Peptide mixture and use thereof as animal feed

Abstract

The present invention relates to a peptide mixture, a method for obtaining a hydrolysate, and the hydrolysate obtained using this method. The invention also relates to the use of the peptide mixture and the hydrolysate as additives in animal feed to improve growth and / or increase physical performance and / or prevent and / or treat stress and / or behavioural disorders.

Description

[0001] Peptide mixture and its use as animal feed

[0002] The present invention relates to a peptide mixture, a method for obtaining a hydrolysate, and the hydrolysate obtained by this method. It also relates to the use of the peptide mixture and the hydrolysate as feed additives to improve growth and / or increase physical performance and / or prevent and / or treat stress and / or behavioral disorders. Therefore, the present invention falls within both the field of biotechnology, more specifically the agri-food industry, and the pharmaceutical field.

[0003] STATE OF THE ART

[0004] The large daily volume of blood generated in slaughterhouses and the low value of its common uses represent an environmental burden. Today, most secondary blood-related waste from slaughterhouses is disposed of.

[0005] However, since residual blood contains a large number of useful components, it can be developed as a raw material for various products. Protein, a major component of blood, can be used as a raw material for fertilizers and / or animal feed. Despite its abundant potential commercial value, these uses are not fully established and are mostly being phased out. Immune proteins and various peptide groups present in cattle blood are physiologically active substances that have recently emerged in the protein material market in recent years. They have an excellent amino acid composition and high nutritional value.

[0006] In this regard, it should be noted that unhydrolyzed animal blood, particularly porcine blood, lacks the biological activities of the hydrolysates, as these activities are carried out by the peptides generated during hydrolysis. Furthermore, various procedures exist for the production of peptides and amino acids through chemical hydrolysis of animal blood proteins, but this process is not sustainable because it involves the use of high concentrations of acids and bases. Moreover, their neutralization results in large quantities of the resulting salt, which must be removed. Additionally, a significant portion of the free amino acids are in the dextrorotatory form, which is not biologically active, and some amino acids, such as tryptophan, are degraded.

[0007] In view of the above, the state of the art shows a need for new approaches and strategies for the use of blood from slaughterhouses, and thus reduce the environmental problem it poses.

[0008] DESCRIPTION OF THE INVENTION

[0009] By conducting trials aimed at recycling pig blood from slaughterhouses, the inventors have observed that the combined administration of peptides (I) - (V), as a food additive, promotes the growth rate, increases swimming performance and allows the prevention and / or treatment of stress and behavioral disorders in the subjects.

[0010] Furthermore, the inventors have developed a method for obtaining a hydrolysate that involves enzymatic hydrolysis of blood from a Sus species, followed by ultrafiltration to obtain bioactive peptides with antioxidant, anti-inflammatory, hypoglycemic, and immunomodulatory activity. This method yields a hydrolysate that can be used in various food and pharmaceutical applications.

[0011] In the examples in this description, it is demonstrated, through the use of in vivo models, that the mixture of peptides and the hydrolysate obtained by the method defined in the claims significantly improves growth performance at a productive level, swimming performance, and stress tolerance capacity, favoring a decrease in aggressiveness (Example 6).

[0012] Peptide blend

[0013] In one aspect, the present invention relates to a mixture of peptides, hereinafter referred to as the “peptide mixture of the invention” or “peptide mixture of the invention”, comprising at least 5 peptides, wherein:

[0014] - a peptide (I) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1 (PDDFNPS);

[0015] - a peptide (II) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 2 (FPPKPKD);

[0016] - a peptide (III) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the GDL sequence;

[0017] - a peptide (IV) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4 (DNPDIPK); and

[0018] - a peptide (V) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 5 (GHLDDLPG).

[0019] As used herein, the term “sequence identity” or “identity” refers to the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences; that is, the proportion of identical nucleotides or amino acids between two nucleotide or polypeptide / protein sequences compared along their entire length. Depending on the number of shared residues between the aligned sequences, a different degree of identity, expressed as a percentage, will be obtained. The degree of identity between two amino acid or nucleotide sequences can be determined by conventional methods, for example, using standard sequence alignment algorithms known in the prior art, such as BLAST. BLAST programs, for example, BLASTN, BLASTX, and TBLASTN, are publicly available on the website of the National Center for Biotechnology Information (NCBI).The expert in the field understands that mutations in the nucleotide sequence of genes that lead to conservative amino acid substitutions in positions not critical for the functionality of the protein are evolutionarily neutral mutations that do not affect its overall structure or functionality.

[0020] In the context of the present invention, a “peptide mixture” or “peptide blend” is understood to mean a combination of two or more different peptides. Furthermore, the term “peptide mixture” or “peptide blend” refers to a combination of peptides and amino acids, whether free or in any bound form. Therefore, the peptide fraction of a composition is the sum of all peptides and amino acids, whether free or in any bound form, present in the composition. Additionally, the peptide and amino acid mixture may be present simultaneously in another matrix, such as an aqueous matrix, like a nutritional composition. In this latter case, the peptide mixture may be referred to as the peptide fraction of that matrix.

[0021] The peptides of the peptide mixture of the invention can be obtained by techniques widely known in the prior art. Examples of protein synthesis techniques include, but are not limited to, chemical or biological synthesis, genetic recombination, or expression of polynucleotides encoding the protein of the invention. The conditions necessary to carry out peptide synthesis are routine practice for a person skilled in the art.

[0022] In a preferred embodiment of the peptide mixture of the invention, said mixture comprises at least 5 peptides, wherein:

[0023] - the peptide (I) comprises SEQ ID NO: 1 ;

[0024] - the peptide (II) comprises SEQ ID NO: 2;

[0025] - the peptide (III) comprises the GDL sequence;

[0026] - the peptide (IV) comprises SEQ ID NO: 4; and

[0027] - the peptide (V) comprises SEQ ID NO: 5.

[0028] In the context of the present invention, a peptide is understood to be a molecule formed by the union of between 3 and 100 amino acids through peptide bonds. Thus, in another preferred embodiment of the peptide mixture of the invention, alone or in combination with one or more of the preceding preferred embodiments, the peptides have a length of 3 to 100 amino acids (including the ends). In a further preferred embodiment, the peptides (I) - (V) have a length of 3 to 20 amino acids (including the ends). In an even more preferred embodiment of the peptide mixture of the invention, the peptides (I) - (V) have a length of 3 to 13 amino acids (including the ends), even more preferably from 3 to 8 amino acids (including the ends).

[0029] In a further preferred embodiment of the peptide mixture of the invention, wherein:

[0030] - the peptide (I) comprises an amino acid sequence of a length of 7 amino acids, with a sequence identity of at least 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 1; - the peptide (II) comprises an amino acid sequence of a length of 7 amino acids, with a sequence identity of at least 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 2;

[0031] - the peptide (III) comprises an amino acid sequence of a length of 3 amino acids, having a sequence identity of at least 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the GDL sequence;

[0032] - the peptide (IV) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4; and

[0033] - the peptide (V) comprises an amino acid sequence of a length of 8 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 5.

[0034] In another preferred embodiment of the peptide mixture of the invention, said mixture comprises at least 5 peptides, wherein:

[0035] - the peptide (I) consists of SEQ ID NO: 1 ;

[0036] - the peptide (II) consists of SEQ ID NO: 2;

[0037] - the peptide (III) consists of the GDL sequence;

[0038] - the peptide (IV) consists of SEQ ID NO: 4; and

[0039] - the peptide (V) consists of SEQ ID NO: 5.

[0040] In another, even more preferred embodiment, the peptide mixture consists of peptide (I), peptide (II), peptide (III), peptide (IV), and peptide (V).

[0041] The peptide mixture of the invention may exhibit hypoglycemic activity. The term "hypoglycemia" (also known as hypoglycemia) refers to a state defined by an abnormally low blood glucose concentration, below 50-60 mg / 100 ml. The hypoglycemic activity of certain molecules is evaluated by their ability to inhibit enzymes, such as, but not limited to, neprisilin and dipeptidyl peptidase-4 (DPP4). Examples of determining whether a molecule or combination of molecules exhibits hypoglycemic activity are included in the "Materials and Methods" section of this description, specifically in the methodology for "Determination of Biological Activities of the Hydrolysate, Its Fractions, and Identified Peptides," as well as in Examples 2 and 3.In another preferred embodiment of the peptide mixture of the invention, alone or in combination with one or more of the preceding preferred embodiments, said mixture inhibits the enzyme neprilysin and / or the enzyme dipeptidyl peptidase-4 by at least 20%. A 20% inhibitory activity is the threshold at which the peptide mixture is considered to have significant enzyme-inhibiting activity. In a more preferred embodiment of the invention, the peptide mixture inhibits the enzyme neprilysin and / or the enzyme dipeptidyl peptidase-4 by at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%. In a most preferred embodiment, the peptide mixture inhibits the enzyme neprilysin and / or the enzyme dipeptidyl peptidase-4 by at least 80%. An 80% inhibitory activity means that the enzyme reaches a high inhibitory activity, with a threshold value of 20% being considered the one at which such inhibition begins to be significant.A higher percentage (%), indicates greater inhibition. In a more preferred embodiment, this mixture inhibits the enzyme neprilysin by 84.7% at a concentration of 1 mg / mL and / or inhibits the enzyme dipeptidyl peptidase-4 (DPP4) by 82.8% at a concentration of 5 mg / mL.

[0042] Methods or techniques for determining the percentage of enzyme inhibition are known in the prior art. In the present invention, selecting two minutes within the linear range of the enzyme kinetics (ARFU = T2 - Ti):

[0043] . . . . . , ARFU negative control - ARFU sample . > > % Inhibition = - - — - - : - x 100 ,

[0044] The percentage of enzyme inhibition can be calculated using the negative control ARFU.

[0045] In the case of the hydrolysate fractions obtained by RP-HPLC, the samples had to be diluted half with double-distilled water compared to the other tests.

[0046] The peptide mixture of the invention may further comprise other bioactive peptides. Thus, in another preferred embodiment of the peptide mixture of the invention, alone or in combination with one or more of the foregoing preferred embodiments, said peptide mixture further comprises one, two, three, four, five, six, seven, eight, or nine peptides selected from the list consisting of:

[0047] - a peptide (VI) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 6 (DNPDIPKLKPDP);

[0048] - a peptide (Vil) comprises an amino acid sequence with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 7 (EDEQKFWGK); - a peptide (VIII) comprises an amino acid sequence with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 8 (DNPDIPKLKPDPV);

[0049] - a peptide (IX) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 9 (GHLDDLPGA);

[0050] - a peptide (X) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 10 (HPDDFNPS);

[0051] - a peptide (XI) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 11 (HHPDDFNPS);

[0052] - a peptide (XII) comprises an amino acid sequence, having a sequence identity of at least 85% with SEQ ID NO: 12 (AHHPDDFNPS);

[0053] - a peptide (XII) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 13 (GHLDDLPGAL);

[0054] - a peptide (XIV) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 3 (DPENFRL); and any combination thereof

[0055] In another preferred embodiment of the peptide mixture of the invention, alone or in combination with each of the preceding preferred embodiments, said mixture further comprises one, two, three, four, five, six, seven, eight or nine peptides selected from the group consisting of:

[0056] - the peptide (VI) comprising SEQ ID NO: 6;

[0057] - the peptide (Vil) comprising SEQ ID NO: 7;

[0058] - the peptide (VIII) comprising SEQ ID NO: 8;

[0059] - the peptide (IX) comprising SEQ ID NO: 9;

[0060] - the peptide (X) comprising SEQ ID NO: 10;

[0061] - the peptide (XI) comprising SEQ ID NO: 11 ;

[0062] - the peptide (XII) comprising SEQ ID NO: 12;

[0063] - the peptide (XII) comprising SEQ ID NO: 13; and

[0064] - the peptide (XIV) comprising SEQ ID NO: 3. In another preferred embodiment of the peptide mixture of the invention, alone or in combination with each of the foregoing preferred embodiments, said mixture further comprises one, two, three, four, five, six, seven, eight or nine peptides selected from the group consisting of:

[0065] - the peptide (VI) comprises an amino acid sequence of a length of 12 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 6;

[0066] %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%

[0067] - the peptide (VIII) comprises an amino acid sequence of a length of 13 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 8;

[0068] - the peptide (IX) comprises an amino acid sequence of a length of 9 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 9;

[0069] - the peptide (X) comprises an amino acid sequence of a length of 8 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 10;

[0070] - the peptide (XI) comprises an amino acid sequence of a length of 9 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 11;

[0071] - the peptide (XII) comprises an amino acid sequence of a length of 10 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 12;

[0072] - the peptide (XII) comprises an amino acid sequence of a length of 10 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 13; and

[0073] - the peptide (XIV) comprises an amino acid sequence of a length of 7 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 3.

[0074] In another preferred embodiment of the peptide mixture of the invention, alone or in combination with each of the preceding preferred embodiments, said mixture further comprises one, two, three, four, five, six, seven, eight or nine peptides selected from the group consisting of:

[0075] - the peptide (VI) consisting of SEQ ID NO: 6;

[0076] - the peptide (Vil) consisting of SEQ ID NO: 7; - the peptide (VIII) consisting of SEQ ID NO: 8;

[0077] - the peptide (IX) consisting of SEQ ID NO: 9;

[0078] - the peptide (X) consisting of SEQ ID NO: 10;

[0079] - the peptide (XI) consisting of SEQ ID NO: 11 ;

[0080] - the peptide (XII) consisting of SEQ ID NO: 12;

[0081] - the peptide (XII) consisting of SEQ ID NO: 13; and

[0082] - the peptide (XIV) consisting of SEQ ID NO: 3.

[0083] In another preferred embodiment of the mixture of the invention, said mixture comprises:

[0084] - the peptide (I) comprising SEQ ID NO: 1 ;

[0085] - the peptide (II) comprising SEQ ID NO: 2;

[0086] - the peptide (III) comprising the GDL sequence;

[0087] - the peptide (IV) comprising SEQ ID NO: 4;

[0088] - the peptide (V) comprising SEQ ID NO: 5;

[0089] - the peptide (VI) comprising SEQ ID NO: 6;

[0090] - the peptide (Vil) comprising SEQ ID NO: 7;

[0091] - the peptide (VIII) comprising SEQ ID NO: 8;

[0092] - the peptide (IX) comprising SEQ ID NO: 9;

[0093] - the peptide (X) comprising SEQ ID NO: 10;

[0094] - the peptide (XI) comprising SEQ ID NO: 11 ;

[0095] - the peptide (XII) comprising SEQ ID NO: 12;

[0096] - the peptide (XII) comprising SEQ ID NO: 13; and

[0097] - the peptide (XIV) comprising SEQ ID NO: 3.

[0098] In a more preferred embodiment of the peptide mixture of the invention, said mixture comprises:

[0099] - the peptide (I) consisting of SEQ ID NO: 1 ;

[0100] - the peptide (II) consisting of SEQ ID NO: 2;

[0101] - the peptide (III) consisting of the GDL sequence;

[0102] - the peptide (IV) consisting of SEQ ID NO: 4;

[0103] - the peptide (V) consisting of SEQ ID NO: 5;

[0104] - the peptide (VI) consisting of SEQ ID NO: 6;

[0105] - the peptide (Vil) consisting of SEQ ID NO: 7;

[0106] - the peptide (VIII) consists of SEQ ID NO: 8;

[0107] - the peptide (IX) consisting of SEQ ID NO: 9;

[0108] - the peptide (X) consisting of SEQ ID NO: 10; - the peptide (XI) consisting of SEQ ID NO: 11;

[0109] - the peptide (XII) consisting of SEQ ID NO: 12;

[0110] - the peptide (XII) consisting of SEQ ID NO: 13; and

[0111] - the peptide (XIV) consisting of SEQ ID NO: 3.

[0112] In another even more preferred embodiment, the peptide mixture consists of peptide (I), peptide (II), peptide (III), peptide (IV), peptide (V), peptide (VI), peptide (Vil), peptide (VIII), peptide (IV), peptide (X), peptide (XI), peptide (XII), peptide (XIII) and peptide (XIV).

[0113] Method of the invention

[0114] In another aspect, the present invention relates to a method for obtaining a blood hydrolysate from a subject of the genus Sus, hereinafter referred to as the “method of the invention”, comprising the steps of: a) hydrolyzing the previously isolated blood sample comprising contacting the sample with an alcalase proteolytic enzyme, and with at least one exopeptidase proteolytic enzyme; and b) subjecting the hydrolysate resulting from step a) to an ultrafiltration process using a molecular exclusion membrane with a pore size equal to or less than 10 kDa.

[0115] In a first step [(step a)] the method of the invention comprises hydrolyzing the previously isolated blood sample comprising contacting the sample with:

[0116] - a proteolytic enzyme alcalase, and with

[0117] - at least one proteolytic enzyme exopeptidase.

[0118] Methods for obtaining and isolating blood samples from slaughterhouses are widely known in the state of the art and their application is routine practice for the expert in the field.

[0119] In the present invention, "subject of the genus Sus" means a member of the genus Sus. Sus is a genus of artiodactyl mammals in the family Suidae, native to Eurasia, which includes domestic pigs and wild boars. This genus belongs to the subfamily Suinae, within the family Suidae, along with other groups such as African warthogs. The subject from which the sample is obtained, to which the present invention refers, may belong to the species, for example, without limitation, Palawan bearded boar (Sus ahoenobarbus), bearded boar (Sus barbatus), Visayan boar (Sus cebifrons), Cebeles warthog (Sus celebensis), Mindoro warthog (Sus oliven), Philippine warthog (Sus philippensis), dwarf warthog (Sus salvanius), domestic pig (Sus scrofa) and Javan warthog (Sus verrucosus).However, in a preferred embodiment of the method of the invention, the subject from which the sample is obtained is a member of the species Sus scrofa. More preferably, it is a member of the subspecies Sus scrofa domesticus.

[0120] Once the blood sample is isolated, it is hydrolyzed using the proteolytic enzyme alcalase and at least one proteolytic enzyme exopeptidase.

[0121] As used in the present invention, the enzyme alcalase is a serine endopeptidase obtained from Bacillus licheniformis, a typical example of the S8 peptidase family, and hydrolyzes proteins with broad selectivity and specificity for peptide bonds. Endopeptidases are a family of proteolytic enzymes that hydrolyze peptide bonds located within a protein molecule, resulting in two fragments of approximately the same or different sizes. Examples of endopeptidases include, but are not limited to, trypsin, chymotrypsin, elastase, thermolysin, pepsin, and alcalase. Furthermore, alcalase also exhibits exopeptidase-type activity, preferentially cleaving large uncharged amino acids at the amino-terminal Pi end, particularly Glu, Met, Leu, Tyr, Lys, and Gln. It also hydrolyzes hydrophobic amino acids at the P2' and P3' positions of the carboxy-terminal end.In another preferred embodiment of the method of the invention, alone or in combination with any of the preceding preferred embodiments, the alcalase enzyme is formulated in the commercial product ALCALASE®. ALCALASE® is a solution whose main component is the alcalase enzyme, which efficiently hydrolyzes proteins to produce peptides or amino acids. This product is marketed by Novonesis (https: / / www.novonesis.com / ) and can be purchased by those skilled in the art through standard sales channels.

[0122] On the other hand, exopeptidases are a family of proteolytic enzymes whose function is to release amino acids or small peptides from the amino or carboxyl ends of a protein or polypeptide. For example, aminopeptidase B releases arginine from the amino terminus. Thus, exopeptidases are divided into two groups: aminopeptidases, which act on the end of the polypeptide chain at the amino terminus, and carboxypeptidases, which act on the end containing the carboxyl group. Examples of exopeptidases include, but are not limited to, aminopeptidase B, leucine aminopeptidase, carboxypeptidase A, and carboxypeptidase B. In another preferred embodiment of the method of the invention, alone or in combination with any of the preceding preferred embodiments, at least one exopeptidase enzyme is formulated in the commercial product PROTANA PRIME®. The PROTANA PRIME® product is a unique blend of exopeptidases that releases free amino acids from proteins.This composition is marketed by Novonesis (https: / / www.novonesis.com / ) and can be purchased by the expert in the field through the usual sales channels.

[0123] In another preferred embodiment of the method of the invention, alone or in combination with any and all of the foregoing preferred embodiments, the alcalase enzyme is formulated in the commercial product ALCALASE® and the at least one exopeptidase enzyme is formulated in the commercial product PROTANA PRIME®.

[0124] In the context of the present invention, the expression “comprises contacting the sample with an alcalase proteolytic enzyme and at least one exopeptidase proteolytic enzyme” implies the condition that other proteolytic enzymes in addition to those mentioned above may be used.

[0125] In the method of the invention, the application of both proteolytic enzymes can be carried out separately, simultaneously, sequentially, or mixed in a single solution. Therefore, in a preferred embodiment of the method of the invention, alone or in combination with one or more of the foregoing preferred embodiments, the sample is contacted sequentially with an alcalase enzyme and at least one exopeptidase.

[0126] In another preferred embodiment of the method of the invention, alone or in combination with one or more of the above preferred embodiments, step a) consists of contacting the sample with an alcalase enzyme and at least one exopeptidase enzyme.

[0127] In the context of the present invention, the expression “consists of contacting the sample with an alcalase enzyme and at least one exopeptidase enzyme” implies that the enzymatic hydrolysis step is carried out only with the proteolytic enzyme types mentioned above, without considering the use of other additional enzymes.

[0128] Protein hydrolysis is typically performed in a reactor, with controlled agitation, pH, temperature, and process time. The blood sample is dissolved or resuspended in water until the pH and temperature stabilize; then, the proteolytic enzyme alcalase and at least one proteolytic enzyme exopeptidase are added to the blood sample, initiating enzymatic hydrolysis.

[0129] In a preferred embodiment of the method of the invention, alone or in combination with one or more of the preceding preferred embodiments, the enzymatic hydrolysis reaction with the alcalase enzyme carried out in step a) is performed at a pH of 6 to 7 and / or for 1 to 3 hours and / or at a temperature between 55 and 70°C. In a more preferred embodiment of the method of the invention, the enzymatic hydrolysis reaction with the alcalase enzyme carried out in step a) is performed at a pH of 6.5, for 2 hours, at a temperature of 65°C. All ranges mentioned herein are inclusive of the extremes.

[0130] In another preferred embodiment of the method of the invention, alone or in combination with one or more of the preceding preferred embodiments, the enzymatic hydrolysis reaction with the at least one exopeptidase enzyme carried out in step a) is performed at a pH of 6 to 7 and / or for a time of 8 to 15 hours and / or at a temperature between 50 and 60°C. In a more preferred embodiment of the method of the invention, the enzymatic hydrolysis reaction with the exopeptidase enzyme carried out in step a) is performed at pH 6.5, for 12 hours, at 55°C. All ranges mentioned herein are inclusive of the extremes.

[0131] To complete the protein hydrolysis, the enzyme can be inactivated by heat, by lowering the pH, or by a combination of both. It can also be removed from the medium by filtration, with the protein then precipitated. In another preferred embodiment of the method of the invention, the method further comprises a step a') following step a), of thermal inactivation of the alcalase and exopeptidase enzymes, which involves subjecting the hydrolysate resulting from step a) to a temperature of 70°C or higher and / or for a period of 2 to 60 minutes. More preferably, the hydrolysate resulting from step a) is subjected to a temperature of 95°C for 10 minutes. All ranges mentioned herein are inclusive of the extremes.

[0132] The method of the invention may comprise a blood sample treatment step using ultrasound prior to enzymatic hydrolysis. Thus, in another preferred embodiment of the method of the invention, alone or in combination with one or more of the preceding preferred embodiments, the method further comprises a blood sample treatment step using ultrasound. Preferably, the ultrasound treatment step is performed for a period of 10 minutes to 4 hours and / or at a frequency between 10 and 100 kHz. More preferably, the ultrasound treatment step is performed at 35 kHz for 1 hour. All ranges mentioned herein are inclusive.

[0133] After step a), a protein hydrolysate (also called peptide extract) is obtained. This hydrolysate is subjected to a second step [(step b)] of separation of the peptides contained therein by ultrafiltration, using a molecular exclusion membrane with a pore size equal to or less than 10 kDa. In another preferred embodiment of the method of the invention, alone or in combination with one or more of the preceding preferred embodiments, step b) is carried out using a molecular exclusion membrane with a pore size of 3 to 10 kDa. More preferably, a molecular exclusion membrane with a pore size of 3 to 5 kDa. Even more preferably, a molecular exclusion membrane with a pore size of 3, 4, or 5 kDa. All ranges mentioned herein are inclusive of the extremes.

[0134] Ultrafiltration is a separation process using a semipermeable membrane that employs pressure or force to force particles, in this case peptides, smaller than the membrane's pore size through it. Larger particles are retained by the membrane.

[0135] Finally, the hydrolysate can be subjected to an atomization process to obtain a fine powder that retains all its beneficial properties, as it is stable for several months at room temperature; or, it can be subjected to a freeze-drying process, which consists of freezing and subsequently dehydrating the product. Thus, in another preferred embodiment of the method of the invention, alone or in combination with any of the preceding preferred embodiments, the hydrolysate is subjected to an atomization or freeze-drying process. The atomization and / or freeze-drying conditions can be determined by routine practice by someone skilled in the art. Atomization is carried out with an atomizer, and freeze-drying with a freeze dryer. In a more preferred embodiment, atomization is carried out with a drying gas flow rate of between 20 and 40 m³ / s. 3and / or an injection temperature of between 115 and 140°C and / or a spray gas flow rate of between 800 and 1200 L / min and / or a pump flow rate of between 1 and 15 mL / min. In a more preferred embodiment, atomization is performed at 30 m 3 / h drying gas flow rate, 120°C injection temperature, 900 L / h spray gas flow rate, and 7.5 mL / min pump flow rate. Under these conditions, the outlet temperature is between 70 and 80°C and the sample collector temperature is between 40 and 50°C. All ranges mentioned in this description include the extremes.

[0136] Hydrolysate obtained by the method of the invention

[0137] In another aspect, the present invention relates to a blood hydrolysate obtainable by the method of the invention, hereinafter referred to as “the hydrolysate obtainable by the method of the invention”.

[0138] Hydrolyzed product of the invention

[0139] In another aspect, the present invention relates to a blood hydrolysate obtainable by the method of the invention, hereinafter referred to as the “hydrolysate of the invention”, wherein said hydrolysate comprises at least 5 peptides, wherein:

[0140] - a peptide (I) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 1;

[0141] - a peptide (II) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 2;

[0142] - a peptide (III) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the GDL sequence;

[0143] - a peptide (IV) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4; and

[0144] - a peptide (V) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 5.

[0145] In another preferred embodiment of this aspect, the hydrolysate of the invention comprises at least 5 peptides, wherein:

[0146] - the peptide (I) comprises SEQ ID NO: 1 ;

[0147] - the peptide (II) comprises SEQ ID NO: 2;

[0148] - peptide (III) comprises the GDL sequence; - peptide (IV) comprises SEQ ID NO: 4; and

[0149] - the peptide (V) comprises SEQ ID NO: 5.

[0150] In the context of the present invention, a peptide is understood to be a molecule formed by the union of between 3 and 100 amino acids through peptide bonds. In another preferred embodiment of the peptide mixture of the invention, alone or in combination with one or more of the preceding preferred embodiments, the peptides are from 3 to 100 amino acids in length (including ends). In a preferred embodiment, the peptides (I) - (V) are from 3 to 20 amino acids in length (including ends). In a further preferred embodiment of the peptide mixture of the invention, the peptides (I) - (V) are from 3 to 13 amino acids in length (including ends), even more preferably from 3 to 8 amino acids (including ends). In a further preferred embodiment of the hydrolysate of the invention:

[0151] - the peptide (I) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1;

[0152] - the peptide (II) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 2;

[0153] - the peptide (III) comprises an amino acid sequence of a length of 3 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the GDL sequence;

[0154] - the peptide (IV) comprises an amino acid sequence of a length of 7 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4: and

[0155] - the peptide (V) comprises an amino acid sequence of a length of 8 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 5.

[0156] In a further preferred embodiment, the hydrolysate of the invention comprises at least 5 peptides, wherein: - peptide (I) consists of SEQ ID NO: 1;

[0157] - the peptide (II) consists of SEQ ID NO: 2;

[0158] - the peptide (III) consists of the GDL sequence;

[0159] - the peptide (IV) consists of SEQ ID NO: 4; and

[0160] - the peptide (V) consists of SEQ ID NO: 5.

[0161] In another, even more preferred embodiment, the peptide mixture consists of peptide (I), peptide (II), peptide (III), peptide (IV), and peptide (V).

[0162] Similar to the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention and the hydrolysate of the invention may exhibit hypoglycemic effects. Thus, in another preferred embodiment of the hydrolysate of the invention, alone or in combination with one or more of the preceding preferred embodiments, said hydrolysate inhibits the enzyme nephlysin and / or the enzyme dipeptidyl peptidase-4 by at least 20%. A 20% inhibitory activity is the threshold at which the hydrolysate is considered to begin having significant enzyme-inhibiting activity. In a more preferred embodiment of the invention, the hydrolysate inhibits the enzyme nephlysin and / or the enzyme dipeptidyl peptidase-4 by at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%. In a more preferred embodiment, the hydrolysate inhibits the enzyme nephlisin and / or the enzyme dipeptidyl peptidase-4 by at least 80%.An 80% inhibitory activity means that the enzyme reaches a high inhibitory level, with a threshold value of 20% being considered the level at which such inhibition begins to be significant. A higher percentage (%) indicates greater inhibition. In a more preferred embodiment, this hydrolysate inhibits the enzyme nephlisin by 84.7% at a concentration of 1 mg / mL and / or inhibits the enzyme dipeptidyl peptidase-4 (DPP4) by 82.8% at a concentration of 5 mg / mL.

[0163] The hydrolysate of the invention may further comprise other components, such as free amino acids, especially essential amino acids, or one or more bioactive peptides. Examples of essential amino acids include, but are not limited to, taurine, asparagine, glutamine, p-alanine, and gamma-aminobutyric acid. Thus, in another preferred embodiment of the hydrolysate of the invention, alone or in combination with any of the preceding preferred embodiments, said hydrolysate further comprises one, two, three, four, five, six, seven, eight, or nine peptides selected from the group consisting of:

[0164] - a peptide (VI) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 6;

[0165] - a peptide (Vil) comprises an amino acid sequence with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 7;

[0166] - a peptide (VIII) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 8;

[0167] - a peptide (IX) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 9;

[0168] - a peptide (X) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 10;

[0169] - a peptide (XI) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 11;

[0170] - a peptide (XII) comprises an amino acid sequence, having a sequence identity of at least 85% with SEQ ID NO: 12;

[0171] - a peptide (XII) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 13; and

[0172] - a peptide (XIV) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 3.

[0173] In another preferred embodiment of the hydrolysate of the invention, alone or in combination with one or more of the foregoing preferred embodiments, said hydrolysate further comprises one, two, three, four, five, six, seven, eight or nine peptides selected from the group consisting of:

[0174] - the peptide (VI) comprising SEQ ID NO: 6;

[0175] - the peptide (Vil) comprising SEQ ID NO: 7;

[0176] - the peptide (VIII) comprising SEQ ID NO: 8;

[0177] - the peptide (IX) comprising SEQ ID NO: 9;

[0178] - the peptide (X) comprising SEQ ID NO: 10;

[0179] - the peptide (XI) comprising SEQ ID NO: 11; - the peptide (XII) comprising SEQ ID NO: 12;

[0180] - the peptide (XII) comprising SEQ ID NO: 13; and

[0181] - the peptide (XIV) comprising SEQ ID NO: 3.

[0182] In a more preferred embodiment of the hydrolysate of the invention, wherein said hydrolysate further comprises one, two, three, four, five, six, seven, eight or nine peptides selected from the group consisting of:

[0183] - the peptide (VI) comprises an amino acid sequence of a length of 12 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 6;

[0184] - the peptide (Vil) comprises an amino acid sequence of a length of 9 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 7;

[0185] - the peptide (VIII) comprises an amino acid sequence of a length of 13 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 8;

[0186] - the peptide (IX) comprises an amino acid sequence of a length of 9 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 9;

[0187] - the peptide (X) comprises an amino acid sequence of a length of 8 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 10;

[0188] - the peptide (XI) comprises an amino acid sequence of a length of 9 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 11;

[0189] - the peptide (XII) comprises an amino acid sequence of a length of 10 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 12;

[0190] - the peptide (XII) comprises an amino acid sequence of a length of 10 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 13; and

[0191] - the peptide (XIV) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 3.

[0192] In another preferred embodiment of the hydrolysate of the invention, alone or in combination with one or more of the foregoing preferred embodiments, said hydrolysate further comprises one, two, three, four, five, six, seven, eight or nine peptides selected from the group consisting of:

[0193] - the peptide (VI) consisting of SEQ ID NO: 6;

[0194] - the peptide (Vil) consisting of SEQ ID NO: 7;

[0195] - the peptide (VIII) consisting of SEQ ID NO: 8;

[0196] - the peptide (IX) consisting of SEQ ID NO: 9;

[0197] - the peptide (X) consisting of SEQ ID NO: 10;

[0198] - the peptide (XI) consisting of SEQ ID NO: 11 ;

[0199] - the peptide (XII) consisting of SEQ ID NO: 12;

[0200] - the peptide (XII) consisting of SEQ ID NO: 13; and

[0201] - the peptide (XIV) consisting of SEQ ID NO: 3.

[0202] In another preferred embodiment of the hydrolysate of the invention, said hydrolysate comprises:

[0203] - the peptide (I) comprising SEQ ID NO: 1 ;

[0204] - the peptide (II) comprising SEQ ID NO: 2;

[0205] - the peptide (III) comprising the GDL sequence;

[0206] - the peptide (IV) comprising SEQ ID NO: 4;

[0207] - the peptide (V) comprising SEQ ID NO: 5;

[0208] - the peptide (VI) comprising SEQ ID NO: 6;

[0209] - the peptide (Vil) comprising SEQ ID NO: 7;

[0210] - the peptide (VIII) comprising SEQ ID NO: 8;

[0211] - the peptide (IX) comprising SEQ ID NO: 9;

[0212] - the peptide (X) comprising SEQ ID NO: 10;

[0213] - the peptide (XI) comprising SEQ ID NO: 11 ;

[0214] - the peptide (XII) comprising SEQ ID NO: 12; - the peptide (XII) comprising SEQ ID NO: 13; and

[0215] - the peptide (XIV) comprising SEQ ID NO: 3.

[0216] In a more preferred embodiment of the hydrolysate of the invention, the hydrolysate comprises:

[0217] - the peptide (I) consisting of SEQ ID NO: 1 ;

[0218] - the peptide (II) consisting of SEQ ID NO: 2;

[0219] - the peptide (III) consisting of the GDL sequence;

[0220] - the peptide (IV) consisting of SEQ ID NO: 4;

[0221] - the peptide (V) consisting of SEQ ID NO: 5;

[0222] - the peptide (VI) consisting of SEQ ID NO: 6;

[0223] - the peptide (Vil) consisting of SEQ ID NO: 7;

[0224] - the peptide (VIII) consisting of SEQ ID NO: 8;

[0225] - the peptide (IX) consisting of SEQ ID NO: 9;

[0226] - the peptide (X) consisting of SEQ ID NO: 10;

[0227] - the peptide (XI) consisting of SEQ ID NO: 11 ;

[0228] - the peptide (XII) consisting of SEQ ID NO: 12;

[0229] - the peptide (XII) consisting of SEQ ID NO: 13; and

[0230] - the peptide (XIV) comprising SEQ ID NO: 3.

[0231] In a further preferred embodiment of the hydrolysate of the invention, the hydrolysate consists of peptide (I), peptide (II), peptide (III), peptide (IV), peptide (V), peptide (VI), peptide (Vil), peptide (VIII), peptide (IV), peptide (X), peptide (XI), peptide (XII), peptide (XIII) and peptide (XIV).

[0232] In another preferred embodiment of the hydrolysate of the invention, alone or in combination with one or more of the preceding preferred embodiments, wherein the peptides of the hydrolysate have a molecular size equal to or less than 10 kDa. More preferably, the peptides of the hydrolysate have a molecular size of between 3 and 10 kDa. Even more preferably, the peptides of the hydrolysate have a molecular size of between 3 and 5 kDa. Even more preferably, a molecular exclusion membrane with a pore size of 3, 4, or 5 kDa. All ranges mentioned herein are inclusive of the extremes.

[0233] In another preferred embodiment of the hydrolysate of the invention, alone or in combination with one or more of the preceding preferred embodiments, the hydrolysate is spray-dried or lyophilized. The peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, and the hydrolysate of the invention may form part of a composition.

[0234] Therefore, another aspect of the present invention relates to a composition, hereinafter referred to as the “composition of the invention”, comprising the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, and the hydrolysate of the invention.

[0235] In a preferred embodiment of the composition of the invention, said composition is a nutritional composition. In another preferred embodiment of the composition of the invention, said composition is a pharmaceutical composition.

[0236] The composition of the invention may be a nutritional composition. In the context of the present invention, the terms “nutritional composition” and “food product” are considered equivalent and may be used interchangeably throughout this description.

[0237] The nutritional composition of the invention can be for animal use / consumption, including a mammal and therefore humans, preferably members of the genus Sus, and refers to any product whose intake beneficially affects the subject by positively favoring its growth, improving its swimming performance and its state of health associated with stress and behavioral disorders in the sense indicated in the present invention.

[0238] In a further preferred embodiment of the nutritional composition of the invention, said nutritional composition is a food or a supplement. Examples of food include, but are not limited to, vegetable products, meat products, snacks, beverages, cereals, bakery products, cookies, dairy products, milk, fermented milk (such as yogurt or cheese), ice cream, butter, margarine, etc. In a preferred embodiment of the food product of the invention, the food is a feed, a beverage (e.g., smoothies, juices, sports nutrition drinks, etc.), a dairy product (e.g., milk, yogurt, cheese, kefir, etc.), or a confectionery product.

[0239] The term “supplement,” in the context of the present invention, refers to products or preparations intended to supplement a person’s normal diet by providing nutrients or other substances with a physiological and / or nutritional effect. Examples of supplements include, but are not limited to, a dietary supplement, a nutritional supplement, a food supplement, etc.

[0240] The composition of the invention may be a pharmaceutical composition. The pharmaceutical composition of the invention may be administered to an animal, including a mammal and therefore to a human, preferably a member of the genus Sus, and refers to any composition whose administration beneficially affects the subject, positively promoting its growth, improving its swimming performance and its health status associated with stress and behavioral disorders in the sense indicated in the present invention.

[0241] In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle and / or excipient.

[0242] The term "excipient" refers to a substance that aids in the absorption of any of the components of the composition, that is, the compositions comprising the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, and the hydrolysate of the invention, or stabilizes said components and / or aids in the preparation of the pharmaceutical composition by giving it consistency or providing flavors that make it more palatable. Thus, excipients could have the function of holding the components together (for example, starches, sugars, or celluloses), sweetening, providing color, protecting the active ingredient (for example, to isolate it from air and / or moisture), filling a tablet, capsule, or any other form of presentation, disintegrating to facilitate the dissolution of the components, etc., without excluding other types of excipients not mentioned in this paragraph.Therefore, the term "excipient" is defined as any substance included in pharmaceutical formulations that is added to the active ingredients or their combinations to facilitate their preparation and stability, modify their organoleptic properties, or determine the physicochemical properties of the pharmaceutical composition and its bioavailability. A "pharmaceutically acceptable" excipient must allow the activity of the compounds in the pharmaceutical composition; that is, it must be compatible with the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, and the hydrolysate of the invention.

[0243] The "galenic form" or "pharmaceutical form" is the arrangement in which the active ingredients and excipients are combined to form a composition or a drug. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

[0244] The "vehicle" or "carrier" is preferably an inert substance, and like the excipient, its purpose is to facilitate the incorporation of other compounds, allow for better dosage and administration, and / or give consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance used in the drug to dilute any of the components of the pharmaceutical composition of the present invention to a specific volume or weight; or, even without diluting said components, it is capable of allowing for better dosage and administration and / or giving consistency and shape to the drug. When the formulation is liquid, the pharmaceutically acceptable vehicle is the diluent.

[0245] Furthermore, the excipient and vehicle must be pharmacologically acceptable, meaning that they are permitted and have been evaluated to ensure they do not cause harm to the recipient. In each case, the presentation of the pharmaceutical composition will be adapted to the type of administration used. Therefore, the composition may be presented as a solution or in any other clinically permitted form of administration and in a therapeutically effective quantity. The pharmaceutical composition of the invention may be formulated in solid (tablets, powders, microgranules (pellets), caplets, pills, capsules, granules, patches, etc.), semi-solid (gels), or liquid (solutions, suspensions, emulsions, syrups, drops, drinkable vials, etc.) forms. In a preferred embodiment of the pharmaceutical composition of the invention, the composition is formulated for oral administration.The form adapted for oral administration refers to a physical state that allows for oral administration.

[0246] In another aspect, the present invention relates to a food additive, hereinafter referred to as the "additive of the invention", comprising the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, and the hydrolysate of the invention.

[0247] In another aspect, the present invention relates to a feed, hereinafter referred to as “feed of the invention”, comprising the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, or the food additive of the invention.

[0248] In another aspect, the present invention relates to a food, hereinafter referred to as the “food of the invention”, comprising the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, or the feed of the invention.

[0249] The food additive of the present invention can be used to prepare food by uniformly mixing the ingredients and then extruding or granulating them. Alternatively, the additive of the present invention can be added by spraying it onto a commercial food product, which would then dry, retaining the additive within the food. These methods are well known to those skilled in the art.

[0250] In a preferred embodiment of the feed or food of the invention, said feed or food is characterized in that the food additive is incorporated into the feed in extruded form.

[0251] In another preferred embodiment of the feed or food of the invention, said feed or food is characterized in that the food additive is incorporated into the feed in granulated, microencapsulated or spray form.

[0252] In another preferred embodiment of the feed of the invention, said feed is intended for feeding pet and / or farm and / or livestock animals. In a more preferred embodiment, the animal is a mammal. In a further preferred embodiment, the animal is a bird. In a further preferred embodiment, the animal is an aquatic animal, more preferably a fish or a mollusk, even more preferably a fish. In a still more preferred embodiment, the fish is a salmon, trout, tilapia, carp, sea bass, gilthead seabream, amberjack, turbot, sole, sturgeon, or eel. Most preferably, the fish is a gilthead seabream.

[0253] The peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate, the composition, the food additive, the feed, and / or the food of the invention possess improved tertiary and biological properties compared to the starting material, making them applicable to various industrial fields. Therefore, in another aspect, the invention relates to the use of the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the food additive of the invention, and the feed of the invention for the manufacture of a food product.

[0254] The peptide mixture, the hydrolysate obtainable by the method of the invention, and the hydrolysate of the invention also possess bioactive properties such as, but not limited to, antioxidant, hypoglycemic, anti-inflammatory, and / or immunomodulatory activity, making them useful for the production of functional foods or as an additive thereto. Therefore, another preferred embodiment of this aspect of the invention relates to the use of the peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, or the feed for the production of a functional food. A “functional food” is understood to be any food that provides health benefits for humans or any other living being beyond basic nutrition, such as improving health by reducing the risk of disease.These functional foods comprising the hydrolysate of the invention, or “functional foods of the invention,” could be, but are not limited to, immunomodulators, antihypertensives (preferably angiotensin-converting enzyme inhibitors), cholesterol-lowering agents, antioxidants, or hypoglycemic agents. In a more preferred embodiment, the functional food of the invention is for the prevention or treatment of stress and / or behavioral disorders, preferably stress-related aggressive behaviors.

[0255] Due to these bioactive properties, another aspect of the invention relates to the use of the peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention for the preparation of a medicament.In another aspect, the invention relates to the peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention for use as a medicament, preferably a veterinary medicament, or alternatively, to the use of the peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention for the manufacture of a medicament, preferably a veterinary medicament. The term “medication” refers to a substance used for the prevention, relief, treatment, or cure of diseases, syndromes, or clinical conditions in animals, including humans.In the context of the present invention, it refers to the peptide mixture, the hydrolysate obtainable by the method of the invention and the hydrolysate, composition, food additive, feed or food of the invention in a therapeutically effective amount.

[0256] In the sense used herein, the term "therapeutically effective amount" refers to the amount of the peptide mixture, hydrolysate obtainable by the method of the invention, and the hydrolysate, composition, feed additive, feed, or food of the invention that produces the desired effect. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, or tolerance of the subject.

[0257] The medicament of the invention can be used alone or in combination with other medicaments or compositions to promote or improve swimming performance in a subject, and to treat and / or prevent stress and / or stress-related aggressive behaviors in a subject. The medicament of the present invention can be used in conjunction with other active ingredients or therapies as a combination therapy. The other active ingredients may be part of the same composition or may be provided by means of a separate composition, and may be administered simultaneously or sequentially.

[0258] In another aspect, the invention relates to the use of the peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, or the food additive of the invention, as animal feed. In a preferred embodiment, the animal is a pet and / or farm and / or crop animal. In a more preferred embodiment, the animal is a mammal. In a further preferred embodiment, the animal is a bird. In a more preferred embodiment, the animal is an aquatic animal, more preferably a fish or a mollusc, and even more preferably a fish. In a still more preferred embodiment, the fish is a salmon, trout, tilapia, carp, sea bass, gilthead seabream, amberjack, turbot, sole, sturgeon, or eel. Most preferably, the fish is a gilthead seabream. The term “aquatic animal” refers to an animal that lives in water for most or all of its life.Aquatic animals can breathe air or extract oxygen from the water through specialized organs called gills, or directly through their skin. It is important to emphasize that when we speak of aquatic animals, we are referring to a group of organisms that require water as their habitat. Some are unable to breathe outside of water, while others can, but still need the aquatic environment to survive. Furthermore, aquatic animals can live in saltwater or freshwater, and only a few species are adapted to move from one ecosystem to another without difficulty. In the present invention, "aquatic animal" refers to a fish or a mollusk. Examples of fish include, but are not limited to: salmon, trout, tilapia, carp, sea bass, gilthead seabream, amberjack, turbot, sole, sturgeon, eel, etc. Examples of mollusks include, but are not limited to: octopus, clam, squid, etc.

[0259] In the following examples, when fermented pig blood is added to feed and then administered to farmed fish, various growth-promoting effects can be observed, such as an increase in total length and weight. These effects can directly lead to economic benefits for fish farmers.

[0260] Thus, in another aspect, the invention relates to the use of the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention, to promote the growth of a subject.

[0261] In other words, in another aspect, the invention relates to the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention, for use in a method for promoting the growth of a subject.

[0262] In other words, another aspect of the present invention relates to a method for promoting the growth of a subject, comprising administering the peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention. The term “subject” or “individual” refers to a human being or a non-human animal, preferably terrestrial, aerial, and / or aquatic animals. The subject to which the present invention refers may be a pet and / or farm and / or crop animal, for example, without limitation, a ruminant such as a lamb, sheep, cow, goat, calf, pig, or horse; poultry such as a turkey, duck, quail, goose, pigeon, chicken, hen, or rooster; fish such as salmon, trout, tilapia, carp, bass, gilthead seabream, amberjack, turbot, sole, sturgeon, and eel; as well as mollusks such as the octopus.

[0263] In a preferred embodiment of the use of the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention to promote the growth of a subject, wherein the subject is a pet and / or farm and / or crop animal. In a more preferred embodiment, wherein the terrestrial animal is a mammal. In a more preferred embodiment, wherein the aerial animal is a bird. In a more preferred embodiment, wherein the animal is an aquatic animal, more preferably a fish or a mollusk, even more preferably a fish. In a still more preferred embodiment, wherein the fish is a salmon, a trout, a tilapia, a carp, a sea bass, a gilthead seabream, amberjack, a turbot, a sole, a sturgeon, or an eel. Most preferably, wherein the fish is a gilthead seabream.

[0264] In another aspect, the invention relates to the use of the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention, to improve the physical performance of a subject.

[0265] In other words, in another aspect, the present invention relates to a method for increasing the physical performance of a subject, comprising administering to the subject the peptide mixture, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention.

[0266] In a preferred embodiment, where physical performance is swimming performance. In a more preferred embodiment, where physical performance is swimming performance and the subject is an aquatic animal.

[0267] In a preferred embodiment of the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention, alone or in combination with any one of the preceding preferred embodiments, for improving the physical performance of a subject, wherein the subject is a pet and / or farm and / or crop animal. In a more preferred embodiment, the animal is a mammal. In a further preferred embodiment, the animal is a bird. In a more preferred embodiment, the animal is an aquatic animal, more preferably a fish or a mollusc, even more preferably a fish. In a still more preferred embodiment, wherein the fish is a salmon, trout, tilapia, carp, sea bass, gilthead seabream, amberjack, turbot, sole, sturgeon, or eel. Most preferably, wherein the fish is a gilthead seabream.

[0268] In the context of the present invention, the expression “improving physical performance” refers to promoting, enhancing, stimulating, or inducing the capacities that allow any individual to perform physical activities efficiently, achieving the best results with less effort. Specifically, “improving swimming performance” refers to promoting, enhancing, stimulating, or inducing swimming performance in an individual; that is, promoting, enhancing, stimulating, or inducing the set of biological elements and processes within the individual (oxygen consumption, metabolic rate, behavior, etc.) that allow them to maintain a stable performance capacity.

[0269] In another aspect, the invention relates to the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention, for use as a veterinary medicinal product, preferably in the prevention and / or treatment of stress and / or behavioral disorders in a subject, preferably stress-related aggressive behaviors.

[0270] In another aspect, the invention relates to the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention, for use as a veterinary medicinal product, in the prevention and / or treatment of stress and / or behavioral disorders in a subject, preferably stress-related aggressive behaviors.

[0271] In another aspect, the invention relates to a method of treating stress and / or behavioral disorders, preferably stress-related aggressive behaviors, comprising administering the peptide mixture, the hydrolysate obtainable by the method of the invention, or the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention.

[0272] In another aspect, the invention relates to the use of the peptide mixture, the hydrolysate obtainable by the method of the invention, or the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention for the manufacture of a veterinary drug.

[0273] In another aspect, the invention relates to the use of the peptide mixture, the hydrolysate obtainable by the method of the invention, or the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention for the prevention and / or treatment of stress and / or behavioral disorders in a subject, preferably stress-related aggressive behaviors.

[0274] The term “treatment” or “treat” as understood in the present invention refers to combating the effects caused as a consequence of the disease or pathological condition of interest in a subject, which includes:

[0275] (i) inhibit the disease or pathological condition, that is, stop its development;

[0276] (i) alleviate the disease or pathological condition, i.e., cause the regression of the disease or pathological condition or its symptoms;

[0277] (iii) stabilize the disease or pathological condition.

[0278] The term “prevention” or “preventing” an infection as understood in the present invention, consists of avoiding the appearance of the disease or clinical condition, that is, preventing the disease or pathological condition from occurring in a subject, in particular, when said subject has a predisposition for the pathological condition, but has not yet been diagnosed with it.

[0279] In a preferred embodiment of the peptide mixture of the invention, the hydrolysate obtainable by the method of the invention, the hydrolysate of the invention, the composition of the invention, the food additive of the invention, the feed of the invention, or the food of the invention, alone or in combination with any one of the foregoing preferred embodiments, for use as a veterinary medicinal product, preferably in the prevention and / or treatment of stress and / or behavioral disorders in a subject, preferably stress-related aggressive behaviors, wherein the subject is a pet and / or farm and / or crop animal. In a more preferred embodiment, the animal is a mammal. In a further preferred embodiment, the animal is a bird. In a most preferred embodiment, the animal is an aquatic animal, more preferably a fish or a mollusc, even more preferably a fish.In an even more preferred embodiment, where the fish is a salmon, trout, tilapia, carp, sea bass, gilthead bream, amberjack, turbot, sole, sturgeon, or eel. Most preferably, where the fish is a gilthead bream.

[0280] DESCRIPTION OF THE FIGURES

[0281] Figure 1. Molecular size distribution of peptides present in pork hydrolysate prior to filtration by <10 kDa

[0282] Figure 2. Profile obtained by SDS-PAGE of the blood hydrolysate filtered through 30 kDa (APP<30) and 10 kDa (APP<10) membrane filters.

[0283] Figure 3. Elution profile of the atomized blood hydrolysate obtained by reversed-phase liquid chromatography (RP-HPLC). The injection volume was 20 pL at a concentration of 40 mg / mL.

[0284] Figure 4. Total ion chromatogram of fraction 1 (F1) collected by RP-HPLC and separated by LC-MS / MS for peptide identification.

[0285] Figure 5. Biological activities of the 5 synthesized peptides, at a concentration of 1 mM, previously identified in fraction 1 (F1) by LC-MS / MS, as well as the synergistic effect of the 5 peptides at 1 mM. Figure 6. Background effect of the diet on swimming performance and O2 consumption in fish exercised with the control feed (CTRL) (A) and with the atomized bioactive hydrolysate (PPH) (B). (C) Maximum metabolic rate (MMR), basal metabolic rate (BMR), and (D) critical rate (Ucrit) values ​​are the mean ± SEM of 5 fish. * indicates statistically significant differences between different groups (Student's test, *P < 0.1, **P < 0.05).

[0286] Figure 7. Effect of the diet, containing the atomized bioactive hydrolysate, on the blood biochemistry of gilthead seabream. Plasma glucose (A, B), triglyceride (C, D), cholesterol (E, F), and cortisol (G, H) levels after 24 and 48 hours post-feeding. * indicates statistically significant differences between fish fed the control diet (CTRL) and the diet with bioactive extract (PPH) at a given time point post-feeding (Student's test, *P < 0.1, **P < 0.05). t indicates statistically significant differences between different time points for the same group (Student's test, t P < 0.1, ttP < 0.05).

[0287] EXAMPLES

[0288] The invention will then be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

[0289] I. Materials and Methods

[0290] Sample preparation, enzymatic hydrolysis and obtaining the final atomized ingredient

[0291] Hygienically collected blood from the slaughterhouse, treated with anticoagulant, is diluted 1:1 with double-distilled water and treated with ultrasound between 10 and 100 kHz, preferably 35 kHz, for between 10 minutes and 4 hours, preferably for 1 hour. Sequential hydrolysis is then performed; first, with an endoprotease enzyme such as Alcalase® at pH 6-7 for 1-3 hours and between 55 and 70°C, preferably 2 hours at pH 6.5 and 65°C, followed by a second hydrolysis with PROTANA PRIME® at pH 6-7 for 8-15 hours at 50-60°C, preferably pH 6.5 at 55°C for 12 hours. Once the hydrolysis is complete, the enzymes are inactivated at a temperature above 70°C for a time between 2 and 60 minutes, preferably at 95°C for 10 minutes.Once the hydrolysate is cooled, the fraction enriched in peptides with a molecular weight of less than 10 kDa and free amino acids is obtained by ultrafiltration through a 10 kDa pore size molecular exclusion membrane. This hydrolysate is then subjected to a spray drying or freeze-drying process.

[0292] Analytical determinations in the hydrolysate

[0293] SDS-PAGE protein electrophoresis

[0294] An SDS-PAGE electrophoresis test was performed to evaluate the size of proteins present in blood hydrolysate previously filtered to 30 and 10 kDa. For sample preparation, an appropriate volume of 62.5 mM Titrate-HCl buffer, pH 6.8, 2% SDS, 20% sorbitol, 5% 2-mercaptoethanol, and 0.002% bromophenol blue was mixed 1:1 with the samples, denatured at 95°C for 5 minutes, and then placed on the gel. Electrophoresis was carried out using a 12% polyacrylamide gel for 30 minutes at 2000 V. The gel was fixed with 40% ethanol and 10% acetic acid for 1 hour and stained with Coomassie blue (Bio-Rad) for 1 hour. Subsequently, MiliQ water was used to decolorize the gel. Finally, the gel was scanned using an image scanner, and the results were processed and analyzed with Imaged v.1.52 software (Wayne Rasband, USA).

[0295] Determination of the concentration of free amino acids

[0296] The determination of the concentration of free amino acids present in the bioactive hydrolysate was carried out following the method described by Aristoy and Toldrá (1991) (Aristoy & Toldrá. 1991. Deproteinization techniques for amino acid analysis in fresh pork muscle and dry-cured ham. Journal of Agricultural and Food Chemistry 39, 792-795) and modified by Flores et al (1997) (Flores et al. 1997. Non-volatile components effects on quality of “Serrano” dry-cured ham as related to processing time. J. Food Sci. 62: 1235-1239). 300 pL of the hydrolysate was homogenized in 0.01 N HCl (1:4; w / v) in a vortex mixer for 8 min and centrifuged at 10,000 g and 4 °C for 20 min. The sample was deproteinized with ethanol and derivatized with phenylisothiocyanate. A 5 mM norleucine solution was used as an internal standard.The chromatographic separation of the derivatized amino acids was performed using a reversed-phase HPLC system equipped with a Waters Pico Tag® C18 column (3.9 x 300 mm; Waters Corp., Milford, MA, USA) at 52°C and a flow rate of 1 mL / min. Elution was controlled at 254 nm. Phase A consisted of 70 mM sodium acetate with 2.5% acetonitrile at pH 6.55. Phase B consisted of acetonitrile:water:methanol in a 45:40:15 ratio. Quantification was performed using the response factors calculated for each amino acid / sample in the series of mixed standards.

[0297] Fractionation of the hydrolysate by RP-HPLC chromatography

[0298] The hydrolysate was fractionated based on the hydrophobicity of its peptides using reversed-phase liquid chromatography (RP-HPLC). Separation was performed on an Agilent 1100 Series chromatograph with a C18 column (4.6 x 250 mm, 5 pm, Waters Co., Milford, MA, USA) in gradient mode. Two phases were prepared: phase A consisted of double-distilled water and 0.1% TFA, and phase B contained 60% acetonitrile and 40% double-distilled water, in the presence of 0.085% TFA. 100 pL of sample at a concentration of 40 mg / mL were injected. For the first two minutes, 100% of phase B was used, and subsequently, 50% of both phases were used until the chromatogram was complete, with a total chromatogram duration of 50 minutes. The fractions were collected every 10 minutes and lyophilized. To carry out the subsequent tests, once lyophilized the samples were resuspended in 500 pL of double-distilled water.

[0299] Analysis and identification of hydrolysate fractions by tandem mass spectrometry

[0300] Peptide sequence identification was performed using a Tims TOF fleX tandem mass spectrometer (Bruker). The sample was loaded onto an Evotip tip and subsequently eluted on an analytical column (PepSep 8 cm x 100 µm, 3 µm; Evosep) in an Evosep One system, using the manufacturer's 30 SDP chromatographic method. The eluted peptides were ionized in an electrospray at 1700 V and 200°C and analyzed in DDA PASEF data-dependent acquisition mode under the following conditions:

[0301] The TIMS conditions were random mode, 1 / K0: 0.7–1.76 Vs / cm²; ramp time: 100 ms; Duty Cycle: 100%; Ramp Rate: 9.42 Hz; Ms Averaging: !; Auto Calibration: off. MS1 scans from 100 to 1700 m / z were obtained in positive ionization mode and PASEF Scan Mode. For the MS2 experiments, up to 10 PASEF ramps were acquired for 1.7 s in high sensitivity mode, with no minimum charge and a maximum charge of 3. System sensitivity was checked with 50 ng of a HELA protein hydrolysate. Synthesis of the selected peptides The peptides were synthesized by the Peptide Synthesis Service of the Pompeu Fabra University (Barcelona, ​​Spain) to the highest certified purity using liquid chromatography-mass spectrometry (LC-MS) analysis.

[0302] Determination of biological activities of the hydrolysate, its fractions and identified peptides.

[0303] Antioxidant activity

[0304] DPPH antioxidant activity was evaluated as the percentage reduction in DPPH radical absorbance in the bioactive hydrolysate samples compared to the control sample. For the assay, 100 µL of the bioactive hydrolysate at an initial concentration of 20 mg / mL were mixed with 500 µL of HPLC-grade ethanol and 125 µL of DPPH reagent (0.02% in ethanol). The resulting mixture was incubated for 60 minutes at room temperature in the dark for subsequent absorbance detection using a UV-Vis spectrophotometer. Ethanol was used as a negative control and BHT (20 mg / mL) as a positive control.

[0305] The iron-reducing activity assay (FRAP) was performed by mixing 140 µL of the bioactive hydrolysate with 140 µL of 200 mM PBS at pH 6.6 and 140 µL of potassium hexacyanoferrate (III) at a concentration of 10 mg / mL. The mixture was then incubated at 50°C for 20 min. To stop the reaction, 200 µL of trichloroacetic acid (100 mg / mL) was added, followed by centrifugation at 200 g for 10 min. The resulting supernatant (400 µL) was mixed with 400 µL of double-distilled water and 80 µL of ferric chloride (1 mg / mL). This mixture was incubated in the dark for 10 min. Absorbance was measured at 700 nm using a UV-Vis spectrophotometer. BHT (20 mg / mL) was used as a positive control (Huang et al. 2006. Antioxidant properties of methanolic extracts from Agrocybe cylindracea, LWT - Food Sci Technol, 39: 379-387).

[0306] ABTS radical scavenging activity was performed by dissolving 7 mM ABTS reagent in 2.45 mM potassium persulfate and allowing it to stand in the dark for 16 h to prepare the ABTS radical reagent solution. The resulting solution was diluted with 50 mM PBS (pH 7.4) to ensure an absorbance of 0.70 (± 0.02) at 734 nm, as measured by a UV-Vis spectrophotometer. Then, 10 pL of the bioactive hydrolysate at an initial concentration of 10 mg / mL was mixed with 990 pL of ABTS radical reagent solution. After 6 min of incubation at room temperature in the dark, the absorbance was read at 734 nm. Ascorbic acid was used as a positive control and PBS as a negative control. The ABTS radical scavenging activity was expressed as Trolox equivalent antioxidant capacity (TEAC) in nmol per mL of bioactive hydrolysate (Re et al. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Radie Biol Med, 26: 1231-1237).

[0307] Hypoglycemic activity through inhibition of DPP-IV and neprilysin enzymes

[0308] The DPP-IV enzyme inhibition assay, for both the hydrolysate and its fractions, was performed using the commercial kit “DPP-IV inhibitor screening” (MAK203, Sigma-Aldrich, Saint Louis, MO, USA). Fifty milliliters (pL) of sample (at 20 mg / mL for the hydrolysate) were mixed with 50 pL of enzyme solution in a 96-well, black, transparent-bottom plate. The mixture was incubated for 10 minutes at 37°C, and then 25 pL of substrate were added to each well. Fluorescence (FLU, A) was measured. e x=360 / A e m=460nm) in a CLARIOstar plate reader (BMG Labtech, Germany) for 30 minutes at 37°C. Sitagliptin was used as a positive control and water as a negative control. The percentage of inhibition was obtained according to the kit protocol.

[0309] The neprilysin enzyme inhibition assay was performed by optimizing the methods described in Spillantini et al. (1986) (Spillantini et al. 1986. In vivo “enkephalinase” inhibition by Acetorphan in human plasma and CSF. European J. Pharmacol. 125: 147-150) and Simonini et al. (2004) (Simonini et al. 2004. Neprilysin levels in plasma and synovial fluid of juvenile idiopathic arthritis patients. Rheumatol Int 25: 336-340). 25 pL of sample and 25 pL of enzyme solution were added to a 96-well black plate with a transparent background and incubated for 10 minutes at 37°C. Subsequently, 50 pL of substrate were added, and fluorescence was measured for 1 h at 37°C using a CLARIOstar plate reader (BMG LABTECH, Ortenberg, Germany). The enzyme solution was prepared by diluting the Human Neprilysin Tag Free enzyme (Aero Biosystems) in water to a concentration of 400 ng / mL.On the other hand, the substrate was prepared by dissolving N-Succinyl-Ala-Ala-Phe-7-amido-4-methylcoumahn in a 50 mM HEPES / KOH buffer at pH 7.4 to obtain a 0.2 mM solution of said substrate. In addition, 0.242 pL of aminopeptidase M per well (Merck) was added to the substrate solution. Thiorphan (1 pM) was used as a positive control and water as a negative control. Finally, the percentage of inhibition was calculated by selecting two minutes within the linear range of the enzyme kinetics (ARFU = T2 - T1): % Inhibition = (ARFU negative control - ARFU sample) / (ARFU sample). > T ^ TT - : - x 100

[0310] ARFU negative control

[0311] In the case of the hydrolysate fractions obtained by RP-HPLC, the samples had to be diluted half with double-distilled water compared to the other tests.

[0312] Determination of anti-inflammatory activity by TACE enzyme inhibition. The assay was performed using the TACE inhibition kit according to the manufacturer's instructions (MAK218, Sigma-Aldrich, St. Louis, MO, USA). Briefly, the assay is based on hydrolyzing the specific FRET substrate to release a fluorescent product that is proportional to the enzymatic activity present. 25 pL of bioactive hydrolysate (at 5 mg / mL final concentration in well) plus 50 pL of TACE enzyme were pre-incubated at 37°C for 5 minutes. Subsequently, 25 pL of substrate were added, and fluorescence was measured at Aex = 318 nm and Aem = 449 nm using a CLARIOstar microplate reader in kinetic mode for 30 minutes at 37°C. GM6001 inhibitor (0.1 mM) was used as a positive control. Activity was expressed as % TACE inhibition.

[0313] Determination of immunomodulatory activity by inhibiting the monoacylglycerol lipase enzyme.

[0314] The monoacylglycerol lipase inhibition assay, used to measure immunomodulatory activity, was performed using the “Monoacylglycerol Lipase / MGL Screening” kit (ab283388, Abeam, Cambridge, UK) according to the manufacturer's instructions. This assay is based on hydrolyzing the substrate (monoacylglycerol), releasing a fluorescent product that is proportional to the enzymatic activity present. Five milliliters of bioactive hydrolysate (at a final concentration of 1 mg / mL per well) were pre-incubated with 90 milliliters of lipase at 37°C for 30 minutes in the dark. Subsequently, 5 milliliters of substrate were added, and fluorescence was measured at Aex = 360 nm and Aem = 460 nm using a CLARIOstar microplate reader in kinetic mode for 30 minutes at 37°C. Activity was expressed as the percentage of monoacylglycerol lipase inhibition.

[0315] Determination of the effect of the hydrolysate on fish

[0316] Fish, feeding trials and diet formulations.

[0317] After the acclimation period at the IATS facilities, juvenile gilthead seabream, with an initial body weight (IBW) of 19.5 g, were tagged in the dorsal musculature with integrated passive transponders (ID-100A 1.25 Nano Transponder; Trovan, Madrid, Spain) and distributed into duplicate 500 L flow tanks (30 fish per tank) for each experimental condition. The fish were maintained under natural photoperiod and temperature conditions at latitude 40°5'N; 0°10'E. Individual animals were weighed and measured at the beginning and end of the trial using an FR-200 FishReader W (Trovan, Madrid, Spain) to assess growth indices, including specific growth rate (SGR) and feed conversion ratio (FCR).

[0318] The feeding trial lasted 10 weeks (July-August 2023), during which the fish were fed using automatic feeders, distributing the feed ration in 3 meals per day (9:00, 12:00, and 15:00 h) close to visual satiation. During the trial, the water temperature and O2 concentration (>75% saturation throughout the trial) were continuously measured via an online environmental monitoring system.

[0319] The two isoproteic, isolipidic, and isoenergetic diets (2 mm pellet size) used for the trial were manufactured by Sparos Lda (Portugal). Both diets contained the same base feed formulation, with the sole exception of the inclusion of porcine blood meal (5%) in the control diet (CTRL), while porcine blood protein hydrolysate (5%) was included in the experimental diet (PPH).

[0320] Sample collection

[0321] At the end of the feeding trial, 16 fish from each experimental group were anesthetized with 1 g / L MS-222 (Sigma, Saint Louis, MO, USA) and used to obtain data for calculating organosomatic indices, such as the viscerosomatic (VSI) and hepatosomatic (HSI), and to collect samples for subsequent analysis. Sample collection was performed at two consecutive events, 24 and 48 hours post-feeding. Blood, liver, and foregut samples were collected for biochemical and transcriptomic analyses at both sampling events, while liver and foregut / hindgut portions for histological analysis were collected only at the first sampling event at 24 h.

[0322] Behavioral monitoring, swimming performance test, and fasting / refeeding test.

[0323] After the feeding trial, the fish were kept separate and fed for one week as previously mentioned. During this time, pre-feeding behavior was monitored by video recording, which began 30 minutes before the daily feeding.

[0324] In parallel with obtaining data on swimming performance and metabolic rate (O2 consumption), 5 fish from each condition were subjected to an exercise test using a swimming tunnel system (dimensions 10 x 10 x 40 cm, Model PA10500, Loligo® Systems, Viborg, Denmark). The analysis included the evaluation of basal and maximum metabolic rate (BMR; MMR) and critical velocity (Ucrit).

[0325] To evaluate the effect of diet on weight loss and regain dynamics, after the period (one week) used for video recording and the swimming test, the remaining fish were subjected to a ten-day fasting phase followed by an eight-day refeeding phase. The weight of the fish was measured before and after refeeding.

[0326] Biochemical and molecular analyses.

[0327] Plasma glucose was determined using the Invitrogen™ Colorimetric Glucose Detection Kit (Invitrogen, EIAGLUC). Plasma cortisol levels were determined using an enzyme immunoassay kit (K003-H1W, Arbor Assays) according to the manufacturer's instructions. Total plasma cholesterol was determined using a cholesterol esterase / cholesterol dehydrogenase reagent (ThermoFisher Scientific). Triglycerides were analyzed using a commercial kit (981786, ThermoFisher Scientific).

[0328] Statistical analysis

[0329] The results were analyzed using one-way ANOVA (P < 0.05) for the composition analysis and bioactivity assays of the hydrolysate. Data on growth performance, organosomatic index, pre-feeding behavior, and blood biochemistry, as well as swimming performance and fasting / refeeding test values, were analyzed using Student's t-test with SigmaPlot v14 (Systat Software Inc., San Jose, CA, USA).

[0330] II. RESULTS Example 1. Obtaining the hydrolysate, study of peptide size and free amino acid content.

[0331] The sequential enzymatic hydrolysis of pig blood using ALCALASE® and PROTANA PRIME® allows the production of small peptides that can potentially be directly absorbed through the intestinal mucosa and thus be responsible for its bioactive properties. Peptide analysis of the hydrolysate by SDS-PAGE electrophoresis confirms the absence of peptides larger than 10 kDa when using filters with a pore size of this size. Furthermore, it demonstrates that the majority of peptides present are smaller than 3 kDa (Figures 1 and 2).

[0332] On the other hand, as shown in Table 1, the hydrolysate is an extract very rich in free amino acids with amounts greater than 20 mg / g of glutamic acid, histidine, asparagine, alanine, valine, leucine, phenylalanine and lysine.

[0333] Table 1. Free amino acid content in the atomized bioactive hydrolysate.

[0334] Example 2. Study of the biological activities of the hydrolysate

[0335] As shown in Table 2, the porcine blood hydrolysate showed antioxidant activity in the three tests performed for ABTS, FRAP and DPPH, obtaining IC50 values ​​of 2.09, 135.05 and 26.73 mg / mL, respectively.

[0336] Table 2. Biological activities of the bioactive hydrolysate.

[0337] Furthermore, as shown in Table 3, the stability of the hydrolysate was verified over time (6 months) under two different storage conditions: at room temperature (20°C) and under refrigeration (4°C).

[0338] Table 3. Stability of the antioxidant activity of the atomized bioactive hydrolysate after being stored at room temperature and refrigerated. ae: Different letters in the same row indicate significant differences (P<0.05). Regarding hypoglycemic activity, the hydrolysate showed inhibition values ​​of 82.78% for the DPP-IV enzyme and 84.72% for the neprilysin enzyme. Both enzymes are found in intestinal tissue, and the importance of this study lies in their participation in the regulation of glucose metabolism by degrading and inactivating peptides such as incretin hormones. Thus, the presence of peptides that inhibit these enzymes could lengthen the half-life of GLP-1 and GIP, improving the glycemic index and, therefore, contributing to blood glucose homeostasis.

[0339] On the other hand, the TACE enzyme (tumor necrosis factor-a converting enzyme) is involved in cell-cell and cell-matrix interactions and plays a significant role in maintaining the immune barrier, as it participates in the activation of Notch signaling between cells. In this case, the hydrolysate achieved an enzyme inhibition value of 50.79%.

[0340] Finally, a 69.08% inhibition of the monoacylglycerol lipase (MAGL) enzyme was achieved. MAGL is an enzyme involved in the transport of endocannabinoid substances into the cell. The endocannabinoid system is one of the most important modulators of the autonomic nervous system, the immune system, and the microcirculation. Therefore, the presence of immunomodulatory peptides capable of exerting a significant effect on this system can contribute to greater animal welfare.

[0341] Example 3. Fractionation of the hydrolysate, identification of peptides and study of their biological activities.

[0342] The hydrolysate was subjected to chromatographic separation into 5 fractions based on its hydrophobicity. The 5 peptide fractions (F1, F2, F3, F4 and F5) were collected, from most polar or hydrophilic to most nonpolar or hydrophobic (Figure 3).

[0343] Table 4 shows the results of biological activities obtained in each of the five separated and collected fractions. Fraction 1 (F1), the most hydrophilic, exhibited the highest antioxidant activity values ​​for FRAP (89.94 pM) and DPPH (17.01%). This fraction also showed the highest inhibition values ​​for the enzymes TACE, DPP-IV, and NEP, with inhibition percentages of 100%, 90.45%, and 84.69%, respectively.

[0344] Table 4. Biological activities of the 5 fractions of the atomized hydrolysate obtained by RP-HPLC.

[0345] Subsequently, analysis using peptidomic techniques revealed the presence of 14 potentially bioactive peptides in this fraction (F1) since they obtained PeptideRanker values ​​greater than 0.5 (Table 5).

[0346] Table 5. Sequences of the most relevant peptides identified in the first fraction (F1) of the atomized hydrolysate collected by RP-HPLC.

[0347] 1 The Peptide Ranker tool was used to predict the bioactive potential of the peptides. Scores close to 1 indicate a higher probability that the peptide is bioactive.

[0348] Finally, from the 14 peptides identified in Table 5, 5 peptides were selected that could have greater hypoglycemic activity based on the Peptide Ranker and their amino acid sequence. The 5 selected peptides were PDDFNPS, FPPKPKD, DNPDIPK, GHLDDLPG, and GDL. Once synthesized, these peptides were subjected to the same biological activity assays described above to demonstrate their bioactivity (Figure 5). All 5 peptides showed significant neprilysin inhibition, up to 60.25% for GDL. Furthermore, they exhibited a significant synergistic effect, as the combination of the 5 peptides at 1 mM inhibited neprilysin by 83.5%. Furthermore, the PDDFNPS peptide also showed a DPP-IV enzyme inhibition value of 42.4% and the synergistic effect of the 5 peptides at 1 mM reached 56.4% inhibition of the activity of said enzyme.

[0349] Example 4. Administration of atomized bioactive hydrolysate as a feed ingredient to gilthead seabream fish and its effects.

[0350] The formulation of this powdered hydrolysate in aquaculture feed and its administration to gilthead seabream has demonstrated, in pilot plant studies, a reduction in the number of aggressive interactions between individuals before ingestion, an increase in critical speed (exhaustion rate) in an exercise test, and a decrease in basal metabolism. This results in favorable fish growth, greater resistance to stress, and weight maintenance after periods without feeding, compared to standard reference feeds.

[0351] As explained previously, the DPP-IV and NEP enzymes are capable of inactivating GLP-1 and GIP, and the inhibition of both enzymes provides significant metabolic benefits. The effect of this dynamic is confirmed by the results obtained in the in vivo test with gilthead seabream, in which the fish fed the experimental diet showed a markedly different response. The prolonged feeling of satiety and the improved nutrient utilization, due to incretin hormones, may have affected the animals' metabolism, as demonstrated during the exercise test by the lower baseline oxygen consumption and the reduced weight loss during the fasting period (Figure 6).

[0352] At the same time, this more energetically favorable state may also have affected the behavioral dynamics of the animals observed during pre-feeding. In fact, the results showed greater aggression among individuals in the control group compared to the group fed the experimental diet. This stressful condition within the tank was also confirmed by the levels of blood stress indicators (Figure 7), which, following the same pattern, showed a clear increase in cortisol and, as a secondary effect, glucose in the control animals compared to the fish fed the experimental diet.

Claims

CLAIMS 1. A mixture of peptides comprising at least 5 peptides, wherein: - a peptide (I) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1; - a peptide (II) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 2; - a peptide (III) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the GDL sequence; - a peptide (IV) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4; and - a peptide (V) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:

5.

2. Peptide mixture according to claim 1, wherein: - the peptide (I) comprises SEQ ID NO: 1 ; - the peptide (II) comprises SEQ ID NO: 2; - the peptide (III) comprises the GDL sequence; - the peptide (IV) comprises SEQ ID NO: 4; and - the peptide (V) comprises SEQ ID NO:

5.

3. Mixture of peptides according to claim 1 or 2, wherein the peptides have a length of 3 to 100 amino acids 4. Peptide mixture according to claim 3, wherein the peptides (I) - (V) have a length of 3 to 20 amino acids.

5. Peptide mixture according to claim 4, wherein the peptides (I) - (V) have a length of 3 to 13 amino acids.

6. Peptide mixture according to claim 5, wherein peptides (I) - (V) have a length of 3 to 8 amino acids.

7. Peptide mixture according to claim 6, wherein: - the peptide (I) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 1; - the peptide (II) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 2; - the peptide (III) comprises an amino acid sequence of a length of 3 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the GDL sequence; - the peptide (IV) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4; and - the peptide (V) comprises an amino acid sequence of a length of 8 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:

5.

8. Peptide mixture according to any one of claims 1 to 7, wherein: - the peptide (I) consists of SEQ ID NO: 1 ; - the peptide (II) consists of SEQ ID NO: 2; - the peptide (III) consists of the GDL sequence; - the peptide (IV) consists of SEQ ID NO: 4; and - the peptide (V) consists of SEQ ID NO:

5.

9. Peptide mixture according to any one of claims 1 to 8, wherein said mixture inhibits the enzyme neprilysin and / or the enzyme dipeptidyl peptidase-4 by at least 20%.

10. Method for obtaining a blood hydrolysate from an animal belonging to the genus Sus, comprising the steps of: a) hydrolyzing the previously isolated blood sample comprising contacting the sample with an alcalase proteolytic enzyme, and with at least one exopeptidase proteolytic enzyme; and b) subjecting the hydrolysate resulting from step a) to an ultrafiltration process using a molecular exclusion membrane with a pore size equal to or less than 10 kDa.

11. Method according to claim 10, wherein said subject is a member of the species Sus scrofa.

12. Method according to claim 11, wherein said subject is a member of the subspecies Sus scrofa domesticus.

13. Method according to any one of claims 10 to 12, wherein in step a), the sample is contacted sequentially with an alcalase enzyme and at least one exopeptidase enzyme.

14. Method according to any one of claims 10 to 13, wherein step a) consists of contacting the sample with an alcalase enzyme and at least one exopeptidase enzyme.

15. Method according to any one of claims 10 to 14, wherein the enzymatic hydrolysis reaction with the alcalase enzyme carried out in step a) is performed at a pH of 6 to 7.

16. Method according to any one of claims 10 to 15, wherein the enzymatic hydrolysis reaction with the alcalase enzyme carried out in step a) is performed for a time of 1 to 3 hours.

17. Method according to any one of claims 10 to 16, wherein the enzymatic hydrolysis reaction with the alcalase enzyme carried out in step a) is performed at a temperature between 55 and 70°C.

18. Method according to any one of claims 10 to 17, wherein the enzymatic hydrolysis reaction with at least one exopeptidase enzyme carried out in step a) is performed at a pH of 6 to 7.

19. Method according to any one of claims 10 to 18, wherein the enzymatic hydrolysis reaction with at least one exopeptidase enzyme carried out in step a) is performed for a time of 8 to 15 hours.

20. Method according to any one of claims 10 to 19, wherein the enzymatic hydrolysis reaction with at least one exopeptidase enzyme carried out in step a) is performed at a temperature between 50 and 60°C.

21. Method according to any one of claims 10 to 20, further comprising a step a') subsequent to step a), of thermal inactivation of the alcalase enzyme and at least one exopeptidase enzyme, comprising subjecting the hydrolysate resulting from step a) to a temperature equal to or greater than 70°C.

22. Method according to any one of claims 10 to 21, further comprising a step a') subsequent to step a), of thermal inactivation of the alcalase enzyme and at least one exopeptidase enzyme, comprising subjecting the hydrolysate resulting from step a) for a time of 2 to 60 minutes.

23. Method according to any one of claims 10 to 22, further comprising a step prior to step a) of treating the blood sample with ultrasound.

24. Method according to claim 23, wherein the prior ultrasound treatment stage is performed for 10 minutes to 4 hours.

25. Method according to claim 24, wherein the prior ultrasound treatment stage is performed at a frequency of between 10 and 100 kHz.

26. Method according to any one of claims 10 to 25, wherein step b) is carried out using a molecular exclusion membrane with a pore size of 3 to 10 kDa.

27. Method according to claim 26, wherein step b) is carried out using a molecular exclusion membrane with a pore size of 3 to 5 kDa.

28. Method according to any one of claims 10 to 27, wherein the hydrolysate is subjected to an atomization or freeze-drying process.

29. Blood hydrolysate obtainable by a method according to any one of claims 10 to 28.

30. Hydrolysate according to claim 29, wherein said hydrolysate comprises at least 5 peptides, wherein: - a peptide (I) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1; - a peptide (II) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 2; - a peptide (III) comprises an amino acid sequence having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the GDL sequence; - a peptide (IV) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4; and - a peptide (V) comprises a sequence of amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:

5.

31. Hydrolyzed according to claim 30, wherein: - the peptide (I) comprises SEQ ID NO: 1 ; - the peptide (II) comprises SEQ ID NO: 2; - the peptide (III) comprises the GDL sequence; - the peptide (IV) comprises SEQ ID NO: 4; and - the peptide (V) comprises SEQ ID NO:

5.

32. Hydrolyzed according to claim 30 or 31, wherein the peptides have a length of 3 to 100 amino acids 33. Hydrolyzed according to claim 32, wherein the peptides (I) - (V) have a length of 3 to 20 amino acids.

34. Hydrolyzed according to claim 33, wherein the peptides (I) - (V) have a length of 3 to 13 amino acids.

35. Hydrolyzed according to claim 34, wherein the peptides (I) - (V) have a length of 3 to 8 amino acids.

36. Hydrolyzed according to any one of claims 29 to 35, wherein: - the peptide (I) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1; - the peptide (II) comprises an amino acid sequence of amino acids of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 2; - the peptide (III) comprises an amino acid sequence of a length of 3 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the GDL sequence; - the peptide (IV) comprises an amino acid sequence of a length of 7 amino acids, having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4; and - the peptide (V) comprises an amino acid sequence of a length of 8 amino acids, with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:

5.

37. Hydrolyzed according to any one of claims 29 to 36, wherein: - the peptide (I) consists of SEQ ID NO: 1 ; - the peptide (II) consists of SEQ ID NO: 2; - the peptide (III) consists of the GDL sequence; - the peptide (IV) consists of SEQ ID NO: 4; and - the peptide (V) consists of SEQ ID NO:

5.

38. Hydrolysate according to any one of claims 29 to 37, wherein said hydrolysate inhibits the enzyme neprilysin and / or the enzyme dipeptidyl peptidase-4.

39. Composition comprising the peptide mixture according to any one of claims 1 to 9, or the hydrolysate according to any one of claims 29 to 38.

40. Composition according to claim 39, wherein the composition is a nutritional composition.

41. Composition according to claim 39, wherein the composition is a pharmaceutical composition.

42. Food additive comprising the peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, or the composition according to any one of claims 39 to 41.

43. I think it comprises the peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, or the composition according to any one of claims 39 to 41.

44. Food comprising the peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, the composition according to any one of claims 39 to 41, the food additive according to claim 42 or the feed according to claim 43.

45. Use of the peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, the composition according to any one of claims 39 to 41, the food additive according to claim 42 or the feed according to claim 43, for the manufacture of a food.

46. ​​Use according to claim 45, wherein the food is a functional food.

47. Use of the peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, the composition according to any one of claims 39 to 41, or the food additive according to claim 42, as animal feed or food.

48. Use of the peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, the composition according to any one of claims 39 to 41, the food additive according to claim 42, the feed according to claim 43, or the food according to claim 44, to promote the growth of a subject, preferably an animal subject.

49. Use according to claim 48, wherein the subject is a pet and / or farm and / or crop animal.

50. Use according to claim 48 or 49, wherein the animal is a mammal.

51. Use according to claim 48 or 49, wherein the animal is a bird.

52. Use according to claim 48 or 49, wherein the animal is an aquatic animal, preferably a fish or a mollusk, more preferably a fish.

53. Use of the peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, the composition according to any one of claims 39 to 41, the food additive according to claim 42, the feed according to claim 43, or the food according to claim 44, to improve the physical performance of a subject, preferably an animal subject.

54. Use according to claim 53, wherein physical performance is swimming performance.

55. Use according to claim 53 or 54, wherein the subject is a pet and / or farm and / or crop animal.

56. Use according to any one of claims 53 to 55, wherein the animal is a mammal.

57. Use according to any one of claims 53 to 55, wherein the animal is a bird.

58. Use according to any one of claims 53 to 55, wherein the animal is an aquatic animal, preferably a fish or a mollusk, more preferably a fish.

59. Peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, the composition according to any one of claims 39 to 41, the food additive according to claim 42, the feed according to claim 43, or the food according to claim 44, for use as a veterinary medicinal product.

60. Peptide mixture according to any one of claims 1 to 9, the hydrolysate according to any one of claims 29 to 38, the composition according to any one of claims 39 to 41, the food additive according to claim 42, the feed according to claim 43, or the food according to claim 44, in the prevention and / or treatment of stress and / or behavioral disorders, preferably stress-related aggressive behaviors in a subject.

61. Peptide mixture, hydrolysate, composition, food additive, feed, or food for use according to claim 59 or 60, wherein the subject is a pet and / or farm and / or crop animal.

62. Peptide mixture, hydrolysate, composition, food additive, feed, or food for use according to claim 61, wherein the animal is a mammal.

63. Peptide mixture, hydrolysate, composition, food additive, feed, or food for use according to claim 61, wherein the animal is a bird.

64. Peptide mixture, hydrolysate, composition, food additive, feed, or food for use according to claim 61, wherein the animal is an aquatic animal, preferably a fish or a mollusc, more preferably a fish.

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