WO2023192711A1 - Compositions et procédés de distribution d'agents bioactifs - Google Patents

Compositions et procédés de distribution d'agents bioactifs Download PDF

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Publication number
WO2023192711A1
WO2023192711A1 PCT/US2023/062076 US2023062076W WO2023192711A1 WO 2023192711 A1 WO2023192711 A1 WO 2023192711A1 US 2023062076 W US2023062076 W US 2023062076W WO 2023192711 A1 WO2023192711 A1 WO 2023192711A1
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Prior art keywords
native
protein
seq
amino acid
lactoglobulin
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PCT/US2023/062076
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English (en)
Inventor
Janine Lin
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Perfect Day, Inc.
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Publication of WO2023192711A1 publication Critical patent/WO2023192711A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C23/00—Other dairy products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/09—Other cheese preparations; Mixtures of cheese with other foodstuffs
    • A23C19/093—Addition of non-milk fats or non-milk proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof

Definitions

  • the present invention relates generally to the use of recombinant milk proteins for delivery of bioactive agents.
  • the present invention relates to recombinant milk proteins with bound bioactive agents and having improved in vivo release of such bioactive agents, to compositions comprising such recombinant milk proteins with bound bioactive agents, and to methods for producing and using such compositions.
  • milk proteins evolved to bind valuable nutrients, such as, for example, minerals and hydrophobic compounds, and thereby to help solubilize, stabilize, and/or protect from degradation or inactivation such nutrients, and to carry them from mother to neonate.
  • strategies may be developed for using milk proteins as high-quality, natural food components that deliver bioactive agents to promote health and treat disorders (see, for example, Chen et al. 2006. Trends Food Sci Technol. 17:272).
  • FIG. 1 is a map of a targeting vector used for production of a recombinant P- lactoglobulin protein having improved in vivo release of bioactive agents, in accordance with representative embodiments of the present invention.
  • a recombinant milk protein with bound bioactive agent wherein the recombinant milk protein comprises a modification compared to a corresponding native milk protein that facilitates cleavage and/or denaturation of the recombinant milk protein in the gastrointestinal tract of a subject, and wherein the modification thereby facilitates in vivo release of the bound bioactive agent from the recombinant milk protein.
  • the modification comprised in the recombinant milk protein with bound bioactive agent may be a modification that introduces a non-native protease recognition or cleavage sequence into the recombinant milk protein that is recognized and/or cleaved by a protease comprised in the gastrointestinal tract of a subject (e.g., a trypsin, a chymotrypsin, an elastase, a carboxypeptidase A, a carboxypeptidase B, a pepsin, a protease produced by a microorganism comprised in the gastrointestinal tract of the subject), and/or may be a modification that eliminates a post- translational modification (PTM) from the recombinant milk protein that is comprised in a corresponding native milk protein, and/or may be a modification that decreases stability of a protein structure of the recombinant milk protein at an acidic pH compared to that
  • PTM post- translational modification
  • the recombinant milk protein according to Paragraph [0009] or [0010] may be a recombinant P-lactoglobulin protein, wherein the modification that facilitates cleavage and/or denaturation of the recombinant P-lactoglobulin protein in the gastrointestinal tract of a subject is a modification that introduces a non-native protease recognition or cleavage sequence in or in the vicinity of a solvent-exposed region of a corresponding native P- lactoglobulin protein.
  • the modification according to Paragraph [0011] may consist of a single modification that creates a non-native protease recognition or cleavage sequence.
  • Such single amino acid substitution may be selected from the group consisting of: I12L, T18K, T18R, I29L, S30K, S36Y, S36L, S36K, K47L, K47P, T49K, Q59R, I72L, I72F, I72W, I72Y, I78L, I78Y, I78W, I78F, A86Y, N90R, Y102F, SHOP, S110L, S110Y, SHOW, S110F, S110K, A111P, A111K, A111L, A111Y, A111W, A111F, T125K, T125F, T125W, T125Y, E127K, E127L, E127W, E127F, E127Y, V128L, D137R, A142P, A142L, H146R, T154K, T154Y, T154L, T154W, T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO
  • the modification according to Paragraph [0011] may consist of two or more modifications that create one or more non-native protease recognition or cleavage sequences.
  • Such two or more modifications may comprise or consist of: two or more amino acid substitutions selected from the group consisting of I12L, T18K, T18R, I29L, S30K, S36Y, S36L, S36K, K47L, K47P, T49K, Q59R, I72L, I72F, I72W, I72Y, I78L, I78Y, I78W, I78F, A86Y, N90R, Y102F, SHOP, S110L, S110Y, SHOW, S110F, S110K, Al IIP, All IK, All IL, A111Y, A111W, All IF, T125K, T125F, T125W, T125Y, E127K, E127L, E127W, E127F, E127Y, V128L, D137R, A142P
  • S116HG, L117M, and Al 18V of native Capra hircus P-lactoglobulin protein S116HG, L117M, and Al 18V of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9); K141Q, A142P, M145G, and H146R of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9); P153L and Q155R of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9); F151L, N152D, P153L, T154K, and L156M of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9).
  • the bioactive agent bound by, and released from, the recombinant milk protein may be any organic compound (e.g., peptide) or inorganic compound that has bioactivity (e.g., a nutraceutical, a hydrophobic compound, a compound that has micro-/biocidal activity, a growth factor, a cytokine, a compound that soothes pain, a compound that reduces inflammation, a compound that has hemostatic activity, a milk protein or fragment thereof, a therapeutic).
  • a nutraceutical e.g., peptide
  • hydrophobic compound e.g., a compound that has micro-/biocidal activity
  • a growth factor e.g., a cytokine
  • a compound that soothes pain e.g., a compound that reduces inflammation
  • a compound that has hemostatic activity a milk protein or fragment thereof, a therapeutic.
  • composition e.g., food product, cosmetic or personal care composition, therapeutic product
  • a composition that comprises a recombinant milk protein with bound bioactive agent according to any of Paragraphs [0009] through [0014].
  • Amino acids can be referred to herein by their single-letter codes, amino acid names, or three-letter codes.
  • the single-letter codes, amino acid names, and three-letter codes are as follows: G - Glycine (Gly), P - Proline (Pro), A - Alanine (Ala), V - Valine (Vai), L - Leucine (Leu), I - Isoleucine (He), M - Methionine (Met), C - Cysteine (Cys), F - Phenylalanine (Phe), Y -Tyrosine (Tyr), W -Tryptophan (Trp), H - Histidine (His), K - Lysine (Lys), R - Arginine (Arg), Q - Glutamine (Gin), N - Asparagine (Asn), E - Glutamic Acid (Glu), D - Aspartic Acid (Asp), S - Serine (Ser), T
  • Amino acid residues are denoted by a first letter for the amino acid, followed by a number that specifies the position of the amino acid in a reference sequence (e.g., any of SEQ ID NOs: 1-9).
  • Amino acid substitutions are denoted by a first letter for the amino acid that is to be replaced, followed by a number that specifies the position of the amino acid to be replaced in a reference sequence (e.g., any of SEQ ID NOs: 1-9), and a second letter that is to be substituted at the position in place of the amino acid that is to be replaced.
  • x, y, and/or z may refer to “x” alone, “y” alone, “z” alone, “x, y, and z", “(x and y) or z", “(x and z) or y”, “(y and z) or x", "x and y” alone, “x and z” alone, “y and z” alone, or "x or y or z”.
  • the term “at least” or “one or more” as used herein refers to one, two, three, four, five, six, seven, eight, nine, ten, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more, or all of the elements subsequently listed.
  • casein refers to a polypeptide that comprises a sequence of at least 20 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a casein natively found in a mammal-produced milk (i.e., a casein that is native to a mammal-produced milk; e.g., a native casein).
  • a casein natively found in a mammal-produced milk i.e., a casein that is native to a mammal-produced milk; e.g., a native casein.
  • caseins include -casein (e.g., amino acids 16 to 224 of UniProt sequence P02666; amino acids 16 to 222 of UniProt sequence Pl 1839 or P33048; amino acids 16 to 226 of P05814), y-casein, K-casein (e.g., amino acids 22 to 190 of UniProt sequence P02668; amino acids 22 to 192 of UniProt sequence P02669 or P02670; amino acids 21 to 182 of UniProt sequence P07498), a-Sl-casein (e.g., amino acids 16 to 214 of UniProt sequence P02662, P04653, or P18626; amino acids 16 to 185 of UniProt sequence P47710), and a-S2-casein (e.g., amino acids 16 to 222 of UniProt sequence P02663; amino acids 16 to 223 of UniProt sequence P04654 or P33049, respectively).
  • -casein e.g., amino acids 16 to 224
  • corresponding native milk protein or “corresponding native P- lactoglobulin protein” as used herein refers to a native milk protein or -lactoglobulin protein, respectively, that is identical to a recombinant milk protein or recombinant P-lactoglobulin protein, respectively, that is compared to the “corresponding native milk protein” or “corresponding native P-lactoglobulin protein”, respectively, except that it does not comprise a modification as provided herein in the recombinant milk protein or recombinant P- lactoglobulin protein, respectively.
  • encoding refers to a polynucleotide that comprises a coding sequence that when placed under the control of appropriate regulatory sequences is transcribed into mRNA that may be translated into a polypeptide.
  • a coding sequence generally starts at a start codon (e.g., ATG) and ends at a stop codon (e.g., UAA, UAG and UGA).
  • a coding sequence may contain a single open reading frame, or several open reading frames (e.g., separated by introns).
  • the term "essentially free of” as used herein refers to the indicated component being either not detectable in the indicated composition by common analytical methods, or to the indicated component being present in such trace amount as to not be functional.
  • the term "functional” as used in this context refers to not materially contributing to properties of the composition comprising the trace amount of the indicated component, or to not having material activity (e.g., chemical activity, enzymatic activity) in the indicated composition comprising the trace amount of the indicated component, or to not having health-adverse effects upon use or consumption of the composition comprising the trace amount of the indicated component.
  • materially contributing refers to the indicated component contributing to an attribute of a composition to such extent that in the absence of the component (e.g., in a reference composition that is identical to the composition except that it lacks the indicated component) the attribute is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% less present/active/measurable.
  • the term "food product” as used herein refers to a composition that can be ingested by a human or an animal for dietary purposes (i.e., without ill health effects but with significant nutritional and/or caloric intake due to uptake of digested material in the gastrointestinal tract), including a domesticated animal (e.g., dog, cat), farm animal (e.g., cow, pig, horse), and wild animal (e.g., non-domesticated predatory animal).
  • the term includes compositions that can be combined with or added to one or more other ingredients to make a food product that can be ingested by a human or an animal.
  • gastrointestinal tract as used herein comprises mouth, esophagus, stomach, small intestine, large intestine, and anus.
  • filamentous fungus refers to an organism from the filamentous form of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • a filamentous fungus is distinguished from a yeast by its hyphal elongation during vegetative growth.
  • fungus refers to organisms of the phyla Ascomy cotas, Basidiomycota, Zygomycota, and Chythridiomycota, Oomycota, and Glomeromycota. It is understood, however, that fungal taxonomy is continually evolving, and therefore this specific definition of the fungal kingdom may be adjusted in the future.
  • fungal host cell refers to a host cell that is obtained from a fungus.
  • the term "homolog” as used herein refers to a protein that comprises an amino acid sequence that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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%, at least 99.5%, or 100%) identical to a sequence of amino acids of a similar length (i.e., a length that is within +/- 20% of the length of the query amino acid sequence) comprised in a reference protein, and that has a functional property that is similar (e.g., is within 50%, within 40%, within 30%, within 20%, or within 10% of) or identical to that of the reference protein.
  • the term includes polymorphic variants, interspecies homologs (e.g., ortholog
  • host cell refers not only to the particular subject cell but to the progeny of such cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the subject cell, but are still included within the scope of the term "host cell” as used herein.
  • identity refers to the nucleotide or amino acid residues that are the same when the two or more polynucleotide or polypeptide sequences, respectively, are aligned for maximum correspondence.
  • the "identity” may exist over a region of the sequences being compared (e.g., over the length of a functional domain) or over the full length of the sequences.
  • a “region” is considered to be a continuous stretch of at least 6, 9, 14, 19, 24, 29, 34, 39, or more nucleotides, or of at least 2, 6, 10, 14, 18, 22, 26, 30, or more amino acids.
  • one sequence acts as a reference sequence to which one or more test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman (1981. Adv Appl Math. 2:482), by the homology alignment algorithm of Needleman & Wunsch (1970. J Mol Biol. 48:443), by the search for similarity method of Pearson & Lipman (1988. Proc Natl Acad Sci USA.
  • milk protein refers to a polypeptide that comprises a sequence of at least 20 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a protein natively found in a mammal-produced milk (i.e., a protein that is native to a mammal-produced milk; e.g., a native whey protein or a native casein).
  • a mammal-produced milk i.e., a protein that is native to a mammal-produced milk; e.g., a native whey protein or a native case
  • mammal-produced milk refers to a milk produced by a mammal.
  • mammals include cow, human, sheep, wild sheep, goat, buffalo, camel, horse, donkey, alpaca, yak, llama, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, and woolly mammoth.
  • the term "native” as used herein refers to what is found in nature in its unmodified state (e.g., a cell that is not genetically modified by a human, and that is maintained under conditions [e.g., level of oxygenation, pH, salt concentration, temperature, and nutrient (e.g., carbon, nitrogen, sulfur) availability] that are not defined by a human).
  • conditions e.g., level of oxygenation, pH, salt concentration, temperature, and nutrient (e.g., carbon, nitrogen, sulfur) availability
  • operably linked refers to an arrangement of elements that allows them to be functionally related.
  • a promoter sequence is operably linked to a protein coding sequence if it controls the transcription of the protein coding sequence
  • a secretion signal sequence is operably linked to a protein if the secretion signal sequence directs the protein through the secretion system of a cell.
  • An "operably linked" element may be in contiguous linkage with another element, or act in trans or at a distance to another element.
  • functions that may be operably linked include control of transcription, control of translation, protein folding, and protein secretion.
  • polymer refers to a molecule that is composed of repeated molecular units that are covalently linked, either directly with each other or via intermediary molecules.
  • polymer network refers to a network of polymers that are crosslinked with each other via covalent bonds.
  • suitable covalent crosslinks include amide bonds (e.g., lactam bridges, native chemical ligation bonds, Staudinger ligation bonds) and disulfide bonds.
  • polynucleotide refers to a polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1,000) nucleotides.
  • the term includes both sense and antisense strands of DNA molecules (e.g., cDNA, genomic DNA, synthetic DNA) and RNA molecules (e.g., mRNA, synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, and/or chemical modifications.
  • a polynucleotide may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels; methylation; substitution of one or more of the naturally occurring nucleotides with an analog; intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates
  • charged linkages e.g., phosphorothio
  • modified nucleotides are described in the art (see, for example, Malyshev et al. 2014. Nature 509:385; Li et al. 2014. J. Am. Chem. Soc. 136:826). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding or other chemical interaction. Such molecules are known in the art and include, for example, molecules in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications may include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" polynucleotides.
  • a polynucleotide may be in any topological conformation.
  • a polynucleotide may be single- stranded, double-stranded, triple- stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.
  • polynucleotide sequence refers to a sequence of nucleotides that are comprised in a polynucleotide or of which a polynucleotide consists.
  • protease refers to a protein that can hydrolyze (i.e., cleave) a peptide bond (e.g., members of enzyme classification groups EC 3.4).
  • polypeptide and protein as used herein can be interchanged, and refer to a naturally-occurring or a naturally not occurring polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100) amino acids.
  • a “polypeptide” or “protein” may have an active structure or lack a functional structure, comprise coded and/or non-coded amino acids, comprise amino acids that occur in nature and/or amino acids that do not occur in nature, comprise chemically modified and/or biochemically modified and/or derivatized amino acids, comprise unmodified and/or modified peptide backbones, and/or be monomeric (i.e., having a single chain) or polymeric (i.e., having of two or more chains, which may be covalently or non-covalently associated).
  • amino acid sequence refers to a sequence of amino acids that is comprised in a “polypeptide” or “protein”, or of which a “polypeptide” or “protein” consists. Amino acids comprised in an amino acid sequence are sequentially numbered by counting from the N-terminus of the polypeptide.
  • a “polypeptide” or “protein may be isolated from naturally occurring systems (e.g., plant or animal lifeforms), synthesized in solution or on solid support, or produced recombinantly.
  • promoter sequence refers to a polynucleotide that directs transcription of a downstream polynucleotide in a cell.
  • a promoter sequence may include necessary nucleotides near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter sequence may also optionally include distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.
  • protease recognition or cleavage sequence or “recognition or cleavage sequence for a protease” as used herein refers to an amino acid sequence in a polypeptide that is preferably recognized by a protease and in which a peptide bond is cleaved by the protease.
  • the general nomenclature of positions in protease recognition or cleavage sequences are defined as described by Schechter & Berger (1967. Biochem Biophys Res Commun. 27(2): 157; 1968. Biochem Biophys Res Commun.
  • recombinant P-lactoglobulin refers to a recombinantly produced polypeptide (i.e., a polypeptide that is produced in a recombinant host cell, or to a polypeptide that is synthesized from a recombinant polynucleotide) that comprises a sequence of at least 20 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a native -lactoglobulin protein (e.g., a native Bos taunts P
  • recombinant host cell refers to a host cell that comprises a recombinant polynucleotide.
  • a recombinant host cell may produce a polynucleotide or polypeptide not found in the native (non-recombinant) form of the host cell, or a recombinant host cell may produce a polynucleotide or polypeptide at a level that is different from that in the native (non-recombinant) form of the host cell. It should be understood that such term is intended to refer not only to the particular subject cell but also to the progeny of such a cell.
  • a recombinant host cell may be an isolated cell or cell line grown in culture, or may be a cell which resides in a living tissue or organism.
  • the term "recombinant polynucleotide” as used herein refers to a polynucleotide that is removed from its naturally occurring environment, or a polynucleotide that is not associated with all or a portion of a polynucleotide abutting or proximal to the polynucleotide when it is found in nature, or a polynucleotide that is operatively linked to a polynucleotide that it is not linked to in nature, or a polynucleotide that does not occur in nature, or a polynucleotide that contains a modification that is not found in that polynucleotide in nature (e.g., insertion, deletion, or point mutation introduced artificially, e.g., by human intervention), or a polynucleotide that is integrated into a chromosome at a heterologous site.
  • a modification that is not found in that polynucleotide in nature
  • a polynucleotide is also considered “recombinant” if it contains a genetic modification that does not naturally occur.
  • an endogenous polynucleotide is considered a "recombinant polynucleotide” if it contains an insertion, deletion, or substitution of one or more nucleotides that is introduced artificially (e.g., by human intervention).
  • Such modification may introduce into the polynucleotide a point mutation, substitution mutation, deletion mutation, insertion mutation, missense mutation, frameshift mutation, duplication mutation, amplification mutation, translocation mutation, or inversion mutation.
  • the term includes a polynucleotide in a host cell’s chromosome, as well as a polynucleotide that is not in a host cell’s chromosome (e.g., a polynucleotide that is comprised in an episome).
  • a recombinant polynucleotide in a host cell or organism may replicate using the in vivo cellular machinery of the host cell; however, such recombinant polynucleotide, although subsequently replicated intracellularly, is still considered recombinant for purposes of this invention.
  • the term "regulatory element” as used herein refers a polynucleotide sequence that mediates, modulates, or regulates expression (e.g., transcription, post-transcriptional events, translation) of a polynucleotide to which the regulatory element is operably linked.
  • secretion signal refers to a peptide that is operably linked to the N-terminus of a protein, and that mediates the delivery of the protein via the intracellular secretory pathway of a host cell in which the protein is produced (i.e., synthesized) to the exterior of the host cell.
  • operable linkage of a recombinant protein with a secretion signal requires removal of a start codon of the polynucleotide sequence encoding the recombinant protein.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • two or more refers to two, three, four, five, six, seven, eight, nine, ten, or more, or all of the elements subsequently listed.
  • whey protein refers to a polypeptide that comprises a sequence of at least 20 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a whey protein natively found in a mammal-produced milk (i.e., a whey protein that is native to a mammal-produced milk; e.g., a native whey protein).
  • a mammal-produced milk i.e., a whey protein that is native to a mammal-produced milk; e.g., a native whey protein
  • Non-limiting examples of whey proteins include a-lactalbumin protein (e.g., amino acids 20-142 of UniProt sequence P00709, P00711, P00712, or P09462), P-lactoglobulin protein (amino acids 17-178 of UniProt sequence P02754, amino acids 19-180 of UniProt sequence P67976 or P02756), lactoferrin protein (e.g., amino acids 20 to 708 of UniProt sequence P24627, D3G9G3, or Q29477; amino acids 20 to 710 of UniProt sequence P02788), transferrin protein (e.g., amino acids 20 to 704 of UniProt sequence Q29443, or W5PF65; amino acids 20 to 698 of UniProt sequence A0A452FJF9 or P02787), serum albumin protein (e.g., amino acids 25 to 607 of UniProt sequence P02769 or P14639; amino acids 19 to 608 of UniProt sequence A0
  • vector refers to a nucleic acid that can carry a polynucleotide sequence to be introduced into a host cell.
  • vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), virus particles (e.g., comprising heterologous polynucleotides), DNA constructs (e.g., produced by cloning or PCR amplification), and linear double-stranded molecules (e.g., PCR fragments).
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
  • Other vectors may be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
  • yeast refers to any organism of the order Saccharomycetales. Vegetative growth of yeast is by budding/blebbing of a unicellular thallus, and carbon catabolism may be fermentative.
  • % by mass refers to a percentage value for a mass as determined in a hydrated composition, such that the composition includes the mass of powder as well as the mass of the hydrating agent, with 100% fixed as the percentage value for the entire hydrated composition.
  • the term refers to a percentage value for a mass as determined relative to the eventual entire hydrated composition (with 100% fixed as the percentage value for that entire eventual hydrated composition).
  • a recombinant milk protein with bound bioactive agent wherein the recombinant milk protein comprises a modification compared to a corresponding native milk protein that facilitates cleavage and/or denaturation of the recombinant milk protein in the gastrointestinal tract of a subject (e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig, human]), and wherein the modification thereby facilitates in vivo release of the bound bioactive agent from the recombinant milk protein.
  • a subject e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig, human]
  • a recombinant milk protein comprising bound bioactive agent according to the above may be administered to a subject (e.g., a human or other animal), may be proteolytically cleaved by one or more proteases comprised in the gastrointestinal tract of the subject, and may thus release the bioactive agent such that it can exert its bioactivity.
  • the rate of release of the bioactive agent may be controlled by the type and/or number of modifications comprised in the recombinant milk protein (e.g., the more modifications, the more proteolytic degradation of the recombinant milk protein, the more release of bioactive compound from the recombinant milk protein).
  • the recombinant milk protein with bound bioactive agent according to the above may provide numerous advantages, such as, for example, providing a suitable vehicle for delivering bioactive agents to a subject and releasing them in the gastrointestinal tract of the subject by a mechanism other than diffusion.
  • the modification comprised in the recombinant milk protein with bound bioactive agent may be a modification that introduces a non-native protease recognition or cleavage sequence into the recombinant milk protein that is recognized and/or cleaved by a protease comprised in the gastrointestinal tract of a subject.
  • the protease comprised in the gastrointestinal tract of the subject may be produced by the subject.
  • Non-limiting examples of proteases produced in the gastrointestinal tracts of subjects include trypsin (e.g., cationic trypsinogen, anionic trypsinogen, mesotrypsin, pancreasin), chymotrypsin (e.g., chymotrypsinogen Bl, chymotrypsinogen B2, caldecrin), elastase (e.g., elastase 2A, elastase 2B, elastase 3A, elastase 3B), carboxypeptidase A (e.g., carboxypeptidase Al, carboxypeptidase A2), carboxypeptidase B (e.g., carboxypeptidase Bl, carboxypeptidase B2), and pepsin.
  • trypsin e.g., cationic tryp
  • the protease comprised in the gastrointestinal tract of the subject may be produced by a microorganism comprised in the gastrointestinal tract (e.g., comprised in the biotome) of the subject.
  • proteases produced by microorganisms comprised in gastrointestinal tracts of subjects include secreted and cell wall-bound extracellular proteases produced by Alistipes putredinis, Anaerotruncus colihominis, Bacillus subtilis, Bacteroides acidofacients, Bacteroides caccae, Bacteroides capillosus, Bacteroides dorei, Bacteroides eggerthii, Bacteroides finegoldii, Bacteroides fragilis, Bacteroides intestinalis, Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides sp, Bacteroides stercosis, Bacteroides thetaiotaomicron,
  • the modification comprised in a recombinant milk protein with bound bioactive agent may, for example, provide for a lysine or arginine amino acid residue at Pl position of a non-native protease recognition or cleavage sequence such that the non-native protease recognition or cleavage sequence is recognized by trypsin.
  • the modification may provide for a leucine, proline, tryptophane, tyrosine, or phenylalanine amino acid residue at Pl position of a non-native protease recognition or cleavage sequence such that the non-native protease recognition or cleavage sequence is recognized by pepsin.
  • the modification may provide for a tryptophane, tyrosine, or phenylalanine amino acid residue at Pl position of a non-native protease recognition or cleavage sequence such that the non-native protease recognition or cleavage sequence is recognized by chymotrypsin.
  • protease recognition or cleavage sequences for proteases comprised in the gastrointestinal tract of mammals produced by mammals or by microorganisms may be deduced from data comprised in peptidase database MEROPS (https://www.ebi.ac.uk/merocs/).
  • the modification comprised in a recombinant milk protein with bound bioactive agent may eliminate a post-translational modification (PTM) from the recombinant milk protein that is comprised in a corresponding native milk protein.
  • PTM post-translational modification
  • the term "post-translational modification”, or its acronym “PTM”, as used herein refers to the covalent attachment of a chemical group to a polypeptide after biosynthesis. PTM may occur on the amino acid side chain of the polypeptide or at its C- or N-termini.
  • Non-limiting examples of PTMs include glycosylation (i.e., covalent attachment to proteins of glycan groups (e.g., monosaccharides, disaccharides, polysaccharides, linear glycans, branched glycans, glycans with galf residues, glycans with sulfate and/or phosphate residues, D-glucose, D-galactose, D-mannose, L-fucose, N-acetyl-D-galactose amine, N- acetyl-D-glucose amine, N- acetyl-D-neuraminic acid, galactofuranose, phosphodiesters, N- acetylglucos amine, N- acetylgalactosamine, sialic acid, and combinations thereof; see, for example, Deshpande et al.
  • glycan groups e.g., monosacc
  • C-linkage i.e., C- glycosylation
  • N-linkage i.e., N-glycosylation
  • O-linkage i.e., O-glycosylation
  • glypiation i.e., addition of a glycosylphosphatidylinositol anchor
  • phosphoglycosylation i.e., linked through the phosphate of a phospho-serine
  • phosphorylation i.e., covalent attachment to proteins of phosphate groups
  • alkylation i.e., covalent attachment to proteins of alkane groups (e.g., methane group in methylation
  • lipidation i.e., covalent attachment of a lipid group (e.g., isoprenoid group in prenylation and isoprenylation (e.g., famesol group in famesylation, geraniol group in geranylation,
  • the modification may make a protease recognition or cleavage sequence (e.g., a native protease recognition or cleavage sequences, or a non-native protease recognition or cleavage sequences) more accessible to a protease comprised in a gastrointestinal tract of a subject, such that the protease can more easily cleave the recombinant milk protein.
  • a protease recognition or cleavage sequence e.g., a native protease recognition or cleavage sequences, or a non-native protease recognition or cleavage sequences
  • the modifications comprised in a recombinant milk protein with bound bioactive agent may decrease stability of a protein structure of the recombinant milk protein at an acidic pH (e.g., pH of less than 7, less than 6.5, less than 6, less than 5.5, less than 5, less than 4.5, less than 4, less than 3.5, less than 3, less than 2.5, less than 2, less than 1.5, or less than 1) compared to that of a corresponding native milk protein.
  • an acidic pH e.g., pH of less than 7, less than 6.5, less than 6, less than 5.5, less than 5, less than 4.5, less than 4, less than 3.5, less than 3, less than 2.5, less than 2, less than 1.5, or less than
  • the modification may effect solvent exposure of a protease recognition or cleavage sequence (e.g., a native protease recognition or cleavage sequences, or a non-native protease recognition or cleavage sequences), such that a protease comprised in a gastrointestinal tract of a subject can cleave the recombinant milk protein.
  • a protease recognition or cleavage sequence e.g., a native protease recognition or cleavage sequences, or a non-native protease recognition or cleavage sequences
  • the modification typically has minimal impact on protein structure of the recombinant milk protein compared to that of the corresponding native milk protein. Such minimal impact may be achieved by creating the modification on a solvent-exposed loop, avoiding changing amino acids in beta-sheet or alpha-helixes, selecting conservative amino acid substitutions (i.e., substitutions of amino acids having similar biochemical properties), and/or selecting amino acid deletions, substitutions, and/or additions that do not create steric hindrances between side chains of amino acids in a three-dimensional conformation of the recombinant milk protein (as determined, for example, by examination using PyMol [Schrodinger, New York, NY] and multi- sequence alignments [e.g., of orthologs of native - lactoglobulin proteins; for example, using MUSCLE (Edgar, 2004, Nucleic Acids Res, 32: 1792-1797)]).
  • the recombinant milk protein according to any of the above may be a recombinant P-lactoglobulin protein, wherein the modification that facilitates cleavage and/or denaturation of the recombinant P-lactoglobulin protein in the gastrointestinal tract of a subject is a modification that introduces a non-native protease recognition or cleavage sequence in or in the vicinity (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids) of a solvent-exposed region of a corresponding native P-lactoglobulin protein.
  • Non- limiting examples of solvent-exposed regions include regions spanning from amino acid 1 to amino acid 14, amino acid 16 to amino acid 20, amino acid 27 to amino acid 31, amino acid 33 to amino acid 36, amino acid 40 to amino acid 41, amino acid 44 to amino acid 72, amino acid 74 to amino acid 79, amino acid 83 to amino acid 103, amino acid 105 to amino acid 117, amino acid 124 to amino acid 139, amino acid 141 to amino acid 146, amino acid 148 to amino acid 155, and amino acid 157 to amino acid 160 of native Bos taunts P-lactoglobulin protein (SEQ ID NO: 1 or 2), native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), Ovis aries musimon P-lactoglobulin protein (SEQ ID NO: 3), and Ovis aries P-lactoglobulin protein (SEQ ID NO: 4), and corresponding regions in homologs.
  • native Bos taunts P-lactoglobulin protein SEQ ID NO
  • Non-limiting examples of suitable modifications that introduce a non-native protease recognition or cleavage sequence in or in the vicinity of a solvent-exposed region of a corresponding native P- lactoglobulin protein are disclosed, for example, in PCT publication WO2021168343, published August 26, 2021, as are methods for producing such modified P-lactoglobulin proteins.
  • the modification may consist of a single modification (e.g., a single amino acid substitution, deletion, or addition) that creates a non-native protease recognition or cleavage sequence.
  • a single modification e.g., a single amino acid substitution, deletion, or addition
  • a suitable single amino acid substitution may be selected from the group consisting of: I12L (e.g., to produce a non-native pepsin recognition or cleavage sequence), T18K (e.g., to produce a non-native trypsin recognition or cleavage sequence), T18R (e.g., to produce a non-native trypsin recognition or cleavage sequence), I29L (e.g., to produce a non- native pepsin recognition or cleavage sequence), S30K (e.g., to produce a non-native trypsin recognition or cleavage sequence), S36Y (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence), S36L (e.g., to produce a non-native pepsin recognition or cleavage sequence), S36K (e.g., to produce a non-native trypsin recognition or cleavage sequence
  • a suitable single amino acid substitution may be selected from the group consisting of: I12L (e.g., to produce a non-native pepsin recognition or cleavage sequence), T18K (e.g., to produce a non-native trypsin recognition or cleavage sequence), T18R (e.g., to produce a non-native trypsin recognition or cleavage sequence), I29L (e.g., to produce a non- native pepsin recognition or cleavage sequence), S30K (e.g., to produce a non-native trypsin recognition or cleavage sequence), S36Y (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence), S36L (e.g., to produce a non-native pepsin recognition or cleavage sequence), S36K (e.g., to produce a non-native trypsin recognition or cleavage sequence
  • a suitable single amino acid substitution may be selected from the group consisting of: I29L (e.g., to produce a non-native pepsin recognition or cleavage sequence), S30K (e.g., to produce a non-native trypsin recognition or cleavage sequence), S36Y (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence), S36L (e.g., to produce a non-native pepsin recognition or cleavage sequence), S36K (e.g., to produce a non-native trypsin recognition or cleavage sequence), R47L (e.g., to produce a non-native pepsin recognition or cleavage sequence), R47P (e.g., to produce a non-native pepsin recognition or cleavage sequence), T49K (e.g., to produce a non-native trypsin recognition or cleavage
  • a suitable single amino acid substitution may be selected from the group consisting of: I29L (e.g., to produce a non-native pepsin recognition or cleavage sequence), S30K (e.g., to produce a non-native trypsin recognition or cleavage sequence), R47L (e.g., to produce a non-native pepsin recognition or cleavage sequence), R47P (e.g., to produce a non- native pepsin recognition or cleavage sequence), T49K (e.g., to produce a non-native trypsin recognition or cleavage sequence), F72L (e.g., to produce a non-native pepsin recognition or cleavage sequence), F72W (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence), F72Y (e.g., to produce a non-native pepsin and chymotis, F
  • a suitable single amino acid substitution may be selected from the group consisting of: I29L (e.g., to produce a non-native pepsin recognition or cleavage sequence), S30K (e.g., to produce a non-native trypsin recognition or cleavage sequence), S36Y (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence), S36L (e.g., to produce a non-native pepsin recognition or cleavage sequence), S36K (e.g., to produce a non-native trypsin recognition or cleavage sequence), R47L (e.g., to produce a non-native pepsin recognition or cleavage sequence), R47P (e.g., to produce a non-native pepsin recognition or cleavage sequence), T49K (e.g., to produce a non-native trypsin recognition or cleavage
  • a suitable single amino acid substitution may be selected from the group consisting of H147R (e.g., to produce a non-native trypsin recognition or cleavage sequence) of native Equus asinus P-lactoglobulin protein (SEQ ID NO: 8), and corresponding single amino acid substitutions in homologs of native Equus asinus P-lactoglobulin protein.
  • the modification may consist of two or more modifications (e.g., two or more amino acid substitutions, deletions, or additions, or combinations thereof) that create one or more non-native protease recognition or cleavage sequences.
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: I12L, T18K, T18R, I29L, S30K, S36Y, S36L, S36K, K47L, K47P, T49K, Q59R, I72L, I72F, I72W, I72Y, I78L, I78Y, I78W, I78F, A86Y, N90R, Y102F, SHOP, SHOE, S110Y, SHOW, S110F, S110K, A111P, A111K, Al HE, A111Y, A111W, All IF, T125K, T125F, T125W, T125Y, E127K, E127L, E127W, E127F, E127Y, V128L, D137R, A142P, A142L, H146R, T154K, T154Y, T154L, T154W, T154F, Q159R, and combinations thereof of native Bos
  • Such two or more amino acid substitutions may comprise or consist of I12L and one or more of: T18K or T18R, I29L, S30K, S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or All IL or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P- lactoglobulin protein (S
  • Such two or more amino acid substitutions may comprise or consist of T18K or T18R and one or more of: I29L, S30K, S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or All IL or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P- lactoglobulin protein (SEQ ID NO:
  • Such two or more amino acid substitutions may comprise or consist of I29L and one or more of: S30K, S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, S 110P or S 110L or S 110Y or S 110W or S 110F or S 110K, Al 1 IP or Al 1 IK or All IL or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of S30K and one or more of: S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or SHOE or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127E or E127W or E127F or E127Y, V128L, D137R, A142P or A142E, H146R, T154K or T154Y or T154E or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of S36Y or S36L or S36K and one or more of: K47E or K47P, T49K, Q59R, I72E or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or SHOE or S110K, A111P or A111K or A111L or A111Y, A111W or A111F, T125K or T125F or T125W or T125Y, E127K or E127E or E127W or E127F or E127Y, V128L, D137R, A142P or A142E, H146R, T154K or T154Y or T154E or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of K47L or K47P and one or more of: T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of T49K and one or more of: Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or All IL or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P- lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of Q59R and one or more of: I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y, A111W or A111F, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of 172L or 172F or 172W or 172Y and one or more of: I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y, A111W or A111F, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of I78L or I78Y or I78W or 178F and one or more of: A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or All IL or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P- lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of A86Y and one or more of: N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y, A111W or A111F, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of N90R and one or more of: Y 102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of Y102F and one or more of: SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or All IL or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P- lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of SHOP or S110L or S110Y or SHOW or S110F or S110K and one or more of: Al IIP or All IK or All IL or A111Y, A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of Al IIP or All IK or All IL or A111Y, A111W or All IF and one or more of: T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of T125K or T125F or T125W or T125Y and one or more of: E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of E127K or E127L or E127W or E127F or E127Y and one or more of: V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of V128L and one or more of: D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of D137R and one or more of: A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of A142P or A142L and one or more of: H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of H146R and one or more of: T154K or T154Y or T154L or T154W or T154F, and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more amino acid substitutions may comprise or consist of T154K or T154Y or T154L or T154W or T154F and Q159R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2).
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: I12L, T18K, T18R, I29L, S30K, S36Y, S36L, S36K, K47L, K47P, T49K, Q59R, I72L, I72F, I72W, I72Y, I78L, I78Y, I78W, I78F, A86Y, N90R, Y102F, SHOP, S110L, S110Y, SHOW, S110F, S110K, A111P, A111K, All IL, A111Y, A111W, All IF, T125K, T125F, T125W, T125Y, E127K, E127L, E127W, E127F, E127Y, V128L, D137R, A142P, A142L, H146R, T154K, T154Y, T154L, T154W, T154F, Q159R, and combinations thereof of native O
  • Such two or more amino acid substitutions may comprise or consist of I12L and one or more of: T18K or T18R, I29L, S30K, S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al 1 IP or Al 1 IK or Al 1 IL or Al 11 Y or Al 11 W or Al 1 IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, O
  • Such two or more amino acid substitutions may comprise or consist of T18K or T18R and one or more of: I29L, S30K, S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hir
  • Such two or more amino acid substitutions may comprise or consist of I29L and one or more of: S30K, S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al 1 IP or Al 1 IK or Al 1 IL or Al 11 Y or Al 11 W or Al 1 IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus L
  • Such two or more amino acid substitutions may comprise or consist of S30K and one or more of: S36Y or S36L or S36K, K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or SHOK, A111P or A111K or A111L or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /i-lacloglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of S36Y or S36L or S36K and one or more of: K47L or K47P, T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /i-lacloglobulin protein (SEQ
  • Such two or more amino acid substitutions may comprise or consist of K47L or K47P and one or more of: T49K, Q59R, I72L or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or SHOE or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127E or E127W or E127F or E127Y, V128L, D137R, A142P or A142E, H146R, T154K or T154Y or T154E or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus Mactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of T49K and one or more of: Q59R, I72E or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or SHOK, A111P or A111K or A111L or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127E or E127W or E127F or E127Y, V128L, D137R, A142P or A142E, H146R, T154K or T154Y or T154E or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /i-lacloglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of Q59R and one or more of: I72E or I72F or I72W or I72Y, I78L or I78Y or I78W or I78F, A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or Al HE or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /i-lacloglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of I72L or I72F or I72W or I72Y and one or more of: 178L or 178 Y or 178 W or 178F, A86Y, N90R, Y102F, S 110P or S 110L or S 110Y or S 110W or S 110F or S 110K, Al 1 IP or Al 1 IK or Al 1 IL or Al 11 Y or Al 11W or Al 1 IF, T125K or T125F or T125W or T125 Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus Mactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of 178L or 178 Y or 178W or I78F and one or more of: A86Y, N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or All IL or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of A86Y and one or more of: N90R, Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, A111P or A111K or A111L or A111Y or A111W or A111F, T125K or T125F or T125W or T125Y, E127K or E127E or E127W or E127F or E127Y, V128E, D137R, A142P or A142E, H146R, T154K or T154Y or T154E or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of N90R and one or more of: Y102F, SHOP or S110L or S110Y or SHOW or S110F or S110K, Al 1 IP or Al 1 IK or Al 1 IL or Al 11 Y or Al 11 W or Al 1 IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of Y102F and one or more of: SHOP or S110L or S110Y or SHOW or S110F or S110K, Al IIP or All IK or All IL or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llacloglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of SHOP or S110L or S110Y or SHOW or S110F or S110K and one or more of: A111P or A111K or A111L or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llacloglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of Al IIP or All IK or All IL or A111Y or A111W or All IF and one or more of: T125K or T125F or T125W or T125Y, E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of T125K or T125F or T125W or T125Y and one or more of: E127K or E127L or E127W or E127F or E127Y, V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of E127K or E127L or E127W or E127F or E127Y and one or more of: V128L, D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of V128L and one or more of: D137R, A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircusMactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of D137R and one or more of: A142P or A142L, H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Macloglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of A142P or A142L and one or more of: H146R, T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of H146R and one or more of: T154K or T154Y or T154L or T154W or T154F, and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /i-lacloglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more amino acid substitutions may comprise or consist of T154K or T154Y or T154L or T154W or T154F and Q159R of native Ovis aries musimon, Ovis aries, or Capra hircus /Llactoglobulin protein (SEQ ID NO: 3, 4, or 9, respectively).
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: I29L, S30K, S36Y, S36L, S36K, R47L, R47P, T49K, F72L, F72W, F72Y, S78L, S78Y, S78W, S78F, D90R, Y102F, Al IIP, A111K, A111L, A111Y, A111W, A111F, T125K, T125F, T125W, T125Y, V128L, T154K, T154Y, T154L, T154W, T154F, and combinations thereof of native Equus caballus - lactoglobulin protein (SEQ ID NO: 5), and corresponding two or more amino acid substitutions in homologs of native Equus caballus /Macloglobulin protein.
  • SEQ ID NO: 5 native Equus caballus - lactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of I29L and one or more of: S30K, S36Y or S36L or S36K, R47L or R47P, T49K, F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11Y or Al 11W or Al 1 IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Llacloglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of S30K and one or more of: S36Y or S36L or S36K, R47L or R47P, T49K, F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al IIP or All IK or All IL or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Llactoglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of S36Y or S36L or S36K and one or more of: R47L or R47P, T49K, F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11 Y or Al 11 W or Al 1 IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Flacloglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of R47L or R47P and one or more of: T49K, F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al IIP or All IK or All IL or Al 11Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Llacloglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of T49K and one or more of: F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or All IK or All IL or Al 11Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus l- lactoglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of F72L or F72W or F72Y and one or more of: S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11Y or Al 11W or Al 1 IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Flacloglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of S78L or S78Y or S78W or S78F and one or more of: D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11Y or Al 11W or Al 1 IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Flacloglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of D90R and one or more of: Y102F, Al IIP or All IK or All IL or Al 11Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus l- lactoglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of Y 102F and one or more of: Al 1 IP or Al 1 IK or Al 1 IL or All 1Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus Llactoglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of Al IIP or All IK or All IL or A111Y or A111W or All IF and one or more of: T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Llactoglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of T125K or T125F or T125W or T125Y and one or more of: V 128L, and T154K or T154Y or T154L or T154W or T154F of native Equus caballus /Flacloglobulin protein (SEQ ID NO: 5).
  • Such two or more amino acid substitutions may comprise or consist of V128L and T154K or T154Y or T154L or T154W or T154F of native Equus caballus fl- lactoglobulin protein (SEQ ID NO:
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: I29L, S30K, R47L, R47P, T49K, F72L, F72W, F72Y, S78L, S78Y, S78W, S78F, D90R, Y102F, Al IIP, All IK, All IL, A111Y, A111W, A111F, T125K, T125F, T125W, T125Y, V128L, T154K, T154Y, T154L, T154W, T154F, and combinations thereof of native Equus asinus /Llactoglobulin protein (SEQ ID NO: 6), and corresponding two or more amino acid substitutions in homologs of native Equus asinus /Flacloglobulin protein.
  • amino acid substitutions selected from the group consisting of: I29L, S30K, R47L, R47P, T49K, F72L, F72W, F72Y, S78L
  • Such two or more amino acid substitutions may comprise or consist of I29L and one or more of: S30K, R47L or R47P, T49K, F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al IIP or All IK or All IL or AlllY or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus Llactoglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of S30K and one or more of: R47L or R47P, T49K, F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11Y or Al 11W or Al 1 IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of R47L or R47P and one or more of: T49K, F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11 Y or Al 11W or Al 1 IF, T125K or T125F or T125W or T125 Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus Llactoglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of T49K and one or more of: F72L or F72W or F72Y, S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11 Y or Al 11 W or Al 1 IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinusL lactoglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of F72L or F72W or F72Y and one or more of: S78L or S78Y or S78W or S78F, D90R, Y102F, Al 1 IP or Al 1 IK or Al 1 IL or Al 11Y or Al 11W or Al 1 IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus /Llactoglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of S78L or S78Y or S78W or S78F and one or more of: D90R, Y102F, Al IIP or All IK or All IL or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of D90R and one or more of: Y102F, AlllP or AlllK or All IL or Al 11Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus /Llacloglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of Y102F and one or more of: Al IIP or All IK or All IL or A111Y or A111W or All IF, T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus /Llacloglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of Al IIP or All IK or All IL or A111Y or A111W or All IF and one or more of: T125K or T125F or T125W or T125Y, V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus Llactoglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of T125K or T125F or T125W or T125Y and one or more of: V128L, and T154K or T154Y or T154L or T154W or T154F of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 6).
  • Such two or more amino acid substitutions may comprise or consist of V128L and T154K or T154Y or T154L or T154W or T154F of native Equus asinus Llactoglobulin protein (SEQ ID NO: 6).
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: I29L, S30K, S36Y, S36L, S36K, R47L, R47P, T49K, V72L, V72F, V72W, V72Y, D78L, D78Y, D78W, D78F, Y102F, T126K, T126F, T126W, T126Y, V129L, S138R, and combinations thereof of native Equus caballus /Llactoglobulin protein (SEQ ID NO: 7), and corresponding two or more amino acid substitutions in homologs of native Equus caballus Llactoglobulin protein.
  • amino acid substitutions selected from the group consisting of: I29L, S30K, S36Y, S36L, S36K, R47L, R47P, T49K, V72L, V72F, V72W, V72Y, D78L, D78Y, D78W, D78F, Y
  • Such two or more amino acid substitutions may comprise or consist of I29L and one or more of: S30K, S36Y or S36L or S36K, R47L or R47P, T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, and S138R of native Equus caballus /Llacloglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of S30K and one or more of: S36Y or S36L or S36K, R47L or R47P, T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, and S138R of native Equus caballus /Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of S36Y or S36L or S36K and one or more of: R47L or R47P, T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, and S138R of native Equus caballus /Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of R47L or R47P and one or more of: T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, and S 138R of native Equus caballus /Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of T49K and one or more of: V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, and S138R of native Equus caballus /Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of V72L or V72F or V72W or V72Y and one or more of D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, and S138R of native Equus caballus /Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of D78L or D78Y or D78W or D78F and one or more of: Y102F, T126K or T126F or T126W or T126Y, V129L, and S138R of native Equus caballus /Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of Y102F and one or more of: T126K or T126F or T126W or T126Y, V129L, and S138R of native Equus caballusMactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of T126K or T126F or T126W or T126Y and one or more of: V129L and S138R of native Equus caballus Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more amino acid substitutions may comprise or consist of V129L and S138R of native Equus caballus /Mactoglobulin protein (SEQ ID NO: 7).
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: I29L, S30K, S36Y, S36L, S36K, R47L, R47P, T49K, V72L, V72F, V72W, V72Y, D78L, D78Y, D78W, D78F, Y102F, T126K, T126F, T126W, T126Y, V129L, S138R, H147R, and combinations thereof of native Equus asinus /Mactoglobulin protein (SEQ ID NO: 8), and corresponding two or more amino acid substitutions in homologs of native Equus asinus /Mactoglobulin protein.
  • SEQ ID NO: 8 native Equus asinus /Mactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of I29L and one or more of: S30K, S36Y or S36L or S36K, R47L or R47P, T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, S138R, and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of S30K and one or more of: S36Y or S36L or S36K, R47L or R47P, T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, S138R, and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of S36Y or S36L or S36K and one or more of: R47L or R47P, T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, S138R, and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of R47L or R47P and one or more of: T49K, V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V 129L, S 138R, and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of T49K and one or more of: V72L or V72F or V72W or V72Y, D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, S138R, and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of V72L or V72F or V72W or V72Y and one or more of: D78L or D78Y or D78W or D78F, Y102F, T126K or T126F or T126W or T126Y, V129L, S138R, and H147R of native Equus asinus Mactoglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of D78L or D78Y or D78W or D78F and one or more of: Y102F, T126K or T126F or T126W or T126Y, V129L, S138R, and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of Y102F and one or more of: T126K or T126F or T126W or T126Y, V129L, S138R, and H147R of native Equus asinus - lactoglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of T126K or T126F or T126W or T126Y and one or more of: V129L, S138R, and H147R of native Equus asinus Mactoglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of V129L and one or more of: S138R and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of S138R and H147R of native Equus asinus /Flacloglobulin protein (SEQ ID NO: 8).
  • Such two or more amino acid substitutions may comprise or consist of K47L, G52D, and D53N of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence KPTPEGD at amino acid position 47 through 53 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence LPTPEDN (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of I72L, A73G, K77E, I78N, A80K, and V81K of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence IAEKTKIPAV at amino acid position 72 through 81 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence LGEKTENPKK (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K83T, I84V, D85N, A86Y, L87Q, N88G, and N90R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence KIDALNEN at amino acid position 83 through 90 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence TVNYQGER (e.g., to produce a non-native pepsin, chymotrypsin, and trypsin recognition or cleavage sequence).
  • SEQ ID NO: 1 or 2 amino acid sequence KIDALNEN at amino acid position 83 through 90 of native Bos taurus P-lactoglobulin protein
  • TVNYQGER e.g., to produce a non-native pepsin, chymotrypsin, and trypsin recognition or cleavage sequence
  • Such two or more amino acid substitutions may comprise or consist of D85N, A86Y, N88D, and N90D of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence DALNEN at amino acid position 85 through 90 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence NYLDED (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of E108G, N109P, SHOP, All IL, E112P, P113S, E114A, Q115E, S116HG, and LI 17M of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1), for example such that amino acid sequence ENSAEPEQSL at amino acid position 108 through 117 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: Q is converted to amino acid sequence GPPLPSAEHGM (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of E108G, N109P, SHOP, A111L, E112P, P113S, E114A, Q115E, S116HG, L117M, and A118V of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 2), for example such that amino acid sequence ENSAEPEQSLA at amino acid position 108 through 118 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 2) is converted to amino acid sequence GPPLPSAEHGMV (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of E127K and D130K of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence EVDD at amino acid position 127 through 130 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence KVDK (e.g., to produce a non-native trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K141Q, A142P, M145G, and H146R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence KALPMH at amino acid position 141 through 146 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence QPLPGR (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of P153L and Q155R of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence PTQ at amino acid position 153 through 155 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence LTR (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of F151L, N152D, P153L, T154K, and L156M of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), for example such that amino acid sequence FNPTQL at amino acid position 151 through 156 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2) is converted to amino acid sequence LDLKQM (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K47L and G52D of native Ovis aries musimon or Ovis aries /i-lacloglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence KPTPEG at amino acid position 47 through 52 of native Ovis aries musimon or Ovis aries / lacloglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence LPTPED (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • SEQ ID NO: 3 or 4 native Ovis aries musimon or Ovis aries /i-lacloglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of I72L, A73G, K77E, I78N, A80K, and V81K of native Ovis aries musimon or Ovis aries /i-lacloglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence IAEKTKIPAV at amino acid position 72 through 81 of native Ovis aries musimon or Ovis aries /Mactoglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence LGEKTENPKK (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K83T, I84V, D85N, A86Y, L87Q, N88G, and N90R of native Ovis aries musimon or Ovis aries /Mactoglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence KIDALNEN at amino acid position 83 through 90 of native Ovis aries musimon or Ovis aries /Mactoglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence TVNYQGER (e.g., to produce a non-native pepsin, chymotrypsin, and trypsin recognition or cleavage sequence).
  • SEQ ID NO: 3 or 4 native Ovis aries musimon or Ovis aries /Mactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of D85N, A86Y, N88D, and N90D of native Ovis aries musimon or Ovis aries /Llacloglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence DALNEN at amino acid position 85 through 90 of native Ovis aries musimon or Ovis aries /Llactoglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence NYLDED (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of E108G, N109P, SHOP, All IL, E112P, P113S, E114A, Q115E, S116HG, L117M, and Al 18V of native Ovis aries musimon or Ovis aries /Llactoglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence ENSAEPEQSLA at amino acid position 108 through 118 of native Ovis aries musimon or Ovis aries /Llactoglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence GPPLPSAEHGMV (e.g., to produce a non- native pepsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of E127K and N130K of native Ovis aries musimon or Ovis aries i- lactoglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence EVDN at amino acid position 127 through 130 of native Ovis aries musimon or Ovis aries Llactoglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence KVDK (e.g., to produce a non-native trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K141Q, A142P, M145G, and H146R of native Ovis aries musimon or Ovis aries /Llacloglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence KALPMH at amino acid position 141 through 146 of native Ovis aries musimon or Ovis ariesL lactoglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence QPLPGR (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of P153L, and Q155R of native Ovis aries musimon or Ovis aries /Llacloglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence PTQ at amino acid position 153 through 155 of native Ovis aries musimon or Ovis aries /Llactoglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence LTR (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of F151L, N152D, P153L, T154K, and L156M of native Ovis aries musimon or Ovis aries /Llacloglobulin protein (SEQ ID NO: 3 or 4, respectively), for example such that amino acid sequence FNPTQL at amino acid position 151 through 156 of native Ovis aries musimon or Ovis aries /Llacloglobulin protein (SEQ ID NO: 3 or 4, respectively) is converted to amino acid sequence LDLKQM (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • SEQ ID NO: 3 or 4 native Ovis aries musimon or Ovis aries /Llacloglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of F72L, A73G, S78N, A80K, and E81K of native Equus caballus or Equus asinus Mactoglobulin protein (SEQ ID NO: 5 or 6 , respectively), for example such that amino acid sequence FAEKTESPAE at amino acid position 72 through 81 of native Equus caballus or Equus asinus /i-lacloglobulin protein (SEQ ID NO: 5 or 6, respectively) is converted to amino acid sequence LGEKTENPKK (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K108G, N109P, A110P, A111L, T112P, P113S, G114A, Q115E, S116HG, and L117M of native Equus caballus or Equus asinus /Macloglobulin protein (SEQ ID NO: 5 or 6, respectively), for example such that amino acid sequence KNAATPGQSL at amino acid position 108 through 117 of native Equus caballus or Equus asinus Mactoglobulin protein (SEQ ID NO: 5 or 6, respectively) is converted to amino acid sequence GPPLPSAEHGM (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of P151L, T154K, and R155Q of native Equus caballus or Equus asinus /i-lacloglobulin protein (SEQ ID NO: 5 or 6, respectively), for example such that amino acid sequence PDLTR at amino acid position 151 through 155 of native Equus caballus or Equus asinus /Macloglobulin protein (SEQ ID NO: 5 or 6, respectively) is converted to amino acid sequence LDLKQ (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of R47L and G52D of native Equus caballus or Equus asinus Mactoglobulin protein (SEQ ID NO: 7 or 8, respectively), for example such that amino acid sequence RPTPEG at amino acid position 47 through 52 of native Equus caballus or Equus asinus />-lacloglobulin protein (SEQ ID NO: 7 or 8, respectively) is converted to amino acid sequence LPTPED (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • SEQ ID NO: 7 or 8 native Equus caballus or Equus asinus Mactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of V72L, A73G, Q74E, D78N, A80K, and V8 IK of native Equus caballus or Equus asinus Mactoglobulin protein (SEQ ID NO: 7 or 8, respectively), for example such that amino acid sequence VAQKTEDPAV at amino acid position 72 through 81 of native Equus caballus or Equus asinus Mactoglobulin protein (SEQ ID NO: 7 or 8, respectively) is converted to amino acid sequence LGEKTENPKK (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of P154L, S155T, and G156R of native Equus caballus or Equus asinus /i-lacloglobulin protein (SEQ ID NO: 7 or 8, respectively), for example such that amino acid sequence PSG at amino acid position 154 through 156 of native Equus caballus or Equus asinus Mactoglobulin protein (SEQ ID NO: 7 or 8, respectively) is converted to amino acid sequence LTR (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • SEQ ID NO: 7 or 8 native Equus caballus or Equus asinus /i-lacloglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of Q152L, P154L, S155K, G156Q, and G157M of native Equus caballus or Equus asinus /3- lactoglobulin protein (SEQ ID NO: 7 or 8, respectively), for example such that amino acid sequence QDPSGG at amino acid position 152 through 157 of native Equus caballus or Equus asinus Mactoglobulin protein (SEQ ID NO: 7 or 8, respectively) is converted to amino acid sequence LDLKQM (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K47L and G52D of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence KPTPEG at amino acid position 47 through 52 of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence LPTPED (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • SEQ ID NO: 9 native Capra hircus P-lactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of I72L, A73G, K77E, I78N, A80K, and V81K of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence IAEKTKIPAV at amino acid position 72 through 81 of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence LGEKTENPKK (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K83T, I84V, D85N, A86Y, L87Q, N88G, and N90R of native Capra hircus P- lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence KIDALNEN at amino acid position 83 through 90 of native Capra hircus P- lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence TVNYQGER (e.g., to produce a non-native pepsin, chymotrypsin, and trypsin recognition or cleavage sequence).
  • SEQ ID NO: 9 native Capra hircus P- lactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of D85N, A86Y, N88D, and N90D of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence DALNEN at amino acid position 85 through 90 of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence NYLDED (e.g., to produce a non-native pepsin and chymotrypsin recognition or cleavage sequence).
  • SEQ ID NO: 9 native Capra hircus P-lactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of E108G, N109P, SHOP, All IL, E112P, P113S, E114A, Q115E, S116HG, L117M, and Al 18V of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence ENSAEPEQSLA at amino acid position 108 through 118 of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence GPPLPSAEHGMV (e.g., to produce a non-native pepsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of K141Q, A142P, M145G, and H146R of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence KALPMH at amino acid position 141 through 146 of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence QPLPGR (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • Such two or more amino acid substitutions may comprise or consist of P153L and Q155R of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence PTQ at amino acid position 153 through 155 of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence LTR (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • SEQ ID NO: 9 native Capra hircus P-lactoglobulin protein
  • Such two or more amino acid substitutions may comprise or consist of F151L, N152D, P153L, T154K, and L156M of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), for example such that amino acid sequence FNPTQL at amino acid position 151 through 156 of native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9) is converted to amino acid sequence LDLKQM (e.g., to produce a non-native pepsin and trypsin recognition or cleavage sequence).
  • SEQ ID NO: 9 native Capra hircus P-lactoglobulin protein
  • the recombinant P-lactoglobulin protein according to any of the above may further comprise a non-native protease recognition or cleavage sequence in or in the vicinity (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids) of a lipid-binding region of a corresponding native P-lactoglobulin protein.
  • Non-limiting examples of lipid- binding regions include regions spanning from amino acid 136 to amino acid 149 of native Bos taurus P-lactoglobulin protein (SEQ ID NO: 1 or 2), native Capra hircus P-lactoglobulin protein (SEQ ID NO: 9), Ovis aries musimon P-lactoglobulin protein (SEQ ID NO: 3), and Ovis aries P-lactoglobulin protein (SEQ ID NO: 4), and corresponding regions in homologs.
  • the bioactive agent bound by, and released from, the recombinant milk protein according to any of the above may be any organic compound (e.g., peptide) or inorganic compound that has bioactivity.
  • suitable bioactive agents include nutraceuticals (i.e., compounds that have physiological benefit or provide protection against disease), hydrophobic compounds, compounds that have micro-/biocidal activity, growth factors, cytokines, compound that soothe pain, compounds that reduce inflammation, compounds that have hemostatic activity, milk proteins or fragments thereof, and therapeutics (i.e., compounds that treat disease; e.g., compounds that have anti-cancer activity).
  • nutraceuticals include prebiotics, probiotics, minerals, pro-vitamins and vitamins, antioxidants, carbohydrates, lipids, and essential and semiessential amino acids.
  • Non-limiting examples of minerals include calcium, phosphorous, potassium, sodium, citrate, chloride, phosphate, sulfate, magnesium, potassium, zinc, iron, molybdenum, manganese, and copper.
  • Non-limiting examples of vitamins include lipid soluble vitamins, water soluble vitamins, thiamin (vitamin Bl), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), cobalamin (vitamin B12), vitamin C, folate, vitamin A, vitamin D2, vitamin D3, vitamin E, vitamin K, and derivatives.
  • Non-limiting examples of antioxidants include fatty acids (e.g., linolenic, linoleic, oleic) and derivatives (e.g., lipoic acid), N-acetyl cysteine, a-tocopherol (e.g., tocopherol comprised in Bos taurus milk), low molecular weight thiols (e.g., low molecular weight thiols comprised in Bos taurus milk), retinol (e.g., retinol comprised in Bos taurus milk), carotenoids (e.g., carotenoids comprised in cow milk, a-carotene, P-carotene, y-carotene, lutein, zeaxanthin, astaxanthin), vitamin E, Azadirachta indica extract, riboflavin, rosemary extract, phenolic diterpenes (e.g., camosol, camosic acid) comprise
  • Nonlimiting examples of carbohydrates include: monosaccharides, such as, for example, glucose, fructose, galactose, and mixtures thereof; disaccharides, such as, for example, maltose, lactose, sucrose, and mixtures thereof; polysaccharides, such as for example, starches (e.g., pectin, com (maize) starch, oat starch, potato starch, sweet potato starch, rice starch, pea starch, wheat starch, azuki starch, green bean starch, kudzu starch, Katakuri starch, arrowroot starch, mung bean starch, sago starch, tapioca starch, plant starch (e.g., starch obtained from any of the plants disclosed herein), and derivatives thereof, and mixtures of two or more thereof.
  • monosaccharides such as, for example, glucose, fructose, galactose, and mixtures thereof
  • disaccharides such as,
  • the starch is a modified starch (e.g., pregelatinized starch (e.g., com, wheat, tapioca), pregelatinized high amylose content starch, pregelatinized hydrolyzed starches (e.g., maltodextrins, corn symp solids, rice symp solids, tapioca symp solids), chemically modified starches such as pregelatinized substituted starches (e.g., octenyl succinate modified starches), alkaline modified starch, bleached starch, oxidized starch, monostarch phosphate, distarch phosphate, phosphated distarch phosphate, acetylated distarch phosphate, acetylated starch, mono starch acetate, acetylated starch, mono starch acetate, acetylated distarch adipate, distarch glycerine, hydroxy propyl
  • the gum is a modified gum (e.g., deacetylated, deacetylated clarified, partially deacetylated, partially deacetylated clarified, and derivatives thereof, and mixtures of two or more thereof)), edible fibers (e.g., acacia fiber, bamboo fiber, barley bran, carrot fiber, cellulose (e.g., wood pulp cellulose), citrus fiber, com bran, soluble dietary fiber, insoluble dietary fiber, oat bran, pea fiber, rice bran, head husks, psyllium husk, konjac, soy fiber, soy polysaccharide, wheat bran, inulin, and derivatives thereof, and mixtures of two or more thereof), and mixtures of two or more thereof; and mixtures of two or more thereof.
  • edible fibers e.g., acacia fiber, bamboo fiber, barley bran, carrot fiber, cellulose (e.g., wood pulp cellulose), citrus fiber, com bran,
  • Non-limiting examples of lipids include fats, oils, monoglycerides, diglycerides, triglycerides, phospholipids, and free fatty acids.
  • oils include plant oils (e.g., sunflower oil, coconut oil, mustard seed oil, peanut oil, camelina sativa oil, canola oil, com oil, cottonseed oil, cuphea oil, flax seed oil, olive oil, palm oil, rapeseed oil, safflower oil, sesame oil, soybean oil, almond oil, beech nut oil, brazil nut oil, cashew nut oil, hazelnut oil, macadamia nut oil, mongongo nut oil, pecan oil, pine nut oil, pistachio nut oil, walnut oil, avocado oil, grape oil), microbe-derived oils, algae-derived oils, fungus-derived oils, marine animal oils (e.g., Atlantic fish oil, Pacific fish oil, Mediterranean fish oil, light pressed fish oil, alkaline treated fish oil, heat treated fish oil, light and heavy brown fish oil, bonito oil, pilchard oil, tuna oil, sea bass oil, halibut oil, spearfish oil
  • plant oils e.g
  • longer chain oils e.g., sunflower oil, corn oil, olive oil, soy oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, canola oil, safflower oil, flax seed oil, palm oil, palm kernel oil, palm fmit oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, engineered sunflower oil that over-expresses oleic acid by 400%) are combined with short-chain triglycerides to produce transesterified fatty acid esters (e.g., to create a specific flavor profile).
  • sunflower oil e.g., corn oil, olive oil, soy oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, canola oil, safflower oil, flax seed oil, palm oil, palm kernel oil, palm fmit oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil,
  • Non-limiting examples of monoglycerides and diglycerides include plant-derived monoglycerides and diglycerides, (e.g., monoglycerides and diglycerides derived from sunflower, coconut, peanut, cottonseed, olive, palm, rapeseed, safflower, sesame seed, soybean, almond, beech nut, Brazil nut, cashew, hazelnut, macadameia nut, mongongo nut, pecan, pine nut, pistachio, walnut, and avocado).
  • the monoglycerides and diglycerides can comprise acyl chains of any free fatty acid known in the art, including acyl chains of any free fatty acid disclosed herein.
  • Non-limiting examples of triglycerides include tributyrin, short-chain triglycerides, short-chain triglycerides comprising three oleic acids; short-chain triglycerides comprising hexanoic acid; short-chain triglycerides comprising hexanoic acid and butyric acid; short-chain triglycerides comprising hexanoic acid and decanoic acid; and short-chain triglycerides comprising one butyric, one hexanoic, and one octanoic acid.
  • Non-limiting examples of phospholipids include lecithin phospholipids (e.g., soy lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, rapeseed lecithin phospholipids, rice bran lecithin phospholipids, com lecithin phospholipids, flour lecithin phospholipids), cardiolipin, ceramide phosphocholines, ceramide phosphoethanolamines, glycerophospholipids, phasphatidicacid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphospingolipids, and phsophatidylserine.
  • lecithin phospholipids e.g., soy lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, rapeseed lecithin phospholipids
  • Non-limiting examples of free fatty acids include butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, pamitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, omega-fatty acids (e.g., arachidonic acid, omega-3-fatty acids, omega-6-fatty acids, omega-7-fatty acids, omega-9-fatty acids), fatty acids with even number of carbons of 4-16 carbons in length, monosaturated acids (particularly with 18 carbons), fatty acids with low interfa
  • Non-limiting examples of essential and semi-essential amino acids include cysteine, methionine, isoleucine, leucine, phenylalanine, tryptophan, and valine.
  • Non-limiting examples of compounds that have micro-Zbiocidal activity include antibiotics, antiseptics, antimicrobial peptides, short-chain triacylglycerides, cannabinoids, copper, non-ionic silver, polyhexamethylene biguanide, iodine, chlorhexidine, benzalconium chloride, triclosan, and benzalkonium chloride.
  • Non-limiting examples of milk proteins or fragments thereof include lactoferrin/lactoferricin protein, lactoperoxidase protein, GMP, casein protein, a-lactalbumin protein, and lysozyme protein.
  • Non-limiting examples of compounds that have anti-cancer activity include all- trans-retinol, all-trans retinoic acid [ATRA], all-trans-retinyl acetate, butyrate, curcumin, epigallocatechin gallate (EGCG), geinistein, 3,3-diindolylmethane (DII), indole-3-carbinol, and resveratrol.
  • the bioactive agent may be bound to the recombinant milk protein according to any of the above (e.g., a recombinant P-lactoglobulin protein according to any of the above) via covalent bonding (e.g., via chemical crosslinking [e.g., thiol-ene click reaction, Schiff base reaction, amide bonding] or enzymatic crosslinking [e.g., crosslinking by activity of a transglutaminase]) or non-covalent bonding (e.g., ionic interaction, hydrophobic interaction, hydrogen bonding).
  • covalent bonding e.g., via chemical crosslinking [e.g., thiol-ene click reaction, Schiff base reaction, amide bonding] or enzymatic crosslinking [e.g., crosslinking by activity of a transglutaminase]
  • non-covalent bonding e.g., ionic interaction, hydrophobic interaction, hydrogen bonding
  • the bioactive agent may be bound to the recombinant milk protein according to any of the above (e.g., a recombinant P-lactoglobulin protein according to any of the above) either directly or indirectly (e.g., via a linker peptide).
  • the bioactive agent may be bound to a site comprised in a solvent-exposed region of the recombinant milk protein (e.g., a solvent-exposed regions of a recombinant P- lactoglobulin protein according to any of the above) or a not-solvent-exposed region of the recombinant milk protein (e.g., a lipid-binding region of a recombinant P-lactoglobulin protein according to any of the above).
  • a solvent-exposed region of the recombinant milk protein e.g., a solvent-exposed regions of a recombinant P- lactoglobulin protein according to any of the above
  • a not-solvent-exposed region of the recombinant milk protein e.g., a lipid-binding region of a recombinant P-lactoglobulin protein according to any of the above.
  • composition that comprises a recombinant milk protein with bound bioactive agent according to any of the above.
  • the composition may comprise between 0.001% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%; between 0.1% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, or 0.2%; between 0.2% and 100%, 95%, 90%, 85%, 80%, 7
  • the composition according to any of the above may be a fluid, semi-solid (e.g., gelatinous), solid, or powder.
  • the powder may comprise a moisture content of less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 3%, or less than 1%; or between 0.1% and 20%, 15%, 10%, 5%, or 1%; between 1% and 20%, 15%, 10%, or 5%; between 5% and 20%, 15%, or 10%; between 10% and 20%, or 15%; or between 15% and 20%.
  • the powder may be used in powder form, or the powder may be reconstituted with a hydrating agent prior to use, or the powder may be mixed with other dry components (e.g., flour, sugar, minerals, pH or ionic strength adjusting agents) before a hydrating agent is added to the mixture.
  • suitable hydrating agents include water, milk (e.g., animal milk, nut milk, plant-based milk), juice (e.g., vegetable juice, fruit juice, other plant juice), brine (e.g., fluid or liquid used to soak beans or legumes), and mixtures thereof.
  • composition according to any of the above may comprise or consist of a recombinant milk protein with bound bioactive agent according to any of the above as a monomer, and/or may comprise a recombinant milk protein with bound bioactive agent according to any of the above as a polymer comprising linked repeated protein monomers, wherein the repeated protein monomers comprise or consist of a recombinant milk protein with bound bioactive agent according to any of the above, or of dimers, trimers, or tetramers of a recombinant milk protein with bound bioactive agent according to any of the above.
  • compositions comprising linked repeated protein monomers that comprise or consist of recombinant milk proteins, and methods for producing such polymers, are disclosed in patent publication US20210235714, filed August 15, 2012.
  • the composition according to any of the above may comprise the recombinant milk protein with bound bioactive agent according to any of the above as a monomer or as a polymer, comprised (e.g., covalently bound, non- covalently bound, encapsulated, physically trapped) in another polymer.
  • Non-limiting examples of suitable polymers in which the recombinant milk protein monomer or polymer according to any of the above may be comprised include a polymer comprising linked repeated monomers of a polysaccharide (e.g., cellulose, cellulose derivatives [e.g., hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose], heparin, hyaluronic acid, pectin, chondroitin sulfate, pullulan, dextrin, dextran), alginate [e.g., calcium alginate], alginate derivatives, chitosan, chitin) or a protein (e.g., collagen, gelatin, elastin, fibrinogen, fibrin, keratin).
  • a polysaccharide e.g., cellulose, cellulose derivatives [e.g., hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethyl cellulose,
  • composition according to any of the above may comprise between 0.001% and 100% (e.g., between 0.001% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01%; between 0.01% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1%; between 0.1% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 1%; between 1% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%; between 10% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%; between 10% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%; between 20% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%,
  • composition according to any of the above may be essentially free of any protein other than the recombinant protein with bound bioactive agent contained in the composition according to any of the above.
  • composition according to any of the above may be essentially free of any recombinant protein other than the recombinant protein with bound bioactive agent contained in the composition according to any of the above.
  • composition according to any of the above may be essentially free of any recombinant milk protein other than the recombinant protein with bound bioactive agent contained in the composition according to any of the above.
  • composition according to any of the above may be essentially free of a component found in a mammal-produced milk (e.g., cow milk, goat milk, sheep milk, human milk, buffalo milk, yak milk, camel milk, llama milk, alpaca milk, horse milk, donkey milk), or may comprise a lower concentration of at least one component found in a mammal- produced milk.
  • a mammal-produced milk e.g., cow milk, goat milk, sheep milk, human milk, buffalo milk, yak milk, camel milk, llama milk, alpaca milk, horse milk, donkey milk
  • Non- limiting examples of components found in mammal-derived milk include lactose, saturated fat, cholesterol, native milk proteins, and native milk lipidsA
  • composition according to any of the above may be essentially free of a component obtained from an animal (i.e., a component that is native to an animal, including animal products [i.e., parts of an animal that are consumables or typically prepared for consumption by humans; e.g., animal meat, animal fat, animal blood], animal byproducts [i.e., products that are typically not consumable by themselves but are the byproducts of slaughtering animals for consumption; e.g., animal bones, animal carcasses, and constituents isolated therefrom], products produced by an animal [e.g., mammal-derived milk, chicken eggs, bee honey], and consumables produced therefrom [e.g., gelatin, rennet, whey proteins extracted from mammal-derived milk, casein extracted from mammal-derived milk, milk lipid extracted from mammal-derived milk, animal lipids, animal proteins]), or comprise 2% or less by mass of such component.
  • an animal i.e., a component that is native to an animal, including animal products
  • composition according to any of the above may have an allergenicity that is lower than that of a corresponding composition (i.e., a composition that is identical to the composition that is compared to the "corresponding composition” except that the “corresponding composition” does comprises a recombinant milk protein with bound bioactive agent that does not have the modification that facilitates cleavage and/or denaturation of the recombinant milk protein as provided herein), such as, for example, an allergenicity of no more than 0%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% relative to that of a comparable composition.
  • a corresponding composition i.e., a composition that is identical to the composition that is compared to the "corresponding composition” except that the “corresponding composition” does comprises a recombinant milk protein with bound bioactive agent that does not have the modification that facilitates cleavage and/or denatur
  • composition according to any of the above may be a food product.
  • the term "food product” as used herein refers to a composition that can be ingested by a human or an animal for dietary purposes (i.e., without ill health effects but with significant nutritional and/or caloric intake due to uptake of digested material in the gastrointestinal tract), including a domesticated animal (e.g., dog, cat), farm animal (e.g., cow, pig, horse), and wild animal (e.g., non-domesticated predatory animal).
  • the term includes compositions that can be combined with or added to one or more other ingredients to make a food product that can be ingested by a human or an animal.
  • Food products comprising milk proteins are desirable, particularly for athletes, as milk proteins have high contents of essential and branched-chain amino acids, which are thought to aide production of muscle tissue.
  • P-lactoglobulin is desirable as a food additive as it has good water binding ability, which property makes P- lactoglobulin suitable for managing water activity of food products.
  • P- lactoglobulin is desirable as a food additive as it has anti-microbial activity, which property makes P-lactoglobulin suitable for extending the shelf life of food products.
  • P- lactoglobulin is desirable as a food additive as it can readily absorb at interfaces, which property makes P-lactoglobulin suitable for producing highly stable dispersions in food products.
  • food products comprising milk proteins with bound bioactive agents are desirable as the milk proteins may solubilize, stabilize, and/or protect from degradation such bioactive agents until they are released by the milk proteins to exert their bioactivity.
  • the food product may be a food product, or may resemble a food product (i.e., may be a “substitute food product”), selected from any of the food product categories defined by the National Health and Nutrition Examination Survey (NHANES).
  • NHANES food product categories include snack foods and gums (e.g., snack bars, crackers, salty snacks from grain products, chewing gums); breads, grains, and pastas (e.g., oat breads and rolls, combread, com muffins, tortillas, flour and dry mixes, biscuits, multi-grain breads and rolls, whole wheat breads and rolls, pastas, rye breads and rolls, cracked wheat breads and rolls, white breads and rolls); beverages (e.g., beers and ales, beverage concentrates, beverages, energy drinks, sports drinks, fluid replacements, soft drinks, carbonated beverages, juices, wines, beers, cocktails, nutrition drinks, nutrition powders, protein-enriched beverages, coffee, tea); sweets and desserts (e.
  • the food product according to any of the above may be a dairy product, a supplemented dairy product (i.e., a conventional dairy product that is supplemented with the recombinant milk protein according to any of the above), or substitute dairy product (i.e., a food product that resembles a conventional dairy product).
  • a supplemented dairy product i.e., a conventional dairy product that is supplemented with the recombinant milk protein according to any of the above
  • substitute dairy product i.e., a food product that resembles a conventional dairy product
  • dairy product refers to milk (e.g., whole milk [at least 3.25% milk fat], partly skimmed milk [from 1% to 2% milk fat], skim milk [less than 0.2% milk fat], cooking milk, condensed milk, flavored milk, goat milk, sheep milk, dried milk, evaporated milk, milk foam), and products derived from milk, including but not limited to yogurt (e.g., whole milk yogurt [at least 6 grams of fat per 170 g], low-fat yogurt [between 2 and 5 grams of fat per 170 g], nonfat yogurt [0.5 grams or less of fat per 170 g], greek yogurt [strained yogurt with whey removed], whipped yogurt, goat milk yogurt, Labneh [labne], sheep milk yogurt, yogurt drinks [e.g., whole milk Kefir, low-fat milk Kefir], Lassi), cheese (e.g., whey cheese such as ricotta; pasta filata cheese such as mozzarella; semi-soft cheese such as Havarti and Muenster;
  • yogurt e.g.,
  • the food product according to any of the above may be an animal meat or animal meat product, a supplemented animal meat or animal meat product (i.e., a conventional animal meat or animal meat product that is supplemented with a recombinant milk protein with bound bioactive agent according to any of the above), or substitute animal meat or animal meat product (i.e., a food product that resembles a conventional animal meat or animal meat product).
  • animal meats and animal meat products include flesh obtained from skeletal muscle or from other organs (e.g., kidney, heart, liver, gallbladder, intestine, stomach, bone marrow, brain, thymus, lung, tongue), or parts thereof, obtained from an animal.
  • the animal meat may be dark or white meat.
  • Non-limiting examples of animals from which animal meat or animal meat product can be obtained include cattle, lamb, mutton, horse, poultry (e.g., chicken, duck, goose, turkey), fowl (e.g., pigeon, dove, grouse, partridge, ostrich, emu, pheasant, quail), fresh or salt water fish (e.g., catfish, tuna, spearfish, shark, halibut, sturgeon, salmon, bass, muskie, pike, bowfin, gar, eel, paddlefish, bream, carp, trout, walleye, snakehead, crappie, sister, mussel, scallop, abalone, squid, octopus, sea urchin, cuttlefish, tunicate), crustacean (e.g., crab, lobster, shrimp, barnacle), game animal (e.g., deer, fox, wild pig, elk, moose, reindeer, caribou,
  • the food product according to any of the above may be an egg or egg product, a supplemented egg product (i.e., a conventional egg or egg product that is supplemented with the recombinant milk protein according to any of the above), or substitute egg or egg product (i.e., a food product that resembles a conventional egg or egg product).
  • eggs or egg products include whole egg (e.g., liquid whole egg, spray-dried whole egg, frozen whole egg), egg white (e.g., liquid egg white, spray-dried egg white, frozen egg white), egg yolk, egg dishes, egg soups, mixtures made with egg whites, mixtures made with egg substitutes, mayonnaise, custard, and salad dressings.
  • Resemblance of a substitute food product provided herein to a conventional food product may be due to any physical, mechanical, chemical/biological, sensory, and/or functional attribute.
  • composition according to any of the above may be a cosmetic or personal care composition.
  • cosmetic or personal care composition refers to a composition that upon application to a body surface (i.e., an exposed area of a human body, such as skin, hair, nail, tooth, and tissues of the oral cavity [e.g., gums]) confers a perceived or actual beautifying or hygienizing effect.
  • Non-limiting examples of cosmetic or personal care compositions include anti-wrinkling treatments (i.e., compositions used for tensioning [e.g., smoothing out of skin, reducing wrinkles in skin, removing fine lines in skin]), antiaging treatments (i.e., compositions used for removing signs of aging [e.g., wrinkles, fine lines, manifestations of photodamage (e.g., sun spots)]), sun protection (i.e., compositions used to protect against UV exposure), anti-burn treatments (i.e., compositions used for soothing bums [e.g., sunburns]), anti-acne treatments (i.e., compositions that are effective in the treatment of acne and/or the symptoms associated therewith), skin cleansers (i.e., compositions used for cleaning skin and/or skin pores [e.g., nose strips for pore cleaning]), anti-dandruff treatments (i.e., compositions used for reducing or eliminating dandruff), antibody odor treatments (i.e.
  • composition according to any of the above may be a therapeutic product that can be used for treating a disorder, disease, or injury in a subject.
  • Non-limiting examples of disorders, diseases, or injuries include cancer, microbial infections or contaminations, viral infections, skin defects (e.g., rashes, sores, abrasions, burns, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers, diabetic ulcers, diabetic foot ulcers, decubitus ulcers, itchy skin), or pain (e.g., acute pain, chronic pain).
  • skin defects e.g., rashes, sores, abrasions, burns, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers, diabetic ulcers, diabetic foot ulcers, decubitus ulcers, itchy skin
  • pain e.g., acute pain, chronic pain
  • a method for producing a composition according to any of the above e.g., a food product according to any of the above
  • the method comprises the step of obtaining a recombinant milk protein with bound bioactive agent according to any of the above.
  • Binding of the bioactive agent may entail covalent bonding (e.g., via chemical crosslinking [e.g., thiol-ene click reaction, Schiff base reaction, amide bonding] or enzymatic crosslinking [e.g., crosslinking by activity of a transglutaminase]) or non-covalent bonding (e.g., ionic interaction, hydrophobic interaction, hydrogen bonding). Binding of the bioactive agent may occur directly to the recombinant milk protein, or indirectly (e.g., via a linker peptide that is covalently or non-covalently attached to the recombinant milk protein).
  • covalent bonding e.g., via chemical crosslinking [e.g., thiol-ene click reaction, Schiff base reaction, amide bonding] or enzymatic crosslinking [e.g., crosslinking by activity of a transglutaminase]
  • non-covalent bonding e.g.
  • composition is a food product (e.g., the food product according to any of the above)
  • a variety of recipes known in the art may be used to prepare the food product.
  • composition comprises or consists of a polymer according to any of the above
  • methods for polymerizing protein monomers known in the art may be used to polymerize the recombinant milk protein according to any of the above (see, for example, patent publication US20210235714, filed October 30, 2020.
  • the bioactive agent may be bound to the recombinant milk protein prior or post polymerization.
  • conditions for polymerization are most suitably selected to not disrupt binding of the bioactive agent to the recombinant milk protein and/or to not denature a tertiary or quaternary structure of the recombinant milk protein.
  • Such conditions may include polymerization under non- or mildly denaturing conditions, such as disclosed, for example, in PCT publication WO2021168343, published August 26, 2021.
  • a method for administering a bioactive agent to a subject comprising administering to the subject a composition according to any of the above.
  • the subject may be a human or other animal (e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig]).
  • a mammal e.g., cow, sheep, goat, rabbit, pig
  • Administering the composition to the subject may occur via any route, including via oral administration.
  • the recombinant vector is constructed using genetic engineering methods known in the art.
  • the recombinant vector comprises an expression construct comprising a protein coding sequence encoding P- lactoglobulin protein (“recombinant P-lactoglobulin protein ORF”; e.g., any of SEQ ID NOs: 1-9); codon-optimized for expression in the host cell, and operably linked to an N-terminal secretion signal sequence (e.g., pre or pre-pro signal peptide of proteins CBH1 or XYN1 for Trichoderma host cell; pre or pre-pro signal peptide of protein GLAA for Aspergillus host cell; pre or pre-pro signal peptide of Saccharomyces cerevisiae alpha mating factor for Pichia pastoris host cell); and under control of a suitable promoter sequence (e.g., promoter sequence of cbhl, xyn
  • the recombinant vector further comprises a polynucleotide that can direct integration of the expression construct into the genome of the host cell (e.g., into the cbhl or egll locus for Trichoderma host cells; into the glaA locus for Aspergillus host cells; into the aoxl locus for Pichia host cells), selection markers for selection of bacterial and/or fungal transformants, and a bacterial origin of replication.
  • the bacterial selection markers and origin of replication are removed from the recombinant vector via restriction enzyme digestion prior to transformation of the recombinant vector into the host cell.
  • site-directed mutagenesis is performed according to the manufacturer’s direction on the recombinant vectors above to introduce the amino acid substitutions listed in Table 1 into the encoded P-lactoglobulin protein, as well as any combination of two or more of such amino acid substitutions.
  • Table 1 Amino acid substitution introduced B-lactoglobulin protein encoded by protein coding sequence of recombinant vector.
  • the recombinant vectors are transformed into the host cell (e.g., Trichoderma reesei, Aspergillus niger, Pichia pastoris I Komagataella phaffii', e.g., strain BG12 [(Biogrammatics, Carlsbad, CA]), and transformants are selected by growth on minimal media or antibiotics for positive selection.
  • the transformants are grown in expression media in 24- well plates, and culture supernatants are harvested for identification of recombinant host cells comprising an integrated copy of the expression construct and secreting a recombinant P-lactoglobulin protein by SDS-PAGE gel analyses.
  • the recombinant host cells are fermented in a stirred fermentation vessel under conditions that permit cell growth and production of the recombinant P-lactoglobulin protein.
  • the fermentation is harvested after at least 100 hours, at a biomass concentration of between about 20 g and about 50 g dry cell weight (DCW) per L.
  • DCW dry cell weight
  • the biomass is removed from the broth by centrifugation at 5,000 x g.
  • the culture supernatant is concentrated over a membrane with suitable molecular weight cutoff.
  • the concentrate retentate is diafiltered over 5 kda MWCO membranes into 50 mM Imidazole, pH 6.8.
  • the concentrated retentate is passed over a Q sepharose FF column.
  • the mobile phase is 50 mM Imidazole, pH 6.8, and the recombinant P-lactoglobulin protein is eluted on a 2M NaCl gradient. The gradient is run from 0-30% over 30 column volumes. Peak fractions are collected and analyzed on RP-HPLC. Peaks containing recombinant P- lactoglobulin protein with a purity of greater than 85% are pooled for final diafiltration into water.
  • the purified recombinant P-lactoglobulin proteins of Example 1 are subjected to protease digestion at 37°C for 0.5 hr, 1 hr, 4 hr, 6 hr, or overnight with pepsin from porcine gastric mucosa, trypsin from human pancreas, or chymotrypsin from human pancreas, all obtained from Sigma (Sigma-Aldrich Chemicals, St. Louis, MO).
  • Pepsin digestion is conducted at pH 1.5 or 3
  • trypsin and chymotrypsin digestions are conducted at pH 6, 7, or 8.
  • the protease digestion procedures are conducted according to experimental details described in Pena-Ramos & Xiong. 2001.
  • the purified recombinant P-lactoglobulin proteins of Example 1 are also subjected to a simulated in vitro digestion system as described in Wroblewska et al. 2016. Food Research International. 83:95, Bossios et al. 2011. Clin Transl Allergy. 1:6, or Benede et al. 2014. Food Research International. 62:1127.
  • a recombinant P-lactoglobulin protein of Example 1 is combined with a weak acid (e.g., 5% acetic acid) or base (e.g., 5% sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide) alone or in combination with an other ingredient (e.g., any of the other ingredients disclosed herein) to a final concentration of between about 2% and about 18% by mass of the recombinant P-lactoglobulin protein, a final pH of between about 4 and about 11, and a final conductivity of between about 10 ms/cm and about 300 mS/cm.
  • a weak acid e.g., 5% acetic acid
  • base e.g., 5% sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide
  • an other ingredient e.g., any of the other ingredients disclosed herein
  • the mixture is heated for 20 minutes at a temperature and a pH at which the recombinant P- lactoglobulin protein is mildly denatured (e.g., at which less than 20% of the recombinant P- lactoglobulin protein is denatured), such that the free thiol group(s) of the recombinant P- lactoglobulin protein are exposed but existing intra-molecular disulfide bonds are not broken (e.g., at between 10°C and 20°C below the Tm of the recombinant P-lactoglobulin protein [as determined in Example 3]).
  • the mixture is then cooled to 21°C or ambient temperature.
  • the polymer is captured by centrifugation (e.g., at 4,000g for 20 min), filtration, solvent extractions, chromatography, or other method.
  • the polymer is dried to a moisture content that still permits shaping. After shaping, the polymer is further dried to set a final form.
  • a cylindrical (10-mm inner diameter, 40-mm length) stainless steel tube is filled with a solution comprising between about 2% and about 18% by mass of a recombinant P- lactoglobulin protein of Experiment 1 at a final pH of between about 4 and about 11.
  • the tube is closed with rubber stoppers, sealed with vinyl electrical tape, and placed vertically in a water bath.
  • the solution is heated as described in Example 2 to obtain mildly denatured recombinant P-lactoglobulin protein.
  • Example 5 Production of Film/Sheet Comprising Recombinant P-Lactoglobulin Protein [0151] A solution comprising between about 20% and about 40% by weight of a recombinant P-lactoglobulin protein of Example 1 is heated as described in Example 3 to obtain mildly denatured recombinant P-lactoglobulin protein.
  • the mildly denatured recombinant P-lactoglobulin protein is optionally combined with a synthetic co-polymer (e.g., polyvinyl acetate [PVAC], polyvinyl alcohol [PVA], polyvinyl pyrollidone [PVP]).
  • a synthetic co-polymer e.g., polyvinyl acetate [PVAC], polyvinyl alcohol [PVA], polyvinyl pyrollidone [PVP]
  • PVAC polyvinyl acetate
  • PVA polyvinyl alcohol
  • PVP polyvinyl pyrollidone

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une protéine laitière recombinante avec un agent bioactif lié et présentant une libération in vivo améliorée de l'agent bioactif, une composition comprenant une telle protéine laitière recombinante avec un agent bioactif lié, et un procédé d'utilisation d'une telle composition.
PCT/US2023/062076 2022-04-02 2023-02-06 Compositions et procédés de distribution d'agents bioactifs WO2023192711A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180271111A1 (en) * 2014-08-21 2018-09-27 Perfect Day, Inc. Compositions comprising a casein and methods of producing the same
WO2021168343A2 (fr) * 2020-02-19 2021-08-26 Perfect Day, Inc. Protéines de lait recombinantes hypoallergéniques et compositions les comprenant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180271111A1 (en) * 2014-08-21 2018-09-27 Perfect Day, Inc. Compositions comprising a casein and methods of producing the same
WO2021168343A2 (fr) * 2020-02-19 2021-08-26 Perfect Day, Inc. Protéines de lait recombinantes hypoallergéniques et compositions les comprenant

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