Compositions and food products comprising a recombinant donkey Βeta-lactoglobulin (BLG)

Abstract

The present invention relates to a hypoallergenic composition comprising a recombinant donkey β-lactoglobulin (BLG). Further provided are food products comprising a recombinant donkey BLG, methods of producing a food product comprising a recombinant donkey BLG, and uses of compositions and food products comprising a recombinant donkey BLG in avoiding milk protein allergies.

Description

[0001] COMPOSITIONS AND FOOD PRODUCTS COMPRISING A RECOMBINANT DONKEY BETA-LACTOGLOBULIN (BLG)

[0002] The present invention relates to a hypoallergenic composition comprising a recombinant donkey p-lactoglobulin (BLG). Further provided are food products comprising a recombinant donkey BLG, methods of producing a food product comprising a recombinant donkey BLG, and uses of compositions and food products comprising a recombinant donkey BLG in avoiding milk protein allergies.

[0003] BACKGROUND TO THE INVENTION

[0004] Dairy products play an important role in human nutrition, offering essential nutrients such as calcium, vitamin D, and high-quality proteins. In addition to these nutritional benefits, dairy products are also highly valued for their functional properties, including thickening, gelling, and foaming, which are important in food processing and product formulation. These properties can, at least in part, be attributed to milk proteins, such as whey proteins. Whey protein is a high-quality protein derived from whey, the liquid portion of milk that separates during cheese production. It is a complete protein, meaning it contains all nine essential amino acids necessary for human health. Whey protein is known for its excellent digestibility and rapid absorption.

[0005] The main component of whey protein is beta-lactoglobulin (BLG). BLG is the predominant whey protein found in the milk of many mammals, including cows, sheep, and goats, but is absent in human milk. It constitutes approximately 50-60% of the total whey protein content and belongs to the lipocalin protein family, known for its ability to bind and transport small hydrophobic molecules. BLG is a complete protein, rich in essential amino acids, particularly branched-chain amino acids, and exhibits excellent functional properties such as foaming, gelling, and emulsifying, making it valuable in food processing.

[0006] However, there is a growing demand for alternatives to cow milk-based dairy products, driven by dietary preferences, lactose intolerance, and allergies to cow's milk protein. In particular, BLG is recognized as a major allergen responsible for allergic reactions in sensitive individuals. These allergic reactions can range from mild symptoms, such as gastrointestinal discomfort and skin rashes, to severe anaphylactic responses, posing significant health risks. As a result, individuals with cow's milk protein allergy (CMPA) must avoid all cow milk-based products, which can limit their dietary options and nutritional intake. The presence of BLG in infant formula can lead to severe allergic reactions in infants with milk protein allergies and it appears desirable to reduce or eliminate BLG in infant formula for this target population of infants.

[0007] Plant-based alternatives, such as soy, almond, and oat milk, have gained popularity as substitutes. Despite their benefits, these plant-based alternatives often fall short in terms of nutritional content, particularly protein quality and quantity. Additionally, plant-based proteins may lack the functional properties of cow's milk protein, such as thickening, gelling, and foaming. Sensory attributes such as flavor, odor, and texture are also often inferior in plant-based products compared to their dairy counterparts.

[0008] To address concerns regarding allergic reactions in infants, extensively hydrolyzed or amino acid-based infant formulas have been created to limit the presence of epitopes triggering allergic reactions. However, these formulas often have sensory characteristics (such as flavor, odor, and texture) that are generally considered inferior to infant formulas containing complete milk proteins. Furthermore, the production of extensively hydrolyzed infant formulas involves enzymatic treatment, which can pose challenges when scaling up for industrial production. Indeed, it is important to maintain the proper conditions to ensure sufficient hydrolysis of the proteins and avoid allergic reactions. Finally, it may be desirable to maintain proteins in a native and non-hydrolyzed form to retain their functional, sensory and nutritional properties that may be of interest in food and nutritional applications.

[0009] Accordingly, there is a need in the art for hypoallergenic alternatives to products containing cow milk protein that can provide similar nutritional and functional benefits without triggering allergic responses. One objective of the present invention is thus to provide hypoallergenic -lactoglobulins for use in human nutrition.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

[0012] 1. A hypoallergenic composition comprising a recombinant donkey p-lactoglobulin (BLG); and one or more elongated variants thereof having an N-terminal elongation with a sequence selected from: A, EA, AEA and / or EAEA.

[0013] 2. The hypoallergenic composition according to embodiment 1, wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, preferably as set forth in SEQ ID NO:1.

[0014] 3. The hypoallergenic composition according to embodiment 1 or 2, wherein the ratio of the one or more N-terminally elongated variants of the recombinant donkey BLG in the composition is at least about 40%, 50%, 60%, 70%, 80% or 90% by weight of total recombinant BLG in the composition.

[0015] 4. The hypoallergenic composition according to any one of embodiments 1 to 3, wherein the composition is free, or essentially free from lactose.

[0016] 5. The hypoallergenic composition according to any one of embodiments 1 to 4, wherein the composition does not comprise milk proteins other than the recombinant donkey BLG or the elongated variants thereof.

[0017] 6. A food product comprising the hypoallergenic composition according to any one of embodiments 1 to 5. The food product according to embodiment 6, wherein the food product is a nutritional formulation (including infant formula), a high protein product (including sport nutrition, high protein medical nutrition product, weight management product, GLP-1 nutrition companion product, protein shakes, high protein bars), a milk powder (including flavored milk powders such as cocoa powders), a malted beverage powder, an instant coffee powder, a nutritional food, a baby food, a fermented food, a yoghurt, a drinking yoghurt, an ice cream, a milk drink, a soup, a sauce, a bar, a gel, a foam, a medical food (including Foods for Special Medical Purposes (FSMPs)), a complete meal replacement, a tube feeding formula, a food supplement, a beverage, a dairy beverage, a beverage whitener, a creamer, a cheese, a dairy tofu, a food emulsion, a dessert, a product that requires the protein to form a heat-set gel, an acid protein fortified beverage, a jelly drink, a protein water, or a heat-set foam extruded food product. The food product according to embodiment 6 or 7, wherein the food product is free, or essentially free from lactose. The food product according to any one of embodiments 6 to 8, wherein the food product does not comprise milk proteins other than the recombinant donkey BLG or the elongated variants thereof. The food product according to any one of embodiments 6 to 9, wherein the food product is free of animal-derived ingredients. An infant formula comprising a recombinant donkey p-lactoglobulin (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1. A milk drink comprising a recombinant donkey p-lactoglobulin (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1. A milk powder comprising a recombinant donkey p-lactoglobulin (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1. A malted beverage powder comprising a recombinant donkey p-lactoglobulin (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1. A high protein food product comprising a recombinant donkey -lactoglobulin (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1. A recombinant protein comprising a recombinant donkey p-lactoglobulin (BLG) as set forth in SEQ ID NO:1 fused to an N-terminal signal peptide. The recombinant protein according to embodiment 16, wherein the N-terminal signal peptide comprises an amino acid sequence as set forth in SEQ ID NO:8. A polynucleotide encoding the recombinant protein according to embodiment 16 or 17. The polynucleotide according to embodiment 18, further comprising a promoter sequence. The polynucleotide according to embodiment 19, wherein the promoter is an inducible promoter. The polynucleotide according to embodiment 20, wherein the inducible promoter is an AOX1 promoter. A vector comprising the polynucleotide according to any one of embodiments 18 to 21. A host cell comprising the polynucleotide according to any one of embodiments 18 to 21 or the vector according to embodiment 22. The host cell according to embodiment 23, wherein the host cell is a yeast cell, in particular wherein the host cell is a Pichia pastoris cell. A method for producing a recombinant donkey p-lactoglobulin (BLG), the method comprising the steps of: a) culturing the host cell of embodiment 23 or 24 under suitable conditions; and b) isolating, and optionally purifying, the recombinant donkey BLG from the culture medium. A method for preparing a food product, the method comprising a) providing a composition of any one of embodiments 1 to 5; and b) mixing the composition with one or more additional ingredients to produce the food product. A composition or food product comprising recombinant donkey p-lactoglobulin (BLG) for use in avoiding an allergic reaction in a subject that has been diagnosed with or is suspected to have a milk protein allergy. A method for avoiding an allergic reaction in a subject that has been diagnosed with or is suspected to have a milk protein allergy, the method comprising feeding or administering to said subject a composition or food product comprising recombinant donkey -lactoglobulin (BLG). A composition or food product comprising recombinant donkey p-lactoglobulin (BLG) for use in reducing, preventing or avoiding the symptoms associated with cow milk allergy or lactose intolerance in a subject. 30. A method for reducing, preventing or avoiding the symptoms associated with cow milk allergy or lactose intolerance in a subject, the method comprising feeding or administering to said subject a composition or food product comprising recombinant donkey p-lactoglobulin (BLG).

[0018] 31. The composition or food product for use according to embodiment 27 or 29 or the method according to embodiment 28 or 30, wherein the composition is the composition according to any one of embodiments 1 to 5; and wherein the food product is the food product according to any one of embodiments 6 to 15.

[0019] 32. The composition or food product for use according to any one of embodiments 27, 29 or 31 or the method according to any one of embodiments 28, 30 or 31, wherein the subject is an infant and wherein the food product is infant formula.

[0020] Accordingly, in one aspect, the invention relates to a hypoallergenic composition comprising a recombinant donkey p-lactoglobulin (BLG); and one or more N-terminally elongated variants thereof, preferably wherein the N-terminally elongated variants are elongated with a sequence selected from: A, EA, AEA and / or EAEA.

[0021] The present invention is based on the observation that donkey beta-lactoglobulin (BLG), unlike bovine BLG, is hypoallergenic. As demonstrated in FIG. 4, both bovine whey protein isolate (WPI) and a commercially available milk drink containing bovine BLG have the potential to cause allergic reactions in humans. In contrast, compositions containing donkey BLG exhibited no or very limited potential for inducing allergic reactions. Consequently, donkey BLG is of particular interest as a supplement for food products, as it can impart the functional, nutritional, and sensory attributes of milk proteins without the associated risk of allergic reactions. In addition, donkey BLG is interesting as it has a significant amount of Leucine which is reported to have several musculoskeletal health benefits. Indeed, leucine is one of the 3 essential branched chain amino acids which are used by skeletal muscle to give energy during exercise. Moreover, leucine is also a key amino acid involved in muscle anabolism. The composition according to the invention is a hypoallergenic composition. The term "hypoallergenic" refers to a substance or product that is designed to minimize the risk of causing an allergic reaction. In the context of the invention, a hypoallergenic composition is formulated to reduce or eliminate the presence of common allergens, thereby making it suitable for individuals with sensitivities or allergies. This is achieved by using ingredients that are less likely to trigger an immune response, such as recombinant donkey p-lactoglobulin (BLG), which has been demonstrated to have lower allergenic potential compared to traditional cow milk proteins. Hypoallergenic compositions are particularly beneficial for individuals with cow milk allergy, as they provide a safe alternative that can be consumed without the risk of adverse reactions.

[0022] Whether or not a composition is hypoallergenic in humans may be determined, without limitation, using a FAST-PASE assay, as described elsewhere herein.

[0023] Preferably, the composition according to the invention is a mixture of a recombinant donkey BLG and at least one N-terminally elongated recombinant donkey BLG variant.

[0024] The recombinant donkey BLG is preferably identical in sequence to a mature form of |3- lactoglobulin as it is found in donkey milk. Two mature forms of BLG exist in donkey milk that are commonly referred to as p-lactoglobu li n-1 (BLG-1) and p-lactoglobu li n-2 (BLG-2).

[0025] In a preferred embodiment, the recombinant donkey BLG is identical in sequence to donkey P-lactoglobulin-1 (BLG-1), having an amino acid sequence as set forth in SEQ ID NO:1:

[0026] SEQ ID NO:1

[0027] TNI PQTMQDLDLQEVAGKWHSVAMAASDI SLLDSEEAPLRVYIEKLRPTPEDNLEI ILREGENKGCAEKKI FAEK TESPAEFKINYLDEDTVFALDSDYKNYLFLCMKNAATPGQSLVCQYLARTQMVDEEIMEKFRRALQPLPGRVQIV PDLTRMAERCRI

[0028] Alternatively, the composition according to the invention may comprise a recombinant donkey BLG that is identical in sequence to donkey p-lactoglobuli n-2 (BLG-2), having an amino acid sequence as set forth in SEQ ID NO:2: SEQ ID N0:2

[0029] TDI PQTMQDLDLQEVAGRWHSVAMVASDI SLLDSESVPLRVYVEELRPTPEGNLEI ILREGANHACVERNIVAQK TEDPAVFTVNYQGERKI SVLDTDYAHYMFFCVGPPLPSAEHGMVCQYLARTQKVDEEVMEKFSRALQPLPGRVQI VQDPSGGQERCGF

[0030] While it is preferred that the recombinant donkey BLG comprised in the composition according to the invention is identical in amino acid sequence to BLG-1 (SEQ ID NO:1) or BLG- 2 (SEQ ID NO:2), the recombinant donkey BLG may also be a variant of mature donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0031] The term "sequence identity" as used herein in the context of amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0032] In certain embodiments, the recombinant donkey BLG is a variant of mature donkey BLG-1 or BLG-2 that differs from SEQ ID NO:1 or SEQ ID NO:2 in up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acid positions. These differences may include amino acid substitutions, deletions, or insertions at one or more of these positions.

[0033] Amino acid substitutions refer to the replacement of one amino acid with another in the protein sequence.

[0034] Insertions refer to the addition of one or more amino acids into the protein sequence. Deletions refer to the removal of one or more amino acids from the protein sequence.

[0035] The skilled person is aware of methods of genetic engineering to manipulate the amino acid sequence of a protein, such as BLG. Theoretically, the recombinant donkey BLG may include one or more insertions at the N- terminal end. However, in embodiments where the recombinant donkey BLG is combined with an N-terminally elongated variant of donkey BLG, the recombinant donkey BLG should not contain any insertions at the N-terminal end. In other words, in compositions comprising both a recombinant donkey BLG and an N-terminally elongated variant thereof, the recombinant donkey BLG remains unmodified at the N-terminal end, without any amino acid insertions or elongations.

[0036] In certain embodiments, the recombinant donkey BLG is a variant of mature BLG-1 or BLG-2 comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acid substitutions compared to SEQ ID NO:1 or SEQ ID NO:2.

[0037] The term "recombinant" in the context of recombinant donkey beta-lactoglobulin (BLG) refers to a form of the protein that is produced through recombinant DNA technology. This involves inserting the gene encoding donkey BLG into a host organism, such as bacteria, yeast, or mammalian cells, which then expresses / proceduces the protein. The host organism's cellular machinery is used to produce the donkey BLG protein, allowing for large-scale production and potential modifications to the protein's structure or amino acid sequence.

[0038] Recombinant donkey BLG can be identical to the natural protein found in donkey milk (SEQ ID NO:1 and 2) or can include specific modifications, such as N-terminal elongations or other amino acid changes, as mentioned herein above. The application of recombinant technology enables the production of large quantities of this protein, eliminating the need for donkey milk as a starting material. Producing recombinant donkey BLG offers advantages over extracting BLG from donkey milk, which is known to be challenging and typically results in limited yields in terms of both quantity and purity of BLG. In addition, the availability of donkey milk remains limited compared to cow's milk. Especially, donkeys produce significantly less milk compared to cows. Additionally, donkeys are not as commonly bred and raised for milk production as cows are. While the recombinant donkey p-lactoglobu li n BLG may be identical in amino acid sequence to BLG-1 or BLG-2 found in natural donkey milk, it may exhibit differences in post-translational modification patterns. Post-translational modifications (PTMs) are chemical changes that occurto a protein after it has been synthesized, and that may influence the protein's structure, function, and stability. Common PTMs include glycosylation, phosphorylation, acetylation, and methylation. These modifications may affect the protein's solubility, immunogenicity, and overall biological activity. In the case of recombinant donkey BLG, variations in PTMs compared to the natural protein may arise due to differences in the expression system or production process. These variations may be leveraged to enhance the hypoallergenic properties, stability, or functionality of the protein, making it even more suitable for use in food products designed for individuals with cow milk allergy or lactose intolerance.

[0039] Thus, in certain embodiments, the recombinant donkey BLG comprises a non-native post- translational modification pattern (PTM pattern). A non-native PTM pattern refers to any PTM arrangement that differs from those found in natural donkey milk BLG.

[0040] Preferably, the recombinant donkey BLG according to the invention is produced in yeast, more preferably Pichia pastoris. Thus, in certain embodiments, the recombinant donkey BLG may have a PTM pattern characteristic of a yeast expression system. In particular, the recombinant donkey BLG may exhibit a PTM pattern characteristic of a P. pastoris expression system. The term "PTM pattern" refers to the specific arrangement and types of post-translational modifications (PTMs) that occur on a protein after it has been synthesized.

[0041] In some embodiments, the recombinant donkey BLG does not comprise any non-native PTMs.

[0042] Preferably, the recombinant donkey BLG comprises a PTM pattern that does not cause allergic reactions in humans. Whether or not a protein having a specific PTM pattern can cause an allergic reaction in humans may be assessed using tests known in the art. For example, a FAST- PASE® assay may be used to test whether a protein having a specific PTM pattern can cause an allergic reaction in humans.

[0043] The FAST-PASE (Fluorescent Allergen Sensitization Test - Passive Sensitization of Effector cells) assay is a functional cell-based diagnostic tool designed to improve the management of allergic diseases. The assay utilizes conditionally immortalized progenitor cells from mice transgenic for the human high-affinity IgE receptor (FCERIO), which can be differentiated into mature mast cells within five days. These Hoxb8 mast cells are passively sensitized with serum from allergic patients, allowing for the accurate measurement of total IgE levels, identification of culprit allergens, and monitoring of allergen-specific immunotherapy (AIT) responses; see Zbaren et al. (J Allergy Clin Immunol 2022 Mar;149(3):1018-1030.ell) for more detail, which is fully incorporated herein by reference.

[0044] In certain embodiments, a recombinant donkey BLG is defined to comprise a PTM pattern that does not cause allergic reactions in humans, if the recombinant donkey BLG fails to induce a reaction in a FAST-PASE assay at concentrations below 0.1, 0.5, 1, 2, 5, or 10 pg / mL.

[0045] In certain embodiments, a composition comprising the recombinant donkey BLG is defined as hypoallergenic, if the recombinant donkey BLG comprised in the composition fails to induce a reaction in a FAST-PASE assay at concentrations below 0.1, 0.5, 1, 2, 5, or 10 pg / mL.

[0046] The composition according to the invention preferably comprises a mixture of a recombinant donkey BLG and one or more N-terminally elongated variant of a donkey BLG. The term "N- terminally elongated donkey beta-lactoglobulin (BLG)" refers to a modified form of donkey BLG, wherein additional amino acids have been added to the N-terminus of the protein.

[0047] The N-terminally elongated variant of donkey BLG may include any N-terminal elongation sequence. This elongation sequence may range from 1 to 20 amino acid residues and may comprise any amino acid sequence.

[0048] In certain embodiments, the N-terminal elongation sequence is a truncated signal peptide. Specifically, the recombinant donkey BLG is preferably produced in a recombinant host. To facilitate purification, a signal peptide may be fused to the N-terminal end of the donkey BLG sequence, promoting the secretion of donkey BLG into the culture medium. During secretion, the N-terminal signal peptide is typically cleaved off. However, incomplete cleavage of the signal peptide may result in N-terminally elongated variants of donkey BLG.

[0049] In a preferred embodiment, the N-terminally elongated donkey BLG comprises one of the following amino acid sequences fused to the N-terminal end of the donkey BLG (in N-to-C direction): Ala (A), Glu-Ala (EA), Ala-Glu-Ala (AEA) or Glu-Ala-Glu-Ala (EAEA; SEQ. ID NO:3). In certain embodiments, the N-terminally elongated donkey BLG comprised in the composition according to the invention is an N-terminally elongated variant of donkey BLG-1 or BLG-2, or of a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0050] In certain embodiments, the N-terminally elongated donkey BLG comprised in the composition according to the invention is an N-terminally elongated variant of donkey BLG-1, or of a sequence variant of donkey BLG-1 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1.

[0051] In certain embodiments, the N-terminally elongated donkey BLG comprised in the composition according to the invention is an N-terminally elongated variant of donkey BLG-1 comprising an N-terminal alanine residue and having a sequence as set forth in SEQ ID NO:4:

[0052] SEQ ID NO:4

[0053] ATNI PQTMQDLDLQEVAGKWHSVAMAASDI SLLDSEEAPLRVYIEKLRPTPEDNLEI ILREGENKGCAEKKI FAE KTESPAEFKINYLDEDTVFALDSDYKNYLFLCMKNAATPGQSLVCQYLARTQMVDEEIMEKFRRALQPLPGRVQI VPDLTRMAERCRI

[0054] In certain embodiments, the N-terminally elongated donkey BLG comprised in the composition according to the invention is an N-terminally elongated variant of donkey BLG-1 comprising the N-terminal sequence motif Glu-Ala (EA) and having a sequence as set forth in SEQ ID NO:5:

[0055] SEQ ID NO:5

[0056] EATNI PQTMQDLDLQEVAGKWHSVAMAASDI SLLDSEEAPLRVYIEKLRPTPEDNLEI ILREGENKGCAEKKI FA EKTESPAEFKINYLDEDTVFALDSDYKNYLFLCMKNAATPGQSLVCQYLARTQMVDEEIMEKFRRALQPLPGRVQ IVPDLTRMAERCRI

[0057] In certain embodiments, the N-terminally elongated donkey BLG comprised in the composition according to the invention is an N-terminally elongated variant of donkey BLG-1 comprising the N-terminal sequence motif Ala-Glu-Ala (AEA) and having a sequence as set forth in SEQ ID NO:6:

[0058] SEQ ID NO:6

[0059] AEATNI PQTMQDLDLQEVAGKWHSVAMAASDI SLLDSEEAPLRVYIEKLRPTPEDNLEI ILREGENKGCAEKKI F AEKTESPAEFKINYLDEDTVFALDSDYKNYLFLCMKNAATPGQSLVCQYLARTQMVDEEIMEKFRRALQPLPGRV QIVPDLTRMAERCRI

[0060] In certain embodiments, the N-terminally elongated donkey BLG comprised in the composition according to the invention is an N-terminally elongated variant of donkey BLG-1 comprising the N-terminal sequence motif Glu-Ala-Glu-Ala (EAEA) and having a sequence as set forth in SEQ ID NO:7:

[0061] SEQ ID NO:7

[0062] EAEATNI PQTMQDLDLQEVAGKWHSVAMAASDI SLLDSEEAPLRVYIEKLRPTPEDNLEI ILREGENKGCAEKKI FAEKTESPAEFKINYLDEDTVFALDSDYKNYLFLCMKNAATPGQSLVCQYLARTQMVDEEIMEKFRRALQPLPGR VQIVPDLTRMAERCRI

[0063] In certain embodiments, the composition according to the invention comprises a recombinant donkey BLG-1 and an N-terminally elongated variant of donkey BLG-1, wherein the N- terminally elongated donkey BLG-1 comprises one of the following amino acid sequences fused to the N-terminal end of the donkey BLG (in N-to-C direction): Ala (A), Glu-Ala (EA), Ala- Glu-Ala (AEA) or Glu-Ala-Glu-Ala (EAEA; SEQ ID NO:3).

[0064] In certain embodiments, the composition according to the invention comprises a recombinant donkey BLG comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1; and one or more N-terminally elongated variants of a donkey BLG comprising or consisting of an amino acid sequence as set forth in SEQ ID NOs: 4-7.

[0065] Further encompassed herein are compositions comprising recombinant donkey BLG-1 and BLG-2, and N-terminally elongated variants of at least one of BLG-1 and / or BLG-2. Also encompassed herein are compositions comprising N-terminally elongated variants of donkey BLG-1 and BLG-2, and at least one of recombinant donkey BLG-1 and / or BLG-2. The recombinant donkey BLG and the N-terminally elongated donkey BLG may be comprised in the composition according to the invention in any ratio.

[0066] In a particular embodiment, the invention relates to the composition according to the invention, wherein the ratio of the one or more N-terminally elongated variants of the recombinant donkey BLG in the composition is at least about 40%, 50%, 60%, 70%, 80% or 90% by weight of total recombinant BLG in the composition.

[0067] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, may comprise approximately 40% by weight of N-terminally elongated donkey BLG and approximately 60% by weight of recombinant donkey BLG.

[0068] In other embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, may comprise approximately 50% by weight of N-terminally elongated donkey BLG and approximately 50% by weight of recombinant donkey BLG.

[0069] In further embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, may comprise approximately 60% by weight of N-terminally elongated donkey BLG and approximately 40% by weight of recombinant donkey BLG.

[0070] In yet another embodiment, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, may comprise approximately 70% by weight of N-terminally elongated donkey BLG and approximately 30% by weight of recombinant donkey BLG.

[0071] In additional embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, may comprise approximately 80% by weight of N-terminally elongated donkey BLG and approximately 20% by weight of recombinant donkey BLG.

[0072] In a specific embodiment, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, may comprise approximately 90% by weight of N-terminally elongated donkey BLG and approximately 10% by weight of recombinant donkey BLG.

[0073] It has been demonstrated in Example 1 that recombinant production of donkey BLG in the yeast Pichia pastoris resulted in approximately 9% by weight of recombinant donkey BLG and approximately 91% by weight of N-terminally elongated donkey BLG.

[0074] Thus, in certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises between 2% and 40% by weight of recombinant donkey BLG, with the remaining part being N-terminally elongated donkey BLG, preferably wherein the N-terminally elongated donkey BLG comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0075] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises between 5% and 30% by weight of recombinant donkey BLG, with the remaining part being N-terminally elongated donkey BLG, preferably wherein the N-terminally elongated donkey BLG comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0076] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises between 5% and 20% by weight of recombinant donkey BLG, with the remaining part being N-terminally elongated donkey BLG, preferably wherein the N-terminally elongated donkey BLG comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0077] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises between 5% and 15% by weight of recombinant donkey BLG, with the remaining part being N-terminally elongated donkey BLG, preferably wherein the N-terminally elongated donkey BLG comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0078] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises between 7.5% and 12.5% by weight of recombinant donkey BLG, with the remaining part being N-terminally elongated donkey BLG, preferably wherein the N-terminally elongated donkey BLG comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0079] The most prevalent forms of donkey BLG in the composition according to the invention are preferably N-terminally elongated donkey BLGs comprising the N-terminal sequence motifs Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0080] Thus, in certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises at least 40% by weight of an N-terminally elongated donkey BLG comprising the N- terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA); and up to 40% by weight of recombinant donkey BLG.

[0081] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises at least 50% by weight of an N-terminally elongated donkey BLG comprising the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA); and up to 30% by weight of recombinant donkey BLG.

[0082] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises at least 60% by weight of an N-terminally elongated donkey BLG comprising the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA); and up to 20% by weight of recombinant donkey BLG.

[0083] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises at least 70% by weight of an N-terminally elongated donkey BLG comprising the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA); and up to 15% by weight of recombinant donkey BLG.

[0084] In certain embodiments, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises at least 80% by weight of an N-terminally elongated donkey BLG comprising the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA); and up to 10% by weight of recombinant donkey BLG. In a particularly preferred embodiment, the protein fraction of the composition according to the invention, in particular the BLG fraction of the composition according to the invention, comprises between 5 and 15% by weight of a recombinant donkey BLG, preferably donkey BLG-1 (SEQ ID NO:1), between 10 and 25% by weight of an N-terminally elongated donkey BLG comprising the N-terminal sequence motif Glu-Ala-Glu-Ala (EAEA), between 60 and 80% by weight of an N-terminally elongated donkey BLG comprising the N-terminal sequence motif Glu-Ala (EA); and up to 10% by weight of other N-terminally elongated donkey BLG variants.

[0085] It is to be understood that the N-terminally elongated donkey BLG variant included in the composition according to the invention is preferably an elongated variant of the recombinant donkey BLG also present in the composition. Thus, the recombinant donkey BLG and the N- terminally elongated variant of donkey BLG are preferably identical in sequence, except for the N-terminal elongation in one variant. This occurs because both variants result from the expression of the same gene but differ in their N-terminal sequences due to variations in the processing of the N-terminal signal peptide.

[0086] Further encompassed herein are composition comprising only non-elongated and / or nontruncated forms of recombinant donkey BLG. In particular, the composition may comprise a recombinant donkey BLG having an amino acid sequence as set forth in SEQ ID NO:1 or 2. Alternatively, the composition may comprise a recombinant variant of donkey BLG-1 or BLG- 2 having an amino acid sequence as set forth in SEQ ID NO: 1 or 2, respectively, but comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acid substitutions.

[0087] Another aspect of the invention relates to a food product, in particular a hypoallergenic food product, comprising recombinant donkey BLG. A food product refers to any substance that is intended for human consumption, whether in its raw, processed, or prepared form. Food products may be consumed directly or used as ingredients in the preparation of other consumable items. The food product according to the invention may be any food product that is suitable for supplementation with a recombinant donkey BLG. Without limitation, the food product may be any one of: a nutritional formulation (including infant formula), a high protein product (including sport nutrition, high protein medical nutrition product, weight management product, GLP-1 nutrition companion product, protein shakes, high protein bars), a milk powder (including flavored milk powders such as cocoa powders), a malted powder, an instant coffee powder, a nutritional food, a baby food, a fermented food, a yoghurt, a drinking yoghurt, an ice cream, a milk drink, a soup, a sauce, a bar, a gel, a foam, a medical food (including Foods for Special Medical Purposes (FSMPs)), a complete meal replacement, a tube feeding, a food supplement, a beverage, a dairy beverage, a beverage whitener, a creamer, a cheese, a dairy tofu, a food emulsion, a dessert, a product that requires the protein to form a heat-set gel, an acid protein fortified beverage, a jelly drink, a protein water, or a heat-set foam extruded food product.

[0088] Thus, in a particular embodiment, the invention relates to the food product according to the invention, wherein the food product is a nutritional formulation (including infant formula), a high protein product (including sport nutrition, high protein medical nutrition product, weight management product, GLP-1 nutrition companion product, protein shakes, high protein bars), a milk powder (including flavored milk powders such as cocoa powders), a malted powder, an instant coffee powder, a nutritional food, a baby food, a fermented food, a yoghurt, a drinking yoghurt, an ice cream, a milk drink, a soup, a sauce, a bar, a gel, a foam, a medical food (including Foods for Special Medical Purposes (FSMPs)), a complete meal replacement, a tube feeding, a food supplement, a beverage, a dairy beverage, a beverage whitener, a creamer, a cheese, a dairy tofu, a food emulsion, a dessert, a product that requires the protein to form a heat-set gel, an acid protein fortified beverage, a jelly drink, a protein water, or a heat-set foam extruded food product.

[0089] In certain embodiments, the food product according to the invention comprises the hypoallergenic composition as defined herein above, i.e., a composition comprising a recombinant donkey BLG, preferably a recombinant donkey BLG-1 comprising or consisting of SEQ ID NO:1, and one or more N-terminally elongated variants thereof, preferably wherein the N-terminally elongated donkey BLG comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA). Thus, in a particular embodiment, the invention relates to a food product comprising the hypoallergenic composition according to the invention.

[0090] However, also encompassed herein are food products comprising any type of recombinant donkey BLG. That is, the food product disclosed herein may comprise recombinant donkey BLG-1 (SEQ ID NO:1) or recombinant donkey BLG-2 (SEQ ID NO:2), or sequence variants of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively. Of note, the variant of BLG-1 or BLG-2 may also be an N-terminally elongated variant as described in more detail elsewhere herein. In a particular embodiments, the food product may comprise a recombinant donkey BLG comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID Nos: 4-7, preferably SEQ ID NO:5 and / or SEQ ID NO:7.

[0091] In a particularly preferred embodiment, the food product is a nutritional formulation.

[0092] A nutritional formulation is a specially designed food product that provides a balanced and concentrated source of essential nutrients, including proteins, carbohydrates, fibers, fats, vitamins, and minerals. These formulations are created to meet specific dietary needs and can be tailored for various purposes such as supporting growth and development, enhancing athletic performance, managing medical conditions, or supplementing daily nutrition. Nutritional formulations can come in various forms, including powders, liquids, bars, and capsules, and are often used in contexts such as infant formula, medical nutrition, sports nutrition, and meal replacements. They are preferably developed based on scientific research to ensure they deliver the required nutrients in appropriate proportions for optimal health and well-being.

[0093] The nutritional formulation may be incomplete nutritional formulation or complete nutritional formulation.

[0094] As used herein, the term "incomplete nutritional formulation" refers to nutritional products that do not contain sufficient levels of macronutrients (protein, fats and carbohydrates) or micronutrients to be sufficient to be a sole source of nutrition for the subject which consumes the nutritional product or the subject to which the nutritional product is being administered. As used herein, the "complete nutritional formulation" refers to nutritional products that contains sufficient levels of macronutrients (protein, fats and carbohydrates) or micronutrients to be sufficient to be a sole source of nutrition for the subject which consumes the nutritional product or the subject to which the nutritional product is being administered.

[0095] Nutritional formulations may be supplemented with recombinant donkey BLG to enhance their protein content and provide a hypoallergenic alternative to traditional dairy proteins. This makes the formulation suitable for individuals with lactose intolerance and / or dairy allergies, expanding its potential consumer base. Additionally, recombinant donkey BLG can improve the texture, solubility, and stability of the nutritional formulation.

[0096] The food product according to the invention is preferably a nutritional formulation in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0097] In a particular embodiment, the food product according to the invention is a nutritional formulation in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0098] In a particularly preferred embodiment, the nutritional formulation is an infant formula.

[0099] As used herein, the expression "infant formula" refers to a foodstuff intended for the particular nutritional use by infants during the first months of life, satisfying by itself the nutritional requirements of this category of person, as defined in Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae. It also refers to a nutritional composition intended for infants, as defined in the Codex Alimentarius (Codex STAN 72-1981) and in Infant Specialties, including Food for Special Medical Purposes (FSMPs). The expression "infant formula" encompasses both "starter infant formula" and "follow-up formula" or "follow-on formula." "Starter infant formula" is designed for use from birth up to the age of six months and is formulated to meet the complete nutritional needs of infants during this critical period of growth and development.

[0100] As used herein, a "follow-up formula" or "follow-on formula" is intended for use from the 6th month onwards. It serves as the principal liquid element in the progressively diversified diet of this category of person, providing essential nutrients to complement the introduction of solid foods. Follow-up formulas are designed to support the continued growth and development of infants as they transition from exclusive milk feeding to a more varied diet. Both starter and follow-up formulas are carefully formulated to provide the appropriate balance of proteins, fats, carbohydrates, vitamins, and minerals necessary for the healthy growth and development of infants. They are manufactured under strict quality control standards to ensure safety and nutritional adequacy.

[0101] Infant formulas are available in different forms to cater to various needs and preferences: Powdered Formula is the most common form of infant formula. It is a dry powder that needs to be mixed with water before feeding. Powdered formula is convenient for storage and transportation and is typically the most economical option.

[0102] Liquid Concentrate Formula is a concentrated liquid that must be diluted with an equal amount of water before feeding.

[0103] Ready-to-Feed Formula is the most convenient form of infant formula, as it comes pre-mixed and ready to use. It does not require any preparation or dilution, making it ideal for on-the-go feeding or for use in situations where clean water may not be readily available.

[0104] Supplementing infant formula with recombinant donkey BLG can provide several significant benefits. Recombinant donkey BLG is a high-quality protein that closely resembles the proteins found in human breast milk, making it an excellent source of essential amino acids necessary for an infant's growth and development. One of the key advantages of recombinant donkey BLG is its hypoallergenic properties and the fact it is devoid of lactose, which make it suitable for infants with lactose intolerance and / or dairy allergies, reducing the risk of allergic reactions and ensuring that these infants receive the necessary proteins for healthy development. Additionally, recombinant donkey BLG may enhance the solubility and stability of the infant formula, ensuring a consistent and easily digestible product that supports optimal nutrient absorption.

[0105] In certain embodiments, the invention relates to an infant formula in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG- 1 (SEQ ID NO:1) or BLG-2 (SEQID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0106] In certain embodiments, the infant formula comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N- terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0107] Preferably, the food product according to the invention is an infant formula in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0108] In a particular embodiment, the invention relates to an infant formula comprising a recombinant donkey p-lactoglobuli n (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1.

[0109] In certain embodiments, the infant formula comprises between 0.5 and 10 g protein per 100 mL of prepared formula, preferably between 0.5 and 5g protein per lOOmL of prepared formula, more preferably between 1 and 3 g protein per 100 mL of prepared formula, wherein some or all of the protein comprised in the infant formula may be a recombinant donkey BLG as defined herein. In certain embodiments, the infant formula may comprise between 0.5 and 10 g of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1 per 100 mL of prepared formula, preferably between 0.5 and 5g of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1 per 100 mL of prepared formula, more preferably between 1 and 3 g of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1 per 100 mL of prepared formula.

[0110] It is preferred herein that the infant formula is a hypoallergenic infant formula. In particular, it is preferred herein that the infant formula is suitable for children or infant with cow's milk protein allergy (CMPA).

[0111] In certain embodiments, the food product according to the invention is a high protein product. A high protein product is a type of food or beverage that is specifically formulated to contain a high concentration of protein, which is essential for muscle repair, growth, and overall health. These products are designed to meet the increased protein needs of individuals such as athletes, bodybuilders, and those seeking to maintain or increase muscle mass. High protein products can come in various forms, including sport nutrition supplements, protein shakes, and high protein bars.

[0112] Supplementing high protein products with recombinant donkey p-lactoglobulin (BLG) can provide several notable benefits. Recombinant donkey BLG is a high-quality protein that delivers a complete profile of essential amino acids, including BCAAs such as leucine, which are crucial for muscle repair, growth, and overall health. Its hypoallergenic properties make it an excellent alternative for individuals with dairy allergies or lactose intolerance, expanding the potential consumer base for high protein products. Additionally, recombinant donkey BLG offers excellent solubility and stability, ensuring that the protein remains effective and easy to incorporate into various product forms such as powders, bars, and drinks.

[0113] In certain embodiments, the invention relates to a high protein product in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG- 1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0114] In certain embodiments, the high protein product comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N- terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0115] Preferably, the food product according to the invention is a high protein product in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0116] In a particular embodiment, the invention relates to a high protein food product comprising a recombinant donkey p-lactoglobuli n (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1.

[0117] In certain embodiments, the high protein product is a sport nutrition product or high protein medical nutrition product or weight management product or GLP-1 nutrition companion product.

[0118] Sport nutrition products are designed to support the dietary needs of athletes and physically active individuals. These products often include high protein content to aid in muscle recovery and performance enhancement. Sport nutrition products can be consumed before, during, or after exercise to provide the necessary nutrients for optimal athletic performance. Sport nutrition products include, without limitation, protein powders, amino acid supplements, preworkout supplements, post-workout supplements, energy gels and chews, electrolyte drinks, creatine supplements, meal replacement shakes, recovery drinks, and protein bars.

[0119] High protein medical nutrition products are designed to support the dietary needs for healthy individuals or individuals having a diseases, disorders or conditions. In particular, high protein supplementation is beneficial in said healthy individuals or individuals having a diseases, disorders or conditions. In particular, the high protein supplementation may support to treat, prevent or reduce the symptoms associated with said diseases, disorders or conditions. For example, individuals with pre-sarcopenia or with sarcopenia may benefit from high protein supplementation as it will support the increase of the muscle mass in said individuals.

[0120] Weight management products are designed to support the dietary needs for individuals who are following a diet or program for maintaining or losing their weight. Such products may support weight loss and weight maintenance, for example by inducing satiety while limiting the carbohydrate intake, and / or alleviate the symptoms associated with their diet, program, weight loss and / or weight maintenance, for example limit muscle mass loss associated with very low caloric diet.

[0121] GLP-1 nutrition companion products are designed to support the dietary needs for individuals who are following or have discontinued a GLP-1 Receptor Agonist treatment. For example, the GLP1 receptor agonist may be selected from the group consisting of albiglutide (Tanzeum), Dulaglutide (Trulicity), Exenatide (Byetta, Bydureon), Liraglutide (Victoza, Saxenda), Lixisenatide (Adlyxin), Semaglutide (Ozempic), Taspoglutide and combination thereof. These individuals often experience significant muscle mass loss while undergoing the treatment, making high protein supplementation highly advantageous. Similarly, individuals who have discontinued the treatment may face challenges in maintaining their weight due to the loss of the appetite suppressing effect of the treatment. In such cases, high protein supplementation can be beneficial by promoting satiety and assisting in weight maintenance post-treatment.

[0122] In certain embodiments, the invention relates to a high protein product which is a sport nutrition product, or high protein medical nutrition product or weight management product or GLP-1 nutrition companion product in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0123] In certain embodiments, the high protein product which is a sport nutrition product or high protein medical nutrition product or weight management product or GLP-1 nutrition companion product comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0124] Preferably, the food product according to the invention is a high protein product which is a sport nutrition product or high protein medical nutrition product or weight management product or GLP-1 nutrition companion product in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0125] In certain embodiments, the food product according to the invention is high protein product which is a protein shake. A protein shake is a liquid beverage that is rich in protein and often consumed as a convenient source of high-quality protein. Protein shakes are commonly used by athletes and fitness enthusiasts to support muscle recovery and growth after workouts or as a meal replacement.

[0126] In certain embodiments, the invention relates to a protein shake in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0127] In certain embodiments, the protein shake comprises a composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N- terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0128] Preferably, the food product according to the invention is a protein shake in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0129] In certain embodiments, the food product according to the invention is a high protein product which is high protein bar. High protein bars are portable, convenient snacks that are formulated to provide a significant amount of protein in a compact form. These bars are often consumed as a quick source of protein on-the-go, making them popular among athletes, busy professionals, and individuals looking to increase their protein intake throughout the day. High protein bars can also include other nutrients such as fiber, vitamins, and minerals to support overall health. In certain embodiments, the invention relates to a high protein bar in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG- 1 (SEQ ID NO:1) or BLG-2 (SEQID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0130] In certain embodiments, the high protein bar comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N- terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0131] Preferably, the food product according to the invention is a high protein bar in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0132] High protein products, including the sport nutrition products, high protein medical nutrition products, weight management products, GLP-1 nutrition companion products, the protein shakes or the high protein bars disclosed herein above, preferably contain a substantial amount of protein to meet the dietary needs of their target consumers. Typically, a high protein product contains at least 5 grams of protein per 100 grams of the final product, preferably at least 11 grams of protein per 100 grams of the final product, more preferably at least 15 grams of protein per 100 grams of the final product, even more preferably at least 20 grams of protein per 100 grams of the final product. This concentration ensures that the product provides a significant source of protein, supporting muscle repair and growth, and contributing to overall nutritional intake. Some high protein products may contain even higher levels of protein, depending on their specific formulation and intended use.

[0133] In certain embodiments, the high protein product according to the invention, including the sport nutrition products, high protein medical nutrition products, weight management products, GLP-1 nutrition companion products, the protein shakes or the high protein bars according to the invention, comprises at least lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, or 20g of recombinant donkey BLG-1 (SEQ ID NO:1) or BLG- 2 (SEQ ID N0:2), or of a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, per 100 g of final product. Preferably, the food product according to the invention is a high protein product, including the sport nutrition products, high protein medical nutrition products, weight management products, GLP-1 nutrition companion products, the protein shakes or the high protein bars according to the invention, comprising at least lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, or 20g of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1, per 100 g of final product.

[0134] Specifically, the high protein product according to the invention is a hypoallergenic high protein product. In particular, the product is hypoallergenic despite the presence of high amount of proteins.

[0135] In certain embodiments, the food product is a milk powder (incl. milk powder and non-dairy milk powder). Milk powder is a type of powdered product that, when mixed with water, resembles milk or a dairy product in appearance, taste, and nutritional profile. It can be made from dairy sources by evaporating the water content from liquid milk, resulting in a dry, powdered form that retains most of the nutritional properties of liquid milk, including proteins, vitamins, and minerals. This traditional milk powder can be reconstituted with water to produce liquid milk or used as an ingredient in various food products such as baked goods, confectioneries, beverages, and processed foods.

[0136] In the context of the invention, the term "milk powder" may also refer to a non-dairy powder that, when mixed with water, mimics the characteristics of milk or a dairy product but does not necessarily contain any milk components. These non-dairy milk powders are typically made from plant-based sources such as soy, almond, coconut, rice, or oats. They are designed to provide a similar taste and nutritional profile to dairy milk powder and are suitable for individuals who are lactose intolerant, allergic to dairy, or following a vegan diet.

[0137] That is, the milk powder according to the invention is preferably a milk powder in which the protein portion comprises or consists of recombinant donkey BLG. Preferably, no other milk proteins are present in the milk powder. The remaining ingredients, such as fats and carbohydrates, may be derived from dairy or may be derived from other sources.

[0138] In certain embodiments, the invention relates to a milk powder or non-dairy milk powder in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0139] In certain embodiments, the milk powder or non-dairy milk powder comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA). Preferably, the food product according to the invention is a milk powder or non-dairy milk powder in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0140] In certain embodiments, the milk powder or non-dairy milk powder may be a flavored milk powder. Flavored milk powders are a type of milk powder or non-dairy milk powder that has been enhanced with additional ingredients to provide specific flavors. Examples include chocolate-flavored milk powder, strawberry-flavored milk powder, and vanilla-flavored milk powder.

[0141] In certain embodiments, the invention relates to a flavored milk powder or flavored non-dairy milk powder in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0142] In certain embodiments, the flavored milk powder or flavored non-dairy milk powder comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0143] Preferably, the food product according to the invention is a flavored milk powder or flavored non-dairy milk powder in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0144] In certain embodiments, the milk powder or non-dairy milk powder may be a cocoa powder or cocoa mix. Cocoa powders are a specific type of flavored milk powder or non-dairy milk powder that includes cocoa as a primary ingredient. These powders are designed to be mixed with water to create a chocolate-flavored beverage. Cocoa powders typically contain a blend of milk powder or non-dairy milk powder, cocoa powder, sugar, and sometimes additional flavorings or nutrients. For example, cocoa powders typically contain a mix of minerals and vitamins.

[0145] Supplementing milk powder or non-dairy milk powder with recombinant donkey |3- lactoglobulin (BLG) can enhance the authenticity of the milk product or non-dairy milk product. Recombinant donkey BLG is a high-quality protein that closely mimics the proteins found in traditional dairy milk, providing a more genuine milk-like taste and texture. Its inclusion can improve the solubility and stability of the milk powder or non-dairy milk powder, ensuring a smooth and consistent beverage when reconstituted with water. Additionally, the hypoallergenic properties of recombinant donkey BLG make the milk powderor non-dairy milk powder suitable for individuals with dairy allergies or lactose intolerance, expanding its appeal to a broader range of consumers.

[0146] In certain embodiments, the invention relates to a cocoa powder in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively. In certain embodiments, the cocoa powder comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N- terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0147] Preferably, the food product according to the invention is a cocoa powder in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0148] In certain embodiments, the milk powder, in particular the cocoa powder, according to the invention comprises at least 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, or 1.5g of recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or of a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, per 100 mL of final product. Preferably, the food product according to the invention is a high protein product comprising at least 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, or 1.5g of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1, per 100 mL of final product.

[0149] An exemplary composition of a chocolate-flavored beverage made from cocoa powder may comprise (per 100 mL of final product):

[0150] Fat: 0.3 g

[0151] - of which saturated fatty acids: 0.2 g

[0152] Carbohydrates: 11.7 g

[0153] - of which sugars: 11.4 g

[0154] Dietary fiber: 0.8 g

[0155] Protein: 1.5 g (which may comprise or consist of recombinant donkey BLG; see above)

[0156] Salt: 0.15 g

[0157] Specifically, the milk powder according to the invention is a hypoallergenic milk powder. In certain embodiments, the food product is a malted beverage powder. Malted beverage powder is a type of powdered drink mix made from malted grains, typically barley, combined with other ingredients such as cocoa, sugar, and milk solids. This powder is designed to be mixed with water or milk (or milk replacements) to create a flavorful and nutritious beverage. Malted beverage powders, such as Milo, are popular for their rich taste and nutritional benefits. They often contain added vitamins and minerals, making them a convenient and enjoyable way to supplement one's diet.

[0158] Supplementing malted beverage powder with recombinant donkey p-lactoglobulin (BLG) may significantly enhance the quality and authenticity of the beverage. Recombinant donkey BLG is a high-quality protein that closely resembles the proteins found in traditional dairy products, providing a more genuine milk-like taste and creamy texture to the malted beverage. Its inclusion may improve the solubility and stability of the malted beverage powder, ensuring a smooth and consistent drink when mixed with water. Additionally, the hypoallergenic properties of recombinant donkey BLG make the malted beverage suitable for individuals with dairy allergies or lactose intolerance, broadening its appeal to a wider range of consumers.

[0159] In certain embodiments, the invention relates to a malted beverage powder in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0160] In certain embodiments, the malted beverage powder comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N- terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0161] Preferably, the food product according to the invention is a malted beverage powder in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1. In certain embodiments, the malted beverage powder, in particular the cocoa powder, according to the invention comprises at least 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6, 1.7g, or 1.8g of recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or of a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, per 100 mL of final product. Preferably, the food product according to the invention is a high protein product comprising at least 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6, 1.7g, or 1.8g of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1, per 100 mL of final product.

[0162] An exemplary composition of a malted beverage made from malted beverage powder may comprise (per 100 mL of final product):

[0163] Fat: 1.7 g

[0164] - of which saturated fatty acids: 0.8 g

[0165] Carbohydrates: 11 g

[0166] - of which sugars: 6.7 g

[0167] Dietary fiber: 0.8 g

[0168] Protein: 1.9 g (which may comprise or consist of recombinant donkey BLG; see above) Salt: 0.06 g.

[0169] Specifically, the milk powder according to the invention is a hypoallergenic milk powder.

[0170] In certain embodiments, the food product is a (non-dairy) milk drink.

[0171] For example, the milk drink may be a synthetic milk drink. A synthetic milk drink is a beverage made from milk proteins and other components that are produced through biotechnological processes rather than being derived from traditional dairy farming. This type of milk is created using techniques such as precision fermentation, where microorganisms like yeast or bacteria are genetically engineered to produce milk proteins such as casein and whey. These proteins are then combined with water, fats, carbohydrates, vitamins, and minerals to create a product that closely resembles traditional cow's milk in taste, texture, and nutritional profile.

[0172] Synthetic milk drinks aim to provide a sustainable and ethical alternative to conventional dairy milk, addressing concerns related to animal welfare, environmental impact, and resource use. They are suitable for individuals who are lactose intolerant, allergic to dairy, or seeking plantbased or cruelty-free options.

[0173] Alternatively, the milk drink may be a non-dairy milk drink. A non-dairy milk drink is a beverage made from plant-based sources that is designed to resemble traditional dairy milk in appearance, taste, and nutritional profile. These drinks are formulated to provide a similar creamy texture and flavor to cow's milk, but without any animal-derived ingredients. Non- dairy milk drinks are suitable for individuals who are lactose intolerant, allergic to dairy, or following a vegan or plant-based diet. Non-dairy milk products may be supplemented with recombinant donkey BLG to closer resemble the protein content and functional properties of traditional dairy milk, enhancing their nutritional value and improving their texture and stability.

[0174] Supplementing a milk drink with recombinant donkey p-lactoglobu li n (BLG) may significantly enhance the quality and authenticity of the beverage. Recombinant donkey BLG is a high- quality protein that closely mimics the proteins found in traditional dairy milk, providing a more genuine milk-like taste and creamy texture. Its inclusion can improve the solubility and stability of the milk drink, ensuring a smooth and consistent beverage. Additionally, the hypoallergenic properties of recombinant donkey BLG make the milk drink suitable for individuals with dairy allergies or lactose intolerance, expanding its appeal to a broader range of consumers.

[0175] In certain embodiments, the invention relates to a milk drink, such as a synthetic milk drink or a non-dairy milk drink, in which the protein portion comprises or consists of recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0176] In certain embodiments, the milk drink, such as the synthetic milk drink or the non-dairy milk drink, comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0177] Preferably, the food product according to the invention is a milk drink, such as a synthetic milk drink or a non-dairy milk drink, in which the protein portion comprises or consists of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0178] In a particular embodiment, the invention relates to a milk drink comprising a recombinant donkey p-lactoglobuli n (BLG), preferably wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1.

[0179] In certain embodiments, the milk drink, such as the synthetic milk drink or the non-dairy milk drink, according to the invention comprises at least 0.2g, 0.4g, 0.6g, 0.8g, lg, 1.2g, 1,4g, 1.6g, 1.8g, 2.0g, 2.2g, 2.4g, 2.6g, 2.8g, or 3g of recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or of a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, per 100 mL of final product. Preferably, the food product according to the invention is a milk drink, such as a synthetic milk drink or a non-dairy milk drink, comprising at least 0.2g, 0.4g, 0.6g, 0.8g, lg, 1.2g, 1,4g, 1.6g, 1.8g, 2.0g, 2.2g, 2.4g, 2.6g, 2.8g, or 3g of a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1, per 100 mL of final product.

[0180] An exemplary composition of a synthetic milk drink may comprise (per 100 mL of final product):

[0181] Fat: 3.0 g

[0182] - of which saturated fatty acids: 1.5 g Carbohydrates: 5.0 g

[0183] - of which sugars: 4.5 g

[0184] Dietary fiber: 0.5 g

[0185] Protein: 3.3 g (which may comprise or consist of recombinant donkey BLG; see above)

[0186] Salt: 0.1 g

[0187] The milk drink according to the invention may further include phosphates, such as dipotassium phosphate, disodium phosphate, or tripotassium phosphate, to enhance stability when mixed with coffee. The addition of phosphates helps prevent curdling and maintains a smooth, consistent texture in the milk drink, even when combined with the acidic components of coffee. This ensures that the milk drink remains visually appealing and palatable.

[0188] The milk drink may further include calcium. Calcium is an essential mineral that plays a crucial role in building and maintaining strong bones and teeth, supporting muscle function, and facilitating nerve transmission. By fortifying the milk drink with calcium, consumers can meet their daily calcium requirements, which is particularly important for individuals who may not get enough calcium from their regular diet, such as children, adolescents, pregnant women, and the elderly.

[0189] Specifically, the milk drink according to the invention is a hypoallergenic drink.

[0190] In certain embodiments, the food product according to the invention is an instant coffee powder comprising a recombinant donkey BLG.

[0191] Instant coffee powder is a type of coffee product made by brewing coffee beans and then removing the water content to create a concentrated, dry powder. This process typically involves brewing the coffee, concentrating the liquid, and then using methods such as spray drying or freeze drying to produce the final powder. Instant coffee powder is designed to dissolve quickly in cold and / or hot water, allowing for the rapid preparation of a cup of coffee without the need for brewing.

[0192] Instant coffee powder may be supplemented with recombinant donkey BLG to enhance its creaminess and mouthfeel, increase its protein content, and provide hypoallergenic properties suitable for individuals with dairy allergies. Additionally, recombinant donkey BLG may improve the stability and solubility of the coffee powder. It may also enhance the foaming and emulsifying properties. This supplementation offers both sensory and nutritional benefits, catering to a wider range of consumer preferences and dietary needs.

[0193] In certain embodiments, the invention relates to an instant coffee powder supplemented with recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0194] In certain embodiments, the instant coffee powder is supplemented with the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA). Preferably, the food product according to the invention is an instant coffee powder comprising a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0195] In certain embodiments, the food product according to the invention is a baby food comprising a recombinant donkey BLG.

[0196] Baby food is a specially formulated food product designed to meet the nutritional needs of infants and young children during their early stages of growth and development. It is typically made from pureed fruits, vegetables, grains, and meats, and is often fortified with essential vitamins and minerals to ensure a balanced diet. Baby food is available in various forms, including jars, pouches, cereals, and snacks, and is designed to be easy to digest and safe for consumption by infants. The texture and consistency of baby food are carefully controlled to accommodate the developmental stages of a baby's eating abilities, transitioning from smooth purees to more textured foods as the child grows.

[0197] Supplementing baby food with recombinant donkey BLG may provide several benefits. Due to its hypoallergenic properties, recombinant donkey BLG is suitable for infants who may be allergic to traditional dairy proteins. This ensures that babies with dairy allergies can still receive the essential proteins needed forgrowth and development. Additionally, recombinant donkey BLG may enhance the protein content of baby food, supporting muscle and tissue development. Its functional properties, such as improved solubility and stability, may also enhance the texture and consistency of baby food, making it more palatable and easier for infants to consume.

[0198] In certain embodiments, the invention relates to a baby food supplemented with recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0199] In certain embodiments, the baby food is supplemented with the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0200] Preferably, the food product according to the invention is a baby food comprising a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0201] In certain embodiments, the food product according to the invention is a food or nutritional supplement comprising a recombinant donkey BLG.

[0202] A food or nutritional supplement is a product intended to provide additional nutrients that may be missing or insufficient in a person's regular diet. These supplements can come in various forms, including tablets, capsules, powders, liquids, and bars. They are designed to deliver specific vitamins, minerals, amino acids, proteins, fibers, fatty acids, or other essential nutrients to support overall health and well-being. Nutritional supplements are often used to address dietary deficiencies, enhance physical performance, support immune function, promote healthy aging, and improve specific health conditions. They are regulated to ensure safety, efficacy, and quality, and are typically consumed in addition to a balanced diet rather than as a replacement for whole foods.

[0203] Food or nutritional supplements may be supplemented with recombinant donkey BLG to provide a high-quality, hypoallergenic protein source. This makes it suitable for individuals with dairy allergies, expanding the range of consumers who can benefit from protein supplementation. Recombinant donkey BLG enhances the protein content, supporting muscle repair and growth, and offers excellent solubility and stability, making it easy to incorporate into various supplement forms like powders, bars, and drinks. Its functional properties improve texture and consistency, while providing essential amino acids crucial for overall health.

[0204] In certain embodiments, the invention relates to a food or nutritional supplement comprising recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0205] In certain embodiments, the food or nutritional supplement comprises the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA). Preferably, the food product according to the invention is a food or nutritional supplement comprising a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0206] In certain embodiments, the food product according to the invention is a creamer comprising a recombinant donkey BLG.

[0207] A creamer is a food product used to add a creamy texture and flavor to beverages such as coffee and tea. Creamers can be dairy-based, made from milk or cream, or non-dairy, made from plant-based ingredients such as soy, almond, coconut, or oat. They are available in liquid, powdered, or concentrated forms and often contain additional ingredients like sweeteners, flavorings, and emulsifiers to enhance taste and consistency. Creamers are designed to dissolve easily in hot or cold beverages, providing a rich and smooth mouthfeel.

[0208] Creamers may be supplemented with recombinant donkey BLG to enhance their nutritional and functional properties. Recombinant donkey BLG is a high-quality protein known for its hypoallergenic properties, making it suitable for individuals with dairy allergies. This allows non-dairy creamers to provide a protein boost without causing allergic reactions. Additionally, recombinant donkey BLG can improve the texture and stability of creamers, ensuring a smooth and consistent product that blends well with beverages.

[0209] In certain embodiments, the invention relates to a creamer supplemented with recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0210] In certain embodiments, the creamer is supplemented with the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N- terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0211] Preferably, the food product according to the invention is a creamer comprising a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0212] In certain embodiments, the food product according to the invention is a tube feeding formula comprising a recombinant donkey BLG.

[0213] Tube feeding, also known as enteral nutrition, is a method of delivering nutrition directly to the stomach or small intestine through a flexible tube. This technique is used for individuals who are unable to consume food orally due to medical conditions, surgeries, or other health issues that impair their ability to eat, swallow, or digest food normally. Tube feeding ensures that patients receive the necessary nutrients, including proteins, carbohydrates, fats, vitamins, and minerals, to maintain their health and support recovery.

[0214] Tube feeding formulas may contain recombinant donkey p-lactoglobuli n BLG to enhance their nutritional and functional properties. Recombinant donkey BLG is a high-quality, hypoallergic protein, making it suitable for patients with dairy allergies or intolerances. This ensures that individuals who are allergic to traditional dairy proteins can still receive essential proteins necessary for their health and recovery.

[0215] In certain embodiments, the invention relates to a tube feeding formula supplemented with recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID N0:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0216] In certain embodiments, the tube feeding formula is supplemented with the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA). Preferably, the food product according to the invention is a tube feeding formula comprising a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0217] In certain embodiments, the food product according to the invention is a medical food, such as a food for special medical purposes (FSMP), comprising a recombinant donkey BLG.

[0218] Medical food and Foods for Special Medical Purposes (FSMPs) are specially formulated food products designed to meet the specific nutritional needs of individuals with medical conditions, diseases, or disorders that require dietary management under medical supervision. These products provide complete or supplemental nutrition when regular food intake is insufficient, impossible, or inappropriate due to medical reasons. Medical foods and FSMPs are developed based on scientific evidence and are subject to strict regulatory standards to ensure their safety, efficacy, and quality. They are tailored to address unique nutritional requirements and play a crucial role in supporting the health and recovery of patients.

[0219] Medical foods or FSMPs may be supplemented with recombinant donkey BLG to provide a high-quality, hypoallergenic protein source that is suitable for individuals with dairy allergies or intolerances. Recombinant donkey BLG offers essential amino acids necessary for muscle maintenance, tissue repair, and overall health, making it an ideal addition to support the nutritional needs of patients with specific medical conditions. Its excellent solubility and stability ensure that the protein remains effective and easy to incorporate into various formulations, while its hypoallergenic properties reduce the risk of allergic reactions. By including recombinant donkey BLG, medical foods and FSMPs may deliver enhanced nutritional value, improved functionality, and cater to a broader range of dietary requirements, ensuring better patient outcomes.

[0220] In certain embodiments, the invention relates to a medical food supplemented with recombinant donkey BLG, such as recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a sequence variant of donkey BLG-1 or BLG-2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, respectively.

[0221] In certain embodiments, the medical food is supplemented with the composition according to the invention, e.g., a composition comprising a recombinant donkey BLG, preferably wherein the recombinant donkey BLG comprises or consists of SEQ ID NO:1, and an N-terminally elongated variant thereof, preferably wherein the N-terminally elongated variant comprises the N-terminal sequence motif Glu-Ala (EA) and / or Glu-Ala-Glu-Ala (EAEA).

[0222] Preferably, the food product according to the invention is a medical food comprising a recombinant protein comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:1.

[0223] In certain embodiments, the composition or food product according to the invention, such as any of the compositions and food products disclosed herein above, may be free, or essentially free, of lactose. A composition or food product is said to be essentially free of lactose if it contains such a minimal amount of lactose that it is unlikely to cause symptoms in individuals who are lactose intolerant. Preferably, the composition or food product contains less than 0.1 grams of lactose per 100 grams or 100 milliliters.

[0224] In certain embodiments, the composition or food product according to the invention, such as any of the compositions and food products disclosed herein above, may not comprise milk proteins other than the recombinant donkey BLG or the elongated variants thereof. That is, the recombinant donkey BLG may be the only milk protein present in the composition or food product according to the invention. In particular, the composition or food product according to the invention may be free or essentially free of casein or whey proteins other than BLG. In certain embodiments, the composition or food product according to the invention, such as any of the compositions and food products disclosed herein above, may be free of animal- derived ingredients. This means that the composition or food product is suitable for individuals who follow a vegan or vegetarian diet, as well as those who have ethical, religious, or health reasons for avoiding animal products.

[0225] In another aspect, the present invention relates to a recombinant protein comprising a recombinant donkey p-lactoglobuli n (BLG) as set forth in SEQ ID NO:1 fused to an N-terminal signal peptide. The protein comprising an amino acid sequence as set forth in SEQ ID NO:1 may be fused to any signal peptide that is suitable for expression and secretion in a host organism.

[0226] A signal peptide is a short, typically hydrophobic amino acid sequence located at the N- terminus of a newly synthesized protein. This peptide sequence directs the nascent protein to the appropriate cellular membrane for translocation, ensuring the protein reaches its correct cellular or extracellular location. Signal peptides are found in both prokaryotic and eukaryotic organisms and play a crucial role in the targeting and secretion of proteins. Signal peptides are beneficial for biotechnological processes because they enable the secretion of the protein product into the culture medium, facilitating easier purification and downstream processing.

[0227] In eukaryotic cells, the signal peptide is recognized and bound by the signal recognition particle (SRP) as the protein is being synthesized by the ribosome. The SRP then facilitates the translocation of the protein-ribosome complex to the endoplasmic reticulum (ER) membrane. Upon reaching the ER membrane, the complex interacts with the SRP receptor, allowing the nascent protein to be inserted into the translocon channel. The protein is then translocated into the ER lumen or integrated into the ER membrane. During or after translocation, the signal peptide is typically cleaved off by signal peptidase enzymes, allowing the mature protein to undergo proper folding and post-translational modifications.

[0228] The signal peptide fused to the N-terminus of SEQ ID NO:1 is preferably a yeast signal peptide, i.e., a signal peptide that facilitates secretion of newly synthesized proteins in yeast cells. In a preferred embodiment, the signal peptide fused to the N-terminus of SEQ ID NO:1 is a signal peptide that facilitates secretion of newly synthesized proteins in the host organisms Pichia pastoris. In Pichia pastoris, a widely used yeast expression system, several signal peptides may be employed to direct the secretion of recombinant proteins into the culture medium, facilitating easier purification and downstream processing. Among the most frequently used signal peptides are the a-mating factor (a-MF) signal peptide from Saccharomyces cerevisiae, known for its high efficiency in protein secretion, and the glucoamylase signal peptide from Aspergillus niger, often used for enzyme production. Other notable signal peptides include the killer toxin signal peptide from Kluyveromyces lactis, the acid phosphatase (PHO1) signal peptide from Saccharomyces cerevisiae, and the invertase (SUC2) signal peptide from Saccharomyces cerevisiae.

[0229] Preferably, the recombinant donkey BLG comprising an amino acids sequence as set forth in SEQ ID NO:1 is fused to an a-mating factor (a-MF) signal peptide having the sequence set forth in SEQ ID NO:8:

[0230] SEQ ID NO:8

[0231] MRFPSI FTAVLFAASSALAAPVNTTTEDETAQI PAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAA KEEGVSLEKREAEA

[0232] Accordingly, in a particular embodiment, the invention relates to the recombinant protein according to the invention, wherein the N-terminal signal peptide comprises an amino acid sequence as set forth in SEQ ID NO:8.

[0233] That is, the recombinant protein, including the signal peptide, may have an amino acid sequence as set forth in SEQ ID NO:9:

[0234] SEQ ID NO:9

[0235] MRFPSI FTAVLFAASSALAAPVNTTTEDETAQI PAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAA KEEGVSLEKREAEATNI PQTMQDLDLQEVAGKWHSVAMAASDI SLLDSEEAPLRVYIEKLRPTPEDNLEI ILREG ENKGCAEKKI FAEKTESPAEFKINYLDEDTVFALDSDYKNYLFLCMKNAATPGQSLVCQYLARTQMVDEEIMEKF RRALQPLPGRVQIVPDLTRMAERCRI In another aspect, the invention relates to a polynucleotide encoding the recombinant protein according to the invention.

[0236] That is, the invention further encompasses polynucleotides encoding a recombinant donkey BLG having an amino acid sequence as set forth in SEQ ID NO:1 fused to an N-terminal signal peptide, preferably a signal peptide having a sequence as set forth in SEQ ID NO:8.

[0237] In certain embodiments, the polynucleotide according to the invention may encode a protein having an amino acid sequence as set forth in SEQ ID NO:9.

[0238] A polynucleotide is a long, linear polymer composed of a sequence of nucleotide monomers, typically linked together by phosphodiester bonds. Each nucleotide consists of a nitrogenous base (adenine, thymine, cytosine, guanine, or uracil), a five-carbon sugar (ribose in RNA or deoxyribose in DNA), and, typically, a phosphate group. Polynucleotides form the backbone of nucleic acids, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), which are essential for storing and transmitting genetic information in living organisms.

[0239] In certain embodiments, the polynucleotide is a DNA molecule and may be part of a gene encoding the recombinant donkey BLG in a host organism.

[0240] In certain embodiments, the polynucleotide may be an RNA molecule encoding the recombinant donkey BLG, such as an mRNA.

[0241] The polynucleotide encoding the recombinant donkey BLG may further comprise regulatory elements, such as promoters or terminators. These regulatory elements play crucial roles in controlling the expression of the recombinant protein. Promoters are DNA sequences that initiate transcription by providing binding sites for RNA polymerase and other transcription factors. Terminators signal the end of transcription, ensuring the proper termination of the RNA transcript. Additionally, the polynucleotide may include enhancers, which are sequences that increase the efficiency of transcription, and silencers, which are sequences that repress transcription. Other regulatory elements that may be included are ribosome binding sites (RBS) for efficient translation initiation, introns for proper RNA splicing, and polyadenylation signals for mRNA stability and export. By incorporating these regulatory elements, the expression of recombinant donkey BLG can be finely tuned to achieve optimal levels of protein production.

[0242] In a particular embodiment, the invention relates to the polynucleotide according to the invention, further comprising a promoter sequence.

[0243] The promoter may be any promoter that is suitable for driving expression of a gene encoding a recombinant donkey BLG in a host organism. The promoter may be an inducible or constitutive promoter.

[0244] An inducible promoter is a type of promoter that initiates gene expression in response to specific external stimuli or environmental conditions, such as the presence of a particular chemical inducer, temperature change, or light exposure. This allows for controlled expression of the recombinant protein, allowing to regulate the timing and level of protein production.

[0245] In contrast, a constitutive promoter is a type of promoter that drives continuous, unregulated expression of the gene under normal cellular conditions, regardless of external factors. Constitutive promoters are useful for maintaining consistent levels of gene expression and protein production.

[0246] By selecting the appropriate promoter, the expression of recombinant donkey BLG can be tailored to meet the specific requirements of the host organism and the desired experimental or production conditions.

[0247] In a particular embodiment, the invention relates to the polynucleotide according to the invention, wherein the promoter is an inducible promoter.

[0248] In certain embodiments, the promoter comprised in the polynucleotide according to the invention is a promoter, preferably an inducible promoter, that drives expression of a recombinant protein is a yeast cell, preferably a Pichia pastoris cell.

[0249] In a particular embodiment, the invention relates to the polynucleotide according to the invention, wherein the inducible promoter is the AOX1 promoter. The A0X1 promoter is a strong, tightly regulated inducible promoter derived from the alcohol oxidase 1 gene in Pichia pastoris. This promoter is induced by methanol, allowing for high- level expression of recombinant proteins when methanol is present in the growth medium. The AOX1 promoter is repressed in the presence of other carbon sources, such as glucose or glycerol, providing a means to control the timing and level of protein expression. This inducible system is particularly advantageous for producing proteins that may be toxic to the host cells if expressed continuously. By using the AOX1 promoter, robust and controlled expression of recombinant donkey BLG can be achieved in Pichia pastoris. The AOX1 promoter is well known in the art and has been described, inter alia, by Oz^elik et al. (Methods Mol Biol, 2019:1923:97- 112), which is fully incorporated herein by reference.

[0250] If the polynucleotide comprises a promoter sequence, the polynucleotide is preferably a DNA molecule. Such DNA molecule may be integrated into the genome of a host organism, such as Pichia pastoris, or may be located in an extrachromosomal element, such as a plasmid or vector.

[0251] Vectors are extrachromosomal elements commonly used in molecular biology. Thus, in another aspect, the invention relates to a vector comprising the polynucleotide according to the invention.

[0252] A vector is a DNA molecule that is used as a vehicle to transfer genetic material into a host cell. Vectors are essential tools in genetic engineering, allowing for the replication, expression, and manipulation of specific genes within a host organism. A vector can facilitate the introduction and stable expression of the recombinant gene in the host organism, enabling the production of the desired protein.

[0253] Particular types of vectors used for such purposes are plasmids. A plasmid is a small, circular, double-stranded DNA molecule that is distinct from the chromosomal DNA of a cell. Plasmids are capable of autonomous replication within a host cell and are commonly used in molecular biology as vectors for gene cloning, expression, and manipulation. They often carry genes that confer advantageous traits, such as antibiotic resistance, and can be engineered to include specific genetic elements, such as promoters, selectable markers, and multiple cloning sites.

[0254] In addition to plasmids, other types of vectors can be used for similar purposes, including Bacterial Artificial Chromosomes (BACs), Yeast Artificial Chromosomes (YACs), and viral vectors. BACs and YACs are larger vectors capable of carrying larger DNA fragments, making them suitable for applications such as genomic library construction and large-scale gene cloning. Viral vectors, such as lentiviruses and adenoviruses, are used for efficient gene delivery and expression in eukaryotic cells.

[0255] Further encompassed herein are host cells comprising a polynucleotide or vector encoding a recombinant donkey BLG. Thus, in a particular embodiment, the invention relates to a host cell comprising the polynucleotide according to the invention or the vector according to the invention.

[0256] The host cell is preferably a host cell that is suitable for the large-scale production of the recombinant donkey BLG. The host cells may be, without limitation, a bacterial cell, such as Escherichia coli, Bacillus subtilis, or Bacillus Licheniformis, a fungal cell, such as cells from Trichoderma reesei, cells from Aspergillus niger, cells from Aspergillus nidulans, or cells from Aspergillus oryzae, a yeast cell, such as Pichia pastoris, Kluyveromyces lactis, Yarrowia lipolytica or Saccharomyces cerevisiae, an insect cell, such as Spodoptera frugiperda (Sf9) or Trichoplusia ni (High Five), a plant cell, a microalgae cell, or a mammalian cell.

[0257] The host cells is chosen based on its ability to produce high yields of recombinant protein, ease of genetic manipulation, scalability of culture conditions, and post-translational modification capabilities. By selecting an appropriate host cell, the production process can be optimized to achieve efficient and cost-effective production of recombinant donkey BLG.

[0258] In the appended examples, Pichia pastoris was utilized for the large-scale production of recombinant donkey BLG. It was demonstrated that the resulting protein, including post- translational modifications introduced by P. pastoris (if any), was hypoallergenic in human cell culture. Thus, in a particular embodiment, the invention relates to the host cell according to the invention, wherein the host cell is a yeast cell, in particular wherein the host cell is a Pichia pastoris cell.

[0259] The polynucleotide, the vector and the host cell according to the invention may be generated using standard molecular biology methods known in the art.

[0260] Further encompassed herein is a method for producing the recombinant donkey BLG. Thus, in another aspect, the invention relates to a method for producing a recombinant donkey |3- lactoglobulin (BLG), the method comprising the steps of: a) culturing the host cell according to the invention under suitable conditions; and b) isolating, and optionally purifying, the recombinant donkey BLG from the culture medium.

[0261] In certain embodiments, the host cell comprises a vector encoding a recombinant donkey BLG. In other embodiments, the host cell is a genetically engineered cell comprising a polynucleotide encoding the recombinant donkey BLG integrated into its genome. Preferably, the host cell for the production of the recombinant donkey BLG is a yeast cell, more preferably a Pichia pastoris cell, and the gene encoding the recombinant donkey BLG is expressed from a plasmid comprising an inducible promoter.

[0262] The skilled person is aware that culture conditions for the expression of a recombinant protein vary between host cells. Additionally, if expression is driven by an inducible promoter, the induction conditions will vary depending on the specific promoter used. However, the skilled person is capable of identifying suitable culture conditions and induction parameters for optimal expression of the recombinant protein in the chosen host cell. This includes adjusting factors such as temperature, pH, nutrient composition, and inducer concentration to achieve efficient and high-yield production of the recombinant donkey BLG. An exemplary protocol for the large-scale production of the recombinant donkey BLG is provided in the appended examples. Another aspect of the invention relates to a method for preparing a food product, the method comprising a) providing a composition according to the invention; and b) mixing the composition with one or more additional ingredients to produce the food product.

[0263] In various embodiments, the method may involve mixing the composition according to the invention or a recombinant donkey BLG with one or more additional ingredients to produce a food product. The food product may be any edible consumer product capable of carrying protein, particularly any of the food products disclosed herein. The skilled person, such as a food scientist, understands that the exact method of mixing the composition or the recombinant donkey BLG with other ingredients depends on the specific type of food product. However, the skilled person would have no difficulty producing any of the food products disclosed herein using the composition according to the invention or the recombinant donkey BLG.

[0264] In another aspect, the present invention relates to methods of using the recombinant donkey BLG disclosed herein in subjects with milk protein allergies. For example, individuals with cow's milk protein allergies must strictly avoid any milk or dairy products, which severely limits their choice of foods. While non-dairy alternatives exist, these options often lack the nutritional profile and functional properties of dairy proteins. The use of recombinant donkey BLG provides a hypoallergenic alternative that closely mimics the nutritional benefits and functional characteristics of cow's milk proteins. This allows individuals with milk protein allergies to enjoy a wider variety of food products, including those that require high-quality protein content, without the risk of allergic reactions. By incorporating recombinant donkey BLG into various food products, nutritionally rich and safe options for those with milk protein allergies can be offered, enhancing their dietary choices and overall quality of life.

[0265] In a particular embodiment, the invention relates to a method for avoiding an allergic reaction in a subject that has been diagnosed with or is suspected to have a milk protein allergy, the method comprising feeding or administering to said subject a composition or food product comprising recombinant donkey p-lactoglobulin (BLG).

[0266] That is, a composition or food product comprising recombinant donkey BLG may be used to avoid allergic reactions in individuals diagnosed with or suspected of having a milk protein allergy. By incorporating recombinant donkey BLG, these food products provide a hypoallergenic protein source, allowing individuals with milk protein allergies to safely consume a wider range of foods without the risk of adverse reactions.

[0267] Milk protein allergy is an adverse immune response to one or more proteins found in milk, such as casein and whey. In cow's milk, p-lactoglobulin (BLG) is one of the primary drivers of milk protein allergies, as confirmed in the appended examples and illustrated in Fig. 4. This condition occurs when the body's immune system mistakenly identifies these proteins as harmful and triggers an allergic reaction. Symptoms of milk protein allergy can include hives, gastrointestinal distress (such as nausea, vomiting, diarrhea, or abdominal pain), respiratory issues (such as wheezing, coughing, or shortness of breath), and in severe cases, anaphylaxis.

[0268] A subject can be diagnosed with a milk protein allergy through a combination of clinical evaluation and diagnostic tests. The process typically involves taking a detailed medical history to assess symptoms such as hives, gastrointestinal distress, respiratory issues, or anaphylaxis upon consuming milk or dairy products. This is followed by allergy testing, which may include skin prick tests to detect immediate allergic reactions and blood tests to measure specific IgE antibodies to milk proteins. An elimination diet, where milk and dairy products are removed from the subject's diet for a period, may be recommended to observe symptom improvement. In some cases, an oral food challenge under medical supervision is conducted to confirm the diagnosis by gradually reintroducing milk products and monitoring for allergic reactions.

[0269] A subject that is suspected of having a milk protein allergy is an individual who exhibits symptoms that may indicate an allergic reaction to milk proteins, but has not yet been definitively diagnosed. These symptoms can include hives, gastrointestinal distress (such as nausea, vomiting, diarrhea, or abdominal pain), respiratory issues (such as wheezing, coughing, or shortness of breath), and in severe cases, anaphylaxis. The suspicion arises based on the correlation of these symptoms with the consumption of milk or dairy products. To confirm the diagnosis, the individual undergoes a series of diagnostic tests and evaluations, as explained above. In certain embodiments, a subject that is suspected of having a milk protein allergy may be a newborn who shows adverse reactions to infant formula.

[0270] In a preferred embodiment, the milk protein allergy in the subject, whether diagnosed or suspected, is specifically a cow's milk protein allergy (CMPA).

[0271] The subject is preferably a human and may be of any age, including newborns, infants, children, adolescents, adults, and the elderly.

[0272] In certain embodiments, the subject is a newborn or an infant. Newborns, also known as neonates, are infants who are within the first 28 days of life. An infant is a young child typically defined as being between the ages of birth and one year. During this critical period, newborns and infants undergo rapid growth and development, and their immune systems are still maturing. As a result, they may be more susceptible to allergic reactions, including milk protein allergies. Identifying and managing milk protein allergies in newborns and infants is essential to ensure their health and well-being. Milk protein allergies may be avoided in newborns and children by feeding them infant formula comprising the recombinant donkey BLG disclosed herein.

[0273] In certain embodiments, the subject is a child. A child is typically defined as an individual between the ages of one and twelve years.

[0274] In certain embodiments, the subject is an adolescent. An adolescent is typically defined as an individual between the ages of 13 and 18 years.

[0275] In certain embodiments, the subject is an adult. An adult is typically defined as an individual who is 18 years of age or older.

[0276] In certain embodiments, the subject is an elderly individual. The elderly are typically defined as individuals who are 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older or 80 years of age or older. An allergic reaction to milk protein may be avoided in the subject by feeding or administering the hypoallergenic composition or food product according to the invention. The recombinant donkey BLG included in these compositions or food products has been demonstrated to be hypoallergenic, in contrast to bovine BLG. Therefore, consuming the hypoallergenic composition or food product according to the invention, which is preferably free of other cow milk components, particularly cow milk proteins, prevents the occurrence of an allergic reaction in subjects with a milk protein allergy, specifically cow's milk protein allergy. Thus, in the context of the present invention, the term "avoid" refers to the prevention or reduction of the likelihood of an allergic reaction in a subject who has been diagnosed with or is suspected to have a milk protein allergy.

[0277] In a particular embodiment, the invention relates to a composition or food product comprising recombinant donkey p-lactoglobulin (BLG) for use in avoiding an allergic reaction in a subject that has been diagnosed with or is suspected to have a milk protein allergy.

[0278] Another aspect of the present invention relates to a method for reducing, preventing or avoiding the symptoms associated with cow milk allergy or lactose intolerance in a subject, the method comprising feeding or administering to said subject a composition or food product comprising recombinant donkey p-lactoglobulin (BLG).

[0279] That is, the composition or food product according to the invention may further by used for reducing, preventing or avoiding the symptoms associated with cow milk allergy or lactose intolerance.

[0280] Cow's milk allergy (CMA), which is used interchangeably herein with cow's milk protein allergy (CPMA), is an immune-mediated adverse reaction to one or more proteins found in cow's milk, such as casein and -lactoglobulin. It is one of the most common food allergies in infants and can manifest through a variety of symptoms; see Giannetti et al. (Nutrients, 2021, 13, 1525) for more detail. The symptoms of cow milk allergy can be categorized into three main types: IgE-mediated, non-lgE-mediated, and mixed IgE and non-lgE-mediated reactions. IgE-mediated reactions are the most common and can affect multiple systems in the body. Skin reactions include urticaria, angioedema, erythema, and pruritus. Cardiovascular symptoms may involve tachycardia, dizziness, and loss of consciousness or fainting. Gastrointestinal reactions can present as oral allergy syndrome, colicky abdominal pain, nausea, vomiting, and diarrhea. Anaphylaxis, a severe and life-threatening systemic hypersensitivity reaction, is also a possible IgE-mediated response. Respiratory reactions, such as asthma and rhinitis, can occur secondary to the ingestion or inhalation of cow milk proteins, leading to wheezing, coughing, and nasal congestion.

[0281] Non-lgE-mediated reactions are caused by different immune mechanisms and typically present with delayed symptoms. These reactions can include food protein-induced enterocolitis, which involves the entire gastrointestinal tract, and food protein-induced enteropathy, which affects only the small bowel. Food protein-induced proctitis / proctocolitis involves the rectum and colon, while food protein-induced pulmonary hemosiderosis (Heiner syndrome) affects the entire respiratory tract, leading to symptoms such as failure to thrive, iron deficiency, and anemia.

[0282] Mixed IgE and non-lgE-mediated reactions involve both humoral and cellular immune mechanisms and can present with acute and chronic symptoms. Eosinophilic gastroenteritis is characterized by ascites, weight loss, edema, and obstruction. Atopic dermatitis presents as a moderate-to-severe rash. Eosinophilic esophagitis involves feeding disorders, reflux symptoms, vomiting, dysphagia, and food impaction.

[0283] The composition or food product according to the invention is preferably used as a replacement for dairy products. In particular, the recombinant donkey BLG provides a high- quality, hypoallergenic protein source that mimics the nutritional benefits, functional properties, and sensory attributes of cow's milk proteins without causing adverse reactions. Accordingly, the composition or food product according to the invention can be used not only for reducing, preventing, or avoiding the symptoms associated with allergies to bovine BLG but also for reducing, preventing, or avoiding the symptoms associated with other milk proteins, as well as lactose intolerance. Lactose intolerance is a clinical syndrome resulting from the inability to properly digest lactose, the sugar found in milk and dairy products. This condition occurs due to a deficiency in lactase, the enzyme responsible for breaking down lactose into glucose and galactose in the small intestine. Lactase deficiency can be primary, which is a normal age-related decline in lactase production, or secondary, resulting from damage to the intestinal lining due to conditions such as gastroenteritis, bowel surgery, or other gastrointestinal disorders. Congenital lactase deficiency, a rare hereditary disorder, results in the complete absence of lactase activity from birth.

[0284] The symptoms of lactose intolerance primarily affect the gastrointestinal tract and typically occur several hours after the consumption of lactose-containing foods. Common symptoms include nausea, vomiting, abdominal pain, bloating, gas, diarrhea, and borborygmi (rumbling stomach sounds). In severe cases, lactose intolerance can lead to dehydration, electrolyte imbalances, and failure to thrive, particularly in infants. The severity of symptoms can vary depending on the amount of lactose consumed and the individual's level of lactase deficiency.

[0285] The composition or food product according to the invention may be used to reduce one or more symptoms associated with cow milk allergy or lactose intolerance, such as any of the symptoms disclosed herein. Reducing these symptoms involves decreasing their severity or frequency. In particular, to achieve this reduction, milk products may be partially or entirely replaced with compositions or food products comprising recombinant donkey p-lactoglobul i n (BLG) in a subject's diet.

[0286] Alternatively or in addition, the composition or food product according to the invention may be used to prevent one or more symptoms associated with cow milk allergy or lactose intolerance, such as any of the symptoms disclosed herein. Preventing these symptoms involves taking proactive measures to stop them from occurring in the first place. In particular, to achieve this prevention, milk products may be replaced with compositions or food products comprising recombinant donkey p-lactoglobulin (BLG) in a subject's diet.

[0287] Alternatively or in addition, the composition or food product according to the invention may be used to avoid one or more symptoms associated with cow milk allergy or lactose intolerance, such as any of the symptoms disclosed herein. Avoiding these symptoms involves completely eliminating exposure to the allergens or lactose that trigger adverse reactions. In particular, to achieve this avoidance, milk products may be entirely replaced with compositions or food products comprising recombinant donkey p-lactoglobulin (BLG) in a subject's diet.

[0288] In a particular embodiment, the invention relates to a composition or food product comprising recombinant donkey p-lactoglobulin (BLG) for use in reducing, preventing or avoiding the symptoms associated with cow milk allergy or lactose intolerance in a subject.

[0289] The composition or food product comprising the recombinant donkey BLG for use in the methods of the invention may be any composition or food product comprising a recombinant donkey BLG, preferably a recombinant donkey BLG-1 (SEQ ID NO:1) or BLG-2 (SEQ ID NO:2), or a variant thereof, as defined elsewhere herein.

[0290] It is, however, preferred that the composition or food product for use in the methods of the invention is any of the compositions or food products disclosed herein. Thus in a particular embodiment, the invention relates to the method according to the invention, wherein the composition is the composition according to the invention; and wherein the food product is the food product according to the invention.

[0291] In certain embodiments, the composition or food product for use in the methods of the invention is free of cow milk; or any components derived from it. Specifically, the composition or food product is preferably free of cow milk proteins, such as caseins and whey proteins, as well as lactose. This ensures that the composition or food product is suitable for individuals with cow milk allergy or lactose intolerance, providing a safe and effective alternative without the risk of triggering adverse reactions.

[0292] Cow milk allergy is one of the most common allergies in infants, posing a significant challenge since infant formula typically contains cow milk protein. Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the subject is an infant and wherein the food product is infant formula. Feeding a food product comprising the recombinant donkey BLG, and preferably free of other components of cow milk, to an infant may avoid an allergic reaction in a subject that has been diagnosed with or is suspected to have a milk protein allergy.

[0293] Alternatively or in addition, feeding a food product comprising the recombinant donkey BLG, and preferably free of other components of cow milk, to an infant may reduce, prevent or avoid the symptoms associated with cow milk allergy or lactose intolerance.

[0294] As used herein, "about," "approximately" and "substantially" are understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. All ranges are inclusive of the endpoints of the range. For example, an amount between 1 and 10 includes both 1 and 10.

[0295] As used in this disclosure and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0296] The words "comprise," "comprises" and "comprising" are to be interpreted inclusively rather than exclusively. Likewise, the terms "include," "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Nevertheless, the compositions and methods disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term "comprising" includes a disclosure of embodiments "consisting essentially of" and "consisting of" the components or steps identified. The terms "at least one of" and "and / or" used respectively in the context of "at least one of X and Y" and "X and / or Y" should be interpreted as "X without Y," or "Y without X," or "both X and Y." Where used herein, the terms "example" and "such as," particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive.

[0297] In the foregoing detailed description of the invention, a number of individual elements, characterizing features, techniques and / or steps are disclosed. It is readily recognized that each of these has benefit not only individually when considered or used alone, but also when considered and used in combination with one another. Accordingly, to avoid exceedingly repetitious and redundant passages, this description has refrained from reiterating every possible combination and permutation. Nevertheless, whether expressly recited or not, it is understood that such combinations are entirely within the scope of the presently disclosed subject matter.

[0298] All technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

[0299] In this specification, a numberof documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF DRAWINGS

[0300] FIG. 1: SDS PAGE analysis of protein produced by Pichia fermentation. M: marker, 1: t=30 h, 2: t=45 h, 3: t= 53h, 4: t=70 h after inoculation, 5: harvest.

[0301] FIG.2: Distribution of donkey BLG forms.

[0302] FIG.3: CD spectroscopy of recombinantly produced donkey BLG.

[0303] FIG.4: Allergenicity test of different BLG variants. Both bovine whey protein isolate (WPI) and a commercially available milk drink containing bovine BLG produced via precision fermentation induce activation of Hoxb8 cells. In contrast, none of the donkey BLG containing samples induced cell activation.

[0304] EXAMPLES

[0305] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0306] Example 1: Production of hypoallergenic donkey BLG

[0307] Plasmids

[0308] Plasmids were constructed for the inducible and constitutive expression of donkey BLG (SEQ ID NO:1, Table 1). Each plasmid included the following components: an inducible promoter (e.g., AOX1 promoter, a sequence encoding a signal peptide derived from a yeast protein sequence (alpha mating factor, SEQ ID NO:2, Table 1), a sequence encoding the wild type donkey BLG variant to be expressed; a yeast transcription terminator sequence (e.g., AOX1, AOD, or CYC1; a bacterial origin of replication from pUC19 to enable replication of the plasmid in E. coli, and a selectable marker cassette (e.g., kanR or zeocinR) to enable selection in bacteria and yeast with antibiotics.

[0309] Table 1: Amino Acid sequence

[0310] Strain Construction

[0311] Prior to transformation, 20 pg of each constructed plasmid was linearized by digestion with the restriction enzyme Sacl. The digested plasmids were then purified and resuspended in 10 pl distilled water. Competent Pichia pastoris cells were prepared as follows: A 500 mL culture of P. pastoris was grown to ODeoo 1.3-1.5 in YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose) at 30 °C. Cells were centrifuged at 1500 x g for 5 min and then resuspended in 500 mL ice cold water. Cells were then centrifuged at 1500 x g for 5 min and then resuspended in 250 mL ice cold water. Cells were centrifuged at 1500 x g for 5 min and then resuspended in 20 mL of ice cold IM sorbitol. Cells were centrifuged at 1500 x g for 5 min and then resuspended in 1 mL ice cold IM sorbitol.

[0312] 80 pL of cells were combined with 10 pg of the linearized plasmid DNA in a chilled 2 mm electroporation cuvette, incubated for 5 min on ice and then subjected to a 1.5 kV pulse (25 pF, 200Q). Immediately afterwards, 1 mL of cold 1:1 YPD:1 M sorbitol was added, and the cells were transferred to a 15 mL sterile culture tube. The cells were then incubated at 30 °C for 1- 2 h without shaking. Finally, the cells were plated onto YPDS agar plates (10 g / L yeast extract, 20 g / L peptone, 20 g / L dextrose, IM sorbitol) containing 100 pg / mL zeocin and grown for two days at 30° C.

[0313] Protein Production

[0314] Single colonies were picked from the agar plates and grown in BMGY medium (Table 2) at 30 °C and 250 rpm until ODeoo 2-6 was reached. Then a 2 L fermentation vessel loaded with 1 L sterile basal salts medium, pH=5.0 containing 4 % glycerol (Table 2) was inoculated with the culture to an ODeoo of 0.2. The entire fermentation was carried out at a temperature of 30 °C. After an initial batch phase, a glycerol fed-batch phase was initiated where a total of 150 g of glycerol feed (Table 2) was fed over the course of 3 h. The fermentation was oxygen limited and the dissolved oxygen levels were kept above 20% by regulating the stirring as well as the aeration to a maximum of 1500 rpm and 2.5 vvm maximum. After consumption of the glycerol, protein production was initiated by slow addition of methanol feed. At this point, the pH was increased to pH=6.0 to improve solubility of the BLG. The speed of the methanol feed was adjusted to keep the DO levels above 20%. Induction phase was carried out for 72 h, after which the fermentation was stopped. During several points, samples were taken to analyze the protein production (FIG.l). Protein purification

[0315] The biomass was separated from the secreted protein by centrifugation at 7250 g for 20 minutes. Then, the supernatant was sterile filtered using a PALL AcroPAK Supor 0.2 pm filter. Subsequently, the filtrate was filtered using a 100 kDa ceramic membrane. The filtrate was then diaf i Ite red / u Itraf i Ite red against 4 volumes of demineralized water using a 3 kDa hollow fiber. The retentate was then flash frozen using dry ice in isopropanol and lyophilized. After lyophilization, the powder was kept at room temperature until further use.

[0316] Analysis

[0317] The protein was analyzed by LC-MS and circular dichroism (CD) spectroscopy for sequence and structural integrity.

[0318] • LC-MS

[0319] Briefly, 5 mg of lyophilized protein were dissolved in 600 pL denaturating and reducing buffer (6.25 M guanidine, 5.2 mM trisodium citrate, 19.2 mM DTT, pH=8.5). The sample was incubated at room temperature for 30 minutes and cleared by centrifugation at 16'000 x g for 10 minutes on a table-top centrifuge. The cleared supernatants were separated on a C4 column (Acquity UPLC Protein BEH C4, 300 A, 1.7 pm, 2.1 mm x 150 mm; Waters Corporation, Milford, MA) using a mobile phase composed of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in acetonitrile / water at 90:10) and the following gradient at 0.5 mL / min: 20, 40, 45, 46.5, 51.4, 95, 95, 20, 20, 95, 95, 20, and 20% buffer B at 0, 3, 5, 6, 14, 17, 18, 18.1, 19, 19.1, 21, 21.1 and 24 min, respectively. The MS signal was recorded from 2 to 24 min in full MS mode on a Thermo Q-Exactive HF (ThermoFisher Scientific, Waltham, MA; heater temperature, 100°C; sheath gas, 53; auxiliary gas, 14; sweep gas, 3; spray voltage, 3.5 kV; capillary temperature, 320°C; S-lens radiofrequency level (arbitrary units), 70; mass range, 800-2,000 m / z; resolution, 15,000; microscans, 5; automatic gain control target, 5e6; maximum injection time, 200 ms). Samples were then analyzed by mass spectrometry using standard ionization parameters.

[0320] • Circular dichroism

[0321] The folding of the recombinantly produced donkey BLG was investigated by means of circular dichroism spectroscopy. A Chirascan™ V100 spectrometer (Applied Photophysics Ltd, Leatherhead, UK) was used to record spectra in the far-UV region (180-260 nm) to determine the secondary structure of the protein. Measurements were performed at wavelengths ranging from 260 down to 180 nm using a bandwidth of 1 nm, with a step of 1 nm and a time- per-point of 1 s. The average of 5 scans recorded at 25°C, corrected by the average of 5 scans of blank solution recorded in the exact same conditions, is reported. The protein concentration was in the range 0.06-0.07 mg / mL at pH 7.0 and a quartz cuvette with a pathlength of 0.5 mm was used. For secondary structure determination, spectra were deconvoluted and fitted using the BeStSel web server (https: / / bestsel.elte.hu / index.php) derived from DSSP (Dictionary of Secondary Structure in Proteins). For determination of the denaturation temperature, the Global3 software was used forcing the fit with one thermal transition event.

[0322] Using LC-MS, it was found that apart from the nature identical sequence (SEQ ID NO:1, Table 1) the protein can contain N-terminal extensions of either alanine (A), alanine-glutamine (EA), two alanines and a glutamine (AEA) or two alanine-glutamine repeats (EAEA). The product distribution is shown in FIG.2.

[0323] CD spectroscopy on samples desalted protein by PD-10 column purification confirmed the structural integrity of the recombinantly produced donkey BLG (FIG.3).

[0324] Example 2: Allergenicity test

[0325] Samples

[0326] Different protein preparations were tested for their allergenicity. For sample preparations of protein powder samples, the powders were dissolved in assay buffer and, if necessary, pH was adjusted to pH=7.0.

[0327] WPI enriched in BLG corresponds to whey protein isolate comprising BLG from cow ( Lacprodan® BLG-100, Aria).

[0328] Donkey BLG PF corresponds to recombinant donkey BLG (SEQ ID NO:1) according to the invention.

[0329] Donkey milk powder corresponds to a commercial donkey milk powder.

[0330] For preparation of the donkey BLG drink, a mixture of 3 % (w / w) recombinant donkey BLG (SEQ ID NO:1), 3 % sucrose and 3 % sunflower oil was mixed in water for 20 minutes. Then, the pH was adjusted to pH=7.0 using 1 N NaOH. Subsequently, an emulsion was produced using an Emulsiflex C5 at 300 bar and the solution was treated by a UHT at 145 °C for 5 s.

[0331] The bovine BLG drink is a commercially available drink comprising recombinant cow (or bovine) BLG: Bored Cow Original Animal-free milk.

[0332] Allergenicity Test

[0333] The allergenicity of the different BLG variants were assessed using the FAST-PASE® assay (as disclosed in Zbaren et al. (J Allergy Clin Immunol 2022 Mar;149(3):1018-1030.ell) . Briefly, the assay was conducted as follows: Hoxb8 mast cells were sensitized with a recombinant monoclonal IgE antibody directed against BLG. Then different BLG samples were added in 8 different concentrations (0, 0.02, 0.1, 0.39 1.56, 6.25, 25, 100 pg / mL) to the sensitized cells. The cells were then stimulated for 30 minutes. Cells were subsequently stained, and activation was assessed by flow cytometry (FIG.4). Positive (anti-lgE, anti-FceRI and a BLG activation control) and negative controls (IgE only, no IgE) were included.

[0334] Example 3: Example of formulations

[0335] Recombinant milk drink

[0336] Table 1

[0337] Malt-containing powdered drink Infant formula powder

Claims

1. 67CLAIMS1. A hypoallergenic composition comprising a recombinant donkey p-lactoglobulin (BLG); and one or more elongated variants thereof having an N-terminal elongation with a sequence selected from: A, EA, AEA and / or EAEA.

2. The hypoallergenic composition according to claim 1, wherein the recombinant donkey BLG comprises an amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, preferably as set forth in SEQ ID NO:1.

3. The hypoallergenic composition according to claim 1 or 2, wherein the ratio of the one or more N-terminally elongated variants of the recombinant donkey BLG in the composition is at least about 40%, 50%, 60%, 70%, 80% or 90% by weight of total recombinant BLG in the composition.

4. The hypoallergenic composition according to any one of claims 1 to 3, wherein the composition is free, or essentially free from lactose; and / or wherein the composition does not comprise milk proteins other than the recombinant donkey BLG or the elongated variants thereof.

5. A food product comprising the hypoallergenic composition according to any one of claims 1 to 4.

6. The food product according to claim 5, wherein the food product is a nutritional formulation (including infant formula), a high protein product (including sport nutrition, high protein medical nutrition product, weight management product, GLP-1 nutrition companion product, protein shakes, high protein bars), a milk powder (including flavored milk powders such as cocoa powders), a malted beverage powder, an instant coffee powder, a nutritional food, a baby food, a fermented food, a yoghurt, a drinking yoghurt, an ice cream, a milk drink, a soup, a sauce, a bar, a gel, a foam, a medical food (including Foods for Special Medical Purposes (FSMPs)), a complete meal replacement,68 a tube feeding formula, a food supplement, a beverage, a dairy beverage, a beverage whitener, a creamer, a cheese, a dairy tofu, a food emulsion, a dessert, a product that requires the protein to form a heat-set gel, an acid protein fortified beverage, a jelly drink, a protein water, or a heat-set foam extruded food product.

7. The food product according to claim 5 or 6, wherein the food product is free, or essentially free from lactose; and / or wherein the food product does not comprise milk proteins other than the recombinant donkey BLG or the elongated variants thereof; and / or wherein the food product is free of animal-derived ingredients.

8. A food product comprising a recombinant donkey p-lactoglobulin (BLG), wherein the recombinant donkey BLG comprises or consists of an amino acid sequence as set forth in SEQ ID NO:1, and wherein the food product is selected from: an infant formula, a milk drink, a milk powder, a malted beverage powder, and a high protein food product.

9. A recombinant protein comprising a recombinant donkey p-lactoglobulin (BLG) as set forth in SEQ ID NO:1 fused to an N-terminal signal peptide, wherein the N-terminal signal peptide comprises an amino acid sequence as set forth in SEQ ID NO:8.

10. A polynucleotide encoding the recombinant protein according to claim 9, preferably wherein the polynucleotide further comprises a promoter sequence, preferably wherein the promoter is an inducible promoter, preferably wherein the inducible promoter is an AOX1 promoter.

11. A vector comprising the polynucleotide according to claim 10.

12. A host cell comprising the polynucleotide according to claim 10 or the vector according to claim 11, preferably wherein the host cell is a yeast cell, in particularwherein the host cell is a Pichia pastoris cell.6913. A method for producing a recombinant donkey p-lactoglobulin (BLG), the method comprising the steps of: a) culturing the host cell of claim 12 under suitable conditions; and b) isolating, and optionally purifying, the recombinant donkey BLG from the culture medium.

14. A method for preparing a food product, the method comprising a) providing a hypoallergenic composition of any one of claims 1 to 4; and b) mixing the composition with one or more additional ingredients to produce the food product.

15. A composition or food product comprising recombinant donkey p-lactoglobulin (BLG) for use in avoiding an allergic reaction in a subject that has been diagnosed with or is suspected to have a milk protein allergy.

16. A composition or food product comprising recombinant donkey -lactoglobulin (BLG) for use in reducing, preventing or avoiding the symptoms associated with cow milk allergy or lactose intolerance in a subject.

17. The composition or food product for use according to claim 15 or 16, wherein the composition is the composition according to any one of claims 1 to 4; and wherein the food product is the food product according to any one of claims 5 to 8.

18. The composition or food product for use according to any one of claims 15 to 17, wherein the subject is an infant and wherein the food product is infant formula.

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