A method of increasing milk production

Abstract

A method of increasing milk yield in lactating mammals, comprising administering a composition comprising at least one casein derived peptide, wherein said lactating mammals are categorized as parity 1, parity 2, parity 3 or parity 4.

Description

P-639212-PCA METHOD OF INCREASING MILK PRODUCTION SEQUENCE LISTING STATEMENT

[0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 21, 2026, is named P-639212-PC-ST26 and is 66,712 bytes in size.BACKGROUND OF THE INVENTION

[0001] The casein protein comprises three fractions, a, P and K, according to their electrophoretic mobility. Casein hydrolysate is the hydrolyzed form of casein which includes, among others, the active beta-casein-derived peptide. It has been established that casein hydrolysate plays a role in immune responses against microbial and fungus infections.

[0002] The global dairy market is estimated at more than $926 billion with an average annual growth rate of 4%. Bovine milk attributes a significant portion of the market whereas plant-based alternatives account for $13.2 billion in global terms and an estimated $13.03 billion is estimated for lactose-intolerant milk. Mammal- or mammalian-produced milk is a very complex fluid that includes several thousand components. Mammal- or mammalian-produced milk includes water, a variety of different lipids, sugar, a variety of different proteins, and a variety of different inorganic salts and compounds.

[0003] In the modem dairy industry, lactating animals in herds go through controlled cycles of milking and pregnancy, as such regimes contribute to a significant increase in milk production. In current management of dairy herds, for example cows and goats, there is a significant overlap between lactation and pregnancy, wherein a “dry period” is imposed between 35 to 75 days prior to parturition by cessation of milking. This regime is set toP-639212-PCcompromise between the need to induce involution, a necessary process for subsequent healthy lactating period, and the requirement for high milk production all year long.

[0004] Milk is an important component of diets for all humans as it is high in essential amino acids. Improving milk production is therefore an important tool for improving the quality of life.

[0005] There is still a great need for an increase in milk production and increasing milk yield.SUMMARY OF THE INVENTION

[0006] In some aspects, disclosed herein is a method for increasing milk yield in lactating mammals, comprising administering a composition comprising at least one casein derived peptide, wherein said lactating mammals are categorized as parity 1, parity 2, parity 3 or parity 4.

[0007] In some related aspects, the lactating mammals are categorized as parity 1. In some further related aspects, the lactating mammals are categorized as parity 2. In some further related aspects, the lactating mammals are categorized as parity 3. In some further related aspects, the lactating mammals are categorized as parity 4.

[0008] In some related aspects, the increase in milk yield does not include a decrease in milk quality, as evident by Energy-corrected milk (ECM).

[0009] In some related aspects, the administration is during lactation. In some further related aspects, the milk yield is increased during the same lactation of administration of said composition comprising at least one casein derived peptide. In some further related aspects, the milk yield is increased during the following lactation of administration of said composition comprising at least one casein derived peptide.

[0010] In some related aspects, the administration is at the beginning of the dry period.P-639212-PC

[0011] In some related aspects, the milk yield is increased, independent of the dry period length.

[0012] In some aspects, the casein derived peptides comprise natural peptide, synthetic peptide, semi-synthetic peptide, or any combination thereof. In some further related aspects, the synthetic peptide is a recombinant peptide.

[0013] In some further relates aspects, the casein derived peptide comprises one or more fragments of P-casein, aS 1 -casein, aS2-casein, K-casein, optimally further comprises amino acids with different lengths or any combination thereof.

[0014] In some further related aspects, the casein derived peptide comprises a casein hydrolysate. In some further related aspects, the casein derived peptide comprises a phosphopeptide. In some further related aspects, the phosphopeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 - SEQ ID NO 26.

[0015] In some related aspects, the method comprises administering between lOpg to lOOOOmg of said casein derived peptides.

[0016] In some related aspects, the administration comprises intramammary infusion to a single teat or a plurality of teats.

[0017] In some aspects, the milk is essentially free of residues and wherein said milk can be used as a raw milk, for dairy production, for breastfeeding, bakery, confectionary, feeding or any combination thereof.

[0018] In some aspects, disclosed herein is a composition comprising at least one casein derived peptide for use in increasing milk yield in lactating mammals, wherein said lactating mammals are categorized as parity 1, parity 2, parity 3 or parity 4.P-639212-PCBRIEF DESCRIPTION OF THE DRAWINGS

[0019] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0020] Fig. 1 is a graphic demonstration for the relative milk production increase from all-parities cows receiving bovine casein hydrolysate (groups A and B) compared to controls;

[0021] Fig. 2 is a graphic demonstration for the relative milk production increase from parities 2 and 3 cows receiving bovine casein hydrolysate (groups A and B) compared to controls;

[0022] Fig.3 is a graphic demonstration for average ECM difference (%) between post-and pre-treatment lactations, groups A and B vs. control in all parities cows;

[0023] Fig. 4 is a graphic demonstration for average ECM difference (%) between post-and pre-treatment lactations, groups A and B vs. control in parities 2 and 3 cows.

[0024] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0025] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understoodP-639212-PCby those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.Method of increasing milk yield

[0026] In some embodiments, disclosed herein is a method for increasing milk yield in lactating mammals, comprising administering a composition comprising at least one casein derived peptide, wherein said lactating mammals are categorized as parity 1, parity 2, parity 3 or parity 4.

[0027] In some embodiments, the increase in milk yield is in comparison to lactating mammals not administered a composition comprising at least one casein derived peptide or administered antibiotics, teat sealant, non-steroidal anti-inflammatory drugs (NSAIDs), antimicrobial peptides, hormones or combination thereof.

[0028] In some embodiments, the mammals are categorized as parity 1, parity 2 parity 3 or parity 4. In one embodiment, the mammals are categorized as parity 1. In another embodiment, the mammals are categorized as parity 2. In another embodiment, the mammals are categorized as parity 3. In another embodiment, the mammals are categorized as parity 4. In another embodiment, the mammals are categorized as parity 1 or parity 2. In another embodiment, the mammals are categorized as parity 1 or parity 3. In another embodiment, the mammals are categorized as parity 2, parity 3 or parity 4.

[0029] A skilled artisan would understand that in mammals, "parity" refers to the number of times an individual has given birth, counting multiple births as one, and usually including stillbirths. It’s often categorized as:Nulliparous: A female that has never given birth.Primiparous: A female that has given birth once (parity 1).P-639212-PCMultiparous: A female that has given birth multiple times (parity 2, 3 and forward).

[0030] In some embodiments, the administration is during lactation.

[0031] In some embodiments, the milk yield is increased during the same lactation of administration of said composition comprising at least one casein derived peptide. In some embodiments, the increase in milk yield is at any stage of lactation.

[0032] In some embodiments, the milk yield is increased during the following lactation of administration of said composition comprising at least one casein derived peptide.

[0033] In some embodiments, the administration is at the beginning of the dry period.

[0034] A skilled artisan would understand that “dry period” means the period before calving that cows are not milked, which is currently about 4 to 13 weeks. The dry period has multiple functions. Main functions are to allow the cow a rest period before birth of the next calf and to maximize milk yield in the next lactation. During the dry period, mammary cells renew at a faster rate than when cows would be milked up to calving. At the beginning of the dry period many dairy farmers administer antibiotics in order to, among other purposes, treat the cow in case of intramammary infections.

[0031] In some embodiments, the milk yield is increased, independent of the dry period length. In one embodiment, the dry off period comprises 28 to 120 days. In another embodiment, the dry off period comprises 28 to 100 days. In another embodiment, the dry off period comprises 28 to 80 days. In another embodiment, the dry off period comprises 28 to 60 days. In another embodiment, the dry off period comprises 28 to 40 days. In another embodiment, the dry off period comprises 40 to 49 days. In another embodiment, the dry off period comprises 50 to 59 days.

[0032] In some embodiments, the milk is essentially free of residues.P-639212-PC

[0033] In some embodiments, the milk can be used as raw milk, for dairy production, for breastfeeding, bakery, confectionary, feeding or any combination thereof. In one embodiment, the milk can be used as raw milk. In another embodiment, the milk can be used for dairy production. In another embodiment, the milk can be used for breastfeeding. In another embodiment, the milk can be used for bakery. In another embodiment, the milk can be used for confectionary. In another embodiment, the milk can be used for feeding.

[0034] In some embodiments, the increase in milk yield does not include a decrease in milk quality, as evident by Energy-corrected milk (ECM).

[0035] A skilled artisan would understand that Energy-corrected milk (ECM) determines the amount of energy in the milk based on the milk composition, specifically protein and butterfat and optionally lactose. Calculating ECM is used to measure the feed efficiency (FE) of a cow’s ability to convert dry matter feed into milk yield (kg).

[0036] In some embodiments, disclosed herein is a composition comprising at least one casein derived peptide for use in increasing milk yield in lactating mammals, wherein said lactating mammals are categorized as parity 1, parity 2, parity 3 or parity 4.

[0037] In some embodiments, the method further comprises administering additional treatments. In one embodiment, the additional treatment comprises antibiotics, hormones, teat sealant or any combination thereof. In another embodiment, the additional treatment comprises antibiotics, hormones and teat sealant. In another embodiment, the additional treatment comprises antibiotics. In another embodiment, the additional treatment comprises hormones. In another embodiment, the additional treatment comprises teat sealant.Casein peptides

[0035] In some embodiments, the method as described comprises administering a composition comprising at least one casein derived peptide.P-639212-PC

[0036] Casein is a protein in non-human mammal’s milk, also found in human mammal’s milk known to include the subgroups aSl, aS2, p and K. Casein is defined according to the amino acid sequences of each of the subgroups aSl, aS2, p and K. In the context of the present disclosure, when referring to casein, it is to be understood as also including acid casein, salts of casein, phosphorous containing casein and rennet casein.

[0037] The term "peptide" as used herein refers to amino acid residues, connected by peptide bonds. A protein sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group. Amino acids, as used herein, refer to naturally occurring and synthetic amino acids, as well as amino acid analogs, and amino acid mimetics, that function in a manner similar to the naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0038] The casein-derived peptide may be a single peptide or a mixture of different peptides which may be independently selected from a naturally occurring peptide, a semisynthetic peptide, a synthetic peptide or a recombinant peptide. It should be further noted that the peptides according to the present disclosure may be produced synthetically, or by recombinant DNA technology, or by any other technology. Methods for producing peptides are well known in the art.

[0039] In some embodiments, the casein-derived peptide may comprise a casein protein breakdown product which occurs when casein protein is cleaved by enzymes or acids to peptide fragments (also known in the art by the term "casein hydrolysate"). A casein hydrolysate is to be understood as the hydrolyzed form of casein (protein). Casein hydrolysate includes, for example, the active beta-, alpha S1-, alpha S2-, kappa-casein-P-639212-PCderived peptide known to those versed in the art. In some embodiments, the casein-derived peptide is or comprises a casein hydrolysate.

[0040] In some embodiments, the casein derived peptide comprises natural peptide, synthetic peptide, semi-synthetic peptide, or any combination thereof. In another embodiment, the casein derived peptide comprises a natural peptide. In another embodiment, the casein derived peptide comprises a synthetic peptide. In another embodiment, the casein derived peptide comprises a semi-synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of natural peptide and synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of natural peptide and semi-synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of synthetic peptide and semi-synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of natural peptide, synthetic peptide and semi-synthetic peptide.

[0041] Natural casein-derived peptides are typically obtained following enzymatic hydrolysis, the enzyme may be any mammal peptidase, such as, without being limited thereto, plasmin, pancreatin, trypsin, chymotrypsin, neutrase, alcalase, pepsin, carboxypeptidase, cathepsin, as well as plant peptidase such as, without being limited thereto, papain, bromelain, as well as enzymes from microorganism source. For example, a naturally occurring casein-derived peptide may be the result of an enzyme activity such as plasmin on casein subunits P-casein, asl- and as2-casein or K-casein. In some embodiments, a casein hydrolysate is obtained by cleavage of the casein protein with trypsin.

[0042] A synthetic peptide may be obtained by any methods known in the art of peptide synthesis including chemical synthesis and recombinant DNA technology. For example, the peptides may be synthesized by using standard solid phase techniques.P-639212-PC

[0043] In one embodiment, the synthetic peptide is a recombinant peptide.

[0044] A semi-synthetic casein-derived peptide may be obtained by chemical hydrolysis of casein, e.g. by prolonged boiling in a strong acid (acid-HVP) or strong base or using a chemical agent such as Cyanogen bromide (CNBr). The casein-derived peptide may also be obtained by molecular engineering, e.g. using recombinant DNA, in molecular techniques known in the art. In such an embodiment, the casein-derived peptide is a recombinant peptide.

[0045] In one embodiment, the recombinant peptide is produced by fermentation, tissue culture or combination thereof. In another embodiment, the recombinant peptide is produced by fermentation. In another embodiment, the recombinant peptide is produced by tissue culture. In another embodiment, the recombinant peptide is produced by a combination of fermentation and tissue culture.

[0046] In one embodiment, the tissue culture comprises mammary gland bovine tissue .

[0047] In some embodiments, the casein derived peptide comprises one or more fragments of P-casein, aS 1 -casein, aS2-casein, K-casein or any combination thereof. In one embodiment, the casein derived peptide comprises one or more fragments of P-casein. In another embodiment, the casein derived peptide comprises one or more fragments of aSl-casein. In another embodiment, the casein derived peptide comprises one or more fragments of aS2-casein. In another embodiment, the casein derived peptide comprises one or more fragments of K-casein. In another embodiment, the casein derived peptide comprises one or more fragments of combination of P-casein, aS 1 -casein, aS2-casein and K-casein.

[0048] In one embodiment, the casein derived peptide further comprises amino acids with different lengths.

[0049] In one embodiment, the casein derived peptide comprises a casein hydrolysate.P-639212-PC

[0050] In one embodiment, the casein derived peptide comprises a phosphopeptide.

[0051] As used herein, the term "phosphopeptide" designates a phosphorylated peptide in form of a conjugated peptide in which the non-peptide portion is a residue of phosphoric acid. The expression "phosphopeptide" or "phosphoserine" designates conjugated serine in which the non-peptide portion is a residue of phosphoric acid.

[0052] In some embodiments, the casein-derived peptide is a single peptide or mixture of a phosphopeptide, namely, which contains a single phosphorous group or is a phosphorus-enriched peptide. In some embodiments, the casein-derived peptide is any phosphoserine, phosphotyrosine, phosphothreonine, and / or phosphohystidine-enriched casein-derived peptides (casein phosphopeptide, CPP) and monovalent cation phosphocaseinates, such as sodium, potassium, calcium or ammonium phosphocaseinates.

[0053] In some embodiments, the casein-derived peptide is a phosphor-peptide.

[0054] The phosphor-peptide may be genetically engineered casein-derived peptides as well as peptidomimetics of casein-derived peptides. For example, phosphorylation of amino acids such as at least one serine residue may be performed by any method as is known in the art. The term "casein-derived peptide" also encompasses peptide fragments or peptidomimetic products obtained from or corresponding to one or more sections of casein protein. The peptidomimetic peptide may be for example a peptoid or a semipeptoid, which are peptide analogs, having, for example, modifications such as, but are not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S-O, O-C-NH, CH2-O, CH2-CH2, S-C-NH, CH-CH or CF-CH, backbone modification and residue modification.

[0055] As used herein, the term “casein-derived peptide” further encompasses any derivatives, analogues, variants or homologues of any of the peptides. The term "derivative"P-639212-PCis used to define amino acid sequences (peptide), with any insertions, deletions, substitutions, and modifications to the amino acid sequences (peptide) that do not alter the activity of the original peptides. By the term “derivative” it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of polypeptides made according to the present invention.

[0056] In some embodiments, the modified, synthetic, semi-synthetic or other types of analogs of the naturally occurring casein-derived peptides are in some embodiments at least 75%, at times 85%, 90%, 95% and even 99% identical (in sequence) to a naturally occurring casein-derived peptide when the two sequences are optimally aligned. Further, any non-naturally occurring casein-derived peptide to be used in accordance with the present disclosure may retain at least part of the biological activity of the naturally occurring casein protein.

[0057] The present disclosure also encompasses homologues of the casein-derived peptide. The term "homologues” is used to define amino acid sequences (peptide) which maintain a minimal homology to the amino acid sequences defined by the invention, e.g. have at least about 65%, at least about 75%, at least about 85%, or at least about 95% overall sequence homology with the amino acid sequence of any of the peptide as structurally defined above, e.g. of a specified sequence.

[0058] In some embodiments, the casein-derived peptide may also include a chemical modification of a naturally occurring peptide, e.g. where one or more amino acids are deleted, substituted or modified, e.g. by removal of a side group, substitution of a side group or the introduction of a chemical group. Without being limited thereto, the chemical modification may include acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative,P-639212-PCmethylation, myristoylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process. When referring to replacement of an amino acid sequence by another, it is likely that the replacement is a conservative substitution. For example, one or more amino acid residues within a casein sequence is substituted by another amino acid of a similar polarity or charge. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Nonetheless, non-conservative substitutions may also take place as long as it does not significantly change the desired (casein like) biological activity of the resulting caseinderived peptide analog.

[0059] A casein-derived peptide in accordance with the present disclosure is characterized by a molecular weight of between about an average 100 to an average 10,000 Dalton (e.g. between 2 to 100 amino acids) at times between about an average 100 to an average 7,000 Dalton and at times between an average 1,000 to an average 5,000 Daltons.

[0060] A casein-derived peptide in accordance with the disclosure is characterized by a length of from 2 to 200, from 2 to 100 amino acids, at times between 4 amino acids to 40 amino acids, at times from 4 amino acids to 30 amino acids, at times 4 amino acids to 10 amino acids, at times between 10 amino acids to 50 amino acids.

[0061] In some embodiments, the casein-derived peptide comprises an amino acid sequence selected form the group consisting of SEQ ID NO. 1 - SEQ ID NO. 26.

[0062] In one embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser-Ser-Ser-Glu (SEQ ID NO:1), wherein at least one Ser residue, at least twoP-639212-PCSer residues or three Ser residues are phosphorylated (phosphorylated serine is denoted herein as Ser(p) or S(p)).

[0063] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser-Ser-Ser-Glu-Glu (SEQ ID NO:2), wherein at least one Ser residue, at least two Ser residues or three Ser residues are phosphorylated.

[0064] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser(p)-Ser(p)-Ser(p)-Glu-Glu (SEQ ID NO:3).

[0065] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as RELEELNVPGEIVES(p)LS(p)S(p)S(p)EESITR (SEQ ID NO:4). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as QMEAESIS(p)S(p)S(p)EEIVPDSVEQK (SEQ ID NO: 5). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as KNTMEHVS(p)S(p)S(p)EESIISNETYK (SEQ ID NO:6). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as KVNELSKNIGS(p)ES(p)TEDQ (SEQ ID NO:7). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as PTLNREQLS(p)TS(p)EENSKKTVD (SEQ ID NO: 8). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as ELEELNVPGEIVES(p)LS(p)S(p)S(p)EESITR (SEQ ID NO:9). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as RELEELNVPGEIES(p)LS(p)S(p)S(p)EESITR (SEQ ID NO: 10). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as QMEAES(p)IS(p)S(p)S(p)EEIVPNS(p)VEQK (SEQ ID NO: 11). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted asP-639212-PCKNTMEHVS(p)S(p)S(p)EESIIS(p)QETYK (SEQ ID NO: 12). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as KVNELSKDIGS(p)ES(p)TEDQ (SEQ ID NO: 13). In another embodiment, the phosphopeptide comprisesamino acid sequence denoted as ESnS(p)QETYKQEKNMAINPSKENLCSTFCKEWRNANEEETSIGS(p)S(p)S(p)EES( p)AEVATEEVKITVDDKHYQKALNEINQFYQKFPGYLQYLYQGPIVLNPWNQVLR NAVPITPTLNREQLS(p)TS(p)EENSKKTVN (SEQ ID NO: 14). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as ELEELNVPGEIES(p)LS(p)S(p)S(p)EESITR(SEQ ID NO: 15).

[0066] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Xi(n)- Ser(P)-Ser(P)-Ser(P)-X2(m)-Lys (SEQ ID NO: 16), wherein at least one of Xi and X2 is independently selected from a positively charged amino acid and wherein each one of n and m is independently selected from 0, 1 and 2.

[0067] In some embodiments, the positively charged amino acid is selected from the group consisting of lysine, arginine and histidine. In some embodiments, the positively charged amino acid is lysine. In some other embodiments, the positively charged amino acid is arginine. In some other embodiments, the positively charged amino acid is histidine.

[0068] According to some embodiments, the formula of SEQ ID NO:16 further comprises a blocking group (also denoted herein as a protecting group) at the C-terminus. In some embodiments, the carboxyl group at the C terminus of the peptide is protected with a protecting group. The protecting group is selected from, but not limited to an amide (i.e., the hydroxyl group at the C terminus is replaced with a primary amine (NH2), secondary amine, or tertiary amine) or ester (i.e. the hydroxyl group at the C terminus is replaced with an ester). According to some embodiments, the blocking group is selected from the groupP-639212-PCconsisting of amide and ester. According to some embodiments, the blocking group is amide.

[0069] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(P)-Ser(P)-Ser(P) (SEQ ID NO: 17). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(P)-Ser(P)-Ser(P)-Lys (SEQ ID NO: 18). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(P)-Ser(P)-Ser(P)-Lys-Lys (SEQ ID NO: 19). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Ser(P)- Ser(P)-Ser(P)-Lys-Lys (SEQ ID NO:20). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys- Ser(P)-Ser(P)-Ser(P)-Lys (SEQ ID NO:21). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Ser(P)-Ser(P)-Ser(P) (SEQ ID NO:22). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser(P)-Ser(P)-Ser(P)-Lys-Lys (SEQ ID NO:23). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser(P)-Ser(P)-Ser(P)-Lys (SEQ ID NO:24). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(p)-Ser(p)-Ser(p)-NH2 (SEQ ID NO:25). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as RELEELNVPGEIVES(p)LS(p)S(p)S(p)EESITRINK (SEQ ID NO:26).

[0070] The casein-derived peptide according to the invention may comprise “L” as well as “D” form residues. While the amino acid residues of the peptide sequences set forth in SEQ ID NOs:l-26 are all in the "L" isomeric form, residues in the "D" isomeric form can substitute any L-amino acid residue so long as the resulting peptide analog retains at least part of the biological activity of the corresponding “L” isomer. One reason for designingP-639212-PCcasein-derived peptides comprising at least one D-amino acid is to increase stability of the peptide to proteolytic degradation.

[0071] In one embodiment, the composition is free of antimicrobials and comprises an acceptable carrier. In another embodiment, the composition is further free from hormones, genetic modified organism, or combination thereof. A skilled artisan would understand that a genetically modified organism (GMO) is an animal, plant, or microbe whose DNA has been altered using genetic engineering techniques.

[0072] In one embodiment, the milk derived protein is measured by UV at a range from 204 to 220 nm.Dosage and administration

[0073] In some embodiments, the methods of the present disclosure comprise administering between lOpg to lOOOOmg of the milk derived protein, per administration. In one embodiment, the methods of the present disclosure comprise administering between lOpg / ml to 500mg of the milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between lOmg to 450mg of the milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between 50mg to 400mg of the milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between 50mg to 70mg of the milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between lOOmg to 350mg of the milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between 150mg to 300mg of the milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between 200mg to 250mg ofP-639212-PCthe milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between 5mg to 30mg of the milk derived protein, per administration. In another embodiment, the methods of the present disclosure comprise administering between 1200mg to 2400mg of the milk derived protein, per administration.

[0074] In one embodiment, the milk derived protein concentration in the composition is between 0.01% to 30%. In another embodiment, the milk derived protein concentration in the composition is 0.01%. In another embodiment, the milk derived protein concentration in the composition is 0.1%. In another embodiment, the milk derived protein concentration in the composition is 1%. In another embodiment, the milk derived protein concentration in the composition is 5%. In another embodiment, the milk derived protein concentration in the composition is 10%. In another embodiment, the milk derived protein concentration in the composition is 10%. In another embodiment, the milk derived protein concentration in the composition is 15%. In another embodiment, the milk derived protein concentration in the composition is 20%. In another embodiment, the milk derived protein concentration in the composition is 25%. In another embodiment, the milk derived protein concentration in the composition is 30%.

[0075] In another embodiment, the administration comprises intramammary infusion to a single teat or a plurality of teats. In another embodiment, the administration comprises intramammary infusion to a single teat. In another embodiment, the administration comprises intramammary infusion to a plurality of teats.

[0076] In some embodiments, the methods of the present disclosure comprise between one to eight administrations to a single teat. In one embodiment, the methods of the present disclosure comprise one administration. In another embodiment, the methods of the present disclosure comprise two administrations. In another embodiment, the methods of the presentP-639212-PCdisclosure comprise three administrations. In another embodiment, the methods of the present disclosure comprise four administrations. In another embodiment, the methods of the present disclosure comprise five administrations. In another embodiment, the methods of the present disclosure comprise six administrations. In another embodiment, the methods of the present disclosure comprise seven administrations. In another embodiment, the methods of the present disclosure comprise eight administrations.

[0077] In some embodiments, the administrations of the present disclosure comprise subsequent administrations. In one embodiment, the administrations are immediately one after the other.

[0078] In some embodiments, the administrations of the present disclosure comprise intervals of from about 1 hour to about 72 hours . In one embodiment, the administrations comprise intervals of about 1 hour. In one embodiment, the administrations comprise intervals of about 4 hours. In one embodiment, the administrations comprise intervals of about 5 hours. In one embodiment, the administrations comprise intervals of about 8 hours. In one embodiment, the administrations comprise intervals of about 10 hours. In one embodiment, the administrations comprise intervals of about 12 hours. In one embodiment, the administrations comprise intervals of about 15 hours. In another embodiment, the administrations comprise intervals of about 16 hours. In another embodiment, the administrations comprise intervals of about 20 hours. In another embodiment, the administrations comprise intervals of about 24 hours. In another embodiment, the administrations comprise intervals of about 25 hours. In another embodiment, the administrations comprise intervals of about 28 hours. In another embodiment, the administrations comprise intervals of about 30 hours. In another embodiment, the administrations comprise intervals of about 35 hours. In another embodiment, theP-639212-PCadministrations comprise intervals of about 36 hours. In another embodiment, the administrations comprise intervals of about 40 hours. In another embodiment, the administrations comprise intervals of about 45 hours. In another embodiment, the administrations comprise intervals of about 50 hours. In another embodiment, the administrations comprise intervals of about 55 hours. In another embodiment, the administrations comprise intervals of about 60 hours. In another embodiment, the administrations comprise intervals of about 65 hours. In another embodiment, the administrations comprise intervals of about 70 hours. In another embodiment, the administrations comprise intervals of about 72 hours.

[0079] In one embodiment, the administration comprises intervals of from about 1 hour to about 24 hours.

[0080] In some embodiments, the administration comprises no intervals.

[0081] In one embodiment, the administration is during the lactating period.

[0082] In one embodiment, the administration comprises continued administrations during the lactating period or during several lactating periods. In one embodiment the administration comprises continued administrations during the lactating period. In another embodiment the administration comprises continued administrations during several lactating periods. In another embodiment the administration comprises continued administrations during two lactating periods. In another embodiment the administration comprises continued administrations during three lactating periods. In another embodiment the administration comprises continued administrations during four lactating periods. In another embodiment the administration comprises continued administrations during five lactating periods. In another embodiment the administration comprises continued administrations during six lactating periods. In another embodiment the administrationP-639212-PCcomprises continued administrations during seven lactating periods. In another embodiment the administration comprises continued administrations during eight lactating periods. In another embodiment the administration comprises continued administrations during nine lactating periods. In another embodiment the administration comprises continued administrations during ten lactating periods.

[0083] In one embodiment, the administration comprises continued administrations during the dry-off period or during several dry-off periods. In one embodiment the administration comprises continued administrations during the dry-off period. In another embodiment the administration comprises continued administrations during several dry-off periods. In another embodiment the administration comprises continued administrations during two dry-off periods. In another embodiment the administration comprises continued administrations during three dry-off periods. In another embodiment the administration comprises continued administrations during four dry-off periods. In another embodiment the administration comprises continued administrations during five dry-off periods. In another embodiment the administration comprises continued administrations during six dry-off periods. In another embodiment the administration comprises continued administrations during seven dry-off periods. In another embodiment the administration comprises continued administrations during eight dry-off periods. In another embodiment the administration comprises continued administrations during nine dry -off periods. In another embodiment the administration comprises continued administrations during ten dry-off periods.EXAMPLES EXAMPLE 1 - BOVINE CASEIN HYDROLYSATE BY INTRAMAMMARY ADMINISTRATION AS A DRY COW THERAPY UNDER FIELD CONDITIONSP-639212-PC

[0084] The objective of this randomized, blinded, controlled study was to assess the association of intramammary administration of bovine casein hydrolysate (bCNH or Bovine CNH) with shortening of the dry period, and with milk yield during the subsequent lactation, as compared to a positive control (antibiotic-treated cows).

[0085] Total 297 Israel Holstein Friesians dairy cows were included in the analysis from seven commercial dairy farms in different climate regions. Eligibility criteria included good general condition, no clinical mastitis or any other intramammary abnormalities.

[0086] There were two bCNH experimental groups, A 1200mg dose, and B 40ml 2400mg dose, and one positive antibiotic treated-control group (conventional dry cow therapy, Nefpenzal® DC). The number of cows included in the analysis in the treatment arms were as follows: A - 106, B - 92, positive control - 99 cows.Materials and methods

[0087] Pregnant dairy cows, primiparous and multiparous at any age, with any milk yield and SCC values, planned for drying-off during the clinical study enrollment period were candidates for inclusion in the study, to receive either bCNH or routine antibiotic treatment at dry-off Cows were excluded if (1) they had less than 4 functioning quarters; (2) received NS AIDs, antimicrobial, hormones, corticosteroids therapy for any reason in the 4 weeks prior to dry-off; (3) physical abnormalities of the udder, quarters or teats were present; (4) clinical signs of disease (other than IMI) or injury that may interfere with response to the treatment; (5) cow was intended for culling; (6) cow’s body condition score (BCS) was less than 2.75 (the protocol dictated BCS>1.0, however to allow for dry-period shortening, only cows with BCS>2.75 were enrolled in the study).

[0088] Cows were milked three times a day.P-639212-PC

[0089] Immediately after last milking during the lactation period, on the treatment day an independent treatment worker treated the allocated subject by intramammary infusion of 20 mL or 40 mL of bCNH. The treatment groups were A, bCNH at a dose of 1200 mg, B, bCNH at a dose of 2400 mg (2x1200 mg simultaneous) and antibiotic-treated (positive) control (Nafpenzal DC®), according to routine dry off treatment.

[0090] Investigators, veterinarians, and monitors were blinded to the treatment assignment during all phases of the study.

[0091] Sample size was calculated for comparisons of milk yield in bCNH treatment groups (four groups) with an antibiotic-treated (positive) control group.

[0092] Cows’ parities distribution for the post-treatment lactation (categorized to three categories: 2, 3, 4 and up), in total and by treatment groups, is presented below in Table 1.Table 1 - Distribution of cows in control and bCNH treatment groups by parity.

[0093] As can be seen in Fig. 1, for 1200mg-dose group (group A) the milk production is similar to that of positive control group (p-value>0.7). For 2400mg-dose group (group B), the increase in milk production is significantly higher than that of control (p-value <0.05), and also significantly higher than that of 1200mg-dose groups (p-value <0.05), demonstrating a dose-response.Fig- 2 demonstrates the average increase in milk production, from cows in parities 2 and 3 receiving bovine casein hydrolysate as a dry-off treatment compared to controls.

[0094] Treatment with 2400 mg bCNH was shown to be beneficial to milk yield increase in the lactation following bCNH treatment at dry-off, by 3.3% more when including all cowsP-639212-PCcompared to positive control (12.1% Vs. 15.4) (p-value 0.052) (Fig.l). When including younger cows only (parities 2 and 3) in the analysis, such milk increase is substantially higher and reaching to 7.3% (18.1% Vs 25.4%) (p-value <0.01) respectively (Fig.2). For the whole lactation period of 305-days, for parties 2 and 3, the increase in milk production with 2400mg bCNH was up to 891.7 kg Vs the control group. Including the contribution of the extra 10 milking days in the pre-treatment lactation, this increase reached 1,127.7 kg.

[0095] No Severe Adverse Events were reported as probably or possibly related to bCNH treatment during the post-treatment dry-off period.

[0096] Higher milk yield increase was demonstrated following treatment with a dose of 2400mg bCNH as compared to a dose of 1200mg, confirming a dose-response.

[0097] Further, it has been demonstrated that the milk increase in young cows is independent to the length, of the dry period, as the dairy cows in the bCNH groups had a mean dry off of 49 days and the dairy cows in the positive control group had a mean dry off of 59 days.

[0098] Figs. 3 and 4 demonstrate the ECM measurements, in all parties (Fig. 3) and in parties 2 and 3 (Fig. 4) before and after treatments, indicating the quality of the milk, as measured by ECM, was not compromised by the treatment.

[0099] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

P-639212-PCCLAIMSWhat is claimed is1. A method of increasing milk yield in lactating mammals, comprising administering a composition comprising at least one casein derived peptide, wherein said lactating mammals are categorized as parity 1, parity 2, parity 3 or parity 4.

2. The method according to claim 1 , wherein said lactating mammals are categorized as parity 1.

3. The method according to claim 1 , wherein said lactating mammals are categorized as parity 2.

4. The method according to claim 1 , wherein said lactating mammals are categorized as parity 3.

5. The method according to claims 1-4, wherein said increase in milk yield does not include a decrease in milk quality, as evident by Energy-corrected milk (ECM).

6. The method according to claims 1-5, wherein said administration is during lactation.

7. The method according to claim 6, wherein said milk yield is increased during the same lactation of administration of said composition comprising at least one casein derived peptide.

8. The method according to claim 6, wherein said milk yield is increased during the following lactation of administration of said composition comprising at least one casein derived peptide.

9. The method according to claims 1-8, wherein said administration is at the beginning of the dry period.

10. The method according to any of the proceeding claims, wherein said milk yield is increased, independent of the dry period length.

11. The method according to claim 9, wherein said dry off period comprises 28 to 120 days.P-639212-PC12. The method according to claim 9, wherein said dry off period comprises 28 to 60 days.

13. The method according to any of the proceeding claims, wherein said casein derived peptides comprises natural peptide, synthetic peptide, semi -synthetic peptide, or any combination thereof.

14. The method according to claim 13, wherein said synthetic peptide is a recombinant peptide.

15. The method according to claim 14, wherein said recombinant peptide is produced by fermentation, tissue culture or combination thereof.

16. The method according to claim 15, wherein said tissue culture comprises mammary gland bovine tissue.

17. The method according to any of the proceeding claims, wherein said casein derived peptide comprises one or more fragments of P-casein, aS 1 -casein, aS2-casein, K-casein, optimally further comprises amino acids with different lengths or any combination thereof.

18. The method according to any of the proceeding claims, wherein said casein derived peptide comprises a casein hydrolysate.

19. The method according to any of the proceeding claims, wherein said casein derived peptide comprises a phosphopeptide.

20. The method according to claim 19, wherein said phosphopeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 - SEQ ID NO 26.

21. The method according to any of the proceeding claims, wherein said composition is free of antimicrobials, immuno-modulators, anti-inflammatory, hormones and comprises an acceptable carrier.

22. The method according to any of the proceeding claims comprising administering between lOpg to lOOOOmg of said casein derived peptides.

23. The method according to any of the proceeding claims, wherein said administration comprises intramammary infusion to a single teat or a plurality of teats.P-639212-PC24. The method according to any of the proceeding claims, wherein said administration comprises between one to eight administrations per teat.

25. The method according to any of the proceeding claims, wherein said administration comprises intervals of from about 1 hour to about 72 hours.

26. The method according to any of the proceeding claims, wherein said administration comprises no intervals.

27. The method according to any of the proceeding claims, wherein said administration comprises continued administrations during the lactating period or during several lactating periods.

28. The method according to any of the proceeding claims, wherein said milk is essentially free of residues and wherein said milk can be used as a raw milk, for dairy production, for breastfeeding, bakery, confectionary, feeding or any combination thereof.

29. The method according to any of the proceeding claims, further comprising administering additional treatments.

30. A composition comprising at least one casein derived peptide for use in increasing milk yield in lactating mammals, wherein said lactating mammals are categorized as parity 1, parity 2, parity 3 or parity 4.

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