RU2780747C2 - Milk mixture with specific peptides of beta-lactoglobulin - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: group of inventions relates to the field of immunology, in particular to compositions for use in induction of oral immune tolerance and/or for use in prevention, and/or reduction in the risk of development of oral immune intolerance, and/or improvement or enhancing oral tolerance to cow milk protein. Inventions are based on the use of Bifidobacterium and hydrolysate of cow milk protein containing an amino acid sequence SEQ ID NO: 5 and at least one peptide having an amino acid sequence SEQ ID NO: 2-4 for the production of a composition for prevention or reduction in the risk of development of oral immune intolerance to milk protein.

EFFECT: inventions allow for obtaining a composition for improvement of induction of oral immune tolerance to cow milk protein.

22 cl, 2 dwg, 10 tbl, 8 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

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

BACKGROUND OF THE INVENTION

Dietary proteins are exposed to the immune system in the gastrointestinal tract. Oral tolerance to these harmless dietary proteins develops as a default immune response. The development of oral immune tolerance is largely evident in infants who are exposed to numerous harmless proteins at an early age when exposed to dietary proteins. When this natural response to harmless proteins is disrupted, a food allergy will occur. It is generally accepted that the prevalence of food allergies has been on the rise in recent decades, especially in Western countries. At an early age, cow's milk allergy is the most common food allergy: currently, 2-5% of infants are allergic to cow's milk, and a higher percentage are at risk of developing an allergy.

For infants allergic to cow's milk protein, infant formulas are available on the market containing highly hydrolyzed proteins (deep protein hydrolyzate) or even free amino acids alone as a source of nitrogen. These formulas contain little or no allergenic proteins or peptides.

Infants born to parents one or both of whom have an atopic disease, or who have one or more siblings with an atopic disease, have a higher risk of developing food protein allergies. For this group, in addition to preferential breastfeeding, hypoallergenic milk formulas containing partial protein hydrolyzate (partially hydrolysed proteins) are available on the market. These partially hydrolyzed proteins have reduced allergenicity. This approach has been confirmed to be effective in preventing sensitization to native proteins present in adapted formula milks. Proteins are usually less hydrolyzed than highly hydrolyzed proteins for infants already allergic. Such milk formulas have the advantage of not only reducing the risk of developing an allergic reaction by preventing sensitization to the protein, but also supporting the natural development of oral immune tolerance to the intact protein. This has the advantage that the native protein can then be introduced into the diet with a reduced risk of allergic reactions.

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

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

EP 0 629 350 discloses the use of non-allergenic whey protein hydrolysates which are said to be capable of inducing tolerance to cow's milk protein.

EP 0 827 697 discloses the use of whey that has been enzymatically hydrolyzed to provide compositions that induce oral tolerance to cow's milk in susceptible mammals. Serum has an immunological detection rate of allergenic proteins ≥ 100 times lower than non-hydrolyzed whey.

WO 00/42863 discloses a hypoallergenic composition for inducing protein tolerance in infants at risk of protein allergy, comprising a non-allergic highly hydrolyzed protein base and/or a free amino acid base, said composition containing at least one tolerogenic peptide of the allergenic protein.

WO 2011/151059 discloses an infant formula with partially hydrolyzed proteins and non-digestible oligosaccharides for use in inducing oral tolerance to native dietary proteins.

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

WO 2017/144730 discloses a strategy to achieve desensitization or induction of tolerance to milk protein allergens, e.g. β-lactoglobulin, in humans or animals, comprising the preparation and use of a composition comprising a purified intact expressed milk protein together with one or more purified peptides from said intact milk protein. squirrel.

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

However, prior art hypoallergenic compositions often provide their effect simply by avoiding the presence of potential allergens - thereby not solving the underlying problem - and/or by requiring the presence of special components such as growth promoters or synthetic or purified peptides. In addition, the peptides identified in the prior art may not be suitable for inducing oral immune tolerance in a significant portion of the human population. Thus, there is still a need for a diet containing a specific hydrolyzate of bovine beta-lactoglobulin with specific peptides and components that further increase oral immune tolerance in subjects at risk of developing or suffering from food allergies, with improved effects on induction of oral immune tolerance to dietary proteins, in particular cow's milk protein.

Thus, the technical problem underlying the present invention is to provide compositions, methods, uses and means to overcome the above disadvantages, in particular to provide improved induction of oral immune tolerance to dietary proteins in humans, in particular in humans at risk of developing food allergies, in particular in infants, and in particular in infants at risk of developing an allergy to cow's milk protein.

SUMMARY OF THE INVENTION

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

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

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

First, a new liquid chromatography-mass spectrometry (LC-MS) method was developed to determine a limited list of specific identical BLG-derived peptides that were naturally present at the highest frequency when mixed with whey protein hydrolyzate, with a focus on the AA region. No. 13-48 mature BLG cattle. The inventors were able to identify a total of thirteen BLG peptides from a minimum of nine amino acids in this region of interest, six of which were identified sequentially across different production batches.

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

Thirdly, the sequences of the identified BLG peptides were examined and found to be recognized by bovine milk-specific human T cell lines derived from various allergic donors and to induce T cell proliferation/activation. Reference is made to Example 4 and its associated Figure 2. Different patterns of T cell reactivity were observed when tested on these different T cell lines, and although not every peptide interacted with every cell line, when taken together (specifically, the BLG peptide of SEQ ID NO:4 alone or the combination of the BLG peptide of SEQ ID NO :1 with one of SEQ ID NO:2, 3 or 5) covered the reactivity of each cell line. These results indicate an improved effect of hydrolysates with these identical BLG-derived peptides in developing oral immune tolerance to whey protein or reducing the risk of developing oral immune tolerance.

As shown in Example 6, the peptides have more MHC class II binding domains and therefore an increased likelihood of their presentation to T cells.

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

In addition, the accompanying experimental part shows that the presence of lactic acid bacteria increases the expression of HLA-DR molecules on the surface of dendritic cells (example 6). Increased HLA-DR expression also indicates improved presentation of peptides to T cells, and hence induction of oral immune tolerance.

LIST OF PREFERRED IMPLEMENTATION OPTIONS

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

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

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

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

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

and the specified composition preferably additionally contains at least one strain of lactic acid bacteria.

2. Use according to claim 1, wherein the protein hydrolyzate comprises:

(i) at least a 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

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

3. A nutritional composition for use according to claim 1 or 2, wherein the composition contains less than 6 μg of allergenic beta-lactoglobulin, preferably less than 3.5 μg of allergenic beta-lactoglobulin per gram of total protein.

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

5. Nutritional composition for use according to one of the preceding claims, containing less than 10 wt.%, preferably less than 6 wt.% peptides or proteins having a size of 5 kDa or more, in terms of total protein.

6. A nutritional composition for use according to one of the preceding claims, wherein at least 1% by weight of the peptides or proteins present in the composition is 1 kDa or more in terms of total protein, preferably at least 5% by weight , more preferably at least 10 wt.% in terms of total protein.

7. A nutritional composition for use according to one of the preceding claims, wherein the composition contains an amount of more than 0.8 μg of allergenic beta-lactoglobulin per gram of total protein.

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

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

10. A nutritional composition for use according to one of the preceding claims, wherein the human is at risk of developing or is allergic to milk protein from milk from species of the genus Bos , Bison , Bubalus or Capra , more preferably from the genus Bos .

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

12. Питательная композиция для применения по одному из предшествующих пунктов, содержащая один или более неперевариваемых олигосахаридов, выбранных из группы, состоящей из фруктоолигосахарида, неперевариваемого декстрина, галактоолигосахарида, ксилоолигосахарида, арабиноолигосахарида, арабиногалактоолигосахарида, глюкоолигосахарида, глюкоманноолигосахарида, галактоманноолигосахарида, маннанолигосахарида, хитоолигосахарида, олигосахарида уроновой acid, sialyloligosaccharide and fucooligosaccharide, and mixtures thereof, preferably fructooligosaccharides.

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

14. A nutritional composition for use according to one of the preceding claims, containing from 10 ⁵ to 10 ¹¹ cfu of a strain of lactic acid bacteria per gram of dry weight and, optionally, at least 2 wt.% indigestible oligosaccharides, calculated on dry weight.

15. Nutritional composition for use according to one of the preceding claims, containing long chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35 wt.% DHA, based on total fatty acids.

16. A nutritional composition for use according to one of the preceding claims, which is an infant formula, second level formula or infant formula.

17. Nutritional composition containing:

a. a strain of lactic acid bacteria belonging to the genus Bifidobacterium ;

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

c. less than 6 micrograms of allergenic beta-lactoglobulin, preferably less than 3.5 micrograms of beta-lactoglobulin, per 1 g of total protein,

d. less than 10% by weight of peptides or proteins having a size of 5 kDa or more, based on total protein, and

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

f. необязательно, один или более неперевариваемых олигосахаридов, выбранных из группы, состоящей из фруктоолигосахарида, неперевариваемого декстрина, галактоолигосахарида, ксилоолигосахарида, арабиноолигосахарида, арабиногалактоолигосахарида, глюкоолигосахарида, глюкоманноолигосахарида, галактоманноолигосахарида, маннанолигосахарида, хитоолигосахарида, олигосахарида уроновой кислоты, сиалилолигосахарида и фукоолигосахарида, и их смеси, предпочтительно fructooligosaccharides, and

g. optionally long chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35 wt% DHA, based on total fatty acids.

18. The nutritional composition of claim 17, wherein the protein hydrolyzate comprises:

(i) at least a 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

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

19. Nutritional composition according to claims 17 or 18, where the amount of allergenic beta-lactoglobulin is more than 0.8 μg per 1 g of protein, and / or where the composition contains more than 1 wt.% peptides or proteins with a size of 1 kDa or more in terms of total protein, more preferably at least 5 wt.%, more preferably at least 10 wt.%.

20. A nutritional composition according to one of claims 17-19, wherein the lactic acid bacteria strain belongs to the species Bifidobacterium breve .

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

22. Nutritional composition according to one of claims 17-21, containing from 10 ⁵ to 10 ¹¹ CFU of lactic acid bacteria per g of dry weight.

23. Nutritional composition according to one of claims 17-22, containing at least 2 wt.% indigestible oligosaccharides, calculated on dry weight.

24. A nutritional composition according to one of claims 17 to 23, which is an infant formula, a second level formula or an infant formula.

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

26. The method of claim 25 wherein the person is an infant or young child, preferably an infant.

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

28. Method:

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

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

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

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

29. Applications of protein hydrolyzate to obtain a nutritional composition for:

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

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

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

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

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention relates to a nutritional composition containing a protein hydrolyzate from mammalian milk,

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

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

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

to improve or enhance oral immune tolerance to milk protein,

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

In a preferred embodiment, the protein hydrolyzate contains:

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

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

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

The present invention also relates to one or more BLG-derived peptides.

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

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

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

to improve or enhance oral immune tolerance to milk protein,

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

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

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

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

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

to improve or enhance oral immune tolerance to milk protein,

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

In one embodiment

induction of oral immune tolerance to milk protein and/or

prevention or treatment of oral immune intolerance to milk protein and/or

reduced risk of oral immune intolerance to milk protein and/or

improvement or enhancement of oral immune tolerance to milk protein

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

The term "improvement or enhancement of oral immune tolerance to milk protein" means that oral immune tolerance to milk protein is improved compared to oral immune tolerance to milk protein prior to administration of the composition of the invention.

A related invention also relates to a nutritional composition comprising:

a. a strain of lactic acid bacteria belonging to the genus Bifidobacterium ;

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

c. less than 6 micrograms of allergenic beta-lactoglobulin, preferably less than 3.5 micrograms of beta-lactoglobulin, per 1 g of total protein,

d. less than 3% by weight of peptides or proteins having a size of 5 kDa or more, based on total protein,

e. at least 50% by weight, preferably at least 95% by weight hydrolyzed whey protein based on total protein,

f. необязательно, один или более неперевариваемых олигосахаридов, выбранных из группы, состоящей из фруктоолигосахарида, неперевариваемого декстрина, галактоолигосахарида, ксилоолигосахарида, арабиноолигосахарида, арабиногалактоолигосахарида, глюкоолигосахарида, глюкоманноолигосахарида, галактоманноолигосахарида, маннанолигосахарида, хитоолигосахарида, олигосахарида уроновой кислоты, сиалилолигосахарида и фукоолигосахарида, и их смеси, предпочтительно fructooligosaccharides, and

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.

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

A nutritional composition in the context of the present invention is defined herein below, and all embodiments are applicable to all aspects of the invention, including composition, composition for use, methods and uses according to the invention.

Peptides/hydrolyzate

The composition of the invention contains at least two peptides consisting of an amino acid sequence corresponding to the consecutive amino acids represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. These peptides are derived from or derived from beta-lactoglobulin and thus are considered to be beta-lactoglobulin derived peptides. In the specification and claims, the amino acid sequences of the BLG peptides of SEQ ID NO:1-5 are identified here below:

SEQ ID NO:1: XIVTQTMKGLDIQKVAGTWYSLAMAAS

SEQ ID NO:2: XIVTQTMKGLDIQKVAGTWYSLAMAASDISLL

SEQ ID NO:3: TMKGLDIQKVAGTWYSLAMAASDISLL

SEQ ID NO:4: TMKGLDIQKVAGTWYSLAMAASDISLLDAQ

SEQ ID NO:5: DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY.

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

The preferred embodiment (i) is based on the ProImmune ProPresent® antigen presentation assay, in which this mixture of peptides was found to cover more HLA-DRB1 alleles tested; preferred option (ii) is based on experiments aimed at T-cell recognition of T-cells in infants allergic to cow's milk, where it was found that these peptides cover the reactivity of each cell line.

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

Hydrolysis can be carried out using a mixture of microbial endopeptidases and exopeptidases using the method described in example 1 of international application WO 2011/151059, incorporated here by reference. Hydrolysates with BLG peptides according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 can preferably be prepared using the formula according to example 1. Preferably, a mixture is used endoprotease and exoprotease. To determine whether any given composition contains one or more BLG-derived peptides, means of identification are described in the experimental portions.

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

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

Although the distribution of peptides by weight is informative, it does not provide any information about the sequence and activity of the peptides present in the formula. Structural information is critical to understanding the overall biological activity that hydrolyzed infant formula may have.

Consistently, several specific peptides derived from the BLG region known to contain T-cell epitopes have been identified in different production batches. Only two studies have previously examined the pHP peptide profile (Wada et al. Peptides 2015 73:101-105; Catala-Clariana et al, Electrophoresis 2013 Jul; 34(13):1886-1894). Although these studies focused on bioactive peptides in general and not specifically on T-cell epitopes, none of the peptides of SEQ ID NO:1-5 identified in this application were found in these previously tested products. . In addition to the profiles of these hydrolyzed protein products, the peptide profile of a non-commercial BLG-based hydrolyzate was determined (Pecquet et al. J Allergy Clin Immunol 2000 Mar; 105(3):514-521). Also in this digest were identified two peptides (AA No. 21-40 and AA No. 25-40) that overlap with the region of interest, but again not the peptides of SEQ ID NO:1-5. At least two of the three studies described above examined hydrolyzates from different manufacturers (Wada and Pecquets) and, as noted above, differences in manufacturing processes may be the main reason for these differences. This is further supported by a recent study in which highly hydrolysed infant formulas from various manufacturers were characterized using peptidomics. The authors have shown that infant formulas from different manufacturers have a different signature based on their peptide profile 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).

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

Preferably, the nutritional composition contains less than 6 micrograms of allergenic beta-lactoglobulin BLG per gram of protein, preferably less than 5 micrograms per gram of protein, more preferably less than 3.5 micrograms per gram of protein. In the context of the invention, the expression "allergenic beta-lactoglobulin" refers to intact or immunogenic BLG and does not include hydrolysed BLG. Preferably, the nutritional composition contains an amount of allergenic beta-lactoglobulin above 0.8 μg per g of protein. Allergic BLG is determined by an ELISA method known in the art, and an amount greater than 0.8 μg per g of protein indicates a partial protein hydrolysate. In comparison, highly hydrolyzed whey protein exhibits less than 0.2 μg of allergenic BLG per gram of protein. Small but significant amounts of allergenic BLG together with hydrolysed BLG or BLG derived peptides, more preferably also in combination with a lactic acid bacteria strain, even more preferably also in combination with lactic acid bacteria and one or more NDO(s) enhance the tolerogenic activity of the composition, however, the amount of BLG is still low enough to reduce the allergenicity of the nutritional composition and for the nutritional composition to be hypoallergenic. From this point of view, it is preferable to use a protein hydrolyzate containing a small amount of allergenic beta-lactoglobulin and containing BLG-derived peptides of the invention compared to synthetic peptides consisting of the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, more preferably consisting of the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 (no allergenic BLG).

lactic acid bacteria

The composition of the present invention preferably contains a strain of lactic acid bacteria species that enhance the oral immune tolerogenic activity of the BLG peptides of the present invention. The bacterial strain is preferably a probiotic. Evidence is provided in examples 5 and 7. It was found that the presence of lactic acid bacteria increases the expression of HLA-DR molecules on the surface of dendritic cells. Increased HLA-DR expression also indicates improved presentation of peptides to T cells and hence induction of oral immune tolerance.

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

The composition preferably contains a strain of lactic acid bacteria belonging to the genus Bifidobacterium , preferably Bifidobacterium breve . B. breve preferably has at least 95% 16S rRNA sequence identity compared to the B. breve type strain ATCC 15700, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846- 849). Suitable strains of B. breve can be isolated from the faeces of healthy breastfed infants. They are usually available on the market from lactic acid bacteria producers, but they can also be directly isolated from faeces, identified, characterized and obtained. In one embodiment, the present composition comprises B. breve selected from the group consisting of B. breve Bb-03 (Rhodia/Danisco), B. breve M-16V (Morinaga), B. breve R0070 (Institute Rosell, Lallemand), B. breve BR03 (Probiotical), B. breve BR92 (Cell Biotech), DSM 20091, LMG 11613, YIT4065, FERM BP-6223 and CNCM I-2219. B. breve can be B. breve M-16V and B. breve CNCM I-2219, most preferably B. breve M-16V. B. breve I-2219 was published in international application WO 2004/093899 and deposited with the National Collection of Cultures of Microorganisms of the Institut Pasteur, Paris, France on May 31, 1999 by Compagnie Gervais Danone. B. breve M-16V has been deposited as BCCM/LMG23729 and is commercially available from Morinaga Milk Industry Co., Ltd.

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

Indigestible oligosaccharides

In a preferred embodiment, the present composition further comprises one or more non-digestible oligosaccharides (NDO). Like lactic acid bacteria, they further increase oral immune tolerance by inducing the properties of BLG protein peptides. Evidence for the enhanced effect is provided in Example 5.

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

The indigestible oligosaccharide is preferably a prebiotic. It is not digested in the intestine by the action of digestive enzymes present in the human upper digestive tract (small intestine and stomach). The indigestible oligosaccharide is fermented by the human gut microbiota. For example, glucose, fructose, galactose, sucrose, lactose, maltose, and maltodextrins are considered digestible. Oligosaccharide starting materials may include monosaccharides such as glucose, fructose, fucose, galactose, rhamnose, xylose, glucuronic acid, GalNac and the like, but they are not part of the oligosaccharides. Неперевариваемый олигосахарид предпочтительно выбирают из группы, состоящей из фруктоолигосахарида, неперевариваемого декстрина, галактоолигосахарида, ксилоолигосахарида, арабиноолигосахарида, арабиногалактоолигосахарида, глюкоолигосахарида, глюкоманноолигосахарида, галактоманноолигосахарида, маннанолигосахарида, хитоолигосахарида, олигосахарида уроновой кислоты, сиалилолигосахарида и фукоолигосахарида, и их смеси, предпочтительно фруктоолигосахаридов. Examples of sialyloligosaccharide are 3-sialyllactose, 6''-sialyllactose, sialyllacto-N-tetraose, disialyllacto-N-tetraose. Examples of fucooligosaccharides are (non)sulfated fucoidan oligosaccharides, 2'-fucosyllactose, 3'-fucosyllactose, lacto-N-fucopentaose I, II, III, LNDH, lactodifucotetraose, lacto-N-difucohexaose I and II.

One suitable type of oligosaccharide is a short chain oligosaccharide which has an average degree of polymerization of less than 10, preferably no more than 8, preferably in the range of 2-7. The short chain oligosaccharide preferably comprises galactooligosaccharides and/or fructooligosaccharides (ie scGOS and/or scFOS). In one embodiment, the composition contains galactooligosaccharides, preferably beta-galactooligosaccharides, preferably transgalactooligosaccharides. Galactooligosaccharides preferably have an average degree of polymerization in the range of 2 to 8, preferably 3 to 7, ie short chain oligosaccharides in the context of the invention. (Trans)galactooligosaccharides are commercially available, for example, under the trade names Vivinal®GOS (Friesland Campina Domo Ingredients, The Netherlands), Bimuno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate55 (Yakult). The composition preferably contains short chain fructooligosaccharides and/or short chain galactooligosaccharides, preferably at least short chain fructooligosaccharides. Fructooligosaccharides may be inulin hydrolyzate products having an average SP in the above (sub)ranges; such FOS products, such as those commercially available as Raftilose P95 (Orafti) or from Cosucra.

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

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

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

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

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

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

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

Other components

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

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

Nutritional composition

Compositions. according to the invention can be used as a nutritional composition, nutritional therapy, nutritional support, as a clinical nutrition, as a food for special medical purposes or as a dietary supplement. The present composition is preferably an enteral (oral) composition. The composition is administered orally or is intended for oral administration to a subject in need thereof, in particular children and infants, including toddlers, preferably children under 6 years of age, preferably infants or young children, usually 0-36 months of age, more preferably infants children 0-12 months, most preferably 0-6 months. Thus, in some embodiments, the present composition is a first level milk formula, a second level milk formula, or an infant formula (also referred to as a third level milk formula), preferably it is a first level milk formula or a second level milk formula, most preferably a first level formula. The term “level 1 formula milk” is clearly defined and constantly monitored by regulatory authorities at the international level. In particular, CODEX STAN 73-1981 "Standard for infant formulas and special medical preparations intended for infants" is generally recognized. He recommends for nutritional value and composition of the mixture, which require the content of 100 ml of cooked milk not less than 60 kcal (250 kJ) and not more than 70 kcal (295 kJ) of energy. The FDA and other regulatory agencies have set nutrient requirements in line with them.

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

In order to meet the caloric requirements of the infant, the present enteral composition preferably contains 50 to 200 kcal/100 ml of liquid, more preferably 60 to 90 kcal/100 ml of liquid, even more preferably 60 to 75 kcal/100 ml of liquid. This calorie content provides an optimal balance between water intake and calories. The osmolarity of this composition is preferably 150 to 420 mOsmol/L, more preferably 260 to 320 mOsmol/L. The low osmolarity aims to reduce gastrointestinal stress.

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

The composition of the invention preferably contains a lipid component, preferably a lipid component suitable for infant formula, as is known in the art. The lipid component of the present composition preferably provides 2.9 to 6.0 g, more preferably 4 to 6 g per 100 kcal of the composition. In liquid form, the composition preferably contains from 2.1 to 6.5 g of lipids per 100 ml, more preferably from 3.0 to 4.0 g per 100 ml. On a dry weight basis, the present Level 1 or Level 2 formula preferably contains 12.5 to 40% by weight lipids, more preferably 19 to 30% by weight.

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

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

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

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

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

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

The digestible carbohydrate component preferably contains at least 60 wt% lactose based on total digestible carbohydrates, more preferably at least 75 wt%, even more preferably at least 90 wt% lactose based on total digestible carbohydrates.

Test people

Target human subjects or population are preferably infants (0-12 months old), preferably infants at risk of developing allergies, preferably milk protein allergy, preferably milk protein from species of the genus Bos , Bison , Bubalus or Capra , more preferably of the genus Bos . most preferably allergic to cow's milk protein, preferably infants allergic to milk protein, preferably milk protein from species of the genus Bos , Bison , Bubalus or Capra , more preferably of the genus Bos , most preferably allergic to cow's milk protein. Infants at risk are preferably determined by a family history, preferably by at least one parent or a history of allergies.

The individual preferably has a particular HLA-DRB1 allele selected from the groups consisting of HLA-DRB1*01:01, HLA-DRB1*04:01, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1 *7:01 and HLA-DRB1*09:01. HLA-DRB1 is the most predominant human class II MHC isotype (>90%), the HLA-DRB1 gene locus is polymorphic, while the HLA-DRA1 gene locus is monomorphic (i.e., the HLA-DRB1 genotype determines the entire HLA-DR molecule) , and the HLA-DRB1 allele is expressed five times higher than its paralogues (HLA-DRB3, -DRB4, or -DRB5) and is present in all humans.

Induction of oral immune tolerance

The invention relates to

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

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

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

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

As described herein above, the subject person or population is preferably the subject infant or population.

In the context of the present invention, the term "oral tolerance" means oral immune tolerance.

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

SYMBOLS FOR FIGURES

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

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

EXAMPLE 1. HYDROLYSED WHEY PROTEIN AND A NEW METHOD FOR IDENTIFICATION OF BETA-LACTOGLOBULIN PEPTIDES

A mixture of acid 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. Sour whey and sweet whey are available on the market. Sweet whey is a by-product of rennet-coagulated cheese and contains caseinoglycomacropeptide (CGMP), while sour whey (also called fermented whey) is a by-product of acid-coagulated cheese 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). A mixture of microbial endopeptidases and exopeptidases was used to hydrolyze these two protein sources using the method described in Example 1 of WO 2011/151059. The hydrolysed protein solution was then spray dried. The size distribution of peptides in this protein hydrolyzate was determined by size exclusion high pressure liquid chromatography known in the art. Briefly, the total surface area of the chromatograms is integrated and divided into mass ranges expressed as a percentage of the total surface area. Mass ranges are calibrated using peptides/proteins of known molecular weight. Whey protein hydrolyzate can be classified as partial or moderate protein hydrolyzate.

The resulting hydrolyzate powder was used as the sole source of protein in infant formulas. Similarly received 10 different batches of milk formula for infants. The amount of beta-lactoglobulin, determined by an ELISA method known in the art, ranged from about 0.8 to 3.5 μg/g of total protein.

To determine the presence of specific sequences in biological samples, mass spectrometry (MS) is the method of choice over the more traditional methods currently used to characterize protein hydrolysates. A recent development in MS called peptidomics allows the characterization of peptide sequences with great sensitivity and specificity (Dallas et al. J Nutr 2015; 145:425-433). This method is able to bridge the gap between understanding the impact of sequence specificity on the biological activity that a protein hydrolyzate may have.

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

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

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

Of the 314 peptides identified by this method, 101 can be attributed to beta-lactoglobulin (BLG), resulting in a coverage of BLG sequences of 90%. Most of the remaining peptides originated from other common whey proteins such as α-lactalbumin and serum albumin. A total of 13 beta-lactoglobulin peptides out of a minimum of 9 AAs were identified in the region of interest (AA Nos. 13-48) (see Table 1 and FIG. 1). Six of them were identified consistently across all 10 batches. The unambiguous identification of peptides was obtained by comparing the characteristics (retention time, peptide mass, and fragmentation spectrum) of the experimental peptide with its synthetic equivalent labeled with a stable isotope. An example is shown in Figure 1, where the fragmentation spectra of the experimental and synthetic peptides show an excellent correlation. Thus, the identity of the six peptides found in all 10 batches was confirmed.

Table 1.

Beta-lactoglobulin peptide sequences identified in 10 different batches of whey-based partially hydrolysed infant formula

Amino acid number in the BLG sequence Subsequence Number of batches in which the peptide was identified SEQID NO AA 1-22 LIVTQTMKGLDIQKVAGTWYSL 7 6 AA 1-25 LIVTQTMKGLDIQKVAGTWYSLAMA 9 7 AA 1-26 LIVTQTMKGLDIQKVAGTWYSLAMAA ten eight AA 1-27 LIVTQTMKGLDIQKVAGTWYSLAMAAS ten 9 AA 1-30 LIVTQTMKGLDIQKVAGTWYSLAMAASDIS 2 ten AA 1-32 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLL ten eleven AA 1-35 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQ 5 12 AA 1-39 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPL 2 13 AA 1-42 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 2 fourteen AA 5-35 QTMKGLDIQKVAGTWYSLAMAASDISLLDAQ 2 fifteen AA 6-32 TMKGLDIQKVAGTWYSLAMAASDISLL ten 3 AA 6-35 TMKGLDIQKVAGTWYSLAMAASDISLLDAQ ten four AA 11-42 DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY ten 5

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

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

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

Table 2.

Donors based on HLA-DRB1 typing information

Donor ID DRB1_1 DRB1_2 P1 *03:01 *03:01 P2 *01:01 *04:01 P3 *01:01 *07:01 P4 *01:01 *04:05 P5 *04:01 *07:01 P6 *13:02 *14:01 P7 *03:01 *09:01 P8 *11:01 *15:01 P9 *03:01 *11:01 P10 *07:01 *15:01 P11 *03:01 *15:01 P12 *01:01 *04:04

Immature monocytic DCs were generated in vitro and matured in the presence of the tested hydrolyzed protein (HP). DCs were collected and lysed to obtain HLA-DR complexes using a specific immunoaffinity method. The peptides were eluted from the HLA-DR complexes and then analyzed by high resolution LC-MS/MS sequencing. The presence of six endogenous relevant proteins (ie ITGAM, ApoB, CLIP, TFRC, FcER2/FcGR2 and LAMP-1/3) was evaluated as a control for this assay. Each donor sample had to express a minimum of 3 relevant proteins in order to qualify for subsequent analysis. The resulting HLA-DR restricted peptide data were compiled and evaluated using sequence analysis software with reference to the Swiss-Prot Human Proteome Database with test element sequences included. The probability of real detection of peptides is described by their expected value ≤0.05. The false positive rate was defined as <1%.

Using the ProPresent® Antigen Presentation Assay, focusing on BLG-derived sequences, 15 relevant peptides with an expected value of ≤0.05 were identified in 5 donor samples (Table 3). These peptides can be further grouped into 2 groups of unique sequences, ie DIQ...DIS (AA nos. 11-30) and AMA...APL (AA nos. 23-39) (Table 4). It is important to note that both groups of sequences overlap with the region of interest (ie, AA#13-48 of mature BLG). This discovery demonstrated that the investigated BLG-derived peptides can be presented by HLA-DR molecules on human DCs when incubated with specific HP.

Table 3

Significant fragments identified from HLA-DR peptide complexes with expected value <0.05

Donor ID DRB1_1 DRB1_2 Fragment Sequence
(mature BLG)
[SEQ ID NO] Amino acid start/
the end expected value P2 *01:01 *04:01 AMAASDISLLDAQSAPL - [16]
- MAASDISLLDAQSAPLR [17] 23-39
24-40 0.043
0.0013 P3 *01:01 *07:01 - MAASDISLLDAQSAPL - [18] 24-39 0.009 P4 *01:01 *04:05 DIQKVAGTWYSLAMAASDI - [19]
- - - - VAGTWYSLAMAASDIS [20]
- - - - - - GTWYSLAMAASDIS [21] 11-29
15-30
17-30 0.000052
0.00021
0.00037 P7 *03:01 *09:01 DIQKVAGTWYSLAMAASDI - - [19]
DIQKVAGTWYSLAMAASDIS - [22]
- - - - VAGTWYSLAMAASDI - - [23]
- - - - VAGTWYSLAMAASDIS - [20]
- - - - - AGTWYSLAMAASDIS - [24]
- - - - - - GTWYSLAMAASDI - - [25]
- - - - - - GTWYSLAMAASDIS - [21] 11-29
11-30
15-29
15-30
16-30
17-29
17-30 0.00019
0.00053
0.002
0.000019
0.0014
0.00023
0.000015 P12 *01:01 *04:04 AMAASDISLLDAQSAPL - [16]
- MAASDISLLDAQSAPL - [18] 23-39
24-39 0.028
0.042

Table 4

The association of HLA-DR alleles in the unique region of mature BLG with significant fragments was determined by an expected value of <0.05

Donor ID DRB1_1 DRB1_2 Unique area
(mature BLG)
[SEQ ID NO] Amino acid start-
the end Number of significant fragments and individual fragment P2 *01:01 *04:01 AMAASDISLLDAQSAPLR [39] 23-40 2
(23-39 and 24-40) P3 *01:01 *07:01 MAASDISLLDAQSAPL [18] 24-39 one
(24-39) P4 *01:01 *04:05 DIQKVAGTWYSLAMAASDIS [22]
VAGTWYSLAMAASDIS [20]
GTWYSLAMAASDIS [21] 11-30 3
(11-29, 15-30 and 17-30) P7 *03:01 *09:01 DIQKVAGTWYSLAMAASDIS [22] 11-30 7
(11-29, 11-30, 15-29, 15-30, 16-30, 17-29, 17-30) P12 *01:01 *04:04 AMAASDISLLDAQSAPL [16] 23-39 2
(23-39, 24-39)

Not all donor antigen-presenting cells bind to peptides. This concerned HLA-DRB1*03:01 in a homozygous P1 donor. Similar arguments can be applied to P6 and P11 donors.

EXAMPLE 3 BINDING OF CLASS II MHC AT IN SILICO EVALUATION

A limitation of the ProPresent® antigen ^presentation assay is the use of a generic anti-HLA antibody rather than a specific antibody for a specific HLA-DRB1 allele (eg, an anti-DRB1*01:01 antibody) to isolate the HLA-DR peptide complex of interest. Therefore, for a donor with heterozygous HLA-DRB1 genotypes, such as *01:01 and *04:01, it is not clear which allele the identified peptide will present.

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

Class II MHC prediction software was used to assess whether fragments of the identified two groups of unique BLG sequences would have high binding affinity to selected HLA-DRB1 alleles of the characterized donors from the ProPresent® assay, i.e. HLA-DRB1 *01:01, *03 :01, *04:01, *04:04, *04:05, *07:01 and *09:01. As shown in Table 5, with an arbitrary percentile rank threshold set at <3% (i.e., 3% top binders), DIQ...DIS fragments (AA #11-30) were predicted to have high binding affinity for 5 HLA alleles. -DRB1, i.e. DRB1 *01:01, *04:01, *04:04, *04:05 and *09:01. On the contrary, it was calculated that only one fragment of AMA...APL (AA no. 23-39) binds to HLA-DRB1 *07:01 with high affinity. Taken together, these results indicate that fragments AA #11-30 were more likely to be presented as T-cell epitopes than fragments AA #23-39. In addition, these data suggest that several common HLA-DRB1 alleles can present fragments derived from the identified two groups of BLG-derived unique sequences.

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

Table 5

In silico evaluation of potential HLA-DRB1 peptide complexes of two identified BLG-derived peptides with percentile rank <3%

HLA-DRB1 allele Predicted 15mer fragment DIQKVAGTWYSLAMAASDIS (SEQ ID NO:25) or AMAASDISLLDAQSAPL (SEQ ID NO:19) [SEQ ID NO] Percentile rank HLA-DRB1*09:01 AGTWYSLAMAASDIS [24] 0.86 HLA-DRB1*04:05 AGTWYSLAMAASDIS [24] 1.38 HLA-DRB1*04:05 VAGTWYSLAMAASDI [23] 1.40 HLA-DRB1*09:01 VAGTWYSLAMAASDI [23] 1.56 HLA-DRB1*04:05 KVAGTWYSLAMAASD [26] 2.26 HLA-DRB1*01:01 AGTWYSLAMAASDIS [24] 2.37 HLA-DRB1*04:04 AGTWYSLAMAASDIS [24] 2.44 HLA-DRB1*09:01 KVAGTWYSLAMAASD [26] 2.53 HLA-DRB1*04:01 AGTWYSLAMAASDIS [24] 2.84 HLA-DRB1*07:01 AASDISLLDAQSAPL [27] 2.39

Table 6

In silico evaluation of potential HLA-DRB1 peptide complexes of a long BLG-derived peptide with <3% percentile rank (upper 3% binders)

HLA-DRB1 allele Predicted 15mer fragment DIQKVAGTWYSLAMAASDIS (SEQ ID NO:25) or AMAASDISLLDAQSAPL (SEQ ID NO:19) [SEQ ID NO] Percentile Rank HLA-DRB1*09:01 GTWYSLAMAASDISL [28] 0.65 HLA-DRB1*09:01 AGTWYSLAMAASDIS [24] 0.86 HLA-DRB1*04:05 GTWYSLAMAASDISL [28] 1.30 HLA-DRB1*09:01 TWYSLAMAASDISLL [29] 1.32 HLA-DRB1*04:05 AGTWYSLAMAASDIS [24] 1.38 HLA-DRB1*04:05 VAGTWYSLAMAASDI [23] 1.40 HLA-DRB1*04:05 TWYSLAMAASDISLL [29] 1.40 HLA-DRB1*09:01 VAGTWYSLAMAASDI [23] 1.56 HLA-DRB1*09:01 WYSLAMAASDISLLD [30] 2.12 HLA-DRB1*09:01 YSLAMAASDISLLDA [31] 2.14 HLA-DRB1*01:01 GTWYSLAMAASDISL [28] 2.18 HLA-DRB1*04:05 KVAGTWYSLAMAASD [26] 2.26 HLA-DRB1*01:01 AGTWYSLAMAASDIS [24] 2.37 HLA-DRB1*07:01 AASDISLLDAQSAPL [27] 2.39 HLA-DRB1*07:01 SDISLLDAQSAPLRV [32] 2.44 HLA-DRB1*04:04 AGTWYSLAMAASDIS [24] 2.44 HLA-DRB1*04:04 GTWYSLAMAASDISL [28] 2.44 HLA-DRB1*04:04 TWYSLAMAASDISLL [29] 2.44 HLA-DRB1*01:01 TWYSLAMAASDISLL [29] 2.46 HLA-DRB1*09:01 KVAGTWYSLAMAASD [26] 2.53 HLA-DRB1*04:04 YSLAMAASDISLLDA [31] 2.57 HLA-DRB1*04:01 GTWYSLAMAASDISL [28] 2.83 HLA-DRB1*04:01 AGTWYSLAMAASDIS [24] 2.84 HLA-DRB1*04:04 WYSLAMAASDISLLD [30] 2.91

Several in silico evaluations confirmed the in vitro results of ProPresent® for HLA-DR peptide complexes. However, we noticed that some in silico predicted complexes did not correlate with those found in the ProPresent® assay, eg DRB1 *04:04 with AGT…DIS. This discrepancy may be explained either by the small size of the study cohort in the ^ProPresent® analysis (n=12), or by the fact that each donor had a different combination of HLA-DRB1 allele types (i.e. inter-individual variability) and/or by the tendency of in silico scores to be overly predictable (which requires a strict limit). Therefore, it is reasonable to combine both in vitro and in silico approaches to identify class II peptide-MHC complexes.

EXAMPLE 4 T-CELL PROLIFERATION ASSAY

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

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

Table 7

Beta-lactoglobulin peptide sequences identified in 10 different batches of whey-based partially hydrolyzed infant formula and tested for T cell proliferation activity

Amino acid number in the sequence Subsequence SEQID NO AA 1-27 LIVTQTMKGLDIQKVAGTWYSLAMAAS 9 AA 1-32 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLL eleven AA 6-32 TMKGLDIQKVAGTWYSLAMAASDISLL 3 AA 6-35 TMKGLDIQKVAGTWYSLAMAASDISLLDAQ four AA 11-42 DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 5

Not all peptides were recognized by each donor, indicating differences between donors. Each donor expressed a different panel of class II MHC molecules. To determine whether and which HLA-DRB1 allele was expressed by the donors of the presented peptides, the same algorithm as described above was used. In silico prediction confirmed that the HLA-DRB1 alleles (*11:01 and *04:04) expressed by donor B are capable of presenting all peptides. Based on the algorithm data, TCL A should only recognize the DIQ...RVY peptide (AA no. 11-42), however, this TCL showed a proliferative response after stimulation with four of the five peptides. A possible explanation could be that the other three peptides were not presented by HLA-DRB1, but by other MHC class II molecules.

In conclusion, examples 1-4 demonstrate that the specific whey protein hydrolyzate contains functional T-cell epitopes. The presence of specific beta-lactoglobulin peptides, which are HLA-DRB1 restricted peptides, has been identified. They are presented and recognized by T cells from a variety of donors, including healthy donors and infants allergic to cow's milk protein. This interaction was confirmed by in silico analysis. Therefore, HP may stimulate the development of oral immunotolerance to the protein.

EXAMPLE 5 A SYNBIOTIC MIXTURE DEMONSTRATES AN EVEN BETTER TOLERANCE-INDUCING EFFECT IN COMPARISON WITH PROBIOTICS OR PREBIOTICS TAKEN SINGLELY

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

To develop oral tolerance, T-cell epitopes in HP subjects must be presented under appropriate conditions. In addition to TCR activation, costimulation and signaling by cytokines play an important role in the generation of Tregs. Pro- or prebiotics play an important role in creating an appropriate environment for this predisposition to Tregs. The preventive effect of synthetic BLG peptides was enhanced when combined with a probiotic or prebiotic diet.

To demonstrate the effect of pro-, pre- and synbiotics, an experiment similar to that described in example 4 of international application WO 2016/148572 was carried out using the same protocol, the same concentration and a mixture of synthetic peptides ("Pepmix"), Pepmix is identified in table 8.

Table 8

Pepmix according to international application WO 2016/148572

Peptide Subsequence 1 (18AA) SEQ ID NO: 33 QKVAGTWYSLAMAASDIS 2 (18AA) SEQ ID NO: 34 WYSLAMAASDISLLDAQS 3 (18AA) SEQ ID NO: 35 AASDISLLDAQSAPLRVY 4 (18AA) SEQ ID NO: 36 LLDAQSAPLRVYVEELKP

A number of diets were compared:

(a) synbiotic diet from example 1 of international application WO 2016/14472 (1 wt.% scFOS/lcFOS in a wt.% ratio of 9:1+2 wt.% 2×10 ⁹ cfu/g Bifidobacterium breve M-16V,

(b) the probiotic diet of example 4 of international application WO 2016/148572 using 2 wt.% 2×10 ⁹ CFU/g Bifidobacterium breve M-16V, and

(c) prebiotic diet containing 1 wt.% scFOS/lcFOS with a weight ratio of 9/1.

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

All groups were pretreated with Pepmix and one of the above diets, sensitized with serum + cholera toxin and challenged with serum.

After serum challenge, the delta of ear edema in mice fed (c) scFOS/lcFOS diet was about 174 µm, in mice fed (b) probiotic diet was about 158 µm, and in mice fed (a) synbiotic diet was about 112 µm. Ear edema in the synbiotic group (a) was statistically significantly lower than in the corresponding scFOS/lcFOS group (c), and there was a trend towards a lower response compared to the probiotic group (b) (p=0.08). These results indicate a further improvement in the effect on the induction of oral immunotolerance:

when using lactic acid bacteria, in particular the Bifidobacterium strain of the species B. breve , and

especially when using lactic acid bacteria in combination with NDOs.

EXAMPLE 6. MHC BINDING DOMAIN IN NATURAL IDENTIFIED HYDROLYSATE PEPTIDES COMPARED WITH BETA-LACTOGLOBULIN SYNTHETIC PEPTIDES

Method: Beta-lactoglobulin peptide sequences that were identified in hydrolysed infant formula (ID_PEP) and synthetic beta-lactoglobulin peptide sequences that were published in international application WO2016/148572 (SYN_PEP, Table 9) were tested in silico with 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 IEDB recommended prediction method and the full set of HLA references were selected in the software. This set of HLA references provides >99% population coverage. The software calculates percentile rank by comparing the binding affinity of the predicted binding domain to a large group of peptides. The higher the affinity, the lower the percentile rank. In this experiment, the top 2% binders were selected.

results

The number of MHC class II binding domains in the identified peptides is higher than in synthetic peptides (see table 9). The software predicted at least one binding domain for all identified peptides and a total of six different domains. For synthetic peptides, four different domains have been predicted. However, all of these domains were derived from the same sequence, namely SYN_PEP_1. For the other two synthetic peptides, MHC class II binding domains were not predicted.

Table 9

Number of MHC class II binding domains per beta-lactoglobulin peptide predicted by IEBD

Peptide Subsequence Number of MHC class II binding domains ID_PEP_1 LIVTQTMKGLDIQKVAGTWYSLAMAAS one ID_PEP_2 LIVTQTMKGLDIQKVAGTWYSLAMAASDISLL 6 ID_PEP_3 TMKGLDIQKVAGTWYSLAMAASDISLL 6 ID_PEP_4 TMKGLDIQKVAGTWYSLAMAASDISLLDAQ 6 ID_PEP_5 DIQKVAGTWYSLAMAASDISLLDAQSAPLRVY 6 SYN_PEP_1 QKVAGTWYSLAMAASDIS four SYN_PEP_2 WYSLAMAASDISLLDAQS 0 SYN_PEP_3 AASDISLLDAQSAPLRVY 0

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

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

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

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

Table 10

Number of MHC class II binding domains per beta-lactoglobulin peptide predicted by IEBD

DCs MFI HLA-DR expression (SD) Immature DCs 11338 (1919.1) LPS-DCs 24676 (9694.4) M16v-DCs 21714 (1516)

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

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

EXAMPLE 8 INFANT MILK FORMULA

Powdered infant formula packaged with instructions to be reconstituted with 40°C water with 3 scoops of powder (13.74 g) in 90 ml of water for a final volume of 100 ml. The product is suitable from birth to 6 months.

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

--->

SEQUENCE LIST

<110> N.V. Nutricia

<120> MILK WITH SPECIFIC BETA-LACTOGLOBULIN PEPTIDES

<130> P6075124PCT1

<150> PCT/NL2018/050281

<151> 2018-04-30

<160> 39

<210> 1

<211> 27

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<220>

<221> misc_feature

<222> (1)..(1)

<223> Haa can be Ile or Leu

<400> 1

Xaa Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser

20 25

<210> 2

<211> 32

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<220>

<221> misc_feature

<222> (1)..(1)

<223> Haa can be Ile or Leu

<400> 2

Xaa Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

20 25 30

<210> 3

<211> 27

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 3

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

1 5 10 15

Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

20 25

<210> 4

<211> 30

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 4

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

1 5 10 15

Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln

20 25 30

<210> 5

<211> 32

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 5

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

1 5 10 15

Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg Val Tyr

20 25 30

<210> 6

<211> 22

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 6

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu

twenty

<210> 7

<211> 25

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 7

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala

20 25

<210> 8

<211> 26

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 8

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala

20 25

<210> 9

<211> 27

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 9

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser

20 25

<210> 10

<211> 30

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 10

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

20 25 30

<210> 11

<211> 32

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 11

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

20 25 30

<210> 12

<211> 35

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 12

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

20 25 30

Asp Ala Gln

35

<210> 13

<211> 39

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 13

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

20 25 30

Asp Ala Gln Ser Ala Pro Leu

35

<210> 14

<211> 42

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 14

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

20 25 30

Asp Ala Gln Ser Ala Pro Leu Arg Val Tyr

35 40

<210> 15

<211> 31

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 15

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

1 5 10 15

Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln

20 25 30

<210> 16

<211> 17

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 16

Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro

1 5 10 15

Leu

<210> 17

<211> 17

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 17

Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu

1 5 10 15

Arg

<210> 18

<211> 16

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 18

Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu

1 5 10 15

<210> 19

<211> 19

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 19

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

1 5 10 15

Ser Asp Ile

<210> 20

<211> 16

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 20

Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

1 5 10 15

<210> 21

<211> 14

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 21

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

1 5 10

<210> 22

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 22

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

1 5 10 15

Ser Asp Ile Ser

twenty

<210> 23

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 23

Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile

1 5 10 15

<210> 24

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 24

Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser

1 5 10 15

<210> 25

<211> 13

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 25

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile

1 5 10

<210> 26

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 26

Lys Val Ala Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp

1 5 10 15

<210> 27

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 27

Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu

1 5 10 15

<210> 28

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 28

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu

1 5 10 15

<210> 29

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 29

Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

1 5 10 15

<210> 30

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 30

Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp

1 5 10 15

<210> 31

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 31

Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala

1 5 10 15

<210> 32

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 32

Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg Val

1 5 10 15

<210> 33

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> Pepmix peptide 1

<400> 33

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

1 5 10 15

Ile Ser

<210> 34

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> Pepmix peptide 2

<400> 34

Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala

1 5 10 15

Gln Ser

<210> 35

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> Pepmix peptide 3

<400> 35

Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg

1 5 10 15

Val Tyr

<210> 36

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> Pepmix peptide 4

<400> 36

Leu Leu Asp Ala Gln Ser Ala Pro Leu Arg Val Tyr Val Glu Glu Leu

1 5 10 15

Lys Pro

<210> 37

<211> 27

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 37

Ile Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser

20 25

<210> 38

<211> 32

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 38

Ile Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

1 5 10 15

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

20 25 30

<210> 39

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> beta-lactoglobulin peptide fragment

<400> 39

Ala Met Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln Ser Ala Pro

1 5 10 15

Leu Arg

<---

Claims (40)

1. Use of a nutritional composition to obtain a product for: - induction of oral immune tolerance to milk protein; and/or - prevention of oral immune intolerance to milk protein; and/or - reducing the risk of developing oral immune intolerance to milk protein; and/or - improvement or enhancement of oral immune tolerance to milk protein, in humans, wherein the nutritional composition comprises a protein hydrolyzate from mammalian milk, preferably milk from species of the genus Bos , Bison , Bubalus , or Capra , more preferably the genus Bos , preferably bovine milk, wherein the protein hydrolyzate comprises at least a peptide having a 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, said composition preferably further comprising one strain of lactic acid bacteria belonging to the genus Bifidobacterium . 2. Use according to claim 1, wherein the composition contains less than 6 μg of allergenic beta-lactoglobulin, preferably less than 3.5 μg of allergenic beta-lactoglobulin per gram of total protein. 3. Application according to any one of paragraphs. 1, 2, where the composition contains at least 50 wt.%, preferably at least 95 wt.% hydrolyzed whey protein in terms of total protein. 4. Application according to any one of paragraphs. 1-3, comprising less than 10 wt.%, preferably less than 6 wt.% peptides or proteins having a size of 5 kDa or more, in terms of total protein. 5. Application according to any one of paragraphs. 1-4, where at least 1 wt.% of the peptides or proteins present in the composition have a size of 1 kDa or more, in terms of total protein, preferably at least 5 wt.%, more preferably at least 10 wt. % in terms of total protein. 6. Application according to any one of paragraphs. 1-5, where the composition contains an amount of allergy-causing beta-lactoglobulin of more than 0.8 μg per 1 g of total protein. 7. Application according to any one of paragraphs. 1-6 wherein the milk protein is from a species of the genus Bos , Bison , Bubalus or Capra , more preferably of the genus Bos , preferably cow's milk protein. 8. Application according to any one of paragraphs. 1-7 where the person is an infant. 9. Application according to any one of paragraphs. 1-8, wherein the human is at risk of developing or allergic to milk protein from milk from species of the genus Bos , Bison , Bubalus , or Capra , more preferably of the genus Bos , and wherein the human is preferably an infant. 10. Application according to any one of paragraphs. 1-9, where the composition contains a strain of lactic acid bacteria belonging to the species Bifidobacterium breve . 11. Application according to any one of paragraphs. 1-10, включающее один или более неперевариваемых олигосахаридов, выбранных из группы, состоящей из фруктоолигосахарида, неперевариваемого декстрина, галактоолигосахарида, ксилоолигосахарида, арабиноолигосахарида, арабиногалактоолигосахарида, глюкоолигосахарида, глюкоманноолигосахарида, галактоманноолигосахарида, маннанолигосахарида, хитоолигосахарида, олигосахарида уроновой кислоты, сиалилолигосахарида и фукоолигосахарида, и их mixtures, preferably fructooligosaccharides. 12. Use according to claim 11, wherein the indigestible oligosaccharides comprise at least two indigestible oligosaccharides selected from the group consisting of fructooligosaccharides and galactooligosaccharides, preferably a mixture of long chain fructooligosaccharides with short chain fructooligosaccharides or with short chain galactooligosaccharides. 13. Application according to any one of paragraphs. 1-12, including long chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35 wt.% DHA, based on total fatty acids. 14. Application according to any one of paragraphs. 1-13, which is infant formula, second level formula, or infant formula. 15. Nutritional composition containing: a. a strain of lactic acid bacteria belonging to the species Bifidobacterium breve ; b. a milk protein hydrolyzate from a species of the genus Bos, Bison, Bubalus or Capra, more preferably of the genus Bos, wherein the milk protein hydrolyzate comprises at least a peptide having the sequence according to SEQ ID NO: 5 and at least one peptide having sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, c. less than 6 micrograms of allergenic beta-lactoglobulin, preferably less than 3.5 micrograms of beta-lactoglobulin, per 1 g of total protein, d. less than 10% by weight of peptides or proteins having a size of 5 kDa or more, based on total protein, and e. at least 50 wt%, preferably at least 95 wt% hydrolysed whey protein based on total protein, f. необязательно, один или более неперевариваемых олигосахаридов, выбранных из группы, состоящей из фруктоолигосахарида, неперевариваемого декстрина, галактоолигосахарида, ксилоолигосахарида, арабиноолигосахарида, арабиногалактоолигосахарида, глюкоолигосахарида, глюкоманноолигосахарида, галактоманноолигосахарида, маннанолигосахарида, хитоолигосахарида, олигосахарида уроновой кислоты, сиалилолигосахарида и фукоолигосахарида, и их смеси, предпочтительно fructooligosaccharides, and g. optionally long chain polyunsaturated fatty acids, preferably docosahexaenoic acid (DHA), more preferably at least 0.35 wt% DHA, based on total fatty acids, wherein the nutritional composition is an infant formula, a second level formula, or an infant formula. 16. A nutritional composition according to claim 15, wherein the amount of allergenic beta-lactoglobulin is more than 0.8 μg per 1 g of protein, and / or where the composition contains more than 1 wt.% of peptides or proteins of 1 kDa or more in terms of total protein, more preferably at least 5 wt.%, more preferably at least 10 wt.%. 17. The nutritional composition according to claim 15 or 16, wherein the indigestible oligosaccharides comprise at least two indigestible oligosaccharides selected from the group consisting of fructooligosaccharides and galactooligosaccharides, preferably a mixture of long chain fructooligosaccharides with short chain fructooligosaccharides or with short chain galactooligosaccharides. 18. Nutritional composition according to any one of paragraphs. 15-17, containing from 10 ⁵ to 10 ¹¹ CFU of lactic acid bacteria per 1 g of dry weight. 19. Nutritional composition according to any one of paragraphs. 15-18, containing at least 2 wt.% indigestible oligosaccharides in terms of dry weight. 20. A method of providing nutrition to a person at risk of developing or suffering from an allergy, more preferably a milk protein allergy, comprising administering to a person a nutritional composition according to any one of paragraphs. 15-19. 21. The method of claim 20 wherein the person is an infant or young child, preferably an infant. 22. Non-therapeutic way: - induction of oral immune tolerance to milk protein; and/or - prevention of oral immune intolerance to milk protein; and/or - reducing the risk of developing oral immune intolerance to milk protein; and/or - improvement or enhancement of oral immune tolerance to milk protein, in humans, comprising administering to the subject a nutritional composition containing a protein hydrolyzate from mammalian milk, preferably milk from species of the genus Bos , Bison , Bubalus , or Capra , more preferably the genus Bos , preferably cow's milk, wherein the protein hydrolyzate contains at least a peptide having the sequence in accordance with SEQ ID NO: 5, and at least one peptide having the sequence in accordance with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and this composition preferably additionally contains one strain of lactic acid bacteria, belonging to the genus Bifidobacterium .

Images