ES2702617B2 - Hydrolysed chicken leg claws, their peptides and uses thereof - Google Patents

Description

DESCRIPTION

Hydrolysed chicken leg claws, their peptides and uses thereof

Field of the Invention

The present invention pertains to the field of protein hydrolysates, in particular it relates to chicken leg claw hydrolysates (GPP), peptides which it already comprises for use in therapy, in particular for the treatment and / or prevention of a condition. in which the incretin system is altered.

Background of the invention

Type 2 diabetes mellitus (DMT2) is one of the fastest growing metabolic diseases worldwide. This endocrine disorder associated with obesity is characterized by alterations in insulin secretion and resistance to it. The first objective in the treatment of DMT2 is to maintain glucose levels within normal physiological ranges. In this sense, incretin hormones: glucagon-like peptide type 1 (GLP-1) and gastric inhibitor polypeptide (GIP) represent a useful strategy for the prevention and / or treatment of DMT2. GLP-1 and GIP are important mediators of glucose homeostasis, being responsible for approximately 50-70% of total insulin secretion after glucose intake. The main strategy is to increase GLP-1 levels instead of GIP levels, because the beta cells of patients with DMT2 have a reduced response to the action of GIP. Accordingly, most incretin-based therapies focus on the use of GLP-1 analog compounds, as well as promoting the secretion of endogenous GLP-1 or the use of dipeptidyl peptidase-4 enzyme inhibitors ( DPP-IV). This enzyme is expressed in different parts of the body, including in the cells of the intestinal wall and in the endothelial cells, although it can also be found soluble in the plasma. The main action of the DPP-IV enzyme is to hydrolyze the GLP-1 hormone, making it inactive, once it is secreted in the plasma, having an average life span of 1-2 minutes. Because the DPP-IV enzyme is responsible for the inactivation of more than 80% of GLP-1 secretion, inhibition of this enzyme represents an alternative to increase levels of active GLP-1 and thus improve homeostasis. of glucose

Peptides with enzyme inhibitory activity are known in the state of the art DPP IV (iDPP-IV activity) obtained in hydrolysates from different protein sources. For example, said peptides have been described in milk hydrolysates (WO 2006/068480) and in egg hydrolysates (WO 2009/128713) and it has been described that said peptides with iDPP-IV activity have at least one proline in their sequence and normally said proline is in position 2 of the N-terminal end.

Surprisingly, the authors of the present invention have developed several GPP hydrolysates and characterized one of them with anti-hyperglycemic activity in vivo. Said hydrolyzate comprises peptides with iDPP-IV activity that do not have the typical pattern of peptides with said activity, that is, they lack proline at position 2 of the N-terminal end.

There are documents in the state of the art that describe the sequence of some of the peptides of the present invention. For example, WO2008 / 063369 describes a LAADDFR sequence peptide and its use as a biomarker for the diagnosis of neurological diseases, such as Alzheimer's. However, it does not describe its use for the treatment and / or prevention of a condition in which the incretin system is altered.

Thus, it is desired to provide new protein hydrolysates that can help in the prevention and / or treatment of the conditions in which the incretin system is altered.

Object of the invention

In a first aspect, the present invention relates to a protein hydrolyzate that has DPP-IV enzyme inhibitory activity in vitro, comprises one or two sequence peptide (s) selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2, and is a protein hydrolyzate of GPP.

In a second aspect, the present invention relates to a medicament, a food supplement or food product comprising a hydrolyzate according to the first aspect of the invention. It also refers to a pharmaceutical composition comprising a hydrolyzate according to the first aspect of the invention and a pharmaceutically acceptable excipient.

In a third aspect, the present invention relates to a hydrolyzate according to the first aspect of the invention for use in therapy.

In a fourth aspect, the present invention relates to a hydrolyzate according to the first aspect of the invention for use in the treatment and / or prevention of a condition in which the incretin system is altered.

In a fifth aspect, the present invention relates to a method for preparing the hydrolyzate of the first aspect of the invention.

In a sixth aspect, the present invention relates to a GPP hydrolyzate obtainable by the method according to the fifth aspect of the invention.

Other objects, features, advantages and aspects of the present application will be apparent to the person skilled in the art from the description and the appended claims.

Brief description of the figures

Figure 1 is a reverse phase HPLC chromatogram on a semi-preparative scale of the active supernatant obtained after centrifugation and ultrafiltration, through a membrane of 3,000 Da pore size, of the "p38 hydrolyzate" obtained by enzymatic hydrolysis with Neutrase ®, 24h, at 25 ° C pH 7 from GPP powder treated at 50 ° C, 1.5 h pH 3, F.1 - F.11 correspond to the eleven (11) collected fractions.

Figure 2A is a semi-preparative scale HPLC chromatogram of the most active fraction (F.3) obtained in the first HPLC separation of the "p38 hydrolyzate" of GPP powder, observed at 214 nm. Specifically, four (4) sub-fractions of F.3 (F.3.1-F.3.4) were collected.

Figure 2B is a reverse phase HPLC chromatogram on a semi-preparative scale of the most active fraction (F.3) obtained in the first HPLC separation of the "p38 hydrolyzate" of GPP powder, observed at 280 nm. Specifically, four (4) sub-fractions of F.3 (F.3.1-F.3.4) were collected.

Figure 3A is a graph showing the evolution in time of the plasma glucose concentration of female Wistar rats fed with standard diet (STD) after administration of 1 mL of water (■) or with cafeteria diet (CAF) after the administration of 1 mL of water (•) or 1 mL of "p38 hydrolyzate" (300 mg of protein / kg of animal weight) (▲) during the glucose tolerance test. Data represent the mean ± ESM for a minimum of six-seven (6-7) animals Different letters indicate significant differences (Anova 2-way, Bonferroni posthoc test).

Figure 3B is a graph showing the area under the curve of the glycemic response of female Wistar rats fed with standard diet (STD) after administration of 1 mL of water (STD + water) or with cafeteria diet (CAF) after administration of 1 mL of water (CAF + water) or 1 mL of "hydrolyzed p38" (300 mg of protein / kg of animal weight) (Hp38) (CAF + Hp38) during the glucose tolerance test. The data represent the mean ± ESM for a minimum of six-seven (6-7) animals, different letters indicate significant differences (Anova 1 way, Bonferroni posthoc test).

Figure 4A is a graph showing the variation in time of the plasma glucose concentration of 7-month-old male Wistar rats fed with standard diet (STD) after administration of 1.5 mL of water (o), of 1 , 5 mL of "p38 hydrolyzate" (300 mg of protein / kg of animal weight) (▲) or 1 mL of vildagliptin (1 mg / kg of animal weight) (□) during the glucose tolerance test The data represent the mean ± ESM for six (6-7) animals, different letters indicate significant differences (Anova 2 way, Bonferroni posthoc test).

Figure 4B is a graph showing the area under the curve of the glycemic response of 7-month-old male Wistar rats fed with standard diet (STD) after administration of 1.5 mL of water (STD + water), of 1 , 5 mL of "hydrolyzed p38" (300 mg of protein / kg of animal weight) (STD + Hp38) or 1 mL of vildagliptin (1 mg / kg of animal weight) (STD + Vildagliptin) during the tolerance test to glucose The data represent the mean ± ESM for six-seven (6-7) animals Different letters indicate significant differences (Anova 1 way, Bonferroni posthoc test).

Figure 5 is a graph showing the area under the curve of the glycemic response of healthy female Wistar rats fed with standard diet (STD) after administration of 1 mL of water (STD + water) or 1 mL of "p38 hydrolyzate "(300 mg of protein / kg of animal weight) (STD + Hp38) during the glucose tolerance test. Data represent the mean ± ESM for a minimum of six-seven (6-7) animals.

Figure 6A is a graph showing the secretion of active GLP-1 from STC-1 cells treated with control (control) or "p38 hydrolyzate" solution (5 mg of lyophilized hydrolyzate / mL) (Hp38) diluted with the same control solution. . Data represent the mean ± ESM (n = 6). * indicates significant differences (T-Student).

Figure 6B is a graph showing the active GLP-1 secretion of ileum tissue segments treated with control (control) or "p38 hydrolyzate" solution (15 mg of lyophilized hydrolyzate / mL) (Hp38) diluted with the same solution. control. Data represent the mean ± ESM (n = 6). * indicates significant differences (T-Student).

Detailed description of the invention

As used in the present application, the singular forms "one / one", "one" and "the" include their corresponding plurals unless the context clearly indicates otherwise. Unless otherwise defined, all The technical terms used herein have the meaning that a person skilled in the art to which this invention belongs usually understands.

In order to facilitate understanding and clarify the meaning of certain terms in the context of the present invention, the following definitions and particular and preferred embodiments thereof, applicable to all embodiments of the various aspects of the present invention are provided:

In the present invention "condition in which the incretin system is altered", refers to any condition harmful to a subject in which the incretin system is altered. The incretin system is formed by the hormones GLP-1 and GIP and the enzyme DPP-IV, and in the present invention the alteration in one, several or all of them is considered an altered incretin system. In particular, it is understood that the incretin system is altered when there is an increase in DPP-IV activity and / or a decrease in the levels of the hormone (s) GLP-1 and / or active GIP (s) and / or a decrease in the response to GLP-1 and / or GIP.

Non-limiting examples of such conditions are diabetes, more particularly type 2 diabetes mellitus, conditions of lower glucose tolerance (IGT), altered fasting plasma glucose conditions, more particularly hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, obesity, overweight and osteoporosis. Thus, in a particular embodiment the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered fasting plasma glucose conditions, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, obesity, overweight, osteoporosis and combinations thereof. Preferably, type 2 diabetes mellitus, IGT, hyperglycemia, obesity, overpressure and combinations thereof, and more preferably type 2 diabetes mellitus, hyperglycemia, overweight, obesity and combinations thereof are selected. The involvement of the incretin system in these disorders is well described in the literature and these disorders are therefore the main objective of the hydrolyzate or peptide according to the present invention.

In the present invention "hyperglycemia" (or hyperglycemia) refers to blood glucose levels higher than those considered standard for a healthy individual (normoglycemia). In particular, it refers to a fasting blood glucose concentration> 110 mg / dl. More particularly, it refers to a fasting blood glucose concentration> 110 mg / dl and> 140 mg / dl after the oral glucose test. This test consists in the determination of plasma glucose 2 hours after the intake of 75 g of glucose in 300 mL of water after a fast night.

In the present invention "obesity" and "overweight" refer to an abnormal or excessive accumulation of fat, higher than those considered standard for a healthy individual, which may be harmful to health. The WHO definition for "obesity" or "overweight", which is applicable to the present invention, indicates that one of the ways to define "obesity" or "overweight" is by determining the body mass index ( BMI, weight of a person divided by their height squared) considering “obesity” when the BMI is equal to or greater than 30 kg / m2 and “overweight” when the BMI is between 25 and 29.9 kg / m2.

"Subject" refers to any member of the Mammalia class, including, without limitation, human beings and nonhuman primates such as chimpanzees and other apes and species of monkeys; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs, and the like. The term does not indicate a specific age or sex. Therefore, adult and newborn subjects, as well as fetuses, whether male or female, are intended for inclusion within the scope of this term. The subject is preferably a human being.

Food supplement (or supplement) ”refers to any food component that provides specific nutritional and / or functional components and does not provide the full required energy value (i.e. generally less than 2000 or 2500 kcal / day) and includes food supplements in the form of powder or tablets, as well as dietary products, such as beverages dietary Also included are ingredients that can be added to the food product before consumption or a preparation that can be consumed as such.

"Food product" refers to any product, solid or liquid, natural or processed, intended to be ingested by a subject. In a preferred embodiment, the food product is a functional food product, which refers to any product, solid or liquid , natural or transformed, intended to be ingested by a subject who, apart from its basic nutritional role from the material and energy point of view, is able to provide a health benefit.

The term "prevention" means the prophylactic administration of the hydrolyzate, peptide, medicament, food supplement, food product and / or pharmaceutical composition to healthy patients to prevent the outbreak of the condition in which the incretin system is altered. Moreover, the term "prevention" means the prophylactic administration of said hydrolysates, peptides, etc. to patients who are in a pre-stage of the condition, especially diabetes, to be treated. In the case of functional food products, in the present invention "prevention" refers to the reduction of the risk of suffering from a condition in which the incretin system is altered.

The present invention relates in a first aspect to a protein hydrolyzate which has an inhibitory activity of the DDP-IV enzyme in vitro, comprises one or two sequence peptide (s) selected from the group consisting of SEQ ID N ° 1 and SEQ ID N 2, and is a GPP protein hydrolyzate. This hydrolyzate is referred to hereafter as the hydrolyzate of the invention or HGPP (from chicken leg claw hydrolyzate).

The hydrolyzate of the present invention also has anti-hyperglycemic activity in vivo (see Examples 6-8) and secretory activity of GLP-1 in vitro (see Example 9). Thus, in a particular embodiment, the first aspect of the invention relates to a protein hydrolyzate that has DDP-IV enzyme inhibitory activity in vitro, has GLP-1 secretory activity in vitro and / or antihyperglycemic activity in vivo, comprises one or two peptide (s) of sequence selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2, and is a GPP protein hydrolyzate.

In another particular embodiment according to any one of the foregoing embodiments, e1HGPP further comprises at least one peptide selected from the group consisting of peptides of sequence SEQ ID No. 3-7, preferably at least one peptide of sequence SEQ ID No. 3. In one Particular embodiment comprises one, several or all of the peptides selected from SEQ ID No. 3-7, more particularly comprising 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7. In a preferred embodiment, the HGP comprises the sequence peptides SEQ ID No. 1 - and / or SEQ ID No. 2, and SEQ ID No. 3 - SEQ ID No. 7.

In another particular embodiment according to any one of the above embodiments, it comprises 1, 2 or 3 peptides selected from SEQ ID NO 8-10. In a preferred embodiment, the HGPP comprises the sequence peptides SEQ ID No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3 -SEC ID No. 10.

The sequences described in the present invention are the following:

I KNOW

The first aspect of the invention also relates to isolated peptides (hereinafter referred to as peptides of the present invention) of sequence SEQ ID No. 1 or SEQ ID No. 2, and a combination of said peptides. Thus, it refers to an isolated peptide whose amino acid sequence is selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2. And it also refers to a combination comprising a peptide of SEQ ID No. 1 and a peptide of SEQ ID No. 2. These peptides have iDDP-IV activity in vitro (see Example 5, Table VI ). In a preferred embodiment, the peptide consists of the sequence SEQ ID No. 1. In another preferred embodiment, the peptide consists of the sequence SEQ ID No. 2. As shown in Table VI , these peptides are the ones with the highest iDDP activity. -IV in vitro have.

In a particular embodiment according to any one of the embodiments of the preceding paragraph, the isolated sequence peptide SEQ ID No. 1 or 2, or the combination thereof is in combination (ie combined) with 1, 2, 3, 4 or 5 peptides selected from the sequences SEQ ID No. 3-7 (preferably at least one peptide of sequence SEQ ID No. 3), and optionally 1, 2 or 3 peptides selected from the sequences SEQ ID No. 8-10. Thus, in a particular embodiment the first aspect of the invention relates to a combination comprising a peptide of sequence SEQ ID No. 1 and / or a peptide of sequences SEQ ID No. 2, and 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, preferably at least one is the sequence peptide SEQ ID No. 3, and optionally comprises 1, 2 or 3 peptides selected from SEQ ID No. 8-10. In a particular embodiment, the combination comprises a peptide of sequence SEQ ID No. 1 and / or a peptide of sequence SEQ ID No. 2, and the peptides of sequence SEQ ID No. 3 - SEQ ID No. 7. In another embodiment In particular, the combination comprises the sequence peptides SEQ ID No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3 -SEC ID No. 10.

As indicated above, it has been described that peptides with iDPP-IV activity have at least one proline in their sequence and usually said proline is in position 2 of the N-terminal end. Surprisingly, the peptides of the present invention do not have such proline in position 2 and in fact, the sequence SEQ ID No. 1 does not comprise any proline. Thus, it was not expected that said peptides had iDPP-IV activity.

Assays for measuring iDDP-IV activity in vitro are widely known to those skilled in the art. Examples of tests that can be carried out to determine it are those shown in the Examples of the present application. Thus, in a particular embodiment, the in vitro iDDP-IV activity is measured following the method of Tulipano et al (Whey protein as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase 4) inhibitors. ”2011. Peptides. 32, 835-838) , as has been carried out in the examples of the present invention.

In a preferred embodiment according to any one of the previous embodiments of the first aspect of the invention, the peptide (s) consists of the sequence (s) given, i.e. SEQ ID No. 1-SEQ ID No. 10.

The hydrolysates, isolated peptides and combinations of peptides of the present invention can be incorporated into foods, food supplements, and into the manufacture of pharmaceutical products. Thus, in a second aspect , the invention relates to a medicament, a food supplement or a food product comprising an HGPP, an isolated peptide and / or a combination of peptides according to any one of the embodiments of the first aspect of the invention.

In a preferred embodiment, the medicament, food supplement or food product comprises an HGPP comprising a peptide of sequence SEQ ID No. 1 or a peptide of sequence SEQ ID No. 2 or a combination of said peptides; and optionally comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, preferably at least one of them being the sequence peptide SEQ ID No. 3. In a particular embodiment according to any one of the embodiments above, the HGPP further comprises 1, 2 or 3 peptides of sequences SEQ ID No. 8-10. Preferably the medicament, food supplement or food product comprises an HGPP comprising peptides SEQ ID No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3 - SEQ ID No. 7, more preferably comprises peptides SEQ ID N ° 1 and / or SEQ ID N ° 2, and SEQ ID N ° 3 - SEQ ID N ° 10.

In another preferred embodiment, the medicament, food supplement or food product comprises a peptide of sequence SEQ ID No. 1 or a peptide of sequence SEQ ID No. 2 or a combination of said peptides; and optionally comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, preferably at least one of them being the sequence peptide SEQ ID No. 3. That is, the medicament, food supplement or product Food comprises a peptide of sequence SEQ ID No. 1 or SEQ ID No. 2, or a combination comprising peptides of sequence SEQ ID No. 1 and sequence SEQ ID No. 2, and optionally comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, preferably at least one of them being the sequence peptide SEQ ID No. 3.

In a particular embodiment according to any one of the above embodiments, the medicament, food supplement or food product further comprises 1, 2 or 3 peptides of sequences SEQ ID NO 8-10. Thus, preferably the medicament, food supplement or food product comprises the peptide SEQ ID No. 1 and / or the peptide SEQ ID No. 2, and the peptides SEQ ID No. 3 - SEQ ID No. 7, more preferably comprises the peptide SEQ ID No. 1 and / or the peptide SEQ ID No. 2, and the peptides SEQ ID No. 3 - SEQ ID No. 10.

Also, the second aspect of the invention relates to a pharmaceutical composition comprising an HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, and a pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition comprises the hydrolysates, isolated peptides and combinations described in the three preceding paragraphs for the drug, food supplement and food product.

Pharmaceutically acceptable excipients are widely known to those skilled in the art, in a particular embodiment they are such as those described in "Remington's Pharmaceutical Sciences Handbook" Mack Pub. Co., NYUSA

The medicament, food supplement and the pharmaceutical composition can be formulated in any desired form of administration, for example, as tablets, capsules, syrups and the like for oral administration, as sterile solutions or suspensions in liquids acceptable for parental administration.

In the food product of the invention, the HGPP or isolated peptide or combination of the invention according to the first aspect of the invention may itself be a functional food product or an ingredient that can be combined with any common food ingredient.

As indicated above, the peptides and hydrolysates of the invention have iDDP-IV activity, whereby medications, food supplements, food products, or pharmaceutical compositions comprising them can be used for the treatment and / or prevention of a condition in which the incretin system is altered. Thus, in a third aspect, the present invention relates to the use of an HGPP, isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for the preparation of a medicament, a food supplement or a food product, in particular for the preparation of a medicine.

The third aspect of the invention also relates to an HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for use in therapy. It also refers to the medicament, food supplement, food product, or pharmaceutical composition according to any one of the embodiments of the second aspect of the invention, for use in therapy.

In a particular embodiment according to any one of the embodiments according to the two preceding paragraphs, the HGPP comprises the peptide of SEQ ID No. 2. More particularly, the HGPP comprises the peptide of SEQ ID No. 2 and the peptide of SEQ ID No. 1. Even more particularly, the HGPP further comprises 1, 2, 3, 4 or 5 peptides selected from SEC ID No. 3-7, preferably at least one of them being the sequence peptide SEQ ID No. 3, and optionally further comprising 1, 2 or 3 peptides of sequences SEQ ID No. 8-10. Preferably, the hydrolyzate comprises peptides SEQ ID No. 1 - SEQ ID No. 7, more preferably comprises peptides SEQ ID No. 1 - SEQ ID No. 10. In another particular embodiment according to any one of the embodiments according to the two paragraphs above, the isolated peptide is the sequence peptide SEQ ID No. 2 and the peptide combination comprises the peptide of SEQ ID No. 2 and the peptide of SEQ ID No. 1; and optionally 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, preferably at least one of them being the sequence peptide SEQ ID No. 3. More particularly, the combination further comprises 1, 2 or 3 peptides selected from SEQ ID No. 8-10. Preferably, the combination comprises peptides SEQ ID No. 1 - SEQ ID No. 7, more preferably comprises peptides SEQ ID No. 1 - SEQ ID No. 10.

In a fourth aspect , the present invention relates to the use of an HGPP, isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for the preparation of a medicament, a food supplement, or a product food for the treatment and / or prevention of a condition in which the incretin system is altered. More preferably, it refers to its use for the preparation of a medicament for the treatment and / or prevention of a condition in which the incretin system is altered.

The fourth aspect of the invention also relates to an HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, for use in the treatment and / or prevention of a condition in which The incretin system is altered.

In addition, the fourth aspect of the invention relates to a method of treating a condition in which the incretin system is altered, which comprises the administration of a therapeutically effective amount of an HGPP, an isolated peptide and / or a combination according to a any of the embodiments of the first aspect of the invention, or of a medicament, food supplement, food product or pharmaceutical composition according to any one of the embodiments described in the second aspect of the invention, to a subject in need thereof. In a preferred embodiment, administration is carried out orally. In another preferred embodiment, the subject is a human.

Also, the fourth aspect of the invention relates to a method of preventing a condition in which the incretin system is altered, which comprises the administration of a prophylactically effective amount of an HGPP, an isolated peptide and / or a combination according to any one of the embodiments of the first aspect of the invention, or of a medicament, supplement food, food product or pharmaceutical composition according to any one of the embodiments described in the second aspect of the invention, to a subject in need thereof. In a preferred embodiment, administration is carried out orally. In another preferred embodiment, the subject is a human.

In a particular embodiment according to any one of the above embodiments according to the fourth aspect of the invention, the HGPP comprises the peptide of SEQ ID No. 1 and / or SEQ ID No. 2. More particularly, the hydrolyzate comprises the peptide of SEQ ID. No. 1 and the peptide of SEQ ID No. 2. In another particular embodiment according to any one of the above embodiments, the hydrolyzate further comprises 1, 2, 3, 4 or 5 peptides selected from SEQ ID No. 3-7, being preferably at least one of them the sequence peptide SEQ ID No. 3, and optionally further comprises 1, 2 or 3, peptides selected from SEQ ID No. 8-10. It preferably comprises peptides SEQ ID No. 1 and / or SEQ ID No. 2, and SEQ ID No. 3- SEQ ID No. 7, and more preferably comprises peptides SEQ ID No. 1 and / or SEQ ID No. 2 , and SEQ ID N ° 3 -SEC ID N ° 10.

In a particular embodiment according to any one of the preceding paragraphs of the fourth aspect of the present invention, the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered plasma glucose conditions fasting, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof. Preferably, type 2 diabetes mellitus, IGT, hyperglycemia, overweight, obesity and combinations thereof, and more preferably type 2 diabetes mellitus, hyperglycemia, overweight, obesity and combinations thereof are selected.

The prevention and / or treatment with HGPP, peptides and / or a combination of the present invention is a great advantage over chemical treatments since food protein hydrolysates are naturally occurring food ingredients that lack toxicity or have a minimal toxicity

The dosage will depend on several factors such as type and severity of the pathological condition to be treated, patient weight and sex, etc. and will be easily determined by the skilled. Preferably, the hydrolysates of the invention are administered at a dose of between 1 mg and 8 g of protein per day and per kg of the subject to which they are administered, preferably between 5 mg and 6 g of protein per day and per kg. As for the isolated peptides, the preferred dose of administration is between 1 mg and 4 g of peptide per kg per day, preferably between 10 mg and 3 g per kg and per day. Thus, in a particular embodiment, the medicament, food supplement, food product or pharmaceutical composition comprises the hydrolyzate and / or peptide or combination of the invention in an amount such that it allows an administration according to the doses mentioned in this paragraph. This embodiment is equally applicable to the second aspect of the invention.

In a fifth aspect , the present invention relates to the method of preparing the HGPP according to any one of the embodiments of the first aspect of the invention (method of the invention).

The method of the present invention comprises the following steps:

a) crush GPP and lyophilize to obtain a powder material with particle size smaller than 2 mm;

b) adjust an aqueous solution of the above powder to a pH between 2-4;

c) heat at 40 ° C to 120 ° C, for 10 to 120 min;

d) cooling the above solution to a temperature between 20 ° C and 55 ° C;

e) adjust the pH of the previous solution to a pH between 6.5 and 7.5;

f) add a proteolytic enzyme selected from the group consisting of:

- proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens identified as EC 3.4.21.62 and 3.4.24.28,

- a Bacillus licheniformis serine protease identified as EC 3.4.21.62,

- a Zinc-dependent metalloprotease from Bacillus amyloliquefaciens identified as EC 3.4.24, and

- a mixture between them,

and perform enzymatic hydrolysis for 1 to 24 hours; Y

g) determine the iDPP-IV activity in vitro and select a hydrolyzate with an iDPP-IV activity greater than 80%.

In a preferred embodiment, the powder of step a) has a humidity of less than 5%.

Surprisingly, the pH treatment of stage b) (referred to hereinafter as "pre-treatment with pH") and the thermal treatment of stage c) (referred to hereinafter in hereinafter as "thermal pre-treatment"), they result in a hydrolyzate with an improved iDPP-IV activity (see Table I ). Also note that these pre-treatments are not excessively expensive and yet result in a large increase in iDPP-IV activity.

Thus, the combination of said steps b) and c) causes an improvement in the iDPP-IV activity of the hydrolyzate obtained by the method of the present invention. In a preferred embodiment according to any one of the above embodiments, step b) is carried out at pH 3. In another preferred embodiment, step c) is carried out at 50 ° C or 100 ° C. More preferably, step b) is carried out at pH 3 and step c) is carried out at 50 ° C or 100 ° C. In another preferred embodiment according to any one of the above embodiments, step c) is carried out for a time of between 70 and 100 minutes.

In another preferred embodiment according to any one of the above embodiments, steps c) and / or f) are carried out under stirring.

In another preferred embodiment according to any one of the above embodiments, the temperature of step d) is between 25 ° C and 50 ° C.

In another preferred embodiment according to any one of the above embodiments, the pH of step e) is pH 7.

In a preferred embodiment of the method of the invention the pH adjustments are made with HCl or NaOH, as appropriate.

In another preferred embodiment according to any one of the above embodiments, the enzymes used in step f) are proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens, and identified as EC 3.4.21.62 and 3.4.24.28; or a zinc dependent metalloprotease from Bacillus amyloliquefaciens identified as EC

3.4.24. Commercial enzymes of these types are the following: Protamex® (Novozyme) which at the date of the present application are the proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens (EC 3.4.21.62 and 3.4.24.28) and Neutrase® (Novozyme) which is a Zinc-dependent metalloprotease from Bacillus amyloliquefaciens (EC 3.4.24). Thus, in a preferred embodiment the proteolytic enzyme is Protamex® or Neutrase®.

In another preferred embodiment according to any one of the above embodiments, the Hydrolysis of step f) is carried out for 2 to 24 hours, preferably for 2 or 24 hours.

In a preferred embodiment according to any one of the above embodiments, the pH of step b) is 3, step c) is carried out at 100 ° C and the enzyme is Protamex®. Preferably, the hydrolysis reaction is carried out at pH 7.50 ° C for 2 hours. As shown in Table I , these conditions result in a hydrolyzate with a DPP-IV inhibition activity of 93.30%.

In another preferred embodiment according to any one of the above embodiments, the pH of step b) is 3, step c) is carried out at 50 ° C, and the enzyme is Neutrase®. Preferably, the hydrolysis reaction is carried out at pH 7, 25 ° C and 24 hours. As shown in Table I , these conditions result in a hydrolyzate with a DPP-IV inhibition activity of 83.22%.

In another preferred embodiment according to any one of the above embodiments, the pH of step b) is 3, step c) is carried out at 100 ° C, and the enzyme is Neutrase®. Preferably, the hydrolysis reaction is carried out at pH 7, 50 ° C and 24 hours. As shown in Table I , these conditions result in a hydrolyzate with a DPP-IV inhibition activity of 100%.

In a particular embodiment according to any one of the above embodiments, step g) is carried out by determining the iDPP-IV activity in vitro according to the method described in Tulipano et al (2011), and the selection of a hydrolyzate with activity> 80 % is made with the original undiluted hydrolyzate.

Thus, in a preferred embodiment according to any one of the aspects of the present invention, a hydrolyzate is considered to show iDPP-IV activity in vitro when the iDPP-IV activity is determined in vitro according to the method described in Tulipano et al (2011) , as is done in the examples of the invention, and the hydrolyzate shows an activity> 80%.

In a particular embodiment, the method comprises an additional step h), in which the hydrolyzate obtained in step g) is dried, preferably by heat in an oven, oven, lyophilizer or atomizer.

In a particular embodiment of the method of the invention, the method consists of steps a) -g) as defined in any of the embodiments described in the preceding paragraphs. In another particular embodiment, the method consists of steps a) -h) as defined in any of the embodiments described in the preceding paragraphs.

As indicated above, the raw material for preparing the hydrolyzate of the invention is GPPs, which are a byproduct of the meat industry. Thus, the method of production of the hydrolyzate of the present invention is a cheap and easy to industrialize method, which allows using a normally discarded industrial by-product.

In a sixth aspect, the present invention also relates to GPP hydrolysates obtainable by any of the methods described in the preceding paragraphs of the fifth aspect. In addition, it refers to medications, food supplements, food products or pharmaceutical compositions that comprise them, their use in therapy, and their use for the treatment of a condition in which the incretin system is altered, as defined in the second, third and fourth aspect of the invention.

Said hydrolysates obtainable according to the methods of the fifth aspect of the invention have iDPP-IV activity in vitro, antihyperglycemic activity in vivo and are capable of inducing GLP-1 secretion.

Lastly, it should be noted that the hydrolyzate of the present invention (according to any one of the embodiments of the first or sixth aspect of the invention) has hypoglycemic activity by means of an in vivo study in which normoglycemic rats were used and to which administered a hydrolyzate of the invention, as shown in Example 8. This hypoglycemic activity is not a desirable activity since it is the decrease in glucose of a healthy subject (person or animal), while the antihyperglycemic activity is the decrease of glucose in a subject with hyperglycemia. It is of great importance that the hydrolyzate of the invention show a therapeutic effect in the subjects with hyperglycemia and not in the healthy ones.

Examples

Specific examples of embodiments of the invention that serve to illustrate the invention are detailed below.

Example 1: Procedure for obtaining GPP powder.

15 kg of GPP Gallus gallus domesticus was washed with water and crushed in a Cato industrial crusher using an 8 mm cutting plate. This crushing was frozen in thin layers (1-1.5 cm) and lyophilized for 5 days. The lyophilisate was ground with a Fagor BV-850 model mixer and the ground obtained was sieved with a 2 mm pore size sieve. A fine powder with particle size <2 mm and a moisture content <5% (GPP powder) was thus obtained. The GPP powder that presented a higher percentage of moisture than the indicated was re-lyophilized or allowed to dry in an oven at 50 ° C for 15 h. Subsequently, it was stored at -20 ° C in closed boats and surrounded by silica gel to avoid an increase in the humidity of the samples.

Example 2. Procedure for obtaining hydrolysates from GPP powder. From the GPP powder, the hydrolysates were obtained following the following process, the conditions of which are summarized in Table I.

Once the GPP powder was obtained, a pre-treatment was carried out with changes in pH and / or temperature as indicated in Table I. 0.1 g of GPP powder was weighed in round bottom plastic tubes, a volume of distilled water was added, thus obtaining a solution with a pH of 7.5 (ie initial pH = 7.5). In those hydrolysates subjected to pH pretreatment, the pH was adjusted to 3 with HCl (0.1 M), obtaining a final volume of 4 mL. Subsequently, the tubes were placed in a water bath at a temperature of 25, 50 or 100 ° C, and left under stirring for 1.5 h.

After this time, the pre-treated samples at 50 or 100 ° C were allowed to cool and all the pre-treated samples were adjusted to pH 7 with 1 M NaOH or 0.1 N HCl. Subsequently, the samples were introduced into an oven for 5 min at 25 or 50 ° C so that the samples reached the desired temperature for hydrolysis and then the enzymatic hydrolysis was carried out.

To carry out said hydrolysis, 0.5 mL of a Neutrase® 0.8 L or Protamex® enzyme solution (dissolved in distilled water) was added to the GPP samples obtained at a GPP enzyme / protein concentration of 0.4 AU. The final reaction volume in all cases was 5 mL, so the volumes were adjusted with distilled water.

Hydrolysis was carried out at 25 or 50 ° C under constant agitation at 250 rpm in orbital, for between 2 and 24 h, as indicated in Table I. After the hydrolysis, the enzymes are heat inactivated in a water bath at 85 ° C for 10 min. The samples were cooled on ice for 10 min and centrifuged at 10,000 xg, 20 min, 4 ° C. The supernatants obtained are those which in the present invention are referred to as "hydrolysates", which were filtered with 0.45 p filters, and frozen at -20 ° C until further analysis.

Table I .- iDPP-IV activity (%) of GPP powder hydrolysates obtained with Neutrase® and Protamex® (0.4 AU) under different hydrolysis and pre-treatment conditions.

Pre-treatment Hydrolysis at pH 7 Hydrolyzed ibition

Ta (° C) pH Enzyme Ta (° C) t ° (h) P-IV (%)

p86 25 7.5 No 0

p103 25 7.5 Neutrase® 25 24 8.39

p39 50 7.5 Neutrase® 25 24 4.40

p102 25 3 Neutrase® 25 24 0.48

p38 50 3 Neutrase® 25 24 3.22

p115 25 7.5 Neutrase® 50 24 0.22

p19 100 7.5 Neutrase® 50 24 7.19

p114 25 3 Neutrase® 50 24 5.24

p16 100 3 Neutrase® 50 24 100

p111 25 7.5 Protamex® 50 2 4.05

p70 100 7.5 Protamex® 50 2 0.49

p110 25 3 Protamex® 50 2 3.30

100 3

Protamex® 50 2

3,30 p68

100 3

Protamex® 50 2

3.30

Example 3: Selection of hydrolysates obtained from GPP powder according to their DPP-IV inhibitory activity

The hydrolysates obtained according to Example 2 were selected according to their ability to inhibit the DPP-IV enzyme, which was determined as described in Example 4.

Only hydrolysates that showed a percentage of inhibition greater than (>) to 80% (marked in bold in Table I ) were selected. These hydrolysates were determined the minimum amount necessary to inhibit 50% of the DPP-IV enzyme (IC50), expressed as pL of hydrolyzate or pg of protein / mL of hydrolyzate ( Table II ). All hydrolysates had an IC50 between 4 and 4.82 pl of hydrolyzate or 270 and 303 pg of protein / mL of hydrolyzate. No significant differences were observed between the IC50, expressed in pg / mL, of the three hydrolysates studied.

Table II IDPP-IV activity represented in IC50 and protein content of GPP powder hydrolysates with greater inhibitions.

IDPP-IV activity

Protein content Hydrolyzed IC 50 IC 50

(mg / mL)

(HL) (Hg / mL)

p16 4.42 297.4 6.83

p38 4.82 302.9 6.24

p68 4.45 300.1 6.62

n = 6 obtained from the measurement of the duplicate activity of each of the hydrolysates

In the absence of significant differences in its activity (expressed as IC50), the hydrolyzate "p38" was selected since in its elaboration, it was the one that required the lowest temperature and therefore the lowest cost from the industrial point of view. with Neutrase® for 24 h at 25 ° C pH 7 with pre-treatment prior to hydrolysis of 50 ° C for 1.5 h at pH 3 (IC50 = 4.82 pL of hydrolyzate or 302.9 pg of protein / mL of hydrolyzate ).

Example 4: Measurement of DPP-IV inhibitory activity of hydrolysates, fractions and peptides

The determination of DPP-IV enzyme inhibitory activity in hydrolysates was performed using the method described by Tulipano et al (2011).

The test was performed in 96-well plates. For which, 15 pL of a swine enzyme solution of DPP-IV (Sigma-Aldrich) in 100 mM Tris HCl buffer pH 8.0 (0.26 mU / well) and 10 pL of the hydrolyzate were introduced into the wells to be analyzed, incubating for 10 min at 37 ° C. Subsequently, 50 pL of the Gly-Pro-pNA chromogenic substrate (final concentration of 0.2mM) (Bachem) was added and the well volume was adjusted to 100 pL with 100 mM Tris HCl buffer, pH 8.0 and the mixture was incubated for 30 min at 37 ° C. The iDPP-IV activity was measured at 405 nm every minute in the cited incubation period. In addition, positive (substrate enzyme) and negative (substrate hydrolyzed) controls were also performed. The validity of the method was verified by testing the compound diprotin A (Ile-Pro-Ne) which is a known inhibitor of this enzyme. The analysis was performed in triplicate.

From the absorbance data, kinetic curves were performed, obtaining a slope. DPP-IV inhibition expressed as a percentage and was calculated according to the following formula:

% inhibition = ((A- (B-C)) / A) x 100

A: slope obtained in the incubation of enzyme and substrate

B: slope obtained in the incubation of enzyme, substrate and hydrolyzate

C: slope obtained in the incubation of the substrate and the hydrolyzate

For the determination of the iDPP-IV activity of the fractions and the peptides the same procedure described above was followed with some variations. The fluorimetric substrate H-Gly-Pro-AMC (Bachem) was used at a final concentration in the well of 0.01 mM and the swine enzyme DPP-IV was supplied by Millipore. The DPPIV activity of the samples was recorded for 30 min at 37 ° C on a fluorimeter using an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

The protein concentration of the hydrolysates to determine the IC50 value was determined by the Kjeldahl method (FIL-IDF. "Milk. Determination of nitrogen content (Kjeldahl method). Standard 20B" .1993. Int. Dairy Fed. Brussels, Belgium ) multiplying the percentage of nitrogen in the sample by 6.25. While the protein concentration of the fractions was determined by the method of bicinconinic acid (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions.

Example 5: Isolation, identification and synthesis of peptides with iDPP-IV activity Analytical and semi -preparative high efficiency liquid chromatography (HPLC) equipment was used, as well as tandem mass spectrometry (MS) equipment that allowed the sequencing of peptides The steps mentioned below were performed.

5.A. Obtaining the soluble fraction of the "p38 hydrolyzate" of GPP powder

Following the procedure described in Example 2, 250 mL of the protein hydrolyzate called "p38 hydrolyzate" was obtained, the supernatant of which was centrifuged again in filtering devices (Centripep, Amicon Inc) with a hydrophilic membrane of 3000 Da pore size. The permeate (fraction less than 3000 Da) obtained was collected, lyophilized and stored at -20 ° C until its subsequent fractionation.

5.B Fractionation by HPLC in reverse phase at semi-preparatory scale

The semipreparative HPLC used was from the Agilent 1260 series (Agilent Technologies) consisting of a quaternary pump, a gradient controller, an injector, an array diode detector, a fraction collector and data acquisition and processing software ( Agilent OpenLab CDS ChemStation Edition for LC & LC / MS systems A.01.04).

The lyophilized permeate obtained in section 5A was dissolved in water at a concentration of 100 mg of lyophilized residue / mL and the peptides were separated by reverse phase chromatography using a C18 column (Europa peptide, 120 A, 25 x 1.0 mm, 5 ^ m; Teknokroma). The solvents used were a mixture of water: trifluoroacetic acid (1000: 1) and acetonitrile: trifluoroacetic acid (1000: 0.8), solvents A and B respectively, and elution was carried out at a flow of 4 mL / min using the following gradients in the order of appearance: 0 to 40% of solvent B in 50 min), 40-90% of B in 3 min, kept at 90% of B for 2 min, 90-100% of B in 1 min . The volume of sample injected was 750 ^ L and the absorbance of the solvent was monitored at 214 nm.

11 different fractions ( Figure 1 ) called F.1-F.11 were collected from the protein hydrolyzate called "p38 hydrolyzate", which were lyophilized and reconstituted in Milli-Q water at different volumes. The protein content of these fractions reconstituted was determined by the method of bicinconinic acid (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions, subsequently diluted to a concentration of 0.20 mg / mL (except for fraction 11 whose protein content was lower at this concentration) and iDPP-IV activity was determined according to the protocol described in Example 4 (see Table 111 ). At the protein concentration tested, all fractions showed a certain iDPP-IV activity although only 4 of them, F.1, F.3, F.8 and F.10, showed inhibitions greater than 80%.

Additionally, those fractions with a percentage of iDPP-IV activity greater than 40% were determined the IC50 ( Table 111 ), thus discarding fraction F.6. The results obtained showed that only 2 of the fractions tested had an IC50 of less than 100 ^ g / mL (F.1 and F.3), with fraction F.3 showing the lowest IC50 value (69 ^ g of protein / mL versus 82.96 g / mL protein for F.3 and F.1, respectively), which indicated that it was the fraction with the highest iDPP-IV activity. Although the F.11 fraction could not be determined by its IC50 due to the small amount of fraction collected and its low protein content, it was discarded as a fraction with good iDPP-IV activity, since according to the results obtained, at a concentration of 90 ^ g protein / mL, only It showed 32.80% of activity, so to reach a 50% inhibition, you would need a higher concentration of protein, greater than 100 ^ g of protein / mL. This value is higher than that shown by fractions F.1 and F.3.

Fraction F.3 corresponded to the eluted peptides between approximately 8.5 and 11.8 min ( Figure 1 ).

Table III Protein content, percentage of activity iDPP-IV and IC50 of the fractions obtained by reverse phase HPLC from the permeate of size <3000 Da obtained from the protein hydrolyzate called "p38 hydrolyzate".

n = 6 per fraction

ND: Not determined

Fraction F.3, which was the fraction with the highest iDPP-IV activity, underwent a second fractionation by reverse phase HPLC on a semi-preparative scale using the same equipment, column and solvents but eluting the sample with a linear gradient of 0- 12% of solvent B in A in 30 minutes and 12% of solvent B in A was maintained for 1 min. To carry out the separation of the peptides contained in this fraction, it was necessary to reconstitute it in Milli-Q water, obtaining a solution of 50 mg of lyophilized residue / mL. In this case, the injection volume was 250 mL and the absorbance of the solvent was monitored at 214 and 280 nm. 4 subfractions were collected from the fraction F.3 (F.3.1, F.3.2, F.3.3 and F.3.4) ( Figure 2 ), which were lyophilized and kept at -20 ° C until further analysis.

In order to determine which of the subfractions collected from fraction F.3 contained the peptides with iDPP-IV activity, these subfractions were determined their inhibitory activity following the method already described in Example 4 and the protein concentration by the BCA method (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions (see Table IV ). In order to carry out these analyzes it was necessary to reconstitute the Milli-Q water fractions and they were all diluted in the same solution until a protein concentration of 0.1 mg / mL in each of them. Three of the four fractions showed inhibitions greater than 50% (F.3.2, F.3.3, F.3.4). These three fractions were also determined the concentration necessary to inhibit 50% of the enzyme. The results of this analysis are shown in Table IV and, as can be seen, fraction F.3.3 showed the lowest IC50 values (26.85 g protein / mL). This subfraction F.3.3 eluted approximately between minutes 11 and 13 ( Figure 2 ), using the separation method described above.

Table IV Protein content, iDPP-IV activity and IC50 of the sub-fractions obtained by semi-preparative HPLC in reverse phase from fraction F.3.

n = 6 per fraction

ND: Not determined

5.C. Identification of peptides with iDPP-IV activity by tandem mass spectrometry (MS / MS)

For the identification of the peptides responsible for the iDPP-IV activity of subfraction F.3.3, obtained by double chromatographic separation of the fraction <3000 Da of the "p38 hydrolyzate", an LTQ Orbitrap Velos mass spectrometer was used (ThermoFisher Scientific) equipped with an ionization source connected online to an Easy-II nano-HPLC (Proxeon, Thermo Fisher Scientific) . For which, subfraction F3.3 was reconstituted in Milli-Q water and diluted with 0.1% trifluoroacetic acid until a protein concentration of 0.1 ^ g / ^ l was achieved. The subfraction peptides were first passed through a C18 EASY-Column pre-column (2 cm in length, ID 100 ^ m, 5 ^ m in particle size; Thermo Fisher Scientific) to eliminate salts that the subfraction could have and separated into a reversed phase C18 nano column (15 cm in length, ID 75 ^ m, 3 ^ m particle; Nikkyo technos Co. LTD) into which 10 ^ L of the diluted fraction was injected. Chromatographic separation was performed with a continuous phase B gradient (0 min 2%, 2 min 2%, 42 min 20%, 72 min 32%, 82 min 50%, 92 min 95%, 102 min 95%, 103 min 2%) using water as mobile phases milli Q water containing 0.1% formic acid (phase A) and acetonitrile containing 0.1% formic acid (phase B) and at a flow rate of 300 nL / min.

All mass spectra were acquired in positive ion mode. The scan (m / z 50-2000) was acquired with a target value of 10,000 in a resolution of 30,000 to 400 m / z and the 10 most intense ions were selected for collision-induced dissociation fragmentation in the LTQ with a target value of 10000 and a collision energy of 35%.

Proteins / peptides were identified in the Proteome Discoverer v.1.4.0.288 (Thermo Fisher) program. All MS and MS / MS spectra were analyzed using the Mascot search engine (v.2.5). Mascot was programmed to search the Gallus_20170224.fasta database (2281 entries) belonging to the Swiss-Prot database and assuming there was no enzymatic digestion. An error of 0.8 Da was allowed for mass of ions of IT-MS / MS fragments and 10.0 ppm for a mass of primary ions of FT-MS. Oxidation of methionine was established as dynamic modifications and the false discovery rate (FDR) and protein probabilities were calculated by Perclorator. Only peptides with a Mascot score greater than 20 and an ACn less than 0.05 were considered. For the relative quantification of the peptide, the precursor ion area of the peptide was used. The peptides that are collected in Table V were identified.

Table V. Peptides identified in subfraction F.3.3

Mass No. Mass

I KNOW THAT. ID. N °:

perimental theoretical aminoac

perimental

1 7 806.88 806.39

2 7 794.96 794.47

3 10 976.02 975.44

4 9 1029.21 1029.59

5 9 1081.20 1081.51

6 7 700.88 700.45

7 8 842.96 842.46

8 9 963.15 963.55

9 9 944.11 944.53

8

989,19

988,57 10

8

989.19

988.57

5.D Synthesis of peptides with iDPP-IV activity

The peptides of the invention were sent to chemically synthesized to Caslo ApS (Lyngby, Denmark) and were obtained prepared with the purity values shown in Table VI .

Once synthesized, their iDPP-IV activity was determined in order to know which or which of the peptides identified in subfraction F.3.3 were responsible for this activity in the fraction. For this, the peptides were reconstituted in Milli-Q water (SEQ ID NO: 1, 2, 3, 5, 6, 7 and 9) or in 1% DMSO (SEQ. ID.No .: 4, 8 and 10 ) depending on its solubility. The iDPP-IV activity of the peptides was determined according to the protocol described in Example 4. Table VI shows the percentages of inhibition shown by the different peptides identified in subfraction F.3.3 at a concentration of 500 ^ M. At the concentration tested, few peptides could inhibit the enzyme, only three of them showed a higher inhibition of 10% (SEQ ID NO: 1,2 and 3). Clearly, SEQ ID No. 1 was the one that showed the highest inhibition with 41.91% at a concentration of 500 ^ M, its IC50 being 818.04 ^ M. The IC50 of peptides SEQ ID N ° 2 and 3, which also showed a certain iDPP-IV activity, could not be determined due to their low solubility at high concentrations. However, the results obtained indicate that their IC50 is greater than 2 mM since at this concentration, these peptides showed inhibition percentages of 41 and 49 (SEQ ID N ° 2 and 3, respectively).

Table VI Characterization of the identified peptides of subfraction F.3.3

Peptide Activida

ureza (%) ^{50 (m M)}

SEC ID iDPP-IV (

1 99.03 41.91 18.04

2 99.41 17.99> 2000

3 95.91 11.88> 2000

4 98.74 3.42 ND

5 99.62 1.95 ND

6 98.98 4.10 ND

7 95.48 6.89 ND

8 97.54 0.00 ND

95,26 0,00 ND 9

95.26 0.00 ND

0,00

ND 10 95.73

0.00

ND

n = 6 per peptide

ND: Not determined

Example 6: Determination of the antihyperglycemic effect of "p38 hydrolyzate" of GPP powder in rats with diet-induced glucose intolerance.

To evaluate the antihyperglycemic effect of the selected hydrolyzate, “p38 hydrolyzate”, an acute study was carried out, with animals that presented diet-induced glucose intolerance by performing a tolerance test to it following the administration of the “hydrolyzate” p38 ".

The animal model used were Wistar rats (Harlan, Barcelona, Spain) obese females of 20 weeks. The animals used in the study were housed individually, with free access to food and water, and with temperature conditions of 22 ° C and cycles of 12 hours of light and 12 hours of darkness. The development of obesity was due to a diet with cafeteria diet (CAF) composed of carrots, bacon and milk with sugar and also with standard feed for 10 weeks. After these 10 weeks, the animals were divided into two groups: the control group, which was treated with water, and the group treated with hydrolyzate. On the day of the experiment at nine in the morning, the treatments were administered to the animals with an intragastric tube (fasted from the previous night). The doses administered were 1 mL of water or 1 mL of hydrolyzate (300 mg of protein / kg of weight of the animal dissolved in water). After 40 minutes, blood was drawn from the tail (extraction at time 0) and then given a dose of glucose (2 g / kg animal weight), also with intragastric tube. Subsequently, blood samples were taken from the tail at 15, 30, 60 and 120 minutes after administration of the glucose dose. Blood samples were quickly centrifuged (2500 xg, 4 ° C, 15 min) to separate the plasma, which was stored at -80 ° C until further analysis.

Prior to the development of obesity and dietary glucose intolerance in these animals. These animals, fed with a standard diet (STD) consisting of feed and water ad libitum, were also tested for glucose tolerance, as previously described, after having administered 1 mL of water or 1 mL of hydrolyzate (300 mg of protein / kg of weight of the animal dissolved in water). Blood was also collected from the tail under the same conditions previously described.

The procedure used in the study was approved by the Ethical Committee of the Rovira i Virgili University.

The antihyperglycemic effect of the "p38 hydrolyzate" was evaluated by quantifying the plasma glucose levels of the animals treated with the hydrolyzate compared to those not treated. Glucose concentrations were determined using the enzymatic colorimetry kit (QCA Glucose oxidase-Peroxidase method). The results obtained were grouped and represented as plasma glucose concentration at different study times (mean ± the standard error of the mean (ESM) for a minimum of 6-7 homogeneous tests).

The kinetics of the different groups of animals were compared in a 2-way analysis of variance (ANOVA) using the Bonferroni test and the difference for values of p <0.05 was considered significant. In addition, the results were also represented by calculating the area under the curve described above. A 1-way variance analysis (ANOVA) was performed using the Bonferroni test at the average of the areas in order to check if there were significant differences between groups. The statistical program SPSS (IBM SPSS Statistics software version 20.0) was used for all statistical analyzes.

Figure 3 A shows the plasma glucose levels observed in healthy rats fed with standard diet (STD) and treated with water and in obese rats fed with cafeteria diet (CAF) and treated with water or with the “hydrolyzed p38”, after of the intake of a dose of 2 g / kg of the animal's glucose weight (glucose tolerance test). At 15 min of glucose intake, an increase in plasma levels of this sugar was observed in all cases, the highest being that obtained in CAF rats treated with water. Treatment with the "p38 hydrolyzate" in CAF rats reduced this glucose peak, being statistically lower than that observed in coffee rats treated with water and similar to that shown by STD rats. As the post-time elapsed administration, plasma glucose levels were decreasing in STD rats until the initial values were recovered, while in the CAF treated with water the original values (glucose intolerance) were not recovered.In the case of CAF rats treated with the "p38 hydrolyzed", glucose levels between 30 and 120 min showed no significant differences with the glucose levels observed in STD rats.

Figure 3 B shows the area below the curve calculated from the curve made with plasma glucose levels obtained during the glucose tolerance test in STD female rats treated with water or CAF treated with water or "p38 hydrolyzate." Thus, the area of glucose under the observed curve was significantly smaller in the CAF animals treated with the "p38 hydrolyzate" than in the CAF group treated with water, with no significant differences observed with the area obtained by STD rats. treated with water

Therefore, in the animal model of diet-induced glucose intolerance, the "p38 hydrolyzate" produced a decrease in plasma glucose peak and tended to normalize glucose values, demonstrating its antihyperglycemic effect.

Example 7: Determination of the antihyperglycemic effect of the "p38 hydrolyzate" of GPP powder in rats with age-induced glucose intolerance.

The possible antihyperglycemic effect of "p38 hydrolyzate" was also tested in another animal model with glucose intolerance, in this case, age-induced.

Male Wistar (Harlan) rats of 7 months (30 weeks) of age g were used, which were fed with standard diet (STD) based on standard feed and water ad libitum. The housing conditions were similar to those described in Example 6. The animals were divided into three groups and treated with 1.5 mL of water, 1.5 mL of hydrolyzate (300 mg of protein / kg of weight of the dissolved animal in water) or 1 mL of vildagliptin (1 mg / mL of water, commercial DPP-IV inhibitor, as a positive control) administered acutely by intragastric tube. Subsequently, they were tested for glucose tolerance and blood was collected at different times. The methodology used is described in Example 6. The results obtained were grouped and represented as plasma glucose concentration at different study times (mean ± ESM for a minimum of 6-7 homogeneous tests).

The kinetics of the different groups of animals were compared in a 2-way analysis of variance (ANOVA) using the Bonferroni test and the difference for values of p <0.05 was considered significant. In addition, the results were also represented by calculating the area under the curve described above. A 1-way variance analysis (ANOVA) was performed using the Bonferroni test at the average of the areas in order to check if there were significant differences between groups. The statistical program SPSS (IBM SPSS Statistics software version 20.0) was used for all statistical analyzes.

Figure 4A shows the curves and Figure 4 B the area of the curves obtained by analyzing the plasma glucose levels of elderly male rats treated with water, "hydrolyzed p38" or vildagliptin. As expected, rats treated with water were shown to be glucose intolerant by not lowering glucose levels after 120 min post-administration. However, rats treated with vildagliptin reversed this effect.

The results show that in this animal model of age-induced glucose intolerance, treatment with the hydrolyzate tended to decrease the plasma glucose curve, normalizing plasma glucose levels at 120 min after ingestion of this sugar by which demonstrates its antihyperglycemic effect in this animal model.

Example 8: Determination of the possible antihyperglycemic effect of the "p38 hydrolyzate" of GPP powder in healthy rats.

In order to rule out whether the hydrolyzate had an antihyperglycemic effect in healthy rats, 8-week-old female Wistar rats fed with standard diet (STD) were used and given 1 mL of water or 1 mL of “p38 hydrolyzate” by intragastric tube. (300 mg of protein / kg of weight of the animal dissolved in water). Subsequently, these animals were tested for glucose tolerance and plasma glucose content was measured. The methodology used in both cases is described in Example 6. The Results obtained were grouped and represented as plasma glucose concentration at different study times (mean ± standard error of the mean (ESM) for a minimum of 6-7 homogeneous tests). The curve obtained was represented and the area under the curve was quantified. An average Student Test analysis was performed in order to verify if there were significant differences between groups using the SPSS statistical program (IBM SPSS Statistics software version 20.0).

Figure 5 shows the area under the glucose curve obtained from the plasma glucose content during this sugar tolerance test, in healthy female rats treated with water or the "p38 hydrolyzate." The area obtained by both treatments It was similar, with no significant differences between them. This result indicates that the administration of "p38 hydrolyzate" has no effect on plasma glucose levels induced by the performance of a glucose tolerance test.

Example 9: Measurement of the ability to induce secretion of GLP-1

In order to elucidate the mechanisms by which the "p38 hydrolyzate" has antihyperglycemic effect in rats with glucose intolerance, in addition to its DPP-IV enzyme inhibitory effect demonstrated in vitro (Tables I and II), the capacity was evaluated of secretion of GLP-1 by the "p38 hydrolyzate" by two studies, one in vitro and one ex vivo.

9.1.- In vitro study

The cell line used was the STC-1, supplied by Dr. B. Wice (University of Washington, St. Louis) with the permission of Dr. D. Hanahan (University of California, San Francisco). This cell line is enteroendocrine from a transgenic mouse tumor (Rindi et al, "Developmentof Neuroendocrine Tumors in the Gastrointestinal Tract of Transgenic Mice." 1990. American Journal of phatology. Vol. 136, No 6). The cells were cultured with DMEM (Dulbecco's Modified Eagle Medium) containing GlutaMAX ™ and 4.5 g / L D-glucose, without sodium pyruvate (Gibco®), supplemented with 17.5% fetal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin (BioWhittaker). The cells were incubated at 37 ° C at 5% CO2 with a humidified atmosphere.

For secretion studies, cells were grown in 24-well plates at a density of 2.0 * 105 cells / well. After two days of growth (density of 80-90% confluence) the secretion study was performed. The cells were washed twice with HEPES buffer (20 mM HEPES, 140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl2, pH 7.4). Subsequently, the "p38 hydrolyzate" in the wells was added to a final concentration of 5 mg of the lyophilized hydrolyzate / mL, dissolved in HEPES buffer with 10 mM glucose and incubated for two (2) hours at 37 ° C at 5% of CO2 with a humidified atmosphere. HEPES with 10 mM glucose was used as a negative control. After incubation, supernatants were collected, which were centrifuged (1000 xg) to remove cell debris and stored at -80 ° C until analyzed. The cells were also collected, which were reconstituted in lysis buffer to extract the total protein from them. The protein content was determined using the BCA method (BCATM Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions. Three replicates from different cell passages were analyzed, including three wells per condition in each replicate. All studies were done between passes 30 and 50.

All the obtained supernatants were determined the concentration of active GLP-1 with the active GLP-1 ELISA kit (Millipore) following the manufacturer's instructions. Concentration levels of active GLP-1 were normalized with the concentration of total protein obtained from cell lysis.

Figure 6A shows the levels of active GLP-1 secreted by STC-1 cells after exposure for 2 hours to HEPES with 10 mM glucose (control) or to the "p38 hydrolyzate dissolved in HEPES with 10 mM glucose. Treatment with the "p38 hydrolyzate" produced a significant increase in the secretion of active GLP-1, being seven times greater than the content of GLP-1 secreted by the cells treated with the control solution.

9.2.- Ex vivo study

Segments of the distal ileum from female Wistar rats were used. To obtain the segments, an incision was made in the ileocecal junction and from that junction, 8 cm of the distal part of the ileum was removed. This segment was cut into 6 parts of 0.75 cm2 in cold HBSS (Hank's balanced salt solution, Termofisher) buffer. These ileum segments were individually placed in 24-well plates and incubated for one hour at 37 ° C and 5% CO2 with a solution of KRBS pH 7.4 (which contained 5 mmol / L KCl, 138 mmol / L NaCl , 4.2 mmol / L NaHCO3, 1.2 mmol / L NaH2PO4, 2.6 mmol / L CaCl2, 1.2 mmol / L MgCl2 and 10 mmol / L HEPES) enriched with 10 mM glucose and 0.1 mM diprotin A, DPP-IV inhibitor, (control) or with the same solution (KRBS glucose diprotin A) but supplemented with the "p38 hydrolyzate" (final concentration of 15 mg of lyophilized hydrolyzate / mL).

After incubation, supernatants were collected, centrifuged (1000 x g) to remove cell debris and stored at -80 ° C until analysis. The supernatants obtained were determined the concentration of active GLP-1 with the active GLP-1 ELISA kit (Millipore) following the manufacturer's instructions. The normalization of the results was carried out taking into account the milligrams of the ileum segment of each well.

Figure 6B shows the levels of active GLP-1 secreted by the ileum segments after 1 hour exposure to "p38 hydrolyzate" and to the control solution, in a medium containing a DPP-IV inhibitor to ensure that the observed effects they are about the secretion of the hormone and not by modulation of said enzyme. Treatment with the "p38 hydrolyzate" stimulated the cells of the ileum to secrete active GLP-1, significantly increasing the levels of active GLP-1 compared to the levels induced by the control solution.

Claims (22)

1. - An isolated peptide of sequence SEQ ID No. 1, for use in the treatment and / or prevention of a condition in which the incretin system is altered. 2. - The peptide for use according to the preceding claim, wherein the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered fasting plasma glucose conditions, hyperglycemia, Metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof. 3. - A combination comprising a peptide of sequence SEQ ID No. 1 and a peptide of sequence SEQ ID No. 2, for use in the treatment and / or prevention of a condition in which the incretin system is altered. 4. - A combination comprising: - a peptide of sequence SEQ ID No. 1, and optionally a peptide of sequence SEQ ID No. 2, and - one, two, three, four or five peptides whose sequence is selected from the group formed by SEQ ID N ° 3-7, for use in the treatment and / or prevention of a condition in which the incretin system is altered. 5. - The combination for use according to claim 3 or 4, further comprising one, two or three peptides whose sequence is selected from the group consisting of SEQ ID No. 8-10. 6. - The combination for use according to any one of claims 3-5, wherein the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered plasma glucose conditions fasting, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof. 7. - A protein hydrolyzate characterized in that it has DPP-IV enzyme inhibitory activity in vitro, comprises a peptide of sequence SEQ ID No. 1, and optionally a peptide of sequence SEQ ID No. 2, and is a protein hydrolyzate of paws claws chicken. 8. - The hydrolyzate according to the preceding claim, which further comprises one, two, three, four or five peptides whose sequence is selected from the group formed by SEQ ID NO. 3-7. 9. - The hydrolyzate according to claim 7 or 8, which further comprises one, two or three peptides whose sequence is selected from the group formed by SEQ ID N ° 8-10. 10. - The hydrolyzate according to any one of claims 7-9, which comprises the sequence peptide SEQ ID No. 2. 11. - A hydrolyzate according to any one of claims 7-10, for use in therapy. 12. - A hydrolyzate according to any one of claims 7 to 10, for use in the treatment and / or prevention of a condition in which the incretin system is altered. 13. - The hydrolyzate for use according to the preceding claim, wherein the condition in which the incretin system is altered is selected from the group consisting of diabetes, type 2 diabetes mellitus, IGT, altered fasting plasma glucose conditions, hyperglycemia, metabolic acidosis, ketosis, arthritis, multiple sclerosis, Graves disease, overweight, obesity, osteoporosis and combinations thereof. 14. - A medicament, food supplement or food product characterized in that it comprises a hydrolyzate according to any one of claims 7-10 and / or a peptide as defined in claim 1 or a combination as defined in any one of the claims 3-5. 15. - A pharmaceutical composition characterized in that it comprises a hydrolyzate according to any one of claims 7-10 and / or a peptide as defined in claim 1 or a combination as defined in any one of claims 3-5, and a pharmaceutically acceptable excipient. 16. - A method for preparing a hydrolyzate according to any one of claims 7 10, characterized in that it comprises the following steps: a) crush chicken leg claws and lyophilize to obtain a powder material with particle size smaller than 2 mm; b) adjust an aqueous solution of the above powder to a pH of 2-4; c) heat at a temperature between 40 ° C and 120 ° C, for 10 to 120 min; d) cooling the previous solution to a temperature between 20 and 55 ° C; e) adjust the pH of the previous solution to a pH between 6.5-7.5; f) add a proteolytic enzyme selected from the group consisting of: - proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens identified as EC 3.4.21.62 and 3.4.24.28, - a Bacillus licheniformis serine protease identified as EC 3.4.21.62, - a Zinc-dependent metalloprotease from Bacillus amyloliquefaciens identified as EC 3.4.24, and - a mixture between them, and perform enzymatic hydrolysis for a time between 1 and 24 h; Y g) determine the iDPP-IV activity and select a hydrolyzate with an iDPP-IV activity greater than 80%. 17. - The method according to claim 16, wherein step b) is carried out at pH 3. 18. - The method according to claim 16 or 17, wherein step c) is carried out at a temperature of 50-100 ° C. 19. - The method according to any one of claims 16 to 18, wherein step c) is carried out for between 70-100 minutes. 20. - The method according to any one of claims 16 to 19, wherein the temperature of step d) is between 25 ° C and 50 ° C 21. - The method according to any one of claims 16 to 20, wherein in step f) the enzyme used is: - proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens identified as EC 3.4.21.62 and 3.4.24.28, or - a Bacillus licheniformis serine protease identified as EC 3.4.21.62. 22. - The method according to any one of claims 16 to 21, comprising an additional stage, step h), wherein the hydrolyzate of step g) is dried.