Compositions and products comprising collagen peptides

Abstract

Described herein is a composition comprising a collagen peptide, the composition comprising at least 0.1 wt% of a peptide comprising an amino acid sequence having two or more GXX repetitive sequences, where each X is independently proline (Pro) or hydroxyproline (Hyp). Related food and feed products and supplements as well as cosmetic products are also described.

Description

Technical Field

[0001] Aspects and embodiments described herein relate to the field of collagen and collagen peptides. In particular, the present disclosure relates to compositions comprising collagen peptides, foods, pet food and feed products and supplements comprising collagen peptides, and cosmetic products comprising collagen peptides. Background Art

[0002] Demand for food and cosmetic products based on natural ingredients is growing. This is particularly because natural ingredients appeal to modern, conscious consumers, are associated with low toxicity, and are widely available from natural sources. Natural ingredients are often obtained from agricultural byproducts, which otherwise have little value. Importantly, natural ingredients often possess various beneficial effects, such as health benefits. For this reason, natural ingredients often impart attractive properties to the various products they are formulated with.

[0003] An example of such a natural product is collagen. Collagen is the main protein of the extracellular matrix (ECM) and is responsible for structural support and cell adhesion. Collagen and collagen hydrolysate (collagen peptides) can be obtained from by-products of the meat and fish industries. Collagen hydrolysate, produced by the hydrolysis of collagen, is a complex active substance composed of a variety of more or less bioactive peptides. Collagen hydrolysate is associated with various health benefits, including positive effects on bone strength, skin elasticity and hydration, hair and nails, muscle mass and strength, and heart health.

[0004] However, collagen and its hydrolysates are complex products containing a variety of collagen peptide sequences, depending on the collagen source and production method. For these reasons, the main active peptides in collagen hydrolysates and their molecular mechanisms remain unclear, making the formulation of products rich in bioactive collagen peptides with increased beneficial effects challenging. Summary of the Invention

[0005] In view of the above, there remains a need to identify specific bioactive components of interest from natural products, such as bioactive peptides from collagen hydrolysates, to develop improved collagen peptide compositions, foods, pet foods, feed products and supplements, cosmetic products, etc., having the beneficial effects conferred by bioactive collagen peptides.

[0006] The present inventors have identified specific collagen peptides that exhibit strong biological effects such as proliferation, differentiation and mineralization of osteoblasts and proliferation, migration and tube formation of endothelial cells.

[0007] Accordingly, aspects and embodiments of the present disclosure address at least some of the problems and needs discussed herein.

[0008] In a first aspect, the present invention relates to a composition comprising collagen peptides, wherein at least 0.1 wt% of the total amount of collagen peptides is a peptide comprising an amino acid sequence having two or more GXX repeating sequences, wherein each X is proline (Pro) or hydroxyproline (Hyp).

[0009] In a second aspect, the present invention relates to a food, pet food or feed product or supplement comprising the composition of the first aspect of the invention.

[0010] In a third aspect, the present invention relates to a cosmetic product comprising the composition of the first aspect of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Effects of collagen hydrolysate on (A) osteoblast proliferation, (B, D) osteoblast differentiation, and (C, E) osteoblast mineralization. Different letters in each column indicate significant differences (P < 0.05).

[0012] Figure 2 Effects of collagen hydrolysate on angiogenesis. Effects of collagen hydrolysate on (A) proliferation activity, (B, C) migration, and (D) tube formation of endothelial cells. Different letters in each column indicate significant differences (P < 0.05).

[0013] Figure 3 : Microarchitecture of the right femur of OVX mice treated with various treatments. (AE) Representative 3D images of trabecular bone of the femur 6 weeks after collagen hydrolysate induction based on micro-CT. Quantitative analysis of (F) bone surface (BS), (G) bone volume percentage (BV / TV), (H) trabecular number (Tb.N), and (I) trabecular spacing (Tb.Sp) in each group. Different letters in each column indicate significant differences (P < 0.05). (J) Representative H&E-stained images showing osteoporosis;

[0014] Figure 4 Genomic analysis of CH-treated OVX mice. (A) Fold changes in gene expression in mice treated with CH compared to the OVX group. (B) Significant terms from KEGG enrichment analysis in CH-treated OVX mice (P value < 0.05). (C) General pathway regulatory network of differentially expressed genes in response to the protective and / or restorative effects of CH in osteoporotic mice. DETAILED DESCRIPTION

[0015] The various features of the aspects and embodiments of the present disclosure are further described below. It should be noted that the headings used in this specification are only used to assist navigation and should not be interpreted as limiting, and the features described in different subsections may be relevant to all aspects and embodiments described herein and can therefore be combined as appropriate.

[0016] Composition

[0017] Provided herein are compositions comprising a peptide comprising an amino acid sequence having two or more GXX repeats, wherein each X is independently proline (abbreviated as Pro or P) or hydroxyproline (abbreviated as Hyp or O).

[0018] The peptides comprising an amino acid sequence having two or more GXX repeating sequences contained in the compositions of the present disclosure are further described in the "Peptides comprising an amino acid sequence having two or more GXX repeating sequences" section.

[0019] In some embodiments, the composition comprises at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt%, or at least 99 wt% of a peptide described herein.

[0020] In some embodiments, optionally in combination with the above minimum amounts, the compositions disclosed herein comprise at most 1 wt%, at most 2 wt%, at most 3 wt%, at most 4 wt%, at most 5 wt%, at most 6 wt%, at most 7 wt%, at most 8 wt%, at most 9 wt%, at most 10 wt%, at most 15 wt%, at most 20 wt%, at most 25 wt%, at most 30 wt%, at most 35 wt%, at most 40 wt%, at most 45 wt%, at most 50 wt%, at most 55 wt%, at most 60 wt%, at most 65 wt%, at most 70 wt%, at most 75 wt%, at most 80 wt%, at most 85 wt%, at most 90 wt%, at most 95 wt%, at most 96 wt%, at most 97 wt%, at most 98 wt% or at most 99 wt% of a peptide as described herein.

[0021] The compositions provided herein may comprise a single type of peptide comprising an amino acid sequence having two or more GXX repeats, wherein each X is independently proline or hydroxyproline, as described herein. The compositions provided herein may also comprise two or more different types of such peptides.

[0022] In addition to one or more peptides of the present invention, the compositions of the present disclosure may also include collagen peptides. Therefore, in some embodiments, the compositions described herein may be referred to as collagen peptide compositions (i.e., compositions comprising collagen peptides) or collagen hydrolysate compositions (i.e., compositions comprising collagen hydrolysate). This is particularly relevant when the peptides described herein are collagen peptides and / or can be obtained by hydrolyzed collagen. Therefore, in some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of collagen peptides.

[0023] Suitable sources of collagen, collagen hydrolysate or collagen peptides are described herein in the section "Peptide comprising an amino acid sequence having two or more GXX repeating sequences".

[0024] In some embodiments, the composition described herein is a collagen peptide composition (or collagen hydrolysate composition) enriched in peptides comprising an amino acid sequence having two or more GXX repeats, wherein each X is independently proline (Pro) or hydroxyproline (Hyp), as described herein. As used herein, "enriched" means that the composition comprises at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% of such peptides described herein relative to the native or natural collagen from which the collagen peptide or collagen hydrolysate is derived.

[0025] In some embodiments, the composition comprising the collagen peptides of the present disclosure is such that the molecular weight of the collagen peptides is 300 to 14,000 Da, preferably 400 to 7,000 Da, more preferably 800 to 3,500 Da, even more preferably 1,200 to 2,300 Da, and most preferably 1,600 to 1,750 Da. Therefore, in some embodiments, the composition comprising the collagen peptides of the present disclosure is such that the molecular weight distribution of the collagen peptides is 300 to 14,000 Da, preferably 400 to 7,000 Da, more preferably 800 to 3,500 Da, even more preferably 1200 to 2,300 Da, and most preferably 1600 to 1,750 Da.

[0026] The term "molecular weight distribution" has been described in the section "Peptides comprising an amino acid sequence having two or more GXX repeats".

[0027] In some embodiments, the composition comprising the collagen peptides of the present invention is such that the molecular weight of the collagen peptide is ≤14,000Da, preferably ≤7,000Da, more preferably ≤3,500Da, even more preferably ≤3,000Da. In some embodiments, the molecular weight can be lower, for example ≤2,300Da or ≤1,750Da. Therefore, in some embodiments, the composition comprising the collagen peptides of the present invention is such that at least 90wt%, at least 91wt%, at least 92wt%, at least 93wt%, at least 94wt%, at least 95wt%, at least 96wt%, at least 97wt%, at least 98wt% or at least 99wt% of the collagen peptides have a molecular weight of ≤14,000Da, preferably ≤7,000Da, more preferably ≤3,500Da, even more preferably ≤3,000Da. In other embodiments, the composition comprising the collagen peptides of the present disclosure is such that at least 90 wt%, at least 91 wt%, at least 92 wt%, at least 93 wt%, at least 94 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt% or at least 99 wt% of the collagen peptides have a molecular weight ≤ 2,300 Da or ≤ 1,750 Da.

[0028] In a preferred embodiment, the composition comprising collagen peptides is such that at least 99% by weight of the collagen peptides in the composition have a molecular weight of ≤14,000 Da, preferably ≤7,000 Da, more preferably ≤3,500 Da, and even more preferably ≤3,000 Da. In other embodiments, the composition comprising collagen peptides is such that at least 99% (w / w) of the collagen peptides in the composition have a molecular weight of ≤2,300 Da or ≤1,750 Da.

[0029] As known to those skilled in the art, various molecular weight fractions of the initial collagen hydrolysate can be obtained by methods and means known in the art, such as ultrafiltration.

[0030] The compositions provided herein can be liquid compositions or powder compositions. In a preferred embodiment, the compositions of the present disclosure are powder compositions.

[0031] The powder compositions disclosed herein can be further processed, for example, by agglomeration, granulation, pressing, etc. In some embodiments, the powder compositions disclosed herein can be further processed by agglomeration. Agglomeration means and methods are known to those skilled in the art, including particle size increase, which is generally used to obtain improved flow properties, easier handling and transportation, improved storage, etc. Therefore, in some embodiments, the powder compositions described herein are agglomerated powder compositions or non-agglomerated powder compositions.

[0032] Typically, the median particle size (D 50 ) is 65 to 100 μm, preferably 70 to 90 μm.

[0033] Typically, the median particle size (D 50 ) is from 100 to 1000 μm, preferably from 110 to 800 μm, even more preferably from 140 to 400 μm, even more preferably from 170 to 250 μm, most preferably from 170 to 200 μm.

[0034] As used herein, “D 50 ” or “median particle size” represents the diameter such that half the sizes are smaller and half are larger. The median particle size is a very stable parameter that can be measured by standard laser diffraction methods, for example using a particle size analyzer such as a Beckman Coulter particle size analyzer using standard software.

[0035] In the case of a liquid composition, the composition may further comprise a solvent. Suitable solvents herein include any solvent or solvent mixture in which the peptides (or peptide salts or peptide mimetics) described herein can be dissolved at a suitable concentration. Non-limiting examples of solvents that can be used in the present invention are water, ethanol, ammonium hydroxide, dimethyl sulfoxide (DMSO), acetic acid, acetonitrile, and dimethylformamide (DMF).

[0036] The compositions described herein can be pharmaceutical compositions, i.e., compositions comprising the peptides described herein and one or more pharmaceutically acceptable ingredients. The pharmaceutical compositions described herein are suitable for administration to humans or animals. As used herein, "pharmaceutically acceptable ingredients" include pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents, and / or excipients.

[0037] The composition described herein may be a nutritional composition, ie, a composition comprising a peptide described herein and one or more nutritional ingredients.

[0038] The compositions described herein can be formulated into compositions suitable for oral administration. Compositions suitable for oral administration include solid dosage forms and liquid dosage forms. Suitable dosage forms include, but are not limited to, capsules, tablets, granules, pills, suspensions, and syrups.

[0039] The compositions described herein can also be formulated into compositions suitable for topical administration. Compositions suitable for topical administration include creams, ointments, lotions, gels, and the like.

[0040] For more information on pharmaceutically acceptable ingredients and oral and topical formulations, see Remington: The Science and Practice of Pharmacy, 23rd ed., Elsevier (2020), which is incorporated herein by reference.

[0041] In another aspect, the pharmaceutical compositions described herein may further comprise more than one therapeutic agent.

[0042] A peptide comprising an amino acid sequence having two or more GXX repeating sequences

[0043] As described above, the compositions of the present disclosure include peptides comprising an amino acid sequence having two or more GXX repeats, wherein each X is independently proline or hydroxyproline. As explained in more detail below, other aspects of the present disclosure relate to foods, pet foods, feed supplements, and products, as well as cosmetics, which also include peptides comprising an amino acid sequence having two or more GXX repeats, wherein each X is independently proline or hydroxyproline. This section describes in more detail these peptides included in the compositions, foods, pet foods, feed supplements, and products, as well as cosmetics, of the present disclosure.

[0044] As used herein, hydroxyproline refers to 4-hydroxyproline (=L-hydroxyproline; IUPAC name: (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid).

[0045] The "GXX repeat sequence" of the peptides disclosed herein may also be expressed as a sequence motif, a sequence pattern, or simply as a sequence. According to the present disclosure, "sequence motif" or "sequence pattern" has the customary and ordinary meaning understood by those skilled in the art. It refers to an amino acid (or nucleotide) sequence that is repeated at several sites in one molecule or several different molecules with a certain degree of variation, and has or is presumed to have or is considered to be associated with the biological significance described herein or to exhibit biological activity. As described in more detail herein, the biological significance or biological activity of the peptides of the present invention is preferably capable of binding to EGFR, promoting PI3K / Akt signaling, and / or inducing osteogenesis and / or angiogenesis effects.

[0046] Osteogenesis and angiogenesis can be measured by well-known methods known to those skilled in the art. For example, osteogenesis can be measured by osteoblast proliferation assay (e.g., as described in Example 1), osteoblast differentiation assay (e.g., as described in Example 2), osteoblast mineralization assay (e.g., as described in Example 3) or gene markers (e.g., Alp, RUNX2, Col1a1, OSX, Bglap, BMP2 or BMP, preferably Alp) expression measurement (e.g., as described in Example 2). Angiogenesis can be measured, for example, by endothelial cell proliferation assay (e.g., as described in Example 4) or endothelial cell migration assay (e.g., as described in Example 4).

[0047] In a preferred embodiment, the two or more GXX repeat sequences of the peptides provided herein are two or three GXX repeat sequences.

[0048] In some embodiments, X is proline. In some embodiments, the GXX repeat sequence is a GX1X2 repeat, wherein X1 and X2 are each proline or hydroxyproline, but not both proline or both hydroxyproline. In other words, in this case, X1 can be proline and X2 can be hydroxyproline; alternatively, X1 can be hydroxyproline and X2 can be proline.

[0049] The GXX repeats of the peptides disclosed herein can be adjacent (i.e., immediately adjacent to each other and not separated by any other amino acid) or non-adjacent (i.e., separated from each other by at least one other amino acid). Preferably, the GXX repeats of the peptides disclosed herein are non-adjacent. Thus, in some embodiments, a peptide is provided, comprising an amino acid sequence having two or more non-adjacent GXX repeats, wherein each X is independently proline or hydroxyproline.

[0050] In embodiments of the disclosed peptides where the GXX repeat sequences are not adjacent, two or more GXX repeat sequences may be separated by at least 1, at least 2, or at least 3 amino acid residues, and optionally by up to 12, 11, 10, 9, 8, 7, or 6 amino acid residues. In some embodiments, two or more GXX repeat sequences are separated by 1 to 12, preferably 1 to 9, and more preferably 2 to 8 amino acids. In some embodiments, two or more GXX repeat sequences are separated by a plurality of amino acids, the plurality of amino acids being a multiple of 3, such as 3, 6, 9, 12, or 15, preferably 3, 6, 9, or 12, more preferably 3, 6, or 9, and most preferably 3 or 6.

[0051] As used herein, "peptide" is understood to refer to a limited number of (usually 2 to 50) amino acid chains. As used herein, "peptide" should be understood to include any kind of peptide, no matter how its source, that is, no matter it is produced by cell-free (in vitro) protein synthesis, purified from natural or engineered producer microorganisms (recombinant protein production), produced by chemical peptide synthesis ("synthetic peptide"), obtained from the hydrolysis of larger proteins (such as collagen) (such as described in more detail elsewhere herein) or produced by other methods. Peptides derived from any of these methods can be further modified by natural processes (such as post-translational processing) or chemical modification techniques, which will be explained in more detail below.

[0052] As used herein, the term "peptide" therefore encompasses synthetic peptides. According to the present disclosure, "synthetic peptide" has the customary and common meaning understood by those skilled in the art. It refers to a peptide produced by a chemical peptide synthesis method. Therefore, in some embodiments, the peptide as described herein can be a synthetic peptide. Synthetic peptides according to the present invention can be prepared or synthesized using conventional methods well known in the art. For example, peptides can be synthesized by conventional solid phase synthesis methods (see, for example, Amblard et al., Methods and protocols of modern solid phase peptide synthesis, Mol Biotechnol 2006, 33: 239-254, which is incorporated herein by reference). The peptides synthesized in this way can be further modified by natural processes (such as post-translational processing) or chemical modification techniques. Such modifications can be inserted into any position of the peptide, including main chain, amino acid side chain and N-terminus or C-terminus. Various types of modifications can occur in a single peptide, or a peptide can include several modifications of a single type. Illustrative but non-limiting examples of modifications are alkylation, acetylation, amidation, acylation, phosphorylation, methylation, demethylation, ADP-ribosylation, disulfide bond formation, ubiquitination, gamma-carboxylation, glycosylation, hydroxylation, iodination, oxidation, pegylation, succinylation, and sulfation. Hydroxylation, particularly of lysine and proline residues, is of interest in the context of the present disclosure because collagen typically contains hydroxyproline and hydroxylysine residues.

[0053] It should be understood that the same advantageous properties discovered by the inventors in the context of the peptides described herein also extend to the salt forms of the peptides and peptide mimetics, which are designed to specifically mimic the peptides. Therefore, throughout this disclosure, salt forms and peptide mimetics are also expressly contemplated and the phrase "peptide, a salt thereof, or a peptide mimetic" may be substituted for the phrase "peptide" throughout this disclosure.

[0054] As used herein, " peptide mimics " is understood to encompass all compounds that contain its basic element (pharmacophore) mimic peptides and retain the ability to interact with the same biological target and produce a biological effect. In the context of the present disclosure, "retention" refers to that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., all) of the initial desired function or activity is retained. Of course, the desired function or activity also can be improved. In some embodiments, peptide mimics comprise a non-naturally occurring amino acid sequence, are essentially composed of a non-naturally occurring amino acid sequence, or are composed of a non-naturally occurring amino acid sequence. In one embodiment, peptide mimics are not naturally occurring and are considered to be artificial. Peptide mimics are typically produced by modifying existing peptides or by designing a similar system for mimicking peptides, such as peptoids and β-peptides. The structure and synthesis of peptide mimetics are described in, for example, William D. Lubell (ed.), Peptidomimetics I and II, Topics in Heterocyclic Chemistry (Book 48), Springer 1st edition, 2017, XVI, page 310, which is incorporated herein by reference. Modifications of existing peptides may be the result of natural processes, such as post-translational processing or chemical modification techniques. In general, peptide mimetics generally refer to compounds containing non-peptide structural elements. In some embodiments, the natural amino acids of the peptides or peptide mimetics according to the present invention are replaced by corresponding non-natural amino acids. As used herein, "corresponding non-natural amino acids" refer to non-natural amino acids as reference natural amino acid derivatives. For example, natural amino acids can be replaced by corresponding β-amino acids, and the amino group of β-amino acids is connected to β-carbon instead of α-carbon. According to some embodiments, the peptides or peptide mimetics of the present invention may further have a targeting portion. Known peptide mimetics can avoid some of the shortcomings associated with natural peptides: for example, stability against proteolysis (duration of activity) and low bioavailability. Certain other properties, such as receptor selectivity or efficacy, can generally be substantially improved.

[0055] Suitable salt forms include, but are not limited to, acid addition salts and base addition salts. As used herein, a "salt" or "salt form" of a peptide refers to a salt that retains the desired function of the peptide. As used herein, a "pharmaceutically acceptable salt" of a peptide refers to a salt that retains the desired function of the peptide and is suitable for administration to humans or animals. In the context of the present disclosure, "retaining" means that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., all) of the original desired function or activity is retained. Of course, the desired function or activity may also be improved. Methods for preparing salts of peptides are known in the art and typically include mixing the peptide with a suitable acid or base, for example, by reacting the product in the form of a free acid or free base with one or more equivalents of a suitable acid or base in a salt-insoluble solvent or medium or in a solvent such as water, followed by removal of the solvent or medium.

[0056] As mentioned above, "peptide" generally refers to a finite number (usually 2 to 50) of amino acid chains. Some more specific lower limits of the peptide lengths disclosed herein are as follows. In some embodiments, the peptides described herein can have a total length of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 or at least 18 amino acids. In a preferred embodiment, the peptides included in the compositions described herein can have a total length of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 or at least 18 amino acids. In a more preferred embodiment, the total length is at least 16, at least 17 or at least 18 amino acids.

[0057] Some more specific upper limits for the length of peptides disclosed herein are as follows. In some embodiments, the peptides described herein may have a total length of at most 30, at most 29, at most 28, at most 27, at most 26, at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, or at most 19 amino acids. In preferred embodiments, the peptides described herein may have a total length of at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, or at most 19 amino acids. In more preferred embodiments, the total length is at most 20 or at most 19 amino acids.

[0058] Thus, in some embodiments, the peptides described herein have a total length of 10 to 25 amino acids, preferably 16 to 20 amino acids. Preferred peptides have a total length of 18 or 19 amino acids.

[0059] In some embodiments, the peptides included in the combinations described herein comprise or consist of the sequence RGXXGXMGXXGLAGXXGE (SEQ ID NO: 1), the sequence AGXXGXTGXAGXXGFXGAV (SEQ ID NO: 2), or a sequence having up to 5 mutations compared to SEQ ID NO: 1 or SEQ ID NO: 2, wherein each X is independently proline or hydroxyproline.

[0060] In the case of sequences having up to 5 mutations compared to SEQ ID NO: 1 or SEQ ID NO: 2, in some embodiments, the mutations are selected from amino acid substitutions, deletions, insertions and / or additions.

[0061] Among amino acid substitutions, deletions, insertions and / or additions, amino acid substitutions are the preferred type of mutation. Amino acid substitutions can be conservative or non-conservative amino acid substitutions, preferably conservative amino acid substitutions.

[0062] Peptides having sequences with up to five mutations compared to SEQ ID NO: 1 or SEQ ID NO: 2 are described above. In some embodiments, these sequences may have up to four, up to three, up to two, or up to one mutation compared to SEQ ID NO: 1 or SEQ ID NO: 2. Similarly, the mutations may be selected from amino acid substitutions, deletions, insertions, and / or additions, preferably amino acid substitutions, more preferably conservative amino acid substitutions.

[0063] In some embodiments, a peptide described herein comprises or consists of the sequence: RGXXGXMGXXGLAGXXGE (SEQ ID NO: 1) or AGXXGXTGXAGXXGFXGAV (SEQ ID NO: 2), wherein each X is independently proline or hydroxyproline.

[0064] In other embodiments, the peptides described herein comprise or consist of a sequence selected from the group consisting of AGPOGPTGPAGPOGFOGAVGAK (SEQ ID NO: 3), AGPOGPTGPAGPOGFPGAVGAK (SEQ ID NO: 4), AGOPGPTGPAGPOGFOGAVGA (SEQ ID NO: 5), AGOPGPTGPAGPOGFOGAV (SEQ ID NO: 6), AGPOGPTGPAGPOGFPGAV (SEQ ID NO: 7), AGPOGPTGPAG POGFOGAVGAKGE (SEQ ID NO: 8), RGPOGPMGPOGLAGPOGE (SEQ ID NO: 9), RGPOGOMGPOGLAGPOGE (SEQ ID NO: 10), RGOPGPMGPOGLAGPOGE (SEQ ID NO: 11), RGPPGPMGPOGLAGPOGE (SEQ ID NO: 12), RGOPGPMGPPGLAGPOGE (SEQ ID NO: 13), RGPOGPMGPPGLAGPOGE (SEQ ID NO: 14), and sequences corresponding to SEQ ID NOs: 3 to SEQ ID NOs. A sequence having up to 5 mutations compared to any one of SEQ ID NOs: 14. In some embodiments, the peptide described herein comprises or consists of a sequence selected from SEQ ID NOs: 3 to 14. "O" represents hydroxyproline.

[0065] In some embodiments, the peptides described herein comprise or consist of the sequence RGPPGPMGPPGLAGPP GE (SEQ ID NO: 15), AGPPGPTGPAGPPGFPGAV (SEQ ID NO: 16), or a sequence having up to 5 mutations compared to SEQ ID NO: 15 or 16. In some embodiments, the peptides described herein comprise or consist of the sequence RGPPGPMGPPGLAGPPGE (SEQ ID NO: 15), the sequence AGPPGPTGPAGPPGF PGAV (SEQ ID NO: 16).

[0066] In some embodiments, the peptide described herein comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 16, and a sequence having up to 5 mutations compared to any one of SEQ ID NO: 3 to SEQ ID NO: 16.

[0067] In the case of a sequence having up to five mutations compared to any one of SEQ ID NOs: 3 to 16, in some embodiments, the mutations are selected from amino acid substitutions, deletions, insertions, and / or additions. Among amino acid substitutions, deletions, insertions, and / or additions, amino acid substitutions are the preferred type of mutation. The amino acid substitutions may be conservative or non-conservative, with conservative amino acid substitutions being preferred.

[0068] Peptides having sequences with up to five mutations compared to any one of SEQ ID NO: 3 to SEQ ID NO: 16 are described above. In some embodiments, these sequences may have up to four, up to three, up to two, or up to one mutation compared to any one of SEQ ID NO: 3 to SEQ ID NO: 16. The mutations may be selected from amino acid substitutions, deletions, insertions, and / or additions, preferably amino acid substitutions, more preferably conservative amino acid substitutions.

[0069] As described in detail in the experimental section, it has been found that the peptides described herein show excellent binding affinity to epidermal growth factor receptor (EGFR) protein. Therefore, in some embodiments, the peptides described herein can be in conjunction with epidermal growth factor receptor (EGFR) protein. Epidermal growth factor receptor (EGFR) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Epidermal growth factor receptor is an ErbB family member receptor, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).

[0070] In a preferred embodiment, the EGFR is human EGFR, also known as HER1. The sequence and structure of human EGFR are well described in the art, for example as reflected in the Protein Data Bank (pdb; www.rcsb.org; entry 11VO) and Uniprot (entry P00533).

[0071] EGFR ligand binding induces the formation of homo- and heterodimers and triggers the activation of downstream signaling pathways, such as the phosphoinositide-3 kinase (PI3K) / Akt pathway (and other pathways), which control various functions including cell proliferation, survival, and migration. Therefore, the PI3K / Akt pathway works downstream of EGFR. Without wishing to be limited to any one theory or mode of action, the inventors have discovered that the peptides described herein can exert their beneficial effects by promoting PI3K / AKT signaling. Therefore, in some embodiments, the peptides described herein can promote PI3K / AKT signaling.

[0072] PI3K / AKT signaling can be measured and quantified using means and methods known to those skilled in the art, such as by measuring the expression of related genes. Gene expression can be assessed by any method known to those skilled in the art. For example, expression can be assessed at the mRNA or protein level by standard assays known to those skilled in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry analysis of protein-derived peptides, or ELISA. PI3K / AKT signaling can also be measured and quantified by assaying phosphorylated AKT protein using anti-phospho-AKT antibodies, such as in a sandwich ELISA assay.

[0073] As previously mentioned, the peptides of the present disclosure may be obtained by any suitable method known to the skilled person. One particularly interesting, but non-limiting, source of the peptides described herein is collagen hydrolysis.

[0074] "Collagen" is the main structural protein of the extracellular matrix of connective tissue in animals and humans. Collagen is composed of amino acid chains that are twisted together to form a triple helix, which in turn forms elongated fibrils (called "collagen helices"). Collagen is primarily found in fibrous tissues such as tendons, ligaments, and skin, but is also present in bones, teeth, corneas, cartilage, intervertebral discs, and blood vessels. The amino acid composition of collagen is atypical for proteins, particularly with respect to its high hydroxyproline content. The most common motifs in collagen amino acid sequences are glycine-proline-X and glycine-X-hydroxyproline, where X is any amino acid other than glycine, proline, or hydroxyproline.

[0075] Collagen can be processed into gelatin by irreversible partial hydrolysis with heat, alkaline solutions or weak acids. Gelatin typically forms gels characterized by a specific gel strength or "Bloom value."

[0076] Gelatin can be further hydrolyzed to produce collagen hydrolysate, which is composed of shorter protein chains or "collagen peptides." In contrast to gelatin, collagen hydrolysate does not form a gel and is generally soluble in water at ambient temperature.

[0077] In some embodiments, the peptides described herein are collagen peptides.

[0078] In some embodiments, the peptides described herein can be obtained from hydrolyzed collagen. This means that the peptides described herein may be present in the collagen, in which case they can be "released" by hydrolyzing the collagen.

[0079] The skilled person will appreciate that in order to obtain more or less purified versions of the peptides described herein from collagen hydrolysates, further separation and purification techniques known to those skilled in the art may be applied, including chromatographic methods and ultrafiltration methods.

[0080] Collagen hydrolysates (collagen peptides) can be formed using different hydrolysis methods, including heat treatment, acid treatment and enzymatic hydrolysis of collagen, and combinations thereof. Enzymatic hydrolysis is preferred. Enzymatic hydrolysis can be performed by proteolytic enzymes known in the art (e.g., pepsin and trypsin). Different enzymes can also be used simultaneously or sequentially.

[0081] Collagen hydrolysate contains peptides of different lengths or molecular weights, which are produced during collagen hydrolysis. In some embodiments, the peptides disclosed herein can be obtained from collagen hydrolysates having a molecular weight of 300 to 14,000 Da, preferably 400 to 7,000 Da, more preferably 800 to 3,500 Da, even more preferably 1,200 to 2,300 Da, and most preferably 1,600 to 1,750 Da. Thus, in some embodiments, the collagen hydrolysate from which the peptides described herein can be obtained is such that the molecular weight distribution of the collagen hydrolysate is 300 to 14,000 Da, preferably 400 to 7,000 Da, more preferably 800 to 3,500 Da, even more preferably 1,200 to 2,300 Da, and most preferably 1,600 to 1,750 Da.

[0082] As used herein, "molecular weight distribution" of molecular weight A to molecular weight B means that at least 90 wt%, at least 91 wt%, at least 92 wt%, at least 93 wt%, at least 94 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt% or at least 99 wt% of the peptides in the collagen hydrolysate have a molecular weight between A and B. In a preferred embodiment, a molecular weight distribution of A to B means that at least 99 wt% of the peptides in the collagen hydrolysate have a molecular weight between A and B.

[0083] The molecular weight distribution of collagen peptides can be determined very accurately and reproducibly, for example by gel permeation chromatography using calibration standards of defined collagen fragments.

[0084] The peptides disclosed herein can be obtained in particular from a collagen hydrolysate having a molecular weight of ≤14,000Da, preferably ≤7,000Da, more preferably ≤3,500Da, or even more preferably ≤3,000Da. In some embodiments, a hydrolysate having a lower molecular weight (e.g., ≤2,300Da or ≤1,750Da) can also be used. Thus, in some embodiments, the collagen hydrolysate from which the peptides described herein can be obtained is such that at least 90wt%, at least 91wt%, at least 92wt%, at least 93wt%, at least 94wt%, at least 95wt%, at least 96wt%, at least 97wt%, at least 98wt% or at least 99wt% of the peptides in the collagen hydrolysate have a molecular weight of ≤14,000Da, preferably ≤7,000Da, more preferably ≤3,500Da, or even more preferably ≤3,000Da. In other embodiments, the collagen hydrolysate from which the peptides described herein can be obtained is such that at least 90 wt%, at least 91 wt%, at least 92 wt%, at least 93 wt%, at least 94 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt% or at least 99 wt% of the peptides in the collagen hydrolysate have a molecular weight ≤ 2,300 Da or ≤ 1,750 Da.

[0085] As known to those skilled in the art, various molecular weight fractions of the initial collagen hydrolysate can be obtained by methods known in the art (eg, ultrafiltration methods).

[0086] In industrial situations, collagen is typically derived from animal skin and / or bones. Hydrolyzed collagen can be obtained starting from a variety of animal collagen sources. In the context of the present disclosure, there is no particular restriction on the source of the collagen material. However, in some embodiments, the hydrolyzed collagen is one or more of hydrolyzed bovine collagen, hydrolyzed porcine collagen, or hydrolyzed fish collagen. In a preferred embodiment, the hydrolyzed collagen is hydrolyzed bovine collagen. Bovine collagen can typically be obtained from cowhide and / or cow bones. Porcine collagen can typically be obtained from pigskin and / or pig bones. Fish collagen can be obtained from various fish by-products (e.g., fish bones, fish scales, and fish skin).

[0087] As mentioned above, the present disclosure also encompasses peptide mimetics and salt forms of peptides. Therefore, on the other hand, peptide mimetics derived from peptides described herein are provided. On the other hand, salt forms of peptides provided herein are provided.

[0088] Food, pet food and feed products and food, pet food and feed supplements

[0089] In another aspect, provided are foods, pet food or feed products or supplements comprising the compositions described herein. In another aspect, provided are foods, pet food or feed products or supplements comprising the peptides described herein.

[0090] A "food product" is typically an edible material (for humans) consisting primarily of one or more macronutrients: protein, carbohydrate, and fat. A food product may also contain one or more micronutrients, such as vitamins or minerals. The term "food product" is used broadly herein to include fluid, semisolid, and solid food products, and therefore also beverages. Beverage types encompassed by the present disclosure include soft drinks, syrups, fruit drinks, dry beverage mixes, and nutritional beverages.

[0091] Examples of food products that can incorporate the peptides of the present disclosure include baked goods, dairy products, convenience foods, high protein beverages, and the like.

[0092] "Pet food" is a food product suitable for pets (such as cats and dogs). Examples of pet food products include dry pet food, such as extruded, cold-pressed, or oven-baked pet food; wet and semi-moist pet food; dental sticks, etc.

[0093] A "feed product" is a food product suitable for domestic animals, preferably livestock.

[0094] Foods, pet foods, and feed supplements are generally intended to correct nutritional deficiencies, maintain adequate intake of certain nutrients, and / or support specific physiological functions. Foods, pet foods, and feed supplements may contain a variety of nutrients and other ingredients, such as vitamins, minerals, amino acids, essential fatty acids, fiber, and various plant and herbal extracts. Foods, pet foods, and feed supplements may be in suitable dosage forms as described elsewhere herein, including capsules, tablets, granules, pills, suspensions, and syrups.

[0095] Foods, pet foods, and feed products containing the peptides described herein may be referred to as "functional" or "fortified" foods, pet foods, and feed products. In general, the foods, pet foods, and feed products, as well as foods, pet foods, and feed supplements provided herein, comprising the peptides or compositions described herein, may also be referred to as nutraceuticals. As used herein, a "nutraceutical" is a food ingredient, food product, beverage, or food, pet food, or feed supplement that is believed to provide a medical or health-related benefit, including the prevention and treatment of disease. Thus, nutraceuticals are often promoted as providing health benefits, rather than simply providing pure nutrition to the consumer.

[0096] In some embodiments, the food, pet food or feed products or supplements disclosed herein comprise at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt%, or at least 99 wt% of a peptide described herein.

[0097] In some embodiments, optionally in combination with the above minimum amounts, the food, pet food or feed products or supplements disclosed herein contain at most 1 wt%, at most 2 wt%, at most 3 wt%, at most 4 wt%, at most 5 wt%, at most 6 wt%, at most 7 wt%, at most 8 wt%, at most 9 wt%, at most 10 wt%, at most 15 wt%, at most 20 wt%, at most 25 wt%, at most 30 wt%, at most 35 wt%, at most 40 wt%, at most 45 wt%, at most 50 wt%, at most 55 wt%, at most 60 wt%, at most 65 wt%, at most 70 wt%, at most 75 wt%, at most 80 wt%, at most 85 wt%, at most 90 wt%, at most 95 wt%, at most 96 wt%, at most 97 wt%, at most 98 wt% or at most 99 wt% of a peptide described herein.

[0098] In some embodiments, the food, pet food or feed products or supplements disclosed herein comprise at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt%, or at least 99 wt% of a composition described herein.

[0099] In some embodiments, optionally in combination with the minimum amounts described above, a food, pet food or feed product or supplement disclosed herein comprises at most 1 wt%, at most 2 wt%, at most 3 wt%, at most 4 wt%, at most 5 wt%, at most 6 wt%, at most 7 wt%, at most 8 wt%, at most 9 wt%, at most 10 wt%, at most 15 wt%, at most 20 wt%, at most 25 wt%, at most 30 wt%, at most 35 wt%, at most 40 wt%, at most 45 wt%, at most 50 wt%, at most 55 wt%, at most 60 wt%, at most 65 wt%, at most 70 wt%, at most 75 wt%, at most 80 wt%, at most 85 wt%, at most 90 wt%, at most 95 wt%, at most 96 wt%, at most 97 wt%, at most 98 wt% or at most 99 wt% of a composition described herein.

[0100] The skilled artisan will appreciate that the appropriate amount herein depends on the type of product. For example, food, pet food, and feed products may typically contain 0.1 to 20 wt%, preferably 0.5 to 15 wt%, of a peptide or composition as described herein. On the other hand, food, pet food, or feed supplements may contain greater amounts.

[0101] In some embodiments, food, pet food and feed products and supplements comprising the collagen peptides of the present disclosure are such that the peptides have the molecular weight and molecular weight distribution described in the "Peptides comprising an amino acid sequence having two or more GXX repeats" section.

[0102] beauty products

[0103] In another aspect, a cosmetic product is provided comprising a composition as described herein. In another aspect, a cosmetic product is provided comprising a peptide as described herein.

[0104] As used herein, a "cosmetic product" (also referred to simply as a "cosmetic product") refers to a product that is intended to be applied to the human body to cleanse, beautify, enhance attractiveness, or alter appearance. In a preferred embodiment, the cosmetic product is a cosmetic product designed for skin care, i.e., a cosmetic product that is intended to be applied to the skin. Such cosmetic skin products can be used for a variety of purposes, including cleansing, exfoliating, protecting, replenishing, or nourishing the skin. In some embodiments, the cosmetic skin product is selected from soap, shampoo, body wash, facial cleanser, toner, serum, moisturizer, eye cream, and balm.

[0105] Examples of cosmetic products, particularly cosmetic skin products, that can incorporate the peptides of the present disclosure include soaps, shampoos, body washes, facial cleansers, toners, serums, moisturizers, eye creams, and balms.

[0106] In some embodiments, the cosmetic products disclosed herein comprise at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt%, or at least 99 wt% of a peptide described herein.

[0107] In some embodiments, optionally in combination with the above minimum amounts, the cosmetic products disclosed herein comprise at most 1 wt%, at most 2 wt%, at most 3 wt%, at most 4 wt%, at most 5 wt%, at most 6 wt%, at most 7 wt%, at most 8 wt%, at most 9 wt%, at most 10 wt%, at most 15 wt%, at most 20 wt%, at most 25 wt%, at most 30 wt%, at most 35 wt%, at most 40 wt%, at most 45 wt%, at most 50 wt%, at most 55 wt%, at most 60 wt%, at most 65 wt%, at most 70 wt%, at most 75 wt%, at most 80 wt%, at most 85 wt%, at most 90 wt%, at most 95 wt%, at most 96 wt%, at most 97 wt%, at most 98 wt% or at most 99 wt% of a peptide as described herein.

[0108] In some embodiments, the cosmetic products disclosed herein comprise at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, at least 96 wt%, at least 97 wt%, at least 98 wt%, or at least 99 wt% of a composition described herein.

[0109] In some embodiments, optionally in combination with the above minimum amounts, the cosmetic products disclosed herein comprise at most 1 wt%, at most 2 wt%, at most 3 wt%, at most 4 wt%, at most 5 wt%, at most 6 wt%, at most 7 wt%, at most 8 wt%, at most 9 wt%, at most 10 wt%, at most 15 wt%, at most 20 wt%, at most 25 wt%, at most 30 wt%, at most 35 wt%, at most 40 wt%, at most 45 wt%, at most 50 wt%, at most 55 wt%, at most 60 wt%, at most 65 wt%, at most 70 wt%, at most 75 wt%, at most 80 wt%, at most 85 wt%, at most 90 wt%, at most 95 wt%, at most 96 wt%, at most 97 wt%, at most 98 wt% or at most 99 wt% of a composition described herein.

[0110] The skilled person understands that the appropriate amount in this context depends on the type of product.

[0111] In some embodiments, a cosmetic product comprising a collagen peptide of the present disclosure is such that the molecular weight and molecular weight distribution of the peptide are as described elsewhere herein in the section "Peptides comprising an amino acid sequence having two or more GXX repeats."

[0112] General information

[0113] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as customary and commonly understood by one of ordinary skill in the art to which this invention belongs and are to be read in light of this disclosure.

[0114] As used herein, "conservative" amino acid substitutions refer to the interchangeability of residues with similar side chains. The following table provides examples of classes of amino acid residues that are conservatively substituted. Substitution of proline for hydroxyproline, or vice versa, can also be considered a conservative substitution.

[0115] acidic residues Asp(D) and Glu(E) basic residues Lys(K), Arg(R), and His(H) Hydrophilic uncharged residues Ser(S), Thr(T), Asn(N) and Gln(Q) Aliphatic uncharged residues Gly(G), Ala(A), Val(V), Leu(L) and Ile(I) Nonpolar uncharged residues Cys(C), Met(M), and Pro(P) Aromatic residues Phe(F), Tyr(Y), and Trp(W)

[0116] Alternative physical and functional classification of amino acid residues:

[0117] Alcohol-containing residues S and T Aliphatic residues I, L, V, and M Cycloalkenyl-related residues F, H, W, and Y hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residues Q, T, K, S, G, P, D, E, and R

[0118] In this document and its claims, the verb "to comprise" and its conjugations are used in its non-limiting sense to mean that items following the word are included or comprised, but items not specifically mentioned are not excluded. Thus, as used herein, the terms "comprise," "include," "contain," and the like are synonymous with "cover," "encompass," or "have," "involve," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0119] Furthermore, the verb "consisting of" can be replaced with "consisting essentially of," meaning that the compositions described herein can include additional components besides those specifically identified, without altering the unique characteristics of the invention. Furthermore, the verb "comprise" can be replaced with "consisting essentially of," meaning that the methods described herein can include additional steps besides those specifically identified, without altering the unique characteristics of the invention.

[0120] Throughout this disclosure, the terms "comprising" and "including" may be replaced with the term "consisting essentially of" or the term "consisting of, respectively.

[0121] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents, unless the context clearly dictates otherwise; for example, "a genetic construct" is understood to refer to more than one genetic construct. Thus, the terms "a" (or "an"), "more than one," and "at least one" can be used interchangeably herein.

[0122] As used herein, "at least" modifies a particular value to mean that particular value or greater. For example, "at least 2" is understood to be the same as "more than 2," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0123] Furthermore, the terms first, second, third, etc. in the description and claims are used to distinguish similar elements and not necessarily to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

[0124] When used in conjunction with a numerical value (e.g. about 10), the expression "about" or "approximately" preferably means that the value may be 10% more or less, preferably 5%, more preferably 1% more or less than the given value (10).

[0125] As used herein, the term "and / or" means that more than one of the stated conditions may appear alone or in combination with at least one of the stated conditions, until all of the stated conditions appear.

[0126] Various embodiments are described herein. Unless otherwise indicated, the embodiments shown herein may be combined. The titles, subtitles, and subheadings used herein are for ease of reading only and are not intended to limit or restrict the present disclosure in any way.

[0127] All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and to the extent the incorporated material is inconsistent with the explicit disclosure herein, in which case the language of the disclosure controls.

[0128] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. In fact, the present invention is in no way limited to the methods and materials described.

[0129] The present invention is further described by the following non-limiting examples.

[0130] Example

[0131] Promoting osteogenesis and angiogenesis using specific collagen peptides by activating the PI3K / AKT signaling pathway in mice General Procedure for Implementing the Embodiments

[0132] Overview: To examine the bioactivity of collagen hydrolysate (CH), MC3T3-E1 cell and EA.hy926 cell viability, alkaline phosphatase activity, Alizarin Red S staining, cell migration, and tube formation were measured. Furthermore, the major active collagen peptides in the collagen hydrolysate were identified by LC-MS / MS and molecular docking. C57BL / 6J mice were randomly divided into sham, ovariectomized (OVX), and CH-treated groups for in vivo studies. Femora were then harvested for microCT examination. Tibial RNA was extracted for gene expression profiling by PCR array. Details are provided below.

[0133] Material

[0134] Collagen bovine bone hydrolysate ( Collagen peptides (peptides) were obtained from PBLeiner (Vilvoorde, Belgium) of the Tessenderlo Group. MC3T3-E1 cell line (CL-0378) and EA.hy926 cell line (CL-0272) were provided by Pronose Life Science Co., Ltd. (Wuhan, China). For osteogenesis, this study used an Alizarin Red S staining kit, an alkaline phosphatase assay kit, and a BCIP / NBT alkaline phosphatase colorimetric kit (Beyotime Biotechnology, Shanghai, China). Matrigel basement membrane matrix (#354234, Corning, USA) was used. The EASYspin Plus Bone Tissue RNA Kit was used (Beijing Aidelai Biotechnology Co., Ltd., Beijing, China). All other chemicals used in this study were analytical reagents.

[0135] Cell culture

[0136] MC3T3-E1 cells were cultured in complete Minimum Essential Medium α (MEM-α, containing L-glutamine, 10% fetal bovine serum (FBS), nucleosides, and antibiotic solution, including 100 units / mL penicillin and 100 units / mL streptomycin). Complete Dulbecco's Modified Eagle's Medium (DMEM, containing 4.5 g / L glucose, L-glutamine, sodium pyruvate, 10% FBS, and AS) was used to culture EA.hy926 cells. Cultures were performed in a cell culture incubator at 37°C with 5% CO2.

[0137] Cell viability assay

[0138] CCK-8 assay was used to determine the viability of all cells. 4 Cells were plated in 96-well plates (100 μL / well) and incubated overnight. In the CH-treated group, MC3T3-E1 cells were further incubated for 72 hours. EA.hy926 cells were treated with CH for 24 hours. Finally, cells were treated with 10% CCK-8 for 2 hours. The cells were analyzed using a microplate reader ( The absorbance at 450 nm was measured by M2 / M2e (California, USA). Cells in the control group were cultured under normal conditions.

[0139] Alkaline phosphatase (ALP) activity and staining assay

[0140] For quantitative ALP measurement, MC3T3-E1 was plated at 5 × 10 3 Cells were seeded at a density of 10 cells / well in a 96-well plate. The cells were then treated by adding sample to the plate. On days 7 and 14 of osteogenesis induction, MC3T3-E1 cells were lysed using RIPA lysis buffer. Alkaline phosphatase activity was measured using an alkaline phosphatase assay kit.

[0141] MC3T3-E1(1×10 5 Cells (100 cells / mL) were seeded in 6-well plates and treated with samples for ALP staining. On days 7 and 14, MC3T3-E1 cells were washed three times with phosphate-buffered saline (PBS). Detection was performed using a BCIP / NBT alkaline phosphatase colorimetric kit according to the manufacturer's instructions.

[0142] mineralization

[0143] After 7, 14, and 21 days of osteoinduction, MC3T3-E1 cells were rinsed twice with PBS. Staining was performed using the Alizarin Red S staining kit for osteogenesis. Calcium deposition was imaged using a microscope. Mineralization was quantified by dissolving MC3T3-E1 cells in 10% cetylpyridinium chloride for 60 minutes in the dark. Absorbance was measured at 570 nm.

[0144] In vitro angiogenesis assay

[0145] EA.hy926 migration was measured by scratch wound healing assay. After 24 h of incubation, cells (5 × 10 5 Cells were seeded in 6-well plates (100 cells / well). When the cells formed a confluent monolayer, a blank area was formed. Subsequently, cells were treated with different samples. Complete medium was used in the control group. The exposed area was imaged under an inverted microscope at time 0 and 6 hours later. The resulting images were further analyzed using ImageJ software.

[0146] EA.hy926 cells were cultured in Matrigel basement membrane matrix for tube formation assay. 96-well plates were pretreated with 50 μL Matrigel at 37°C for 30 minutes. EA.hy926 cells in logarithmic growth phase were then seeded in 96-well plates (5 × 10 4 EA.hy926 cells were treated with 1000 μg / mL of the sample for 12 hours at 37° C. Angiogenesis was imaged using an inverted microscope (Leica, DM 1L LED).

[0147] Peptide identification

[0148] Peptides in CH were separated using a Thermo-Dionex Ultimate 3000 HPLC system at a flow rate of 0.250 μL / min for 60 minutes. A Thermo LTQ-Orbitrap Velos pro mass spectrometer was connected in series to the system. The capillary column used was a fused silica capillary column (75 μm inner diameter, 150 mm length; Upchurch, Oak Harbor, Washington) filled with C18 resin (300A, 5 μm; Varian, Lexington, Massachusetts). The mobile phase was 0.1% (v / v) formic acid and acetonitrile containing 0.1% (v / v) formic acid.

[0149] Molecular docking

[0150] AlphaFold2 was used to construct the 3D structure of the identified peptide using MMseqs2 (Mirdita et al., ColabFold:making protein folding accessible to all, Nature Methods 2022, 19 (6): 679-682). Molecular docking studies were performed using the HPEPDOCK server (huanglab.phys.hust.edu.cn / hpepdock, version: 2021-11-13) (Zhou et al., HPEPDOCK:a web server for blind peptide–protein dockingbased on ahierarchical algorithm, Nucleic Acids Research 2018, 46 (W1): W443-W450). In short, the crystal structure of EGFR (1IVO) was obtained from the Protein Data Bank (www.rcsb.org). The receptor and ligand were adjusted and optimized. Specifically, after removing the water molecules of all bound ligands and EGFR, the missing hydrogen and charge were corrected by AutoDockTools-1.5.7. The compound score "docking score" is calculated to indicate the affinity between the ligand and the binding pocket of the receptor. The interaction force and the interaction site of the ligand and receptor were also evaluated using the protein-ligand interaction instrument (PLIP) (plip-tool.biotec.tu-dresden.de) (Adasme et al., PLIP 2021: expanding the scope of the protein-ligand interaction profiler to DNA and RNA, Nucleic Acids Research 2021, 49 (W1): W530-W534).

[0151] animal

[0152] Female C57BL / 6J mice, 7 weeks old, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and were adaptively fed for 1 week before surgery. Bilateral ovariectomy was performed, and mice in the sham group were not ovariectomized. After surgery, the mice were fed to recover for 1 week. 30 mice were randomly divided into the following different groups (n=6 per group): sham group (saline treatment), OVX group (saline treatment), OVX-CH group (CH treatment, 50, 100, 200 mg / kg / day). Mice received peptide (in saline) or saline by gavage every day. The Institutional Animal Care and Use Committee of Beijing Tiantan Hospital, Capital Medical University, approved the animal experimental protocol. All procedures were performed under inhalation anesthesia, and all experiments were conducted in an ethical and humane manner to minimize animal suffering.

[0153] Microcomputed tomography (micro-CT) analysis

[0154] MicroCT scanning was performed using a high-resolution microCT scanner (SkyScan 1276, Bruker, Karlsruhe, Germany) with an image pixel size of 6.03 μm, a voltage of 55 keV, and a current of 200 μA to analyze changes in bone mineral density and bone morphometric parameters. Bone image reconstruction was performed using the 3D Creator software provided with the instrument as described previously. At the distal end of the femur, a region of interest (ROI) of trabecular bone was selected 0.5 mm below the growth plate. Morphometric parameters were also measured.

[0155] Histological and histomorphometric analysis

[0156] Femurs were decalcified in 10% EDTA (pH 7.4), embedded in paraffin, and sectioned along the coronal plane. Sections were stained with hematoxylin and eosin (H&E). Histological images were digitized using a pathology slide scanner (WISLEAP WS-10, Jiangsu, China).

[0157] Quantitative real-time PCR

[0158] Bone tissue RNA was extracted using the EASYspin Plus Bone Tissue RNA Kit according to the manufacturer's instructions. Gene expression profiles were established using an osteoporosis PCR array and an angiogenesis PCR array (Woji Gene, Shanghai, China). Data were analyzed using Woji Gene software. Omicshare tools ( www.omicshare.com / tools ) was used to determine the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Results were considered to be significantly different when the P value was less than 0.05.

[0159] Statistical analysis

[0160] Analysis of variance (ANOVA) was used to characterize significant differences in multiple comparisons. Statistical significance of regression coefficients was obtained using the software package SPSS 22.0 (SPSS, Inc., Chicago, IL, USA).

[0161] Example 1: Collagen peptides promote osteoblast proliferation

[0162] To characterize the osteoblast proliferation-promoting activity of collagen peptides, MC3T3-E1 cells were treated with different concentrations of collagen hydrolysate (CH) for 72 hours. Figure 1 As shown in A, CH significantly increased osteoblast proliferation in a dose-dependent manner.

[0163] Example 2: Collagen peptides stimulate differentiation activity in osteoblasts

[0164] Osteoblast differentiation is conducive to bone formation. It is well known that alkaline phosphatase is an important biomarker synthesized in the early stage of osteoblast differentiation (Lu et al., Mechanical loading induced expression of bonemorphogenetic protein-2, alkaline phosphatase activity, and collagen synthesis in osteoblastic MC3T3-E1 cells, Chinese medical journal 2012, 125(22): 4093-4097). In this study, we investigated the effect of CH on the osteogenic differentiation of MC3T3-E1 cells by evaluating alkaline phosphatase activity. Figure 1 As shown in B and D, on day 7, the control group produced less purple precipitate, while the CH group produced more granular precipitate. This difference became more pronounced on day 14. Alkaline phosphatase activity results showed that all samples increased alkaline phosphatase activity. CH strongly stimulated MC3T3-E1 cells to release more alkaline phosphatase, promoting differentiation. Therefore, CH is expected to play a key role in promoting early osteogenic differentiation of MC3T3-E1 cells.

[0165] Example 3: Collagen peptides stimulate mineralization of osteoblasts

[0166] The development of mineralized calcium nodules is a key indicator for evaluating osteogenic differentiation (Wang Zq et al., MicroRNAs: important mediators of ossification, Chinese Medical Journal 2012, 125(22): 4111-4116). Based on the analysis of Alizarin Red S staining results, the in vitro mineralization status of MC3T3-E1 cells was qualitatively evaluated. Compared with the control group, treatment of MC3T3-E1 cells with CH for 21 days significantly increased mineral deposition ( Figure 1 C and 1E). Interestingly, CH-treated cells showed greater red intensity compared with the other groups, indicating that CH-treated cells were more differentiated than the other groups.

[0167] Example 4: Collagen peptides promote angiogenesis

[0168] Regarding in vitro angiogenesis, cell viability studies have shown that collagen hydrolysate can stimulate endothelial cell proliferation in a dose-dependent manner ( Figure 2 A). Scratch wound healing assay showed that CH could increase endothelial cell migration in a dose-dependent manner. The CH concentration that could best promote cell migration was 1000 μg / mL ( Figure 2 B and 2C). We then investigated the effects of CH on endothelial cell tube development. Figure 2 As shown in D, CH showed a significant stimulatory effect on tube development.

[0169] Example 5: Identification of peptide sequences by mass spectrometry

[0170] Collagen hydrolysate is composed of a variety of collagen peptides with well-defined amino acid sequences. To identify the effective amino acid sequences of collagen peptides responsible for osteogenesis and angiogenesis in CH, the amino acid sequences of the collagen peptides were identified. The main peptide sequences are shown below, where "O" represents hydroxyproline (Table 1).

[0171] Table 1: Amino acid sequences of bovine collagen hydrolysates with less than 3K components

[0172]

[0173]

[0174] Example 6: Molecular docking of collagen peptides

[0175] From the amino acid sequences of the collagen peptides in Example 5, we obtained 19 amino acid sequences that shared overlapping peptide sequences. To screen for collagen peptides with anti-osteoporosis properties, the 19 identified peptides were docked with the EGFR protein. Two peptides (Pep-1: RGPPGPMGPPGLAGPPGE (SEQ ID NO: 15) and Pep-2: AGPPGPTGPAGPPGFPGAV (SEQ ID NO: 16) were able to bind to EGFR with lower Hpepdock scores (Table 2).

[0176] Table 2: Molecular docking of peptide sequences from collagen hydrolysate

[0177] sequence SEQ ID NO Peptidoglycan Hpepdock score RMSD GKEGPVGLPGIDGRPGPIGPA 41 0.465411 -193.217 1.907 GAPGLPGPRGIPGPVGA 42 0.888364 -191.386 1.661 AGPPGPTGPAGPPGFPGAV 16 0.862544 -222.732 1.386 PGPAGPAGPR 43 0.873845 -166.136 2.044 GLPGSRGERGLPGVA 44 0.564212 -193.073 1.544 RGPPGPMGPPGLAGPPGE 15 0.817219 -226.688 1.774 GPAGPAGPIGPV 45 0.867215 -159.002 1.859 GSDGSVGPVGPAGPIGS 38 0.603386 -184.194 1.262 VGPAGPNGFAGPA 20 0.81301 -194.194 1.724 LTGPIGPPGPA 46 0.438276 -165.127 0.976 KGPSGEPGTAGPPGTPGPQGLL 47 0.371679 -190.793 1.618 GPPGAPGLPGPPGF 48 0.968244 -193.635 1.581 GPIGSRGPSGPPGPDGNK 49 0.670141 -202.566 1.676 GFPGDRGLPGPVGAL 50 0.872564 -215.928 1.683 GFPGADGVAGPKGPAGE 51 0.663011 -181.877 1.407 GISVPGPMGPSGPRGLPGPPGAPGPQ 52 0.434399 -224.761 1.846 GFPGTPGLPGFKG 53 0.918474 -207.630 1.348 GIPGEFGLPGPA 54 0.789572 -203.428 1.412 GPIGSAGPPGFPGAPGPKGE 55 0.860301 -200.666 1.249

[0178] We also analyzed the docking interactions between Pep-1 and Pep-2 and EGFR. Pep-1 forms numerous hydrophobic interactions, hydrogen bonds, and salt bridges with EGFR, including numerous hydrogen bonds. Furthermore, several key amino acid residues in the EGF-EGFR interaction site, including Leu382, Gln384, His409, Phe412, Val417, and Ile438, were also key interaction sites in this study.

[0179] Example 7: Collagen peptides rescue bone microstructure in ovariectomized (OVX) mice

[0180] It was found that the trabeculae in the OVX group were sparse and had large gaps ( Figure 3 B). Trabecular bone loss was observed in the OVX group compared to the sham group. In addition, a significant improvement in trabecular microarchitecture was observed in the collagen hydrolysate group. Figure 3 A to E depict exemplary micro-CT images of trabecular bone microarchitecture in the right femur, which support the above findings: trabecular bone density, thickness, and continuity were enhanced after collagen hydrolysate treatment.

[0181] Next, if Figure 3 As shown in Figures F to I, increases in bone surface area (BS), bone volume fraction (bone volume to total volume ratio; BV / TV), trabecular number (Tb.N), and trabecular spacing (Tb.Sp) occurred in the model group. Trabecular bone quality in the right femur treated with collagen hydrolysate significantly improved, alleviating the severe symptoms observed in the model group.

[0182] After surgery (bilateral ovariectomy), OVX model mice developed osteoporosis, as evidenced by empty spaces in trabecular bones and pyknotic nuclei in osteocytes. Subsequently, osteoporosis was further characterized by histological evaluation ( Figure 3 J). CH was observed to significantly reduce the incidence of osteoporosis, as demonstrated by these representative H&E staining images.

[0183] Example 8: Potential signaling pathways affected by collagen peptides in osteoporosis

[0184] To investigate the possible therapeutic mechanism of collagen peptides in ovariectomized (OVX) osteoporosis mice, we characterized the differentially expressed genes related to osteoporosis and angiogenesis in mouse bone by PCR array. Compared with the sham group, slightly more DEGs were upregulated than downregulated in OVX mice.

[0185] Differential gene classification ( Figure 4 A) Shows that compared with the OVX group, CH treatment significantly enhanced the expression of several genes involved in bone regeneration and BMPs. While the expression of most cytokine, growth factor, and receptor genes was decreased in the OVX group, CH upregulated these genes. Furthermore, CH significantly upregulated the expression of angiogenesis-related genes, including those associated with ECM and adhesion molecules.

[0186] To explore the possible mechanism of CH, KEGG enrichment analysis was performed on DEGs to find gene pathways. Figure 4 As shown in B, each pathway enriched in the CH group has a smaller P value and a larger gene percentage, indicating a higher confidence level. In addition, these pathways are closely related to PI3K-AKT, a key pathway for inhibiting osteoporosis.

[0187] Next, we further analyzed the KEGG enrichment results, which showed a pathway diagram of differentially expressed genes in response to the restorative effect of CH on osteoporosis mice ( Figure 4 C). These results indicate that CH stimulates the regulation of growth factor-related genes and ECM synthesis-related genes in OVX mice through activation of the PI3K / AKT pathway.

[0188] in conclusion

[0189] This study demonstrated that collagen peptides simultaneously stimulated osteoblast proliferation, differentiation, and mineralization, as well as endothelial cell proliferation and migration. These findings suggest that some active peptides from collagen hydrolysates possess both osteogenic and angiogenic activities. In summary, this study demonstrated that collagen peptides can alleviate osteoporosis by improving bone mass, reducing osteoporotic bone loss, and promoting angiogenesis. Two novel peptides, RGPPGPMGPPGLAGPPGE (SEQ ID NO: 15) and AGPPGPTGPAGPPGFPGAV (SEQ ID NO: 16), were identified based on their interaction with EGFR and demonstrated their bioactivity. Genomic data suggest that the potential mechanism by which CH containing these active peptides may inhibit osteoporosis is through activation of the PI3K / AKT signaling pathway by regulating various growth factors and genes involved in ECM synthesis, thereby promoting bone formation and angiogenesis.

Claims

1. A composition comprising collagen peptide, wherein: The composition comprises at least 0.1 wt% of a peptide comprising an amino acid sequence having two or more GXX repeating sequences, wherein each X is independently proline (Pro) or hydroxyproline (Hyp).

2. The composition according to claim 1, wherein The total length of the peptide comprising an amino acid sequence having two or more GXX repeating sequences is 10 to 25 amino acids, preferably 16 to 20 amino acids.

3. The composition according to claim 1 or 2, wherein The total length of the peptide comprising an amino acid sequence having two or more GXX repeating sequences is 18 or 19 amino acids.

4. The composition according to any one of claims 1 to 3, wherein The peptide comprising an amino acid sequence having two or more GXX repeating sequences comprises or consists of the following sequence Composition: sequence RGXXGXMGXXGLAGXXGE (SEQ ID NO: 1), sequence AGXXGXTGXAGXXGFXGAV (SEQ ID NO: 2), or a sequence having up to 5 amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 1 or SEQ ID NO: 2, wherein each X is independently proline (Pro) or hydroxyproline (Hyp).

5. The composition according to any one of claims 1 to 4, wherein The peptide comprising an amino acid sequence having two or more GXX repeating sequences comprises or consists of the following sequence Composition: A sequence selected from SEQ ID NO: 3 to SEQ ID NO: 16, and a sequence having up to 5 mutations compared to any one of SEQ ID NO: 3 to SEQ ID NO:

16.

6. The composition according to any one of claims 1 to 5, wherein The peptide comprising an amino acid sequence having two or more GXX repeating sequences can bind to epidermal growth factor receptor (EGFR) protein.

7. The composition according to claim 6, wherein The peptide comprising an amino acid sequence having two or more GXX repeating sequences can promote PI3K / AKT signal transduction.

8. The composition according to any one of claims 1 to 7, wherein At least 99% of the collagen peptides in the composition are collagen peptides with a molecular weight of ≤3,000 Da.

9. A food or feed product or supplement comprising the composition of any one of claims 1 to 8.

10. A cosmetic product comprising the composition according to any one of claims 1 to 8.

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