays were performed in which the peptide mixture was found to cover more of the tested HLA-DRB1 alleles; preferred embodiment (ii) is an experiment based on T cell recognition of T cells from milk-allergic infants in which the peptides were found to cover the reactivity of each cell line.

[0128] The peptide is preferably provided by a protein hydrolysate (i.e., hydrolyzed protein) derived from mammalian milk, preferably from milk of species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from milk of species of *Bos*, and most preferably from cow's milk (*Bos Turus*). The amino acid sequences of BLG in species of *Bos* and *Buffalo* are similar, and SEQ ID NOs: 9, 11, 3, 4, and 5 are available in hydrolysates of milk proteins from these species. In species of *Capus* and *Buffalo*, 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 are available in hydrolysates of milk proteins from these species. In a preferred embodiment, the peptide is derived from whey protein. The nutritional composition preferably comprises at least 50% by weight, more preferably at least 70% by weight, and even more preferably at least 95% by weight, of hydrolyzed whey protein based on total protein. Suitable sources are mixtures of acidic whey protein and demineralized sweet whey protein. Acidic whey and sweet whey are commercially available. Sweet whey is a byproduct of rennet curdling and contains casein glycomacropeptide (CGMP), while acidic whey (also known as sour whey) is a byproduct of sour curdling and does not contain CGMP. Suitable sources of whey protein are demineralized whey (Deminal, Friesland Campina, the Netherlands) and / or whey protein concentrate (WPC80, Friesland Campina, the Netherlands). Whey protein preferably comprises acidic whey, more preferably at least 50% by weight, and more preferably at least 70% by weight, based on total whey protein. Compared to sweet whey protein, acidic whey has an improved amino acid profile.

[0129] Hydrolysis can be performed using a mixture of microbial endopeptidases and exopeptidases, employing the method described in Example 1 of WO 2011 / 151059 (incorporated herein by reference). Hydrolysates containing 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 preferably be prepared using the formulation of Example 1. Preferably, a mixture of endopeptidases and exopeptidases is used. Methods for identification are described in the Experimental section to determine whether any given composition contains one or more BLG-derived peptides.

[0130] The composition preferably comprises less than 10% by weight, preferably less than 6% by weight, of peptides or proteins having a size of 5 kDa or more, based on total protein. In one embodiment, the composition comprises more than 0.3% by weight, preferably more than 0.5% by weight, more preferably more than 1% by weight, and even more preferably more than 1.5% by weight, of such peptides having a size of 5 kDa or more, based on total protein. Preferably, more than 1% by weight of peptides or proteins having a size of 1 kDa or more, more preferably at least 5% by weight, and even more preferably at least 10% by weight, based on total protein. More preferably, more than 1% by weight of peptides or proteins having a size of 3 kDa or more, more preferably at least 5% by weight, and even more preferably at least 10% by weight, based on total protein.

[0131] The size distribution of peptides in protein hydrolysates can be determined using high-performance liquid chromatography with molecular sieves, a method known in the art. An example is described in 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. In short, the total surface area of ​​the chromatogram 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. Preferably, the protein hydrolysate containing said peptides is characterized by comprising 64 to 89% by weight peptides with a molecular weight less than 1000 Da, 10 to 30% by weight peptides with a molecular weight of 1000 to 5000 Da, and 1 to 6% by weight peptides or proteins with a molecular weight greater than 5 kDa, all based on total protein.

[0132] While the peptide weight distribution provides information, it does not offer any information about the sequence and activity of the peptides present in the formulation. Structural information is crucial for understanding the overall biological activity that hydrolyzed infant formula may possess.

[0133] Consistently, several specific peptides derived from regions of BLG known to contain T-cell epitopes have been identified in different production batches. Previously, only two studies investigated the peptide profiles of pHP (Wada et al. Peptides 2015 73:101-105; Catala-Clariana et al, Electrophoresis 2013 Jul; 34(13):1886-1894). While these studies generally focused on bioactive peptides rather than specifically on T-cell epitopes, the peptides containing SEQ ID NO:1-5 identified in this application were not found in these previously tested products. In addition to the profiles of these hydrolyzed protein products, peptide profiles of non-commercially available BLG-based hydrolysates have also been identified (Pecquet et al J Allergy Clin Immunol 2000 Mar; 105(3):514-521). Two peptides (AA#21-40 and AA#25-40) were also identified in this hydrolysate, overlapping in situ with the target region, but peptides with SEQ ID NO:1-5 were not identified. In at least two of the three studies reported above, hydrolysates from different manufacturers (Wada and Pecquets) were investigated, and as mentioned above, the main reason for these differences is likely the difference in preparation methods. Recent studies further confirm this, in which peptidomics were used to characterize deeply hydrolyzed infant formulas from different manufacturers. The authors demonstrated that infant formulas from different manufacturers have different characteristics based on their peptide profiles, and therefore may have different effects in clinical trials (Lambers et al. Food Sci Nutr 2015 Jan; 3(1):81-90. Hochwallner et al, 2017, Allergy 72:416-425).

[0134] In a preferred embodiment of the invention, the composition comprises at least 10 mcg (micrograms), more preferably 10 to 5000 mcg, more preferably 20 to 2000 mcg, more suitable 30 to 500 mcg, and particularly preferably 50 to 250 mcg of total β-lactoglobulin-derived peptides / g of total protein, wherein the β-lactoglobulin-derived peptides consist of the amino acid sequences 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 the amino acid sequences 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 a therapeutically effective amount.

[0135] Preferably, the nutritional composition contains less than 6 μg of allergenic β-lactoglobulin (BLG) / g protein, more preferably less than 5 μg / g protein, and more preferably less than 3.5 μg / g protein. In the context of this invention, the term "allergenic β-lactoglobulin" refers to intact or immunogenic BLG, excluding hydrolyzed BLG. Preferably, the nutritional composition contains more than 0.8 μg of allergenic β-lactoglobulin / g protein. Allergenic BLG was determined using an ELISA method known in the art; amounts exceeding 0.8 μg / g protein indicate partial protein hydrolysis. For comparison, extensively hydrolyzed whey protein showed less than 0.2 μg of allergenic BLG / g protein. Small but significant amounts of allergenic BLG, together with hydrolyzed BLG or BLG-derived peptides, more preferably in combination with strains of lactic acid-producing bacteria, and even more preferably in combination with lactic acid-producing bacteria and one or more NDOs, enhance the tolerogenicity of the composition, while the amount of BLG remains sufficiently low to reduce the allergenicity of the nutritional composition and make the nutritional composition hypoallergenic. From this perspective, it is preferable to use a proteolytic product containing a small amount of allergenic β-lactoglobulin and the BLG-derived peptide of the present invention, rather than consisting of the amino acid sequences 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 a synthetic peptide (free of allergenic BLG) consisting of the amino acid sequences represented by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0136] Lactic acid bacteria

[0137] The compositions of the present invention preferably comprise strains of lactic acid-producing bacteria that enhance the oral tolerance-inducing ability of the BLG peptides of the present invention. The bacterial strains are preferably probiotics. Examples 5 and 7 provide evidence. The presence of lactic acid-producing 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, thereby improving oral immune tolerance induction.

[0138] Suitable lactic acid-producing bacteria include strains of the genus *Bifidobacteria* (e.g., *Bifidobacterium breve*, *Bifidobacterium longum*, *Bifidobacterium infantis*, *Bifidobacterium bifidum*), strains of the genus *Lactobacillus* (e.g., *Lactobacillus acidophilus*, *Lactobacillus paracasei*, *Lactobacillus johnsonii*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Lactobacillus lactis*) and strains of the genus *Streptococcus* (e.g., *Streptococcus thermophilus*). *Bifidobacterium breve* and *Bifidobacterium longum* are particularly suitable lactic acid-producing bacteria.

[0139] The composition preferably comprises a strain of lactic acid-producing bacteria belonging to the genus *Bifidobacterium* (preferably *Bifidobacterium breve*). Preferably, the 16S rRNA sequence of *Bifidobacterium breve* has at least 95% identity with the 16S rRNA sequence of the model strain of *Bifidobacterium breve* ATCC 15700, more preferably, at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44: 846-849). Suitable strains of *Bifidobacterium breve* can be isolated from the feces of healthy, breastfed infants. These strains are typically commercially available from lactic acid bacteria manufacturers, but can also be isolated, identified, characterized, and produced directly from feces. According to one embodiment, the composition of the present invention contains a selection of Bifidobacterium breve: Bifidobacterium breve Bb-03 (Rhodia / Danisco), Bifidobacterium breve M-16V (Morinaga), Bifidobacterium breve R0070 (Institute Rosell, Lallemand), Bifidobacterium breve BR03 (Probiotical), Bifidobacterium breve BR92 (CellBiotech), DSM 20091, LMG 11613, YIT4065, FERM BP-6223, and CNCM I-2219. The Bifidobacterium breve may be Bifidobacterium breve M-16V and Bifidobacterium breve CNCM I-2219, with Bifidobacterium breve M-16V being the most preferred. Bifidobacterium breve I-2219 is disclosed in WO 2004 / 093899 and deposited by Compagnie Gervais Danone on May 31, 1999, at the National Collection of Microorganisms, Institute of Pasteur, Paris, France. Bifidobacterium breve M-16V is deposited as BCCM / LMG23729 and is commercially available from Morinaga Milk Industry Co., Ltd.

[0140] In the context of this invention, lactic acid-producing bacteria can be present in the composition at any suitable concentration, preferably at a therapeutically effective amount or "amount for therapeutic effectiveness". Preferably, the strain of lactic acid-producing bacteria is present at 10 4 -10 13 CFU / g dry weight of the composition, preferably 10 5 -10 11 cfu / g, optimal value 10 6 -10 10 The amount of cfu / g is included in the composition of the present invention.

[0141] Non-digestible oligosaccharides

[0142] In a preferred embodiment, the compositions of the present invention further comprise one or more non-digestible oligosaccharides [NDO]. Similar to lactic acid-producing bacteria, these NDOs further enhance the oral immune tolerance-inducing properties of the BLG protein peptides. Evidence of this enhancement is provided in Example 5.

[0143] Advantageously and most preferably, the non-digestible oligosaccharide is water-soluble (according to the method disclosed in L. Prosky et al., J. Assoc. Anal. Chem 71:1017-1023, 1988), and preferably has a degree of polymerization (DP) of 2 to 200. The average DP of the non-digestible oligosaccharide is preferably below 200, more preferably below 100, even more preferably below 60, and most preferably below 40.

[0144] Non-digestible oligosaccharides are preferably prebiotics. They are not digested in the intestines by digestive enzymes present in the upper digestive tract (small intestine and stomach). Non-digestible oligosaccharides are fermented by the human gut microbiota. For example, glucose, fructose, galactose, sucrose, lactose, maltose, and maltodextrin are considered digestible. Oligosaccharide raw materials may include monosaccharides such as glucose, fructose, fucose, galactose, rhamnose, xylose, glucuronic acid, GalNac, etc., but these are not part of oligosaccharides. Non-digestible oligosaccharides are preferably selected from fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabino-oligosaccharides, arabinogalacto-oligosaccharides, oligodextrose, glucomannan oligosaccharides, galacto-oligosaccharides, mannose, chitosan oligosaccharides, uronic acid oligosaccharides, sialic acid oligosaccharides, and fucose oligosaccharides and mixtures thereof, with fructooligosaccharides being preferred. Examples of sialic acid oligosaccharides are 3-sialylactose, 6'-sialylactose, sialyl-N-tetrasaccharide, and disialialyl-N-tetrasaccharide. Examples of fucose oligosaccharides are (un)sulfated fucoidan oligosaccharides, 2'-fucosyllactose, 3'-fucosyllactose, lacto-N-fucopentose I, II, III, LNDH, lactodifucotetrasaccharide, and lacto-N-difucohexasaccharide I and II.

[0145] A suitable type of oligosaccharide is a short-chain oligosaccharide having an average degree of polymerization of less than 10, preferably at most 8, and more preferably in the range of 2-7. The short-chain oligosaccharide preferably comprises galactooligosaccharides and / or fructooligosaccharides (i.e., scGOS and / or scFOS). In one embodiment, the composition comprises galactooligosaccharides, preferably β-galactooligosaccharides, and more preferably trans-galactooligosaccharides. Preferably, the galactooligosaccharides have an average degree of polymerization in the range of 2-8, more preferably 3-7, i.e., the galactooligosaccharides are short-chain oligosaccharides in the context of this invention. (Trans)galactooligosaccharides can be marketed, for example, under trade names... GOS (Friesland Campina Domo Ingredients, Netherlands), Bimuno (Clasado), Cup-oligo (Nissin Sugar), and Oligomate 55 (Yakult) are obtained. The composition preferably comprises short-chain fructooligosaccharides and / or short-chain galactooligosaccharides, preferably at least short-chain fructooligosaccharides. The fructooligosaccharides can be inulin hydrolysates with an average DP within the aforementioned (sub)range; such FOS products are available, for example, from Raftilose P95 (Orafti) or commercially available from Cosucra.

[0146] Another suitable type of oligosaccharide is long-chain fructooligosaccharides (lcFOS), which have an average degree of polymerization of 10 or higher, typically in the range of 10-100, preferably 15-50, and most preferably 20 or higher. A specific type of long-chain fructooligosaccharide is inulin, such as Raftilin HP.

[0147] The compositions of the present invention may comprise a mixture of two or more types of non-digestible oligosaccharides, most preferably a mixture of two non-digestible oligosaccharides. In the case where the NDO comprises a mixture of two different oligosaccharides or is composed of a mixture of two different oligosaccharides, one oligosaccharide may be a short-chain oligosaccharide as defined above, and the other oligosaccharide may be a long-chain oligosaccharide as defined above. Most preferably, the short-chain oligosaccharide and the long-chain oligosaccharide are present in a short-chain to long-chain weight ratio ranging from 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, and most preferably about 9:1.

[0148] In one embodiment, the composition comprises at least two types of fructooligosaccharides and / or galactooligosaccharides. Suitable mixtures include mixtures of long-chain fructooligosaccharides with short-chain fructooligosaccharides or short-chain galactooligosaccharides, most preferably mixtures of long-chain fructooligosaccharides with short-chain fructooligosaccharides.

[0149] The compositions of the present invention preferably contain 0.05 to 20% by weight of the non-digestible oligosaccharides based on the dry weight of the compositions of the present invention, more preferably 0.5 to 15% by weight, even more preferably 1 to 10% by weight, and most preferably 2 to 10% by weight. When the compositions of the present invention are in liquid form, they preferably contain 0.01 to 2.5% by weight of non-digestible oligosaccharides based on 100 ml, more preferably 0.05 to 1.5% by weight, even more preferably 0.25 to 1.5% by weight, and most preferably 0.5 to 1.25% by weight.

[0150] In one embodiment, the NDO mixture does not contain any detectable amount of acidic oligosaccharides.

[0151] When the non-digestible oligosaccharides are a mixture, the average values ​​of their respective parameters are used to define this invention.

[0152] The combination of NDO and lactic acid-producing bacteria as defined above is also referred to as a "synbiotic." It is believed that the presence of therapeutically effective amounts of NDO and lactic acid-producing bacteria together can further improve oral immune tolerance induction properties. Bifidobacteria (preferably Bifidobacterium breve strains) with fructooligosaccharides. See Example 5.

[0153] Other ingredients

[0154] The composition may further comprise long-chain polyunsaturated fatty acids (LC-PUFAs). LC-PUFAs are fatty acids in which the acyl chain has a length of 20 to 24 carbon atoms (preferably 20 or 22 carbon atoms) and wherein the acyl chain contains at least two unsaturated bonds between the carbon atoms in the acyl chain. More preferably, the composition of the present invention comprises at least one LC-PUFA selected from: eicosapentaenoic acid (EPA, 20:5n3), docosahexaenoic acid (DHA, 22:6n3), arachidonic acid (ARA, 20:4n6), and docosapentaenoic acid (DPA, 22:5n3), preferably DHA, EPA, and / or ARA. Such LC-PUFAs have a further beneficial effect on reducing the risk of allergies.

[0155] The preferred content of LC-PUFA in the composition of the present invention is no more than 15% by weight of total fatty acids, preferably no more than 10% by weight, and even more preferably no more than 5% by weight. Preferably, the composition of the present invention contains at least 0.2% by weight of total fatty acids, preferably at least 0.25% by weight, more preferably at least 0.35% by weight, and even more preferably at least 0.5% by weight of LC-PUFA, and more preferably DHA. The composition of the present invention preferably contains ARA and DHA, wherein the weight ratio of ARA / DHA is preferably 0.25 or more, preferably 0.5 or more, more preferably 0.75-2, and even more preferably 0.75-1.25. The weight ratio is preferably less than 20, more preferably 0.5 to 5. The amount of DHA is preferably 0.2% by weight or more of total fatty acids, more preferably 0.3% by weight or more, more preferably at least 0.35% by weight, and even more preferably 0.35-0.6% by weight.

[0156] Nutritional composition

[0157] The compositions of the present invention can be used as nutritional compositions, nutritional therapies, nutritional supports, as medical foods, as foods for special medical purposes, or as nutritional supplements. The compositions of the present invention are preferably enteral (oral) compositions. The compositions are given orally to subjects who require them, particularly children and infants, including toddlers, preferably children up to 6 years of age, preferably infants or toddlers typically 0-36 months of age, more preferably infants 0-12 months of age, and most preferably infants 0-6 months of age. Therefore, in some embodiments, the compositions of the present invention are infant formula, follow-up formula, or toddler formula (also referred to as growing milk), preferably infant formula or follow-up formula, and most preferably infant formula. The term "infant formula" is internationally and uniformly defined and controlled by regulatory agencies. In particular, CODEX STAN 73-1981 "Standard For Infant Formula and Formulas For Special Medical Purposes Intended for Infants" is widely accepted. It recommends nutritional value and formulation composition, requiring that the prepared milk contain no less than 60 kcal (250 kJ) and no more than 70 kcal (295 kJ) of energy per 100 ml. The FDA and other regulatory agencies have established nutritional requirements based on this.

[0158] Preferably, the enteric composition (preferably a nutritional composition) of the present invention is intended to provide daily nutritional needs for humans, particularly for feeding humans, especially infants including toddlers, and preferably carries a risk of milk protein allergy, preferably to milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably from species of *Bos*, and most preferably to bovine milk protein allergy. In one embodiment, the milk protein allergy involves milk from a specific mammal species, wherein the hydrolyzed milk proteins contained in the composition are from the same mammal species, preferably both from the genus *Bos*.

[0159] To meet the calorie requirements of infants, the enteric composition of the present invention preferably contains 50 to 200 kcal / 100 ml of fluid, more preferably 60 to 90 kcal / 100 ml of fluid, and even more preferably 60 to 75 kcal / 100 ml of fluid. This calorie density ensures an optimal ratio between water and calorie consumption. The molar osmolality of the composition of the present invention is preferably 150 to 420 mOsmol / L, more preferably 260 to 320 mOsmol / L. The low molar osmolality aims to reduce gastrointestinal pressure.

[0160] Preferably, the enteric composition of the present invention is in liquid form, preferably with a viscosity of less than 35 mPa·s, more preferably less than 6 mPa·s, as measured in a Brookfield viscometer at a shear rate of 100 s⁻¹ at 20°C. Suitably, the enteric composition of the present invention is in powder form, preferably reconstituteable with water to form a liquid, or in the form of a liquid concentrate diluted with water. When the enteric composition of the present invention is in liquid form, the preferred daily volume is about 80 to 2500 ml, more preferably about 450 to 1000 ml / day.

[0161] The compositions of the present invention preferably contain a lipid component, more preferably a lipid component known in the art suitable for infant nutrition. The lipid component of the compositions of the present invention preferably provides 2.9 to 6.0 g, more preferably 4 to 6 g / 100 kcal composition. When the composition is in liquid form, it preferably contains 2.1 to 6.5 g lipid / 100 ml, more preferably 3.0 to 4.0 g / 100 ml. Based on dry weight, the infant formula or subsequent formula of the present invention preferably contains 12.5 to 40% by weight of lipids, more preferably 19 to 30% by weight.

[0162] In addition to the β-lactoglobulin-derived peptides of the present invention, the compositions of the present invention may also contain other protein-like substances. In the context of the present invention, other “proteins” or “protein-like substances” or “protein equivalents” include proteins, peptides, free amino acids, and partially or extensively hydrolyzed proteins.

[0163] Preferably, other protein components—other than β-lactoglobulin-derived peptides—are non-allergenic or hypoallergenic, such as free amino acids and hydrolyzed proteins. As other protein components, i.e., in addition to β-lactoglobulin-derived peptides, the compositions of the present invention preferably contain free amino acids, hydrolyzed whey protein, and more preferably partially hydrolyzed whey protein.

[0164] The compositions of the present invention preferably contain less than 1% by weight of intact mammalian (bovine) milk protein. The compositions may contain other protein components in intact, partially hydrolyzed, and / or extensively hydrolyzed forms, selected from free amino acids, hydrolyzed whey protein, and proteins from other sources (such as soybean, pea, rice, collagen, etc.).

[0165] The compositions of the present invention preferably contain at least 50% by weight of protein components derived from non-human milk, more preferably at least 90% by weight, based on the dry weight of total protein.

[0166] The compositions of the present invention preferably contain 4 to 25%, more preferably 5 to 20%, more preferably 7 to 16%, and most preferably 7 to 12% protein, based on total calories. When the compositions of the present invention are in liquid form, they preferably contain 0.5 to 6.0 g, more preferably 0.8 to 3.0 g, and even more preferably 1.0 to 2.5 g of protein per 100 ml. The compositions of the present invention preferably contain at least 7.0% by weight, more preferably at least 8.0% by weight, and most preferably at least 9% or at least 10% by weight of protein, based on the dry weight of the total composition. Preferably, the compositions of the present invention contain up to 40% by weight, more preferably up to 15% by weight, and most preferably up to 20% by weight of protein, based on the dry weight of the total composition.

[0167] The composition may contain digestible carbohydrates. Typically, digestible carbohydrates known in the art to be suitable for use in infant nutritional compositions are used, such as those selected from digestible polysaccharides (e.g., starch, maltodextrin), digestible monosaccharides (e.g., glucose, fructose), and digestible disaccharides (e.g., lactose, sucrose). Lactose and / or maltodextrin are particularly suitable. In one embodiment, the composition does not contain lactose.

[0168] The digestible carbohydrate component preferably contains at least 60% by weight lactose, more preferably at least 75% by weight, and even more preferably at least 90% by weight lactose, based on total digestible carbohydrates.

[0169] human subjects

[0170] The target human subjects or population are preferably infants (0-12 months), preferably infants at risk of developing allergies, preferably with milk protein allergy, preferably with milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably with milk proteins from species of the genus *Bos*, most preferably with bovine milk protein allergy, and preferably infants suffering from milk protein allergy, wherein the milk protein allergy is preferably with milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably with milk proteins from species of the genus *Bos*, and most preferably with bovine milk protein allergy. Infants at risk are preferably identified by family history, preferably with at least one parent having an allergy or a history of allergies.

[0171] Humans preferably possess specific HLA-DRB1 alleles selected from 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 prevalent human MHC class II isotype (>90%). The HLA-DRB1 locus is polymorphic, while the HLA-DRRA1 locus is monomorphic (i.e., the HLA-DRB1 genotype determines the entire HLA-DR molecule). The expression of the HLA-DRB1 allele is 5 times higher than that of its paralogous genes (HLA-DRB3, -DRB4, or -DRB5), and it is present in all humans.

[0172] Oral induction of immune tolerance

[0173] This invention relates to

[0174] - To induce oral immune tolerance to milk proteins in human subjects or populations, preferably milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably milk proteins from species of the genus *Bos*, and most preferably bovine milk proteins, or

[0175] - For the prevention or treatment of oral immune intolerance to milk proteins in human subjects or populations, preferably milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably milk proteins from species of the genus *Bos*, and most preferably bovine milk proteins, or

[0176] - To reduce the risk of oral immune intolerance to milk proteins in human subjects or populations, preferably milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably milk proteins from species of the genus *Bos*, and most preferably bovine milk proteins, or

[0177] - To improve or enhance oral immune tolerance to milk proteins in human subjects or populations, preferably milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably milk proteins from species of the genus *Bos*, and most preferably bovine milk proteins.

[0178] As described above, the human subjects or populations are preferably infant subjects or groups.

[0179] In the context of this invention, the term "oral tolerance" refers to oral immune tolerance.

[0180] In a preferred embodiment, the present invention relates to a nutritional composition as defined above for the prevention of oral immune intolerance to bovine milk protein in infant subjects or groups at risk of developing milk protein allergy, preferably to milk proteins from species of the genus Bosnia, Buffalo, Buffalo or Caprus, more preferably to milk proteins from species of the genus Bosnia, and most preferably to bovine milk protein allergy. In a preferred embodiment, the present invention relates to a nutritional composition as defined above for reducing the risk of oral immune intolerance to milk proteins in infant subjects or populations at risk of developing milk protein allergy (preferably milk proteins from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, more preferably milk proteins from species of the genus *Bos*, and most preferably bovine milk protein allergy), wherein a portion of the mammalian protein hydrolysate has at least 1% by weight, more preferably at least 5% by weight, and even more preferably at least 10% by weight of peptides with a molecular weight of 1 kDa or more and / or more than 0.8 μg allergenic BLG / g total protein. Attached Figure Description

[0181] Figure 1 Comparison of fragmentation spectra of experimental record (B) and its synthetic equivalent (A) of a representative peptide (SEQ ID NO:3; shown above). The mass difference between the fragment ions of the experimental and synthetic peptides corresponds to the presence of a stable isotopically labeled lysine in the synthetic peptide, indicated in italics in the sequence (+8 Da). * indicates dehydrated fragments of the corresponding β-ion.

[0182] Figure 2 T cell responses to stimulation with identified peptides. Three different donor bovine milk-specific T cell lines (TCLs) 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). Bovine milk protein (CMP) served as a control. A stimulation index ≥2 was considered significant.

[0183] Example 1: A new method for hydrolyzing whey protein and identifying β-lactoglobulin peptides

[0184] A mixture of acidic whey protein and demineralized sweet whey protein (protein weight ratio 77:23) was dissolved in water (purified by reverse osmosis) and then hydrolyzed under specific conditions. Acidic and sweet whey are commercially available. Sweet whey is a byproduct of rennet crepe production and contains casein glycomacropeptide (CGMP), while acidic whey (also known as sour whey) is a byproduct of sour crepe production and does not contain CGMP. Suitable sources of acidic whey protein are demineralized whey (Deminal, Friesland Campina, the Netherlands) and whey protein concentrate (WPC80, Friesland Campina, the Netherlands). Both protein sources were hydrolyzed using a mixture of microbial endopeptidases and exopeptidases, as described in Example 1 of WO 2011 / 151059. The hydrolyzed protein solution was then spray-dried. The peptide size distribution in the protein hydrolysate was determined using high-performance liquid chromatography with molecular sieves, a method known in the art. In short, the total surface area of ​​the chromatogram is integrated and divided into mass ranges, expressed as a percentage of the total surface area. Mass ranges are calibrated using peptides / proteins with known molecular weights. Whey protein hydrolysates can be classified as partially or moderately hydrolyzed.

[0185] The resulting hydrolysate powder was used as the sole protein source in infant formula. Ten different batches of infant formula were produced in a similar manner. The amount of β-lactoglobulin, as determined by an ELISA method known in the art, ranged from approximately 0.8 to 3.5 μg / g total protein.

[0186] To determine the presence of specific sequences in biological samples, mass spectrometry (MS) is the preferred method, contrasting with more traditional techniques currently used to characterize protein hydrolysates. Recent developments in MS, known as peptidomics, allow for the characterization of peptide sequences with high sensitivity and specificity (Dallas et al. J Nutr 2015; 145:425-433). This technique can close the gap between understanding the influence of sequence specificity on the potential biological activities of protein hydrolysates.

[0187] The samples were prepared essentially according to the description by Butré et al., with the addition of reduction and alkylation steps (Butré et al. Anal Bioanal Chem 2014; 406: 5827-5841). All chemicals were obtained from Sigma Aldrich. Briefly, partially hydrolyzed protein (pHP) batches were diluted to 0.5% (v / v) with 50 mM ammonium bicarbonate, then the peptides were reduced with 4 mM DTT and alkylated with 8 mM iodoacetamide. The mixture was clarified by centrifugation at 20,000 × g for 10 min and diluted to 0.1% (v / v) with 0.1 M acetic acid.

[0188] All samples were analyzed using nanofluidic chromatography on an Agilent 1200 HPLC system (Agilent Technologies) coupled online to an LTQ Velos mass spectrometer (Thermo Fisher Scientific). The liquid chromatography section of the system was operated essentially as described above (14). Peptides were captured at 5 μl / min in 100% solvent A (0.1 M acetic acid in aqueous solution) on a 2-cm capture column (100 μm inner diameter, filled with Aqua C18, 5 μm resin (Phenomenex)) and eluted at approximately 100 nl / min in a gradient of 10% to 40% solvent B (0.1 M acetic acid in 8:2 (v / v) acetonitrile / water) over 90 min. The eluent was sprayed through a standard coated emission tip (New Objective) docked to the analytical column. The mass spectrometer operated in data correlation mode, automatically switching between MS and MS / MS. After accumulating to the target value of 3000, a full-scan mass spectrum (m / z 300 to 1200) was obtained by scaling the scan rate. After accumulating to the target value of 10,000, the five strongest ions with a threshold above 500 were selected for collision-induced collisions at 35% of the normalized collision energy.

[0189] 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 retrieving individual peak lists of CID fragmentation spectra from databases containing selected bovine whey and casein using Mascot (version 2.4.1, Matrix Science). Enzymes were not specified, and missed cleavage was not permitted. The tolerance for precursor ion mass was set to 0.2 Da, and the tolerance for product ion mass was set to 0.5 Da. Carbamidomethylation (C) was set as a fixed modification.

[0190] Of the 314 peptides identified by this method, 101 were assigned to β-lactoglobulin (BLG), resulting in 90% sequence coverage of BLG. Most of the remaining peptides were derived from other abundant whey proteins, such as α-lactalbumin and serum albumin. Within the target region (AA#13-48), a total of 13 β-lactoglobulin peptides with a minimum of 9 AAs were identified (see Table 1 and...). Figure 1Of these, six were consistently identified in all 10 batches. Clear peptide identification can be achieved by comparing the properties (retention time, peptide mass, and fragmentation spectrum) of the experimental peptide with its stable isotope-labeled synthetic equivalent. Figure 1 Examples are shown where the fragmentation spectra of experimental and synthetic peptides exhibit good correlation. Using this method, the identities of six peptides found in all 10 batches were confirmed.

[0191] Table 1. Sequences of β-lactoglobulin peptides identified in 10 different batches of partially hydrolyzed whey-based infant formula.

[0192]

[0193]

[0194] Similar analyses were performed on multiple batches of Nutrilon pepti, an infant formula containing extensively hydrolyzed whey protein, commercially available for dietary management of cow's milk allergy. The BLG peptides listed in Table 1 were not present in Nutrilon pepti.

[0195] Example 2: In vitro identification of HLA-DR restricted peptides

[0196] As described in (Lamberth et al, Sci Transl Med 201711; 9(372):10.1126 / scitranslmed.aag1286.), the identification of BLG-derived peptides presented by human DCs was performed using ProImmune. Briefly, peripheral blood mononuclear cell samples were obtained from 12 HLA-DR genotype healthy adult donors. Donors were selected based on the 11 common HLA-DRB1 alleles (Table 2). In this study, HLA-DRB1 was given priority because the HLA-DR molecule is the most prevalent human MHC class II isotype (>90%) (Sturniolo et al. Nat Biotechnol 1999; 17:555-561.), and the HLA-DRB1 locus is polymorphic, while the HLA-DRA1 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 expression of the HLA-DRB1 allele is 5-fold higher than that of its paralogs (HLA-DRB3, -DRB4, or -DRB5), and it is present in all individuals (O'Leary et al. Nucleic Acids Res 20164; 44(D1):D733-45.).

[0197] Table 2: Donors with HLA-DRB1 typing information

[0198] 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

[0199] Immature mononuclear cell-derived dendritic cells (DCs) were generated in vitro and matured in the presence of the tested hydrolysate (HP). DCs were collected and lysed using a specific immunoaffinity assay to obtain the HLA-DR complex. Peptides were eluted from the HLA-DR complex and then analyzed by high-resolution sequencing LC-MS / MS. The presence of six endogenous related proteins (ITGAM, ApoB, CLIP, TFRC, FcER2 / FcGR2, and LAMP-1 / 3) was assessed as controls for this assay. Each donor sample must express at least three related proteins for subsequent analysis. Data on the obtained HLA-DR-restricted peptides were compiled and evaluated using sequence analysis software referencing the Swiss-Prot human proteome database and the included test sequences. The probability of a peptide being a true discovery was described by its expected value being ≤0.05. The false discovery rate was determined to be <1%.

[0200] use Antigen presentation assays, focusing on BLG-derived sequences, identified 15 relevant peptides with expected values ​​≤0.05 in five donor samples (Table 3). These peptides were further subdivided into two distinct sequence groups: DIQ…DIS (AA#11-30) and AMA…APL (AA#23-39) (Table 4). Importantly, both sequence groups overlapped with the target region (i.e., AA#13-48 of mature BLG). This finding indicates that target BLG-derived peptides can be presented by HLA-DR molecules on human DCs when incubated with specific HP.

[0201] Table 3. Important fragments with expected values ​​<0.05 identified from peptide-HLA-DR complexes.

[0202]

[0203] Table 4. HLA-DR allele associations on unique regions of mature BLG with important segments defined by expected values ​​<0.05.

[0204]

[0205]

[0206] Not all donor antigen-presenting cells bind to the peptide. This is related to HLA-DRB1*03:01 in homozygous donor P1. Similar reasoning applies to donors P6 and P11.

[0207] Example 3: Computer Evaluation of MHC Class II Integration

[0208] The limitation of antigen presentation assays is that they use generic anti-HLA-DR antibodies, rather than specific antibodies against a particular HLA-DRB1 allele (e.g., anti-DRB1*01:01 antibody), to isolate the target peptide-HLA-DR complex. Therefore, for donors with HLA-DRB1 heterozygous genotypes (e.g., *01:01 and *04:01), it is uncertain which allele will present the identified peptide.

[0209] Therefore, the identified HLA-DR-restricted peptides were input into the IEDB MHC class II binding prediction software (http: / / tools.iedb.org / mhcii / ) for calculation to assess the binding of the identified peptides to selected HLA-DRB1 alleles, as described in (Wang P, et al. PLoS Comput Biol 2008 4;4(4):e1000048.;Wang et al, BMC Bioinformatics 2010,11:568-2105-11-568.). The default IEDB recommended prediction method was selected. For each peptide sequence (15mer long), a percentile ranking was obtained by comparing the peptide's score with the scores of 5 million random 15mers selected from the Swiss-Prot database. A lower percentile ranking indicates a higher affinity of the peptide for a specific MHC class II allele, with IEDB recommending selection based on the top 10% of consistent percentile rankings. To ensure more rigorous computer evaluation, selection is based on the top 3% of consistent percentile rankings.

[0210] Using MHC class II prediction software, we assessed whether fragments from two unique sequence sets of identified BLG possessed high affinity for binding to [the target gene]. Selected HLA-DRB1 alleles from the donors characterized in the 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, with an arbitrary percentage threshold of <3% (i.e., the top 3% of high-binding fragments), the DIQ...DIS (AA#11-30) fragment was predicted to bind five HLA-DRB1 alleles (i.e., DRB1*01:01, *04:01, *04:04, *04:05, and *09:01) with high affinity. In contrast, only one AMA...APL (AA#23-39) fragment was estimated to bind HLA-DRB1*07:01 with high affinity. In summary, these findings suggest that fragments from AA#11-30 are more likely to be presented as T-cell epitopes compared to fragments from AA#23-39. Furthermore, the data indicate that several common HLA-DRB1 alleles can present fragments derived from two identified BLG-derived unique sequence sets.

[0211] These unique sequence sets of BLG (AA#11-30 and #23-39) overlap and can be linked into a single sequence (i.e., AA#11-39). This linked sequence is highly correlated with a persistently identified peptide (AA#11-42; DIQ...RVY) in the tested HLA. Therefore, sequence AA#11-42 was included in the MHC class II prediction software. Importantly, the predicted complex of AA#11-42 further confirmed the prediction results of the two in vitro identified unique sequences (Table 6), indicating that both unique sequences of BLG can originate from the AA#11-42 peptide present in the tested formulation. Furthermore, a portion of AA#11-42 was predicted to have high affinity for the same identified HLA-DRB1 allele set (i.e., DRB1*01:01, *04:01, *04:04, *04:05, *07:01, and *09:01). In summary, computer evaluation confirmed the in vitro finding that BLG-derived peptides can bind to common HLA-DRB1 alleles.

[0212] Table 5. Computer evaluation of potential peptide-HLA-DRB1 complexes for two identified BLG-derived peptides at a percentage level of <3%.

[0213]

[0214] Table 6. Computer evaluation of potential peptide-HLA-DRB1 complexes for BLG-derived long peptides with a percentage grade <3 (top 3% of the binder).

[0215]

[0216] Multiple computer evaluations have confirmed the in vitro efficacy of the peptide-HLA-DR complex. Discovery. However, the inventors observed that some computer-predicted complexes are related to... The complexes found in the assays were unrelated, for example, DRB1*04:04 and AGT...DIS. This difference can be explained by... Determination of a small cohort (n=12) of test subjects, each donor having a different combination of HLA-DRB1 allele types (i.e., inter-individual variation) and / or a tendency for over-prediction in computer assessment (thus requiring strict cutoff values), necessitates a cautious approach that combines in vitro and computer methods to identify peptide-MHC class II complexes.

[0217] Example 4: T-cell proliferation assay

[0218] Synthetic peptides were obtained using the JPT technique. Only peptides containing at least nine amino acids (AAs) were considered, as this is the minimum size for binding to MHC class II molecules and subsequent T cell recognition (Holland et al., Front Immunol 2013 Jul 1; 4:172.). The same synthetic peptides (using the JPT technique) identified in all batches of HP in Example 1 were dissolved in dimethyl sulfoxide (Sigma Aldrich) at a concentration of 5.3 mM. These peptides were tested on milk-specific T cell lines (TCLs) from three different donors. These peptides were tested on milk-specific T cell lines (TCLs) from three infant donors diagnosed with milk protein allergy, aged <1 year, 7.5 months, and 6 years. These TCLs were previously generated and had been shown to recognize epitopes in the target region (AAs #13-48 of mature BLGs). Proliferation was measured as previously described (Ruiter et al., Clin Exp Allergy 2006, 36(3):303-310). The stimulation index (SI, the ratio between allergen / peptide-stimulated T cell proliferation and non-stimulated T cell proliferation) was calculated, and an SI ≥ 2 was considered positive.

[0219] To confirm that the peptides identified in *Helicobacter pylori* (HP) are recognized by T cells, synthetic peptides identical to these identified peptides were tested on bovine milk-specific human TCLs. Due to overlap among the identified peptides, five peptides identified in all batches were tested (Table 7), with differences between them exceeding 9 Å. All tested peptides were able to induce proliferation (…). Figure 2However, each donor exhibited a different recognition pattern. This was also seen in the computer-predicted data (data not shown). All five peptides induced TCL B proliferation, indicating that the TCL recognizes the overlapping portions of the peptides (AA#11-27) or multiple T cell epitopes in that region. Furthermore, TCL A also showed a proliferative response after stimulation with several peptides. Since the peptide LIV...AAS (AA#1-27) did not induce TCL A proliferation, while the peptide LIV...SLL (AA#1-32) did, the region containing AA#28-32 (DISLL) is essential for the TCL A response. Surprisingly, TCL C recognized the peptide LIV...AAS (AA#1-27), while the longer peptide LIV...SLL (AA#1-32), containing the same sequence and five other AAs, did not induce a proliferative response, suggesting that longer peptides are not always presented better.

[0220] Table 7. Sequences of β-lactoglobulin peptides identified and tested for T-cell proliferation activity in 10 different batches of partially hydrolyzed whey-based infant formula.

[0221] Amino acid numbering in the BLG sequence sequence SEQ ID NO AA 1-27 LIVTQTMKGLDIQKVAGTWYSLAMAAS 9 AA 1-32 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLL 11 AA 6-32 TMKGLDIQKVAGTWYSLAMAASDISLL 3 AA 6-35 TMKGLDIQKVAGTWYSLAMAASDISLDAQ 4 AA 11-42 DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 5

[0222] Not all peptides are recognized by every donor, indicating differences between donors. Each donor expresses a different MHC class II molecule. The same algorithm described above was used to determine whether the HLA-DRB1 allele expressed by the donor presents the peptide and which donor's HLA-DRB1 allele presents the peptide. Computer prediction confirmed that the HLA-DRB1 allele expressed by donor B (*11:01 & *04:04) presents all peptides. According to the algorithm data, TCL A should only recognize the peptide DIQ...RVY (AA#11-42); however, after stimulation with four of the five peptides, the TCL exhibited a proliferative response. A possible explanation is that the other three peptides are not presented by HLA-DRB1, but by other MHC class II molecules.

[0223] In summary, Examples 1 through 4 demonstrate that specific whey protein hydrolysates contain functional T-cell epitopes. Specific β-lactoglobulin peptides, which are HLA-DRB1-restricted peptides, were identified. These peptides were presented to and recognized by T cells from various donors (including healthy donors and infants with bovine milk protein allergy). This interaction was confirmed by computer analysis. Therefore, HP can stimulate oral immune tolerance to the protein.

[0224] Example 5: Synbiotic blends showed further improved tolerance compared to probiotics or prebiotics alone. Induction effect

[0225] Several published studies support the idea that dietary antigens (including peptides) in the intestinal lumen can be absorbed, subsequently captured and presented by intestinal antigen-presenting cells (i.e., antigen sampling mechanisms) without disrupting the intestinal barrier. In healthy individuals, at least two distinct mechanisms work sequentially to sample dietary antigens: microfold / M cell-mediated transcytosis and goblet cell-associated antigen delivery. Both pathways supply dietary antigens to CD103+ DCs in the intestinal lamina propria (which in turn can imprint gut-homing molecules on T and B cells), promote the differentiation of IgA-producing plasma cells in the gut, and generate intestinal Tregs, which are key players in a state of low reactivity to imported antigens known as oral tolerance. This suggests that peptides in *Helicobacter pylori* (HP) can be orally absorbed, then captured and presented by intestinal CD103+ DCs, ultimately leading to the development of functional Tregs.

[0226] To induce oral tolerance, T-cell epitopes within the test hepatocytes need to be presented under appropriate conditions. Besides TCR activation, co-stimulation and cytokine signaling transduction play important roles in Treg production. Probiotics or prebiotics play a crucial role in creating a suitable environment for Treg production. Combining a diet with probiotics or prebiotics enhances the preventative effect of synthesized BLG peptides.

[0227] To demonstrate the effects of probiotics, prebiotics, and synbiotics, experiments similar to those described in Example 4 of WO 2016 / 148572 were conducted using the same protocol, concentrations, and a mixture of synthetic peptides (“Pepmix”), the identified pepmixes of which are shown in Table 8.

[0228] Table 8. Pepmix according to WO 2016 / 148572

[0229]

[0230] A comparison of some diets:

[0231] (a) Synbiotic diet of Example 1 of WO 2016 / 14472 (1 wt% scFOS / lcFOS in a weight ratio of 9:1 + 2 wt% 2x10 9 cfu / g Bifidobacterium breve M-16V,

[0232] (b) Containing 2% by weight of 2x10 9 The probiotic diet of Example 4, cfu / g Bifidobacterium breve M-16V WO 2016 / 148572, and

[0233] (c) A prebiotic diet containing 1% by weight of scFOS / lcFOS in a weight ratio of 9 / 1.

[0234] Short-chain (sc-) and long-chain (lc-) fructooligosaccharides (FOS) were commercially obtained from Raftilose P95 (Orafti) and Raftiline HP, respectively.

[0235] All groups were pretreated with pepmix and one of the diets above, sensitized with whey + cholera toxin, and challenged with whey.

[0236] Following whey stimulation, the delta ear swelling in mice fed a (c) scFOS / lcFOS diet was approximately 174 μm, in mice fed a (b) probiotic diet was approximately 158 μm, and in mice fed a (a) synbiotic diet was approximately 112 μm. Ear swelling in the synbiotic group (a) was statistically significantly lower than that in the corresponding scFOS / lcFOS group (c), and showed a lower trend toward response compared to the probiotic group (b) (p = 0.08). These results suggest a potential for further improvement in oral immune tolerance induction.

[0237] - When using lactic acid-producing bacteria, especially Bifidobacterium strains from the Bifidobacterium breve species, and

[0238] - Especially when lactic acid-producing bacteria are used in combination with NDO.

[0239] Example 6: Binding of natural, identified peptides from hydrolysis products to MHC in synthetic β-lactoglobulin peptides. Domain Comparison

[0240] Methods: The sequences of β-lactoglobulin peptides identified in hydrolyzed infant formula (ID_PEP) and those of synthetic β-lactoglobulin peptides disclosed in WO2016 / 148572 (SYN_PEP, Table 9) were tested 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 used the IEDB-recommended prediction method and a complete HLA reference set. This HLA reference set provided >99% population coverage. The software calculated percentile rank by comparing the binding affinity of the predicted binding domains to a large set of peptides. Higher affinity resulted in a lower percentile rank. In this experiment, the top 2% of bindings were selected.

[0241] result

[0242] The number of MHC class II binding domains was higher in the identified peptides than in the synthetic peptides (see Table 9). The software predicted at least one binding domain and a total of six distinct domains for all identified peptides. For the synthetic peptides, four distinct domains were predicted. However, all of these domains originated from a single sequence, SYN_PEP_1. The other two synthetic peptides were predicted to lack MHC class II binding domains.

[0243] Table 9. Number of MHC class II binding domains predicted by IEBD for each β-lactoglobulin peptide

[0244]

[0245] The identified peptides contain a higher number of MHC class II binding domains than the synthetic peptides. In other words, more T cell epitopes can be derived from the identified peptides rather than the synthetic peptides. Therefore, the identified peptides are more likely to be presented to T cells. This presentation to T cells is essential for the induction of oral immune tolerance.

[0246] Example 7: HLA-DR expression on mature dendritic cells of lactic acid-producing bacteria

[0247] Methods: Monocytes (CD14+ cells) were cultured for 7 days with GM-CSF and IL-4 to generate immature dendritic cells (DCs). After 7 days, immature DCs were washed and incubated with culture 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-cyanin 7-labeled anti-human HLA-DR antibody to determine HLA-DR expression on the cell surface. Samples were analyzed using FACSDIVA software on a BD FACS Canto flow cytometer. HLA-DR expression is expressed as mean fluorescence intensity (MFI).

[0248] Results: Maturation of Bifidobacterium breve M-16V (M16v-DC) increased HLA-DR expression to a level similar to that of LPS (LPS-DC, see Table 10).

[0249] Table 10. HLA-DR expression in different dendritic cell subtypes

[0250] DC HLA-DR expression MFI (SD) Immature DC 11338(1919,1) LPS-DC 24676(9694,4) M16v-DC 21714(1516)

[0251] DC = dendritic cells, MFI = mean fluorescence intensity, SD = standard deviation

[0252] Bifidobacterium breve M-16V increases the expression of HLA-DR molecules on the surface of dendritic cells. Increased HLA-DR expression indicates increased peptide presentation to T cells.

[0253] Example 8: Infant Formula

[0254] A powdered infant formula has the following instructions on its packaging: It should be reconstituted with water at 40°C by adding 3 scoops of powder (13.74g) to 90ml of water, resulting in a final volume of 100ml. This product is suitable for use from birth to a maximum of 6 months.

[0255] Each 100ml of infant formula contains 66kcal, 1.5g protein (hydrolyzed whey protein from Example 1), 3.3g fat (vegetable oil, single-cell oil, and fish oil, containing 0.4% by weight DHA and 0.35% by weight ARA based on total fatty acids), 7.2g digestible carbohydrates (primarily lactose), 0.8g non-digestible oligosaccharides (a mixture of 9 / 1 w / w scGOS and lcFOS), and approximately 3 x 10^6 kcal. 7 It contains cfu / g of Bifidobacterium breve M-16V, and vitamins, minerals, trace elements and other micronutrients according to international guidelines for infant formula.

Claims

1. Use of two peptides in the preparation of a nutritional composition, said nutritional composition for use in... - Inducing oral immune tolerance to milk proteins; and / or - Reduce the risk of developing oral immune intolerance to milk proteins; and / or - Improves or enhances oral immune tolerance to milk proteins. The nutritional composition comprises the two peptides, wherein the sequence of the first peptide is SEQ ID NO: 1, and the sequence of the second peptide is SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

5. The nutritional composition described herein is used in human subjects.

2. The use according to claim 1, wherein the composition further comprises at least one lactic acid-producing strain.

3. The use according to claim 1 or 2, wherein the composition comprises less than 6 μg of allergenic β-lactoglobulin / g total protein.

4. The use according to claim 1 or 2, wherein the composition comprises at least 50% by weight of hydrolyzed whey protein based on total protein content.

5. The use according to claim 1 or 2, wherein, based on total protein, the nutritional composition comprises less than 10% by weight of peptides with a size of 5 kDa or more.

6. The use according to claim 1 or 2, wherein, based on total protein, at least 1% by weight of the peptides present in the composition have a size of 1 kDa or greater.

7. The use according to claim 1 or 2, wherein the composition comprises an amount of more than 0.8 μg of allergenic β-lactoglobulin / g total protein.

8. The use according to claim 1 or 2, wherein the peptide is derived from mammalian milk protein, said milk protein being derived from the genus *Bovine* (…). Bos ), Bison ( Bison ), buffalo ( Bubalus ) or Capricornus ( Capra ) of the species.

9. The use according to claim 1 or 2, wherein the human subject is an infant.

10. The use according to claim 1 or 2, wherein the human subject is at risk of developing or has milk protein allergy, the milk being derived from a species of the genus *Bos*, *Buffalo*, *Buffalo*, or *Capus*, and wherein the human subject is an infant.

11. The use according to claim 1 or 2, wherein the composition comprises a substance belonging to the genus Bifidobacterium (Bifidobacterium). Bifidobacterium Lactic acid-producing strains.

12. The use according to claim 1 or 2, wherein the composition comprises a species belonging to Bifidobacterium breve (… Bifidobacterium breve ) species of lactic acid producing strains.

13. The use according to claim 1 or 2, wherein the composition comprises one or more non-digestible oligosaccharides selected from the following: fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinogalactooligosaccharides, arabinogalacto-oligosaccharides, glucose oligosaccharides, glucomannan oligosaccharides, galactomannan oligosaccharides, mannose oligosaccharides, chitosan oligosaccharides, uronic acid oligosaccharides, sialic acid oligosaccharides, and fucose oligosaccharides.

14. The use according to claim 13, wherein the composition comprises fructooligosaccharides and galactooligosaccharides.

15. The use according to claim 13, wherein the non-digestible oligosaccharide comprises a mixture of long-chain fructooligosaccharides and short-chain fructooligosaccharides or short-chain galactooligosaccharides.

16. The use according to claim 1 or 2, wherein the composition comprises a long-chain polyunsaturated fatty acid.

17. The use according to claim 1 or 2, wherein the composition comprises docosahexaenoic acid (DHA), wherein the DHA comprises at least 0.35% by weight based on total fatty acids.

18. The use according to claim 1 or 2, wherein the composition is an infant formula.

19. A nutritional composition comprising: a. Belongs to the genus Bifidobacterium ( Bifidobacterium Lactic acid-producing strains; b. Two peptides derived from milk proteins, said milk proteins being derived from species of the genera *Bos*, *Buffalo*, *Buffalo*, or *Capus*, wherein the sequence of the first peptide is SEQ ID NO: 1, and the sequence of the second peptide is SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

5. c. Less than 6 μg of allergenic β-lactoglobulin per gram of total protein. d. Based on total protein, less than 10% by weight of peptides or proteins with a size of 5 kDa or greater, and e. Based on total protein, at least 50% by weight of hydrolyzed whey protein. f. Optionally, one or more non-digestible oligosaccharides selected from the following: fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabino-oligosaccharides, arabinogalacto-oligosaccharides, glucose-6-hydroxy-2-ethylhexano ... g. Optional, long-chain polyunsaturated fatty acids.

20. The nutritional composition of claim 19, wherein the two peptides are derived from milk proteins, the milk proteins being derived from species of the genus Bovine.

21. The nutritional composition of claim 19, comprising less than 3.5 μg of allergenic β-lactoglobulin per gram of total protein.

22. The nutritional composition of claim 21, wherein the amount of allergenic β-lactoglobulin is greater than 0.8 μg per gram of protein, and / or wherein the composition comprises more than 1% by weight of a peptide or protein of size 1 kDa or greater based on total protein.

23. The nutritional composition according to claim 19, 20, 21 or 22, wherein the lactic acid-producing strain belongs to Bifidobacterium breve (Bifidobacterium). Bifidobacterium breve )kind.

24. The nutritional composition according to claim 19, 20, 21 or 22, wherein the non-digestible oligosaccharide comprises fructooligosaccharides and galactooligosaccharides.

25. The nutritional composition according to claim 19, 20, 21 or 22, wherein the non-digestible oligosaccharide comprises a mixture of long-chain fructooligosaccharides and short-chain fructooligosaccharides or short-chain galactooligosaccharides.

26. The nutritional composition according to claim 19, 20, 21 or 22, wherein each gram of dry weight contains 10 5 Up to 10 11 CFU (caffeine-producing bacteria) 27. The nutritional composition according to claim 19, 20, 21 or 22, comprising at least 2% by weight of non-digestible oligosaccharides based on dry weight.

28. The nutritional composition according to claim 19, 20, 21 or 22, wherein it is an infant formula.

29. Use of the nutritional composition according to claim 19, 20, 21 or 22 for preparing a product for providing nutrition to human subjects who are at risk of developing or who have milk protein allergy.

30. The use according to claim 29, wherein the human subject is an infant or young child.

31. The use according to claim 3, wherein, based on total protein, the nutritional composition comprises less than 10% by weight of protein with a size of 5 kDa or more.

32. The use according to claim 4, wherein, based on total protein, the nutritional composition comprises less than 10% by weight of protein with a size of 5 kDa or more.

33. The use according to claim 3, wherein, based on total protein, at least 1% by weight of the protein present in the composition has a size of 1 kDa or more.

34. The use according to claim 4, wherein, based on total protein, at least 1% by weight of the protein present in the composition has a size of 1 kDa or more.

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