WO2025046004A1

WO2025046004A1 - Method for the purification of a recombinant milk protein

Info

Publication number: WO2025046004A1
Application number: PCT/EP2024/074177
Authority: WO
Inventors: Arthur Maurits Christiaan Janse; Ferdinand Antoine SPROS; Niels Theodorus Margaretha VAN MOSSEVELDE
Original assignee: Vivici BV
Current assignee: Vivici BV
Priority date: 2023-08-29
Filing date: 2024-08-29
Publication date: 2025-03-06
Anticipated expiration: 2026-02-28

Abstract

The present invention relates to recombinant milk proteins, compositions comprising such recombinant milk proteins, and to methods for producing such recombinant milk proteins and compositions. In particular, the present invention relates to recombinant milk proteins purified by cationic exchange resin, resulting in recombinant milk proteins with low (poly)sugar levels.

Description

Method for the purification of a recombinant milk protein

Field of the invention

Background of the invention

Milk is a popular source of nutrition. It comprises high-quality protein, essential minerals, and vitamins. In addition, milk comprises proteins with advantageous functional characteristics that permit production of a wide variety of derivative products (e.g. yogurt, cheese, cream, butter), and that are useful for industrial applications (e.g. production of polymers, therapeutics, household products).

There is widespread concern over the impact of conventional milk production via animal husbandry on animal welfare and the environment, and the potential danger of contaminating products derived from animal husbandry. Moreover, milk comprises components (e.g., lactose, allergens, saturated fats, cholesterol) that can cause unhealthy reactions in humans but that are not easily removed or avoided in the course of conventional, animal-based production processes.

These concerns have fuelled development of alternatives to conventional, animal-derived milk products. Increasingly, alternatives to animal-derived milk are produced from components (e.g., proteins, lipids) that are produced recombinantly (e.g., using microbial host cells). However, these efforts have to date fallen short on matching flavour and nutritional profile of milk and dairy products, and on recreating proteins that have identical or similar functionalities as native milk proteins. Therefore, there exists a need for proteins that have identical or similar, if not superior, attributes as native milk proteins.

The use of recombinant components in milk products also poses new problems. One such problem is that the use of large amounts of recombinant components may be impacted by other, sometimes undesired, components that are simultaneous produced by the recombinant host cells from which the recombinant components are obtained, and that potentially co-purify with the recombinant component. Such undesired component are carbohydrates such as sugars. Products high in sugar contribute additional calories to the diet without providing any additional nutrients, and are linked to weight gain and metabolic syndrome, a condition that increases the risk of diabetes and heart disease. Therefore, there exists a need for recombinant milk proteins with reduced levels of unwanted sugars. It is an object of the present invention to provide improved recombinant milk proteins, especially a recombinant p- lactoglobulin protein, specifically with low sugar content.

Summary of the invention

The invention relates to a method for the purification of a recombinant milk protein, comprising subjecting a liquid containing the recombinant milk protein to cationic exchange chromatography to provide the purified recombinant milk protein. The invention further relates to a purified recombinant milk protein obtainable by a method according to the invention.

The invention further relates to a composition, preferably a composition obtainable by a method according to the invention, comprising 80 wt.% or more, according to Dumas factor 6.29, recombinant milk protein and 0.01 to 5 wt.% of sugars.

The invention further relates to a food product comprising a purified recombinant milk protein obtainable by a method according to the invention, or comprising a composition according to the invention.

The invention further relates to a method for the preparation of a food product comprising contacting a food product with a purified recombinant milk protein obtainable by a method according to the invention, or with a composition according to the invention.

A preferred recombinant milk protein is a p-lactoglobulin.

Description of the invention

The inventors have established that when subjecting a liquid containing a recombinant milk protein to cationic exchange chromatography, the amount of unwanted sugars can substantially be lowered. It has been demonstrated in the examples herein that for several recombinant milk proteins, the amount of unwanted sugars could substantially be reduced by cationic exchange chromatography using various cationic resins.

Accordingly, in a first aspect, there is provided for a method for the purification of a recombinant milk protein, comprising subjecting a liquid containing the recombinant milk protein to cationic exchange chromatography to provide the purified recombinant milk protein. The method is herein referred to as the method according to the invention, or the method.

In the embodiments herein, the liquid containing the recombinant milk protein may be any liquid known to the person skilled in the art comprising a recombinant milk protein, such as a tissue culture broth or supernatant of a tissue culture wherein the recombinant milk protein has been produced.

In the embodiments herein, the liquid containing the milk protein may comprise about 5 to about 60 wt.% of total sugars on dry weight. In the embodiments herein, the liquid containing the milk protein may comprise 5 to 60 wt.% of total sugars on dry weight. In the embodiments herein, the liquid containing the milk protein may comprise about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,17, 18, 19, 20, 21 ,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 to about 60 wt.% of total sugars on dry weight. In the embodiments herein, the liquid containing the milk protein may comprise 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,17, 18, 19, 20, 21 ,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 to 60 wt.% of total sugars on dry weight.

In the embodiments herein, the purified recombinant milk protein may comprise from about 0.01 to about 5 wt.% of sugars on dry weight and preferably more than 80 wt.% of the recombinant milk protein as measured according to Dumas factor 6.29. In the embodiments herein, the purified recombinant milk protein may comprise from 0.01 to 5 wt.% of sugars on dry weight. In the embodiments herein, the purified recombinant milk protein may comprise from 0.01 to 5 wt.% of sugars on dry weight and preferably more than 80 wt.% of the recombinant milk protein as measured according to Dumas, factor 6.29. In the embodiments herein, the purified recombinant milk protein may comprise more than 80 wt.% of the recombinant milk protein as measured according to Dumas, factor 6.29.

In the embodiments herein, the purified recombinant milk protein may comprise on dry weight from about 0.01 to about 1 wt.% mannans, and/or from about 0.01 to about 0.1 wt.% glucans, and/or from about 0.01 to about 0.1 wt.% glucosamines. In the embodiments herein, the purified recombinant milk protein may comprise on dry weight from 0.01 to 1 wt.% mannans, and/or from 0.01 to 0.1 wt.% glucans, and/or from 0.01 to 0.1 wt.% glucosamines.

In the embodiments herein, the purified recombinant milk protein may comprise on dry weight less than about 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% total sugars. In the embodiments herein, the purified recombinant milk protein may comprise on dry weight less than 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% total sugars.

In the embodiments herein, the “total sugars” may comprise one or more of arabinan, galactan, glucan, glucosamine, mannan, and xylan.

In the embodiments herein, a purified recombinant milk protein may comprise on dry weight less than about 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% mannans. In the embodiments herein, a purified recombinant milk protein may comprise on dry weight less than 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% mannans.

In the embodiments herein, a purified recombinant milk protein may comprise on dry weight less than about 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% glucans. In the embodiments herein, a purified recombinant milk protein may comprise on dry weight less than 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% glucans.

In the embodiments herein, a purified recombinant milk protein may comprise on dry weight less than about 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% glucosamines.

In the embodiments herein, a purified recombinant milk protein may comprise on dry weight less than 15 wt.%, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 wt.% glucosamines. In the embodiments herein, when the purified recombinant milk protein is a p-lactoglobulin, the p- lactoglobulin may comprise on dry weight less than about 0.4 wt.% mannans, less than about 0.01 wt.% glucans on dry weight, and less than about 0.01 wt.% glucosamines on dry weight. Preferably, such purified p-lactoglobulin comprises less than about 0.6 wt.% total sugars. More preferably, such purified p-lactoglobulin comprises less than 0.6 wt.% total sugars on dry weight.

In the embodiments herein, the recombinant milk protein may be produced in any host cell known to the person skilled in the art to be suitable for the production of the recombinant milk protein. Exemplary hosts include fungi, such as filamentous fungi, as well as bacteria, yeast, algae, plant, insect, and mammalian cells.

In the embodiments herein, when the host cell is a yeast cell, the yeast host cell may be a host cell selected from the group consisting of Pichia pastoris (also known as Komagataella phaffii), Kluyveromyces lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida giabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia methanoiica, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, and Candida albicans species. In some embodiments, the yeast cell is a Saccharomycete.

In the embodiments herein, when the host cell is a filamentous fungal cell, the filamentous fungal host cell may be a host cell selected from the group consisting of Aspergillus spp. and Trichoderma spp, such as Aspergillus niger, Aspergillus oryzae and Trichoderma reesei.

In some embodiments, the host cell may be selected from Pichia pastoris, Kluyveromyces lactis and Aspergillus niger. In some embodiments, the host cell is selected from Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae, Trichoderma reesei, and Aspergillus niger. In some embodiments the host cell is selected from Pichia pastoris and Aspergillus niger. In some embodiments the host cell is selected from Pichia pastoris, Kluyveromyces lactis and Saccharomyces cerevisiae. In some embodiments the host cell is Pichia pastoris.

In the embodiments herein, when the host cell is a bacterial host cell, the bacterial host cell may be a host cell selected from the group consisting of Lactococcus lactis, Bacillus subtilis and Escherichia coli. Other host cells include bacterial host such as, but not limited to, Lactococci sp., Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glutamicum, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum and cyanobacteria such as Synechocystis , such as Synechocystis sp. 6803.

In the embodiments herein, the recombinant milk protein may be comprised of a plurality of recombinant proteins, such as p-lactoglobulin proteins, produced separately by one or more different host cells of one or more different microbial species, genera, species or strains. Combining modified recombinant proteins produced by different species may provide certain advantages, for example, to achieve a complement of modified recombinant proteins to achieve a particular desired functionality of the composition or to achieve desired production volumes.

In the embodiments herein, the recombinant milk protein may be comprised of one or more recombinant proteins, such as p-lactoglobulin proteins, expressed by a first host cell and one or more recombinant proteins, such as p-lactoglobulin proteins, expressed by a second host cell to form the plurality of recombinant proteins. In some embodiments, the second host cell may be a different species to the first host cell or the second host cell may be a different strain of the same species as the first host cell. In some embodiments, the composition may further comprise one or more recombinant proteins expressed by a third and/or fourth host cell, and so on.

Host cells comprising genetic constructs, such as expression constructs, as disclosed herein and/or as known by the person skilled in the art, may be used in methods well known in the art (see e.g. Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of proteins disclosed herein. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polynucleotide or protein disclosed herein. The expressed recombinant proteins, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).

Expression of a target protein can be provided by an expression vector, a plasmid, a nucleic acid integrated into the host genome or other means. For example, in some embodiments the host cell comprises a polynucleotide, e.g. an expression vector that comprises a sequence encoding a p- lactoglobulin protein (x) and may additionally comprise one or more of (a) a promoter element, (b) a signal peptide sequence, (c) a leader sequence, (d) a processing site and (e) a terminator element. The promoter (a) and leader sequence (c) may be, or may be derived from, any suitable yeast, fungal, bacterial or mammalian promoter or leader sequence.

In some embodiments herein, the host cell may comprise a polynucleotide, such as an expression vector, comprising a processing site located between a leader sequence and the sequence encoding a mature dairy protein. In some embodiments herein, the processing site may be a KEX processing site. In some embodiments herein, the processing site may have the sequence KREA (SEQ ID NO: 33), KREAEA (SEQ ID NO: 32), KR or KREAEAM (SEQ ID NO: 29).

Expression vectors that can be used for expression of the recombinant milk protein include those containing an expression cassette with elements (a), (b), (c), (d) and/or (e) as depicted here above. In some embodiments, the signal peptide sequence (b) need not be included in the vector. In some embodiments, a signal peptide may be part of the native signal sequence of the protein, for instance, the protein may comprise a native signal sequence as depicted in bold in Table 1. In some embodiments, the vector may comprise a polynucleotide encoding a protein sequence according to any of the sequences in Table 1 . In some embodiments, the vector may comprise a polynucleotide encoding a mature protein sequence, as exemplified in Table 1 , with a heterologous signal sequence. In general, the expression cassette is designed to mediate the transcription of the coding sequence when integrated into the genome of a cognate host microorganism or when present on a plasmid or other episomal replicating vector maintained in a host cell.

To aid in the amplification of the vector prior to transformation into the host microorganism, a replication origin (f) may be contained in the vector. To aid in the selection of microorganism stably transformed with the expression vector, the vector may also include a selection marker (g). The expression vector may also contain a restriction enzyme site (h) that allows for linearization of the expression vector prior to transformation into the host microorganism to facilitate the expression vectors stable integration into the host genome. The expression vector may contain integration sequences (i) homologous to genomic sequences of the host cell that enable or aid integration of a fragment of the expression vector or the entire expression vector into the genome of the host cell. In some embodiments the expression vector may contain any subset of the elements (a) to (i). Other expression elements and vector element known to one of skill in the art can be used in combination or substituted for the elements described herein. For example, because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. These might include one or more p-lactoglobulin proteins, one or more different dairy proteins (for example, one or more caseins, a-lactalbumin or lactoferrin proteins), and/or one or more non-dairy proteins. Gram positive bacteria (such as Lactococcus lactis and Bacillus subtilis), yeasts and filamentous fungi may be used to secrete target proteins into the media, and gram-negative bacteria (such as Escherichia coli) may be used to secrete target proteins into periplasm or into the media. In some embodiments, the bacterially-expressed proteins expressed may not have any post-translational modifications (PTMs), which means they are not glycosylated and/or may not be phosphorylated.

Recombinant milk proteins may be expressed and produced in Lactococcus lactis both in a nisin- inducible expression system (regulated by PnisA promoter), lactate-inducible expression system (regulated by P170 promoter) or other similar inducible systems, as well as a constitutively expressed system (regulated by P secA promoter), wherein both are in a food-grade selection strain, such as NZ3900 using vector pNZ8149 (lacF gene supplementation/rescue principle). The secretion of functional proteins may be enabled by the signal peptide of Usp45 (SP(usp45)), the major Secdependent protein secreted by L. lactis.

Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987).

In some embodiments where it is desirable that the recombinant protein composition is substantially free of aspartyl protease-like activity, host cells having functional knockout or replacement of the endogenous pep4 gene in the host cell may be used. Suitable methods for achieving functional knockout or gene replacement in a host cell are well known in the art, and include CRISPR/CAS9 technology, mutation of the coding sequence to modify the amino acid sequence of the protein encoded by the gene to render it non-functional, or by deletion and/or insertion of nucleotides into the coding sequence. Alternatively, the promoter expressing the pep4 gene can be replaced by another promoter with low regulated expression.

In some embodiments, the functional knockout/gene replacement of pep4 and insertion of an expression cassette for the one or more recombinant milk proteins may be accomplished simultaneously using CRISPR-CAS9 technology. An example is described herein in the examples. Briefly, CRISPR-CAS9 may be used to integrate a fragment containing the one or more recombinant milk proteins and deleting the pep4 gene in the host cell. An "all in one" expression vector may be used, which expresses both CAS9 and guide RNA (gRNA) targeting the pep4 gene sequence. In various embodiments the CAS9 gene and the pep4 gRNA are under the control of separate promoters. Preferably, the plasmid contains a selectable marker, such as an antibiotic selectable marker and an autonomously replicating sequence to select and maintain the plasmid in the cell after transformation to the host cell.

Compositions comprising the plurality of recombinant milk proteins may be produced by culturing a host cell expressing the protein using standard methods well known in the art. The cell culture biomass may then be centrifuged to remove solid matter and the liquid phase subjected to filtration. In the embodiments herein, the recombinant milk protein may be any recombinant milk protein known to the person skilled in the art, such as but not limited to a p-lactoglobulin, a lactoferrin, or an a- lactalbumin. A preferred recombinant milk protein is p-lactoglobulin. Another preferred recombinant milk protein is lactoferrin. Another preferred recombinant milk protein is a-lactalbumin.

In the embodiments herein, a lactoferrin protein preferably is an iron-binding glycoprotein present in various secretory fluids including milk, with antimicrobial, antiviral, and immune-modulating properties and can be a lactoferrin protein from any mammalian species, e.g., an ape, baboons, bear, buffalo, camel, cat, chimpanzee, cow, dog, donkey, echidna, elephant, fox, gibbons, goat, gorilla, guinea pig, horse, human, lemur, lion, macaque, mandrill, monkey, mountain goat, mouse, opossum, orangutan, panda, pig, rabbit, rat, sheep, squirrel, tiger, wallaby, whale, wolf, or woolly mammoth lactoferrin protein. A lactoferrin protein can also be a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wildtype lactoferrin protein. A nucleic acid encoding a lactoferrin protein can encode a protein that is at least 80% (e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to a wildtype lactoferrin protein. a-Lactalbumin is of high nutritional quality as it is rich in essential amino acids, which are crucial for growth and development. It has a high proportion of tryptophan, an amino acid that supports the synthesis of serotonin and melatonin, aiding in sleep regulation and mood improvement. a-Lactalbumin provides enhanced mineral absorption as it is particularly high in calcium and zinc binding capabilities, facilitating better absorption of these vital minerals. This is particularly beneficial in promoting bone growth and a robust immune system. a-Lactalbumin provides immunological benefits as it contains components that can help boost the immune system. Its bioactive peptides can enhance antimicrobial activity, which is critical in reducing the risk of infections in infants. a-Lactalbumin, being derived from milk, is typically easier to digest compared to other proteins. This makes it especially suitable for infant formulas as it is gentle on an infant’s developing digestive system. a-Lactalbumin displays similarity to human breast milk, so its inclusion in formula helps to mimic the protein composition of breast milk more closely than formulas without it. This similarity can be important for the acceptance and tolerance of formula in some infants. a-Lactalbumin may lead to reduction in allergic reactions: Its structure and properties might help reduce the risk of developing allergies compared to formulas based on cow's milk proteins, which are structurally different from the proteins found in human milk. In addition, a-lactalbumin supports cognitive development: The high levels of tryptophan in a-lactalbumin not only support better sleep patterns but are also crucial for brain development and function, potentially aiding in improved cognitive outcomes for infants. Due to these benefits, a-lactalbumin is a valuable component in infant formulas and other nutritional products aimed at mimicking the qualities of human breast milk. p-lactoglobulin is the major whey protein in the milk of many mammals. In bovine milk it accounts for approximately 10 - 15% of total milk proteins and about 50 - 54% of whey protein.

Bovine p-lactoglobulin is expressed as a precursor protein comprising a 16 amino acid N-terminal signal peptide (referred to herein and elsewhere as the "full-length" p-lactoglobulin protein), which is cleaved to form a mature 162 amino acid protein. There are two primary variants of bovine p-lactoglobulin - variants A and B and a lesser variant - variant C. Sequences for both the mature and full-length forms of bovine p-lactoglobulin variants A, B and C, and wild-type full length and mature forms of p- lactoglobulin from other species are presented in Table 1 .

Preferred p-lactoglobulins include proteins comprising an amino acid sequence having at least about 70% sequence identity to the sequence of a wild-type (native) p-lactoglobulin (either full length or mature p-lactoglobulin lacking a signal sequence, but preferably the mature sequence), but particularly any wild-type bovine, ovine, caprine, buffalo, equine, donkey or reindeer p-lactoglobulin sequence, including any sequence of SEQ ID NO: in Table 1 . In some embodiments the amino acid sequence of such variants may comprise a truncation or an elongation at the N-terminus and/or the C-terminus relative to the wild-type sequence, for example, truncations or elongations of from about 1 to about 20 amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, variants or modified p-lactoglobulin proteins may contain from 1 to 20 amino acid insertions, deletions, and/or substitutions (collectively) with respect to the wild-type sequence. Such proteins may be referred to herein as "elongated p-lactoglobulin proteins". In some embodiments the variants or modified p-lactoglobulin proteins may comprise one or more post-translational modifications that differ to a wild-type p-lactoglobulin protein, including glycosylation and or phosphorylation at one or more residues. An N-terminal elongation may have a sequence comprising or consisting of EA, or two or more repeats of EA, for example three or more repeats of EA, four or more repeats of EA, or five or more repeats of EA. For example, the N-terminal elongation may have a sequence comprising or consisting of EA, EAEA (SEQ ID NO: 22), EAEAEA (SEQ ID NO: 23), EAEAEAEA (SEQ ID NO: 24), EAEAEAEAEA (SEQ ID NO: 25, REAEAM (SEQ ID NO: 26), REAEAEAM (SEQ ID NO: 27), or REAEAEAEAM (SEQ ID NO: 28), KREAEAM (SEQ ID NO: 29), KREAEAEAM (SEQ ID NO: 30), or KREAEAEAEAM (SEQ ID NO: 31).

In the embodiments herein, the recombinant milk protein may be a mixture of heterogenous milk proteins. In the embodiments herein, the recombinant milk protein may be a plurality of recombinant p- lactoglobulin proteins heterogeneous in amino acid sequence, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18 or 20 recombinant p -lactoglobulin proteins of differing amino acid sequence and/or of differing elongation.

The p-lactoglobulins, elongated p-lactoglobulins and mixes thereof as set forward in WO2022/269549 are preferred p-lactoglobulins and are herein incorporated by reference. Accordingly, the recombinant p-lactoglobulins comprising or consisting of an amino acid sequence having at least about 70% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 61 , 71 , 72, 73, and 74 as set forward in WO2022/269549 are preferred p-lactoglobulins herein.

In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation, In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 3%, preferably at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and/or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

In the embodiments herein, a specifically preferred p-lactoglobulin is an elongated p-lactoglobulin wherein at least 45% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and/or wherein at least 5% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

As set forth in WO2022/269549, the p-lactoglobulins may comprise a truncation in the N-terminal part of the protein, such as a p-lactoglobulin lacking an L, LI, LIV, LIVT, LIVTQ or LIVTQT. Such truncation at the N-terminal part of the protein may be present together with the EA or EAEA N-terminal elongation. In the embodiments herein, the recombinant milk protein may comprise non-native post-translational modification modulating e.g. the glycosylation and/ or phosphorylation of the recombinant milk protein, as set forward in WO2020219596A1 , which is herein incorporated by reference.

Accordingly, the recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, S21 , S27, S30, S36, T49, T76, T97, S110, S116, T125, S150, N152, and T154 of Bos taurus p-lactoglobulin, and having non-native glycosylation on one or more of such amino acid residues. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises amino acid residue N 152 of Bos taurus p-lactoglobulin, and having non-native N-glycosylation on such amino acid residue. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, S21 , S27, S30, S36, T49, T97, SI 10, SI 16, T125, S150, and T154 of Bos taurus p-lactoglobulin, and having non- native O-glycosylation on one or more of such amino acid residues. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, Y20, S21 , S27, S30, S36, Y42, T49, T76, T97, Y99, Y102, SI 10, SI 16, T125, S150, and T154 of Bos taurus p-lactoglobulin, and having non-native phosphorylation on one or more of such amino acid residues. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from K8, K14, R40, K47, K60, K69, K70, K75, K77, K83, K91 , K100, K101 , R124, K135, K138, K141 , and R148 of Bos taurus p-lactoglobulin, and having non-native methylation on one or more of such amino acid residues. The recombinant milk protein of the invention can be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C66, C106, C119, C121 , and C160 of Bos taurus p-lactoglobulin, and having non-native palmitoylation on one or more of such amino acid residues. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from K8, K14, K47, K60, K69, K70, K75, K77, K83, K91 , K100, K101 , K135, K138, and K141 of Bos taurus p-lactoglobulin, and having non-native sumoylation on one or more of such amino acid residues. The recombinant milk protein may be a recombinant p- lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C66, C106, C119, C121 , and C160 of Bos taurus p-lactoglobulin, and having non-native nitrosylation on one or more of such amino acid residues. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from Y20, Y42, Y99, and Y102 of Bos taurus p-lactoglobulin, and having nonnative tyrosine nitration on one or more of such amino acid residues. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from F151 of Bos taurus p-lactoglobulin, and having non-native glypiation on such amino acid residue. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C160 of Bos taurus p-lactoglobulin, and having non-native farnesylation on such amino acid residue. The recombinant milk protein may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C160 of Bos taurus p- lactoglobulin, and having non-native geranylgeranylation on such amino acid residue.

In the embodiments herein, the recombinant milk protein may have an attenuated or essentially eliminated allergenicity, such e.g. the recombinant milk protein as set forward in WO2021168343, which is herein incorporated by reference. Accordingly, the recombinant p-lactoglobulins comprising or consisting of an amino acid sequence having at least about 70% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10 of WO2021168343 are preferred p-lactoglobulins herein.

In the embodiments herein, the recombinant milk protein may be a fusion protein, such as the fusion proteins as set forward in WO2022031941 , which is herein incorporated by reference. Such fusion protein may comprise different milk proteins, or repeats of milk different and identical milk proteins, with or without linkers between the protein units.

In the embodiments herein, the recombinant milk protein may be an egg replacer as set forward in WO2020219595, which is herein incorporated by reference.

In the embodiments herein the liquid comprising the recombinant milk protein may be essentially eliminated from esterase activity, as set forward in W02020081789, which is herein incorporated by reference.

In the embodiments herein, the recombinant milk protein is subjected to cation exchange chromatography, herein also depicted as cationic exchange chromatography. Recombinant milk proteins according to the invention are purified on the basis of their surface charge or hydrophobicity/hydrophilicity. Cation exchange chromatography is a form of ion exchange chromatography (IEX), which can be used to separate molecules based on their net surface charge. Cation exchange chromatography, more specifically, uses a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges. Cation exchange chromatography is based on reversible interactions between positively charged molecules (mobile phase) and immobilized ion exchange groups of negative charge (stationary phase), wherein a positively charged molecule binds to the separation resin at low ionic strength and can be eluted with a salt or pH gradient. Prior to cationic exchange chromatography, the stationary phase is generally equilibrated. Once equilibrated, the negatively charged ions in the stationary phase will be attached to exchangeable ions of opposite charge, such as Na⁺. After equilibration, the stationary phase is generally washed with a wash buffer to elute out all impurities, wherein the pH of the wash buffer is equal to the pH of the sample comprising a recombinant protein, which generally is a pH below the isoelectric point (pl) of the recombinant milk protein of interest. At a pH below the pl of a recombinant milk protein of interest, the recombinant protein generally carries a net positive charge, and binds the negatively charged stationary phase. The stationary phase can then be washed to elute out all non-desired proteins. An elution buffer can be used to separate the recombinant milk protein of interest from other proteins. However, in some embodiments, the pH of the elution buffer may above the isoelectric point (pl) of the recombinant milk protein of interest.

In the embodiments herein, the cationic exchange chromatography may comprise a matrix comprising or consisting of dextran, cellulose, acrylic, methacrylic or sephacel. In addition, the matrix further comprises cationic exchange residues, such as sulfonate, sulfopropyl, phosphate, carboxylate, or carboxymethyl. In a preferred embodiment, the matrix comprises an agarose matrix wherein dextran chains are covalently coupled to the agarose matrix, wherein ion exchange residues are attached to the dextran through chemically stable ether bonds. Dextran can increase the exposure of target molecules to the charged groups. Dextran molecules according to the invention are flexible enough to allow passage of charged recombinant milk proteins.

In the embodiments herein, a cationic exchange chromatography material may comprise carboxylic acid-functionalized resins or sulphonic acid-functionalized resins, preferably sulphonic acid- functionalized resins. The backbone of such sulphonic acid-functionalized resins may be cellulose, polystyrene, styrene-divinylbenzene (example Mitsubishi R160 M), agarose (examples Cytiva SP Sepharose FF, Cytiva SP Sepharose XL, Bioworks 40S), or methacrylate (examples Resindion Relisorb SP400, Chromalite MS/M (sulphopropyl methacrylate). In a preferred embodiment, the backbone of sulphonic acid-functionalized resins comprises Cytiva Sepharose SP XL agarose.

Accordingly, in the embodiments herein, the cationic exchange chromatography material may comprise a matrix selected from the group consisting of a dextran, cellulose, acrylic, methacrylic, sephacel, and within the matrix a cationic exchange residue selected from the group consisting of sulfonate, sulfopropyl, phosphate, carboxylate, and carboxymethyl.

In the embodiments herein, a preferred cationic ion exchange resin is Cytiva Sepharose SP XL. Another preferred cationic exchange resin is Relisorb SP400.

In the embodiments herein, the recombinant milk protein may bind electrostatically to the matrix whereafter the bound recombinant milk protein is eluted with a buffer, preferably a buffer comprising a compound selected from the group consisting of sodium chloride, hydrochloric acid, citric acid, lactic acid, succinic acid, acetic acid, and phosphate. The pH of the elution buffer may be at or below the IEP of the recombinant milk protein.

In some embodiments, the recombinant milk protein is eluted with a buffer wherein the pH is higher than the isoelectric point of the recombinant milk protein, such as a pH of 6.0.

In an embodiment, the recombinant milk protein is a p-lactoglobulin and the elution buffer comprises citric acid and sodium chloride and has a conductivity of about 50mS/cm and a pH of about 6.0. In the embodiments herein, the recombinant milk protein may be purified to a purity of greater than 30%, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97 or greater than 99% relative to other components comprised in the fermentation broth or preparation, or to at least 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold greater abundancy relative to other components comprised in the fermentation broth, or to a purity of greater than 30%, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, or greater than 99% by weight.

In a second aspect, there is provided for a purified recombinant milk protein obtainable by a method of the first aspect. The features of this aspect are preferably the features as set forward in the first aspect. In the embodiments of this aspect, the recombinant milk protein may be any recombinant milk protein as defined elsewhere herein and preferably is a p-lactoglobulin or a lactoferrin. A preferred recombinant milk protein is a p-lactoglobulin as defined elsewhere herein. A preferred recombinant milk protein is a lactoferrin as defined elsewhere herein. A preferred recombinant milk protein is a-lactalbumin as defined elsewhere herein.

In a third aspect, there is provided for a composition comprising 80 wt.% or more, according to Dumas factor 6.29, recombinant milk protein and 0.01 to 5 wt.% of sugars. The features of this aspect are preferably the features as set forward in the first and second aspects. In the embodiments of this aspect, the composition is preferably obtainable by or obtained by a method of the first aspect.

In the embodiments of this aspect, the recombinant milk protein may be any recombinant milk protein as defined elsewhere herein and preferably is a p-lactoglobulin or a lactoferrin. A preferred recombinant milk protein is a p-lactoglobulin as defined elsewhere herein. A preferred recombinant milk protein is a lactoferrin as defined elsewhere herein. A preferred recombinant milk protein is a-lactalbumin as defined elsewhere herein.

In the embodiments of this aspect, the composition may be a powder having a dry matter content of at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, or at least 98%.

In the embodiments of this aspect, the amount of mannans may be from 0.01 to 1 wt.%, and/or the amount of glucans may be from 0.01 to 0.1 wt.%, and/or the amount of glucosamines may be from 0.01 to 0.1 wt.%.

In a fourth aspect, there is provided for a food product comprising a purified recombinant milk protein as defined in the second aspect, or comprising a composition as defined in the third aspect. The features of this aspect are preferably the features as set forward in the first, second, and third aspects.

In the embodiments of this aspect, the recombinant milk protein may be any recombinant milk protein as defined elsewhere herein and preferably is a p-lactoglobulin or a lactoferrin. A preferred recombinant milk protein is a p-lactoglobulin as defined elsewhere herein. A preferred recombinant milk protein is a lactoferrin as defined elsewhere herein. A preferred recombinant milk protein is a-lactalbumin as defined elsewhere herein. This aspect also provides for a method for the preparation of a food product comprising contacting a food product with a purified recombinant milk protein as defined in the second aspect, or with a composition as defined in the third aspect. In the embodiments herein, the food product may comprise at least about 1 %, 1.5%, 2%, or 2.5% of the recombinant recombinant milk protein protein by weight, such as from about 1 to about 50%, about 1 to about 45%, about 1 to about 40%, 1 to about 35%, about 1 to about 30%, or about 1 % to about 25% of the recombinant milk protein by weight, and useful ranges may be selected from between any of these values (for example, from about 1 % to about 20%, or about 1 % to about 16%, 1 % to about 15%, 1 % to about 14%, or about 1 % to about 12%, or about 1 % to about 10%, or about 2% to about 20%, or about 2% to about 16%, 2% to about 15%, 2% to about 14%, or about 2% to about 12%, or about 2% to about 10%, about 4% to about 20%, or about 4% to about 16%, 4% to about 15%, 4% to about 14%, or about 4% to about 12%, or about 4% to about 10%, about 5% to about 20%, or about 5% to about 16%, 5% to about 15%, 5% to about 14%, or about 5% to about 12%, or about 5% to about 10%).

When the food product comprises a composition as disclosed herein, the composition of the recombinant milk protein may be mixed with one or more additional ingredients to produce a food product. In various embodiments, the method may comprise mixing the composition or the recombinant milk protein with one or more additional ingredients to produce a food product. The food product may be any edible consumer product which is able to carry protein.

In various embodiments, the food product may comprise at least about 1 %, 1.5%, 2%, or 2.5% total protein by weight. In various embodiments, the food product may comprise from about 1 to about 50%, about 1 to about 45%, about 1 to about 40%, 1 to about 35%, about 1 to about 30%, or about 1 % to about 25% total protein by weight, and useful ranges may be selected from between any of these values (for example, from about 1 % to about 20%, or about 1 % to about 16%, 1 % to about 15%, 1 % to about 14%, or about 1 % to about 12%, or about 1 % to about 10%, or about 2% to about 20%, or about 2% to about 16%, 2% to about 15%, 2% to about 14%, or about 2% to about 12%, or about 2% to about 10%, about 4% to about 20%, or about 4% to about 16%, 4% to about 15%, 4% to about 14%, or about 4% to about 12%, or about 4% to about 10%, about 5% to about 20%, or about 5% to about 16%, 5% to about 15%, 5% to about 14%, or about 5% to about 12%, or about 5% to about 10%).

In the embodiments herein, the food product may be a fermented food, a yoghurt, a soup, a sauce, a bar, a gel, a foam, a nutritional formulation, a beverage, a beverage whitener, a cheese, a dairy tofu, a food emulsion or a dessert. In various embodiments the food product may be a yoghurt, drinking yoghurt, a bar, a gel, a foam, a nutritional formulation, medical food, dairy beverage, a product that requires the protein to form a heat-set gel, an acid protein fortified beverage, a jelly drink, a protein water, a foam, a heat-set foam extruded food product, or a food emulsion. In various embodiments the food product is free of animal-derived ingredients. In various embodiments the food product is considered suitable for those on a vegan diet. The food product may comprise one or more additional sources of protein. In various embodiments the additional source of protein is a non-dairy source of protein. Liquid nutritional compositions may include a medical beverage. A beverage may include a sports beverage, dairy beverage, or a yoghurt beverage.

In the embodiments herein, the food product may have one or more characteristics of a dairy food product. In various embodiments the food product has one or more characteristics of a dairy food product selected from the group comprising: appearance, consistency, firmness, organoleptic properties, density, stiffness, structure, viscosity, texture, elasticity, storage stability, heat stability, acidheat stability, coagulation, binding, leavening, aeration, foaming capacity, foam stability, foam overrun, behaviour when whipped, creaminess, gelling structure and emulsification. In various embodiments the organoleptic properties are taste, aroma, mouthfeel, in-mouth creaminess, appearance, colour, grittiness, sandiness, and smoothness.

In the embodiments herein, the food product may contain nutrients that include vitamins and minerals. The recommended daily requirements of vitamins and minerals can be specified for various population subgroups. See for instance, Dietary Reference Intakes: RDA and Al for vitamins and elements, United States National Academy of Sciences, Institute of Medicine, Food and Nutrition Board (2010) tables recommended intakes for infants 0-6, 6-12 months, children 1 -3, and 4-8 years, adults males (6 age classes), females (6 age classes), pregnant (3 age classes) and lactating (3 age classes). Concentrations of essential nutrients in the liquid nutritional composition can be tailored in the exemplary serve size for a particular subgroup or medical condition or application so that the nutrition and ease of delivery requirements can be met simultaneously.

Nutrient content can be assessed using analytical methods known in the art, including but not limited to AOAC International reference methods AOAC 990.03 and AOAC 992.15, electrophoresis (e.g., SDS- PAGE), liquid column chromatography, immunochemical tests, or on-chip electrophoresis (e.g., using the Agilent Protein 80 kit and the Agilent 2100 Bioanalyzer) for determination of type and/or content of proteins and amino acids. Alternatively, chemical/biological attributes can be calculated from the nutrient contents of ingredients.

In the embodiments herein, the pH of the food product may be adjusted using food-safe acidic or basic additives. In various embodiments, the pH of the protein containing food product may be adjusted to about pH 3 to about pH 8, for example about pH 3.3 to about pH 8, about pH 4 to about pH 8, about pH 4 to about pH 7, or about pH 4 to about pH 6.8, or about pH 5 to about pH 7, or about pH 5 to about pH 6.8. In various embodiments, the pH of the protein containing food product may be adjusted to about pH 6.8. pH may be measured by equilibrating samples to 25°C and measuring using a pH probe (EC620132, Thermo Scientific) after calibrating using standards at pH 4, 7, and 10 (Pronalys, LabServ). Other methods of measuring pH will be apparent to a skilled worker.

In the embodiments herein, the food product may be administered to a subject to maintain or increase muscle protein synthesis, maintain or increase muscle mass, prevent or increase loss of muscle mass, maintain or increase growth, prevent or decrease muscle catabolism, prevent or treat cachexia, prevent or treat sarcopenia, increase rate of glycogen resynthesis, modulate blood sugar levels, increase insulin response to raised blood glucose concentration, increase satiety, increase satiation, increase food intake, increase calorie intake, improve glucose metabolism, increase rate of recovery following surgery, increase rate of recovery following injury, increase rate of recovery following exercise, increase sports performance, and/or provide nutrition.

In the embodiments herein, the food product may comprise at least about 0.1 % fat by weight, such as about 0.1 %, or about 0.5%, or about 1 %, or about 3%, or about 5%, or about 10% fat by weight. In various embodiments, the protein containing food product may comprise from about 0.1 % to 40% fat by weight, and useful ranges may be selected from between any of these values (for example, from about 0.1 % to about 40%, or about 0.5% to about 40%, or about 1 % to about 40%, or about 3% to about 40%, or about 5% to about 40%, or about 10% to about 40%, or about 15% to about 40%, or about 20% to about 40%, or about 0.1 % to about 35%, or about 0.5% to about 35%, or about 1 % to about 35%, or about 3% to about 35%, or about 5% to about 35%, or about 10% to about 35%, or about 15% to about 35%, or about 20% to about 35%, or about 0.1 % to about 30%, or about 0.5% to about 30%, or about 1 % to about 30%, or about 3% to about 30%, or about 5% to about 30%, or about 10% to about 30%, or about 15% to about 30%, or about 20% to about 30%, or about 0.1 % to about 20%, or about 0.5% to about 20%, or about 1 % to about 20%, or about 3% to about 20%, or about 5% to about 20%, or about 10% to about 20%, or about 15% to about 20%).

In embodiments herein, the food product may comprise at least about 0.1 % carbohydrate by weight, such as about 0.1 %, or about 0.5%, or about 1 %, or about 3%, or about 5%, or about 10% fat by weight. In various embodiments, the protein containing food product may comprise from about 0.1 % to 40% carbohydrate by weight, and useful ranges may be selected from between any of these values (for example, from about 0.1 % to about 40%, or about 0.5% to about 40%, or about 1 % to about 40%, or about 3% to about 40%, or about 5% to about 40%, or about 10% to about 40%, or about 15% to about 40%, or about 20% to about 40%, or about 0.1 % to about 35%, or about 0.5% to about 35%, or about 1 % to about 35%, or about 3% to about 35%, or about 5% to about 35%, or about 10% to about 35%, or about 15% to about 35%, or about 20% to about 35%, or about 0.1 % to about 30%, or about 0.5% to about 30%, or about 1 % to about 30%, or about 3% to about 30%, or about 5% to about 30%, or about 10% to about 30%, or about 15% to about 30%, or about 20% to about 30%, or about 0.1 % to about 20%, or about 0.5% to about 20%, or about 1 % to about 20%, or about 3% to about 20%, or about 5% to about 20%, or about 10% to about 20%, or about 15% to about 20%). Advantageously, the method of the first aspect allows for the preparation of a low-sugar or sugar-free food product comprising a fermentatively derived p-lactoglobulin. Low-sugar or sugar-free refers to from 0.01 % to 10% sugar by weight of the food product, such as from 0.01 % to 9%, or from 0.05% to 8%, or from 0.1 % to 6%, or from 0.2% to 5%, or from 0.5% to 4%.

In embodiments herein, the food product, preferably a medical beverage, may comprise at least about 10 kcal per 100 ml. of the food product. In various embodiments, the protein containing food product may comprise from about 10 to about 400 kcal per 100 mL of the food product, and useful ranges may be selected from between any of these values (for example, from about 10 to about 400, 10 to about

350, or about 10 to about 300, or about 10 to about 300, or about 10 to about 250, or about 10 to about

200, or about 10 to about 150, or about 10 to about 100, or about 50 to about 400, or about 50 to about

350, or about 50 to about 300, or about 50 to about 300, or about 50 to about 250, or about 50 to about

200, or about 50 to about 150, or about 50 to about 100, or about 100 to about 400, or about 100 to about 350, or about 100 to about 300, or about 100 to about 300, or about 100 to about 250, or about 100 to about 200, or about 100 to about 150, or about 150 to about 400, or about 150 to about 350, or about 150 to about 300, or about 150 to about 300, or about 150 to about 250, or about 200 to about 400, or about 200 to about 350, or about 200 to about 300, or about 200 to about 350).

In the embodiments herein, the food product may be in a bar or other solid moulded form. The bar may further comprise one or more additional ingredients selected from one or more sweeteners, one or more additional protein sources, one or more stability enhancers (such as glucose syrup, glycerine, plasticisers (such as glycerine), one or more lipids and one or more lecithins.

Table 1 : Overview of sequences

Figure legends

Figure 1 , Relative composition of samples obtained after purification of raw material (first bar from the left) by means of anion exchange chromatography (second bar) and cation exchange chromatography (bars 3-5) clearly demonstrating the superior result obtained with cationic exchange chromatography.

Figure 2. Analysis by SDS PAGE of the p-lactoglobulin A in various steps of purification comprising Cation Exchange Chromatography (CEX) in example 2. Samples were 10-times diluted and loading buffer was added before being loaded on the gel. After electrophoresis, the gel was stained with Sypro ruby.

Definitions

"Sequence identity" is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S„ et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S„ et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, Wl. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninevaline, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; lie to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

A “nucleic acid molecule” or “polynucleotide” (the terms are used interchangeably herein) is represented by a nucleotide sequence. A “polypeptide” is represented by an amino acid sequence. A “nucleic acid construct” is defined as a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of the peptide or polypeptide in a cell or in a subject.

“Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production/expression of the peptide or polypeptide of the invention in a cell and/or in a subject. “Operably linked” may also be used for defining a configuration in which a sequence is appropriately placed at a position relative to another sequence coding for a functional domain such that a chimeric polypeptide is encoded in a cell and/or in a subject.

“Expression” is construed as to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification and secretion.

A “control sequence” is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein.

The term "transformation" refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e. DNA exogenous to the cell). When the cell is a bacterial cell, as is intended in the present invention, the term usually refers to an extrachromosomal, self-replicating vector which harbors a selectable antibiotic resistance.

An “expression vector” may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide of the invention in a cell and/or in a subject. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene. It is related to the binding site identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

A “polypeptide” or “protein" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

The term “recombinant polypeptide” or “recombinant protein” as used herein refers to a polypeptide that is produced in a cell of a different species or type as compared to the species or type of cell that produces the polypeptide in nature, or that is produced in a cell at a level at which it is not produced in nature.

The term "heterogeneous" as used herein with reference to a plurality of recombinant proteins means that the plurality of recombinant proteins comprises at least two or two or more, three or more, four or more, five or more, six or more, or seven or more proteins of differing amino acid sequence.

The term "mature" as used herein with reference to a protein refers to the protein, or amino acid sequence of the protein, after cleavage of the signal sequence. The term "full length" as used herein with reference to a protein refers to the protein, or amino acid sequence of the protein, comprising the signal sequence. Examples of mature and full-length proteins are provided in Table 1 herein.

The term "wild-type" as used herein with reference to proteins or polynucleotides refers to a protein or polynucleotide having an amino acid or nucleotide sequences that is the same as that expressed naturally. This term includes all naturally occurring variants of a particular protein, for example, all naturally occurring variants of p-lactoglobulin. Furthermore, this term includes both full length proteins and mature proteins and polynucleotides that encode wild-type full length and mature protein. The term is generally synonymous with the term "native".

The term “Dumas method” as used herein is similar to the well-known Kjeldahl method and refers to an analytical method to establish protein content by determining the nitrogen content of a sample, consisting of combusting a sample of known mass to a temperature between 800 and 900°C in the presence of oxygen, which leads to the release of carbon dioxide, water and nitrogen. The gases are then passed over special columns (such as potassium hydroxide aqueous solution) that absorb the carbon dioxide and water. A column containing a thermal conductivity detector at the end is then used to separate the nitrogen from any residual carbon dioxide and water and the remaining nitrogen content is measured. The instrument must first be calibrated by analysing a material that is pure and has a known nitrogen concentration. The measured signal from the thermal conductivity detector for the unknown sample can then be converted into a nitrogen content (see e.g. Maubois and Lorient; Dairy proteins and soy proteins in infant foods nitrogen-to-protein conversion factors; Dairy Sci. & Technol. (2016) 96:15-25). For the group of whey proteins, a factor of 6.29 is the scientifically acceptable calculation factor.

Sequence identity herein of a polynucleotide, polynucleotide construct or of a polypeptide is preferably at least 70%. Preferably at least 70% is defined as preferably at least 70%, more preferably at least 71 %, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81 %, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 98%, more preferably at least 99%, or most preferably 100% sequence identity. In case of 100% sequence identity, the polynucleotide or polypeptide has exactly the sequence of the depicted SEQ ID NO:. Sequence identity is preferably determined over the entire length of the subject sequence.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

In this document and in its claims, the verbs "to comprise", “to contain”, and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist of’ may be replaced by “to consist essentially of’ meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; the additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

Further embodiments

Further embodiments of the invention are listed here below.

1. A method for the purification of a recombinant milk protein, comprising subjecting a liquid containing the recombinant milk protein to cationic exchange chromatography to provide the purified recombinant milk protein.

2. A method for the purification of a recombinant milk protein according to embodiment 1 , wherein the purified recombinant milk protein comprises 0.01 to 5 wt.% of sugars on dry weight and more than 80 wt.% of the recombinant milk protein as measured according to Dumas factor 6.29. A method for the purification of a recombinant milk protein according to embodiment 1 or 2, wherein the purified recombinant milk protein comprises on dry weight from 0.01 to 1 wt.% mannans, and/or from 0.01 to 0.1 wt.% glucans, and/or from 0.01 to 0.1 wt.% glucosamines. A method for the purification of a recombinant milk protein according to any of the preceding embodiments, wherein the liquid containing the recombinant milk protein comprises 5 to 50 wt.% of sugars on dry weight. A method for the purification of a recombinant milk protein according to any one of the preceding embodiments, wherein the recombinant milk protein is produced by a microorganism selected from the group consisting of a species of Pichia, Kluyveromyces, Saccharomyces, Trichoderma, and Aspergillus. A method for the purification of a recombinant milk protein according to any one of the preceding embodiments, wherein the recombinant milk protein is a p-lactoglobulin, an a-lactalbumin or a lactoferrin, preferably an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and/or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation. A method for the purification of a recombinant milk protein according to any one of the preceding embodiments, wherein the cationic exchange chromatography comprises a matrix selected from the group consisting of a dextran, cellulose, acrylic, methacrylic, sephacel, and within the matrix a cationic exchange residue selected from the group consisting of sulfonate, sulfopropyl, phosphate, carboxylate, and carboxymethyl. A method for the purification of a recombinant milk protein according to any one of the preceding embodiments, wherein the recombinant milk protein binds electrostatically to the matrix whereafter the bound recombinant milk protein is eluted with a buffer, preferably a buffer comprising a compound selected from the group consisting of: sodium chloride, hydrochloric acid, citric acid, lactic acid, succinic acid, acetic acid, and phosphate. A purified recombinant milk protein obtainable by a method according to any one of embodiments 1 to 8. A composition, preferably a composition obtainable by a method according to any one of embodiments 1 to 8, comprising 80 wt.% or more, according to Dumas factor 6.29, recombinant milk protein and 0.01 to 5 wt.% of sugars, preferably 0.02 to 2 wt. % of sugars. A composition according to embodiment 10, wherein the recombinant milk protein is a p- lactoglobulin, an a-lactalbumin or a lactoferrin. A composition according to embodiment 10 or 11 , wherein the recombinant milk protein is an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and/or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation, A composition according to anyone of embodiments 10 to 12, which is a powder having a dry matter content of 80 to 98%. 14. A composition according to any one of embodiments 10 to 13, wherein the amount of mannans on dry weight is from 0.01 to 1 wt.%, and/or the amount of glucans on dry weight is from 0.01 to 0.1 wt.%, and/or the amount of glucosamines on dry weight is from 0.01 to 0.1 wt.%.

15. A food product comprising a purified recombinant milk protein according to embodiment 9, or comprising a composition according to any one of embodiments 10 to 14.

16. A method for the preparation of a food product comprising contacting a food product with a purified recombinant milk protein according to embodiment 9, or with a composition according to any one of embodiments 10 to 14.

17. A food product according to embodiment 15, wherein the recombinant milk protein is a p- lactoglobulin, an a-lactalbumin or a lactoferrin.

18. A food product according to embodiment 17, wherein the recombinant milk protein is a an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and/or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

Examples

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention.

As demonstrated in the examples here below, purification of fermentatively produced p-lactoglobulin (BLG) using cationic exchange resin (CEX) results in markedly improved results in terms of (poly)sugar removal as compared to anionic exchange resin (AEX).

Materials and Methods

The chemicals used are Bentonorit CA-1 (Norit) activated carbon, Dicalite BF (Dicalite). Acids and bases, sodium acetate, sodium chloride, di-sodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O, sodium hydroxide were from Merck.

Example 1 ; Comparative example of purification of fermentatively produced B-lactoqlobulin A using anionic exchange resin (AEX)

Results and discussion p-lactoglobulin A was produced using fermentation as disclosed in WO 2022/269549, Example 1. WO 2022/269549 is herein incorporated by reference.

A 12.0 kg fermentation broth was obtained after fermentation during 7 days at a temperature of 30°C. The broth was diluted with water (-80%) and centrifuged at 4500 rpm, 4°C for 30 minutes in a Sorvall Bios 16 centrifuge. The almost clear supernatants were extra clarified on a standard paper filter (Pall) and a Nalgene Rapid flow filtration bottle and the filtrates were stored frozen until the next process step. The clarified and sterile filtered supernatants were purified using a PALL 200/180 column filled with Cytiva Q Sepharose beads. For equilibration and washing, standard phosphate buffers (Na2HPO4 and NaH2PO4) were applied, while for elution NaCI was added to the buffers and finally NaOH (1 M) was used as sanitizing agent.

The eluate was concentrated to 1000 g and dialyzed to a conductivity of 4 mS/cm in a Millipore unit using a 5 kD MWCO polyethersulphone ultra filtration membrane. After the sterile filtration step using a Nalgene filtration bottle, the concentrate was filled in Lyogard freeze dry trays, stored frozen and subsequently freeze dried. A total of 148 g powder with the following properties was obtained:

The sugars analysis was as follows:

The results demonstrate that purification of p-lactoglobulin A using anionic exchange chromatography resulted in a product wherein minor amounts of sugars were removed. Remaining amount of total carbohydrates was 46% of the starting material.

Example 2; Purification of B-lactoqlobulin A using Cation Exchange Chromatography (CEX)

Fermentation broth (10 kg) was collected from two fermentations which were fermented as disclosed in WO 2022/269549 for 7 days at a temperature of 30°C. At the end of the fermentation, the pH of the broth was brought down from the fermentation pH of 6 towards pH 4 and cooled down to 4°C. Before the start of the recovery, the broth was well mixed for at least 20 minutes before solid liquid separation. This unit operation was performed in a Sorvall Lynx 6000 centrifuge during 1 hour. The biomass was not washed and the slight turbid supernatant was further clarified by filtration using dead-end filters. This filtration step was performed in two steps by standard paper filters (Pall). A total amount of 16669 g of clarified supernatant was obtained. For the subsequent ion exchange step, the clarified liquid was concentrated and dialyzed with demi water using an ultrafiltration unit with a 5 kD MWCO Millipore polyethersulphone membrane, until a conductivity of 8-10 mS/cm was reached. The amounts of retentate and permeate were 2967 g and 14704 g, respectively.

The main purification step was performed on an chromatography system equipped with a XK50 column. The column was filled with Cytiva Sepharose SP XL resin. The resin was first equilibrated with a sodium acetate buffer (20 mM, pH 4, conductivity 10 mS/cm), after which the feed was loaded onto the column. After binding, the bound protein was washed with the same buffer as applied during equilibration. Elution was performed with a 20 mM sodium acetate buffer at pH 4 and a conductivity of 48 mS/cm (with NaCI). After this step, an equilibration was performed.

As elution was done by sodium chloride, a dialysis and concentration step were performed using a 10 kD MWCO Koch membrane unit. A total amount of 3361 g of eluate was obtained. During dialysis, a decrease in conductivity until ~3 mS/cm was achieved. A total of 825 g of washed retentate was obtained.

Subsequently, 20 g/kg activated carbon (CA-1 , Norit) was added in batch at ambient temperature during 1 hr. The activated carbon was removed by paper filters (Pall) after the centrifugation step. Finally, the product was dried in a freeze drier (Christ Alpha 2-4 L-D Plus freeze drier), and the product was dry in 3 days at 0.05 mbar and -80°C. The product had the following properties:

The sugars analysis was as follows:

The results demonstrate that purification of p-lactoglobulin A using cationic exchange chromatography resulted in a product wherein significant amounts of sugars were removed. The remaining amount of total carbohydrates was 1 .9% of what was present in the starting material. Glucans and glucosamines were completely removed.

Additionally, the integrity of the p-lactoglobulin A was confirmed at various points in the process using SDS-page analysis (see Figure 2 and the table here below). The results demonstrate that the molecular weight and therefore the integrity of the molecule remains unchanged throughout the process of purification of p-lactoglobulin A using Cation Exchange Chromatography (CEX).

Example 3; Purification of B-lactoqlobulin B using Cation Exchange Chromatography (CEX)

Following the same process route as outlined in Example 2, also p-lactoglobulin variant B (LGB) from two different fermentations was recovered, ending up with the following product compositions (numbered LGB1 and LGB2):

The sugars analysis for sample LGB1 was as follows:

The sugars analysis for sample LGB2 was as follows:

The results demonstrate that purification of p-lactoglobulin B using cationic exchange chromatography resulted in a product wherein nearly all sugars were removed. The remaining amount of total carbohydrates was 2% of what was present in the starting material. Glucans and glucosamines were completely removed.

A graphic representation of the results obtained in examples 1 to 3, is provided in Figure 1.

Example 4; Comparative example on alternative p-lactoglobulin A purification methods, namely ultrafiltration or precipitation.

1 . Ultrafiltration

In this experiment, a 10 kDa PES membrane from Synder was used for the purification of the same p- lactoglobulin A as used in Example 2. The results in the table here below demonstrate that both p- lactoglobulin A and sugars are retained in the retentate and consequently ultrafiltration was not successful in contrast to when using a cationic exchange resin as e.g. in Example 2 herein.

2. Precipitation

In this experiment, the same p-lactoglobulin A as used in Example 2 was precipitated with sodium hexametaphosphate at pH 4. After that the precipitate was separated from the liquid by centrifugation and the pellet was redissolved in water at neutral pH. In this redissolved phase, still 9% exopolysaccharides are present on dry weight. This means that a reduction in sugar content was achieved but far less than when using a cationic exchange resin as e.g. in Example 2 herein.

Example 5; Purification of lactoferrin using Cation Exchange Chromatography (CEX)

Sugars from the waste stream of Example 2 were used to artificially create a non-pure lactoferrin sample. Thus, lactoferrin was mixed with a portion of the high-sugar waste stream from the chromatography step of Example 2. The resulting mixture was used for chromatographic purification using a chromatography system equipped with a XK50 column filled with Relisorb SP400 resin from Resindion. Further conditions were identical to the conditions in Example 2, albeit that different buffers were used as the pl of lactoferrin is higher than that of p-lactoglobulin B (pl lactoferrin ~9). The feed, equilibration, and wash buffers were pH 7 (phosphate buffer). For the elution buffer a high salt buffer was chosen (phosphate buffer + NaCI, ~50 mS/cm). The results are summarized in the table here below and demonstrate that purification of lactoferrin using cationic exchange chromatography resulted in a product wherein nearly all sugars were removed.

Claims

2. A method for the purification of a recombinant milk protein according to claim 1 , wherein the purified recombinant milk protein comprises 0.01 to 5 wt.% of sugars on dry weight and more than 80 wt.% of the recombinant milk protein as measured according to Dumas factor 6.29.

3. A method for the purification of a recombinant milk protein according to claim 1 or 2, wherein the purified recombinant milk protein comprises on dry weight from 0.01 to 1 wt.% mannans, and/or from 0.01 to 0.1 wt.% glucans, and/or from 0.01 to 0.1 wt.% glucosamines.

4. A method for the purification of a recombinant milk protein according to any of the preceding claims, wherein the liquid containing the recombinant milk protein comprises 5 to 50 wt.% of sugars on dry weight.

5. A method forthe purification of a recombinant milk protein according to any one of the preceding claims, wherein the recombinant milk protein is produced by a microorganism selected from the group consisting of a species of Pichia, Kluyveromyces, Saccharomyces, Trichoderma, and Aspergillus.

6. A method forthe purification of a recombinant milk protein according to any one of the preceding claims, wherein the recombinant milk protein is a p-lactoglobulin, an a-lactalbumin or a lactoferrin, preferably an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N-terminal elongation and/or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation.

7. A method forthe purification of a recombinant milk protein according to any one of the preceding claims, wherein the cationic exchange chromatography comprises a matrix selected from the group consisting of a dextran, cellulose, acrylic, methacrylic, sephacel, and within the matrix a cationic exchange residue selected from the group consisting of sulfonate, sulfopropyl, phosphate, carboxylate, and carboxymethyl.

8. A method forthe purification of a recombinant milk protein according to any one of the preceding claims, wherein the recombinant milk protein binds electrostatically to the matrix whereafter the bound recombinant milk protein is eluted with a buffer, preferably a buffer comprising a compound selected from the group consisting of: sodium chloride, hydrochloric acid, citric acid, lactic acid, succinic acid, acetic acid, and phosphate.

9. A purified recombinant milk protein obtainable by a method according to any one of claims 1 to 8.

10. A composition, preferably a composition obtainable by a method according to any one of claims 1 to 8, comprising 80 wt.% or more, according to Dumas factor 6.29, recombinant milk protein and 0.01 to 5 wt.% of sugars, preferably 0.02 to 2 wt. % of sugars.

11. A composition according to claim 10, wherein the recombinant milk protein is a p-lactoglobulin, an a-lactalbumin or a lactoferrin.

12. A composition according to claim 10 or 11 , wherein the recombinant milk protein is an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N- terminal elongation and/or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,

13. A composition according to any one of claims 10 to 12, which is a powder having a dry matter content of 80 to 98%.

14. A composition according to any one of claims 10 to 13, wherein the amount of mannans on dry weight is from 0.01 to 1 wt.%, and/or the amount of glucans on dry weight is from 0.01 to 0.1 wt.%, and/or the amount of glucosamines on dry weight is from 0.01 to 0.1 wt.%.

15. A food product comprising a purified recombinant milk protein according to claim 9, or comprising a composition according to any one of claims 10 to 14.

16. A method for the preparation of a food product comprising contacting a food product with a purified recombinant milk protein according to claim 9, or with a composition according to any one of claims 10 to 14.

17. A food product according to claim 15, wherein the recombinant milk protein is a p-lactoglobulin, an a-lactalbumin or a lactoferrin.

18. A food product according to claim 17, wherein the recombinant milk protein is a an elongated p-lactoglobulin wherein at least 40% of the relative amount of p-lactoglobulin has an EAEA N- terminal elongation and/or wherein at least 3% of the relative amount of p-lactoglobulin has an EA N-terminal elongation,