Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
  • A23J3/342—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of collagen; of gelatin
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/18—Peptides; Protein hydrolysates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/56—Materials from animals other than mammals
  • A61K35/60—Fish, e.g. seahorses; Fish eggs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/01—Hydrolysed proteins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/01—Hydrolysed proteins; Derivatives thereof
  • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
  • A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
  • B01D15/361—Ion-exchange
  • B01D15/363—Anion-exchange
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2200/00—Function of food ingredients
  • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
  • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2250/00—Food ingredients
  • A23V2250/54—Proteins
  • A23V2250/542—Animal Protein
  • A23V2250/543—Fish protein
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2300/00—Processes
  • A23V2300/28—Hydrolysis, degree of hydrolysis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to a composition of fish skin (s) collagen peptides.
• the invention relates in particular to such a peptide composition obtained by enzymatic hydrolysis of fish skin collagen (s).
• the invention also relates to such a composition for use as a medicament.
• the invention thus relates to the use of such a composition for the curative or preventive treatment of a pathology affecting the human or animal body.
• the invention particularly relates to such a composition for its use as a medicament in the treatment of a gastrointestinal candidiasis in humans or animals and / or in the treatment of intestinal inflammation in humans or animals. animal.
• the invention also relates to such a composition for use as a medicament in a microbiota stimulating treatment.
• FR 2 720 067 discloses a peptide powder obtained by hydrolysis by papain of a collagen-rich raw material derived from the skin or skeleton of fish, molluscs or crustaceans. In the peptide powder obtained, 38% of the peptides have a molecular weight of between 10,000 Da and 50,000 Da.
• the peptide composition of FR 2 720 067 has a wide range of molecular weights, particularly greater than 10% of peptides with a molecular weight greater than 10,000 Da.
• the peptide composition of FR 2 720 067 is heterogeneous in peptide size and has a water-soluble peptide fraction of only 80% to 90%. Such high molecular weight peptide powders higher than 10,000 Da are not perfectly water soluble. They are also not totally absorbable by the digestive tract and therefore pose the problem of their bioavailability.
• FR 2 720 067 also describes the use of such a peptide powder obtained from live fish in the great depths in the curative treatment of inflammation of the tendons of the joints of the lower limbs (knees, balls and hooves) of horses race. Obtaining such a composition is problematic. It requires the removal of abyssal fish. In addition, such a powder is limited in its use in the treatment of joint inflammation.
• the invention therefore aims to overcome these disadvantages.
• the aim of the invention is to propose a novel peptide composition resulting from an enzymatic hydrolysis of skin skin (s) collagen (s), said peptides being water-soluble, ie 100% water-soluble, and totally absorbable by the digestive tract.
• the invention also aims to propose a novel peptide composition formed by enzymatic hydrolysis of skin collagen (x) of temperate water fish (s).
• the invention also aims at providing such a peptide composition that can be used as a medicament.
• the invention aims in particular to provide such a peptide composition resulting from an enzymatic hydrolysis of skin collagen (x) of temperate water fish (s) and may be used as a drug.
• the aim of the invention is to propose such a peptide composition which at the same time has a distribution of their apparent molecular weights extending over a narrow range of between one hundred and a few thousand daltons, ie excluding peptides from high apparent molecular weights, and also exhibiting a low proportion of apparent low molecular weight peptides.
• the object of the invention is to propose such a peptide composition that can be used as a medicament in the treatment of intestinal candidiasis.
• the invention also aims at providing such a peptide composition that can be used as a medicament in the treatment of inflammatory digestive diseases, including inflammatory bowel disease (IBD).
• IBD inflammatory bowel disease
• the invention also aims to propose such a peptide composition that can be used to promote the balance and maintenance of intestinal flora (or microbiota).
• the invention also aims to propose the use of such a peptide composition resulting from an enzymatic hydrolysis of collagen of skin (s) of fish (s) of temperate waters as a dietary supplement.
• the invention relates to a peptide composition having an aminogram in which:
• the glycine is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 20.0% and 24.5%;
• the hydroxyproline is in a molar amount such that the ratio of this quantity to the sum of the molar amounts of the amino acids in the composition is between 6.0% and 12.0%, in particular between 7.0% and 11, 0%;
• the proline is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 10.6% and 14.6%, especially between 11.6% and 13.6%; %, in particular between 12.1% and 13.1%, preferably of the order of 12.6%; the peptide composition exhibiting, in an exclusion chromatography analysis, in which each peptide of the peptide composition is eluted with a retention time representative of the apparent molecular weight of that peptide, an elution curve (c ') that is, a chromatogram elution curve) peptides having a value of area under the curve (i.e., an area value representative of the mass quantity of peptides) corresponding to the peptides of apparent molecular weight less than 1400 Da such that the ratio of this area value to the total area under the curve (corresponding to all the peptides of the composition) is less than 40%, in particular less than 38%, preferably between 30% and 38%, in particular between 30% and 35%
• a filtration column of dimensions 300 x 7.8 mm comprising a stationary phase formed of a silica gel with a porosity of 5 ⁇ ;
• the column being maintained at a temperature of 40 ° C .;
• aminogram means the list of free amino acids forming, by peptide linking (or binding), the sequence of a peptide or the peptide sequences of a mixture of peptides. Such an aminogram is obtained by analysis - in particular by global analysis or by sequential analysis - of the amino acids constituting a peptide or a mixture of peptides.
• the nature and amount of the constituent amino acids of the peptides of the composition according to the invention are determined by any overall analysis method known in itself to those skilled in the art.
• this analysis is carried out in accordance with ISO 13903: 2005 by assaying free and total amino acids by means of an amino acid analyzer or by means of high performance liquid chromatography (HPLC) equipment. Hydroxyproline is assayed by continuous flow analysis and colorimetric detection.
• the aminogram obtained by global analysis of the composition according to the invention is representative of the amino acid composition of collagen of skins of temperate water fish.
• the invention thus relates to such a peptide composition resulting from an enzymatic hydrolysis of collagen from skins of temperate water fish by a cysteine protease of plant origin - in particular a protease of the class EC 3.4.22.2-.
• protease cysteine provides a composition of peptides which are water-soluble - i.e., 100% water soluble - and which are fully absorbable through the digestive tract and have an apparent molecular weight distribution that extends over a narrow range between one hundred and a few thousand daltons, ie excluding high apparent molecular weight peptides, and also having a low proportion of apparent low molecular weight peptides.
• a separation of the peptides from the peptide composition according to the invention is carried out as a function of their apparent molecular weight by subjecting the peptide composition to an analytical separation step by liquid chromatography on a silica gel filtration column ( BioSep-SEC-S2000, Phenomenex, Peck, France) porous and high density in silanol groups.
• a solution comprising (A) ultra-pure water supplemented with trifluoroacetic acid (0.1% by volume) and (B) acetonitrile (A / B; 75/25; / v).
• the gel filtration column is maintained at a temperature of 40 ° C during the analysis.
• the flow rate of the mobile phase in the stationary phase is 0.6 mL / min.
• the volume of peptide composition to be analyzed introduced at the top of the gel filtration column is 25 ⁇ L ⁇ and the detection is carried out continuously by absorbance at the wavelength of 214 nm.
• a chromatogram is obtained on which each peak is characterized by a value of time or retention time (expressed in minutes following the introduction of the mixture to be analyzed at the top of the column) determined at the maximum value of peak absorbance.
• the apparent molecular weight of each peptide corresponding to this retention time value is determined at the maximum absorbance value of each peak by means of a predetermined calibration curve obtained by analysis under the same chromatographic conditions as described above. above-peptides of apparent molecular weight determined.
• a calibration curve is produced by analyzing, under the same chromatographic conditions, a mixture of peptides / reference proteins and apparent molecular weight known and between 100 Da and 30 kDa.
• the chromatogram representing the variation of absorbance at 214 nm during the chromatographic analysis of the peptide composition according to the invention determines the proportion of peptides with an apparent molecular weight of less than 1400 Da by evaluating the ratio of the value of the area extending under the curve-that is, the sum of the areas extending under the peaks of the curve-corresponding to peptides of apparent molecular weight less than 1400 Da over the value of the total area extending under the entire curve-that is, the sum of the areas extending under each peak of the curve-and corresponding to all the peptides of the peptide composition .
• the invention therefore relates to a composition of peptides derived from an enzymatic hydrolysis of collagen from skins of temperate water fish by a cysteine protease of plant origin, each peptide of the composition having an amino acid number of between 2 and a few tens, preferably between 2 amino acids and 100 amino acids.
• the composition according to the invention has an aminogram in which the glycine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 20.0% and 22.4%, especially between 20.0% and 21.9%, in particular between 20.4% and 21.4%.
• the composition according to the invention has an aminogram in which the glycine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 22.4%. and 24.9%, especially between 22.9% and 24.4%, in particular between 23.0% and 24.0%.
• the composition according to the invention has an aminogram in which hydroxyproline is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 7.0% and 9.0%, in particular between 7.5% and 8.5%, preferably between 7.7% and 8.5%.
• the composition according to the invention has an aminogram in which the hydroxyproline is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 9.5. % and 11.5%, in particular between 10.0% and 11.0%, preferably of the order of 10.5%.
• the composition according to the invention has an aminogram in which the glutamic acid is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 8.0. % and 13.0%.
• the composition according to the invention has an aminogram in which the glutamic acid is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 8.0. % and 10.0%, in particular between 8.5% and 9.5%, preferably between 9.0% and 9.5%.
• the composition according to the invention has an aminogram in which the glutamic acid is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 10, 5% and 12.5%, in particular between 11.0% and 12.0%, preferably in the order of 11.6%.
• the composition according to the invention has an aminogram in which the arginine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 6.9%. and 10.9%, between 7.9% and 9.9%, in particular between 8.0% and 9.0%.
• the composition according to the invention has an aminogram in which the alanine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 7.3%. and 11.5%, especially between 8.0% and 10.0%, in particular between 8.1% and 9.6%.
• the composition according to the invention has an aminogram in which the aspartic acid is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 3.1. % and 7.1%, especially between 4.1% and 6.1%, in particular between 4.6% and 5.6%, preferably between 5.0% and 5.5%.
• the composition according to the invention has an aminogram in which the lysine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 1.5% and 5.5%, especially between 2.5% and 4.5%, in particular between 3.0% and 4.0%, preferably between 3.1% and 3.6%.
• the composition according to the invention has an aminogram in which the serine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 1.5% and 5.5%, especially between 2.5% and 4.5%, in particular between 3.0% and 4.0%, preferably between 3.2% and 3.6%.
• the composition according to the invention has an aminogram wherein threonine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0.7% and 4.7%, especially between 1.7% and 3.7%, in particular between 2.2% and 3.2%, preferably between 2.4% and 2.8%.
• the composition according to the invention has an aminogram in which the leucine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0.6% and 4.6%, especially between 1.6% and 3.6%, in particular between 2.1% and 3.1%, preferably between 2.4% and 2.9%.
• the composition according to the invention has an aminogram in which the phenylalanine is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0.3% and 4.3%, especially between 1.3% and 3.3%, in particular between 1.8% and 2.8%, preferably between 1.8% and 2.4%.
• the composition according to the invention has an aminogram in which the valine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0% and 4, 0%, especially between 1.0% and 3.0%, in particular between 1.5% and 2.5%, preferably between 1.8% and 2.5%.
• the composition according to the invention has an aminogram in which the isoleucine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0% and 3%. , 5%, especially between 0.5% and 2.5%, in particular between 0.9% and 2.0%, preferably between 0.9% and 1.6%.
• the composition according to the invention has an aminogram in which the hydroxylysine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0% and 3%. , 5%, especially between 0.5% and 2.5%, in particular between 1.0% and 2.0%, preferably of the order of 1.5%.
• the composition according to the invention has an aminogram in which the histidine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0% and 3%. , 3%, especially between 0% and 2.3%, in particular between 0.5% and 1.5.
• the composition according to the invention has an aminogram in which the methionine is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0% and 2, 5%, especially between 0% and 2.0%, in particular between 0.5% and 1.8%, preferably between 0.7% and 1.6%.
• the composition according to the invention has an aminogram in which the tyrosine is in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0% and 1, 5%, especially between 0% and 1.0%, in particular between 0% and 0.9%, preferably between 0.2% and 0.8%.
• the composition according to the invention has an aminogram in which cysteine and cystine are together in molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 0. % and 2%, especially between 0% and 1.0%, in particular between 0% and 0.5%, preferably of the order of 0.03%.
• composition according to the invention has an aminogram in which the essential amino acids (lysine, methionine, phenylalanine, threonine, valine, leucine and isoleucine and histidine) are between 15% and 20% together. %.
• At least 90% -particularly at least 95% -peptide of the peptide composition have an apparent molecular weight of less than 15 000 Da (Dalton), in particular between 200 Da and 15,000 Da, of preferably between 200 Da and 14 000 Da, in particular between 200 Da and 13 000 Da.
• at least 90% of the peptides of the composition have an apparent molecular weight of between 200 Da and 12,000 Da.
• the peptides of the composition according to the invention have a narrow apparent molecular weight distribution extending over a narrow range of between one hundred and a few thousand daltons, that is to say excluding high molecular weight peptides exhibiting more than 100 amino acids, and also having a low proportion of apparent low molecular weight peptides.
• the peptides of the composition have a value of average apparent molecular weight of between 2500 Da and 3600 Da, especially between 2700 Da and 3600 Da.
• Each peptide of the peptide composition according to the invention having a mass contribution in the composition the value of the average apparent molecular weight of peptides of the composition corresponds to the average of each value, called the weighted value, of the apparent molecular weight of each peptide. weighted by a value representative of the mass contribution of each peptide in the composition.
• the representative value of the mass contribution of each peptide or group of peptides in the composition is expressed as a percentage of the value of the area under the curve corresponding to said peptide or to said group of peptides on the value of the total area under the curve. corresponding to all the peptides of the composition.
• the value of the weighted apparent molecular weight of a group of peptides corresponding to the same peak of a chromatogram corresponds to the value of the apparent molecular weight found at the (maximum) peak of this peak of the chromatogram, multiplied by the ratio of the value of the area extending under the curve of this peak over the (total) area extending under the curve of the chromatogram.
• "Area extending under the curve” or "area under the curve” or “area extending under the peak” or “Area under the peak” means the area of the surface extending between the curve describing the peak of the chromatogram and the baseline of the chromatogram.
• the area under one of the peaks of the chromatogram extends between two minima of the chromatogram curve framing a peak (or maximum) of the chromatogram curve.
• the peptide composition according to the invention exhibits during its analysis by exclusion chromatography in which each peptide of the peptide composition is eluted with a retention time representative of the apparent molecular weight of this peptide, a representative elution curve (i.e. a chromatogram) of the peptides having an area value under this curve (i.e., an area value representative of the mass quantity of peptides) corresponding to peptides with an apparent molecular weight greater than 10,000 Da, in particular between 10,000 Da and 50,000 Da, such as the ratio of this area value over the total area under the curve (corresponding to all the peptides of the composition) is less than 15%, especially less than 10%. According to certain particular embodiments, this ratio is between 2.5% and 8.5%.
• the peptide composition according to the invention may be free of any high molecular weight peptide. According to these other particular embodiments, the peptide composition according to the invention is formed of water-soluble peptides.
• the peptide composition according to the invention exhibits during its analysis by exclusion chromatography in which each peptide of the peptide composition is eluted with a retention time representative of the apparent molecular weight of this peptide, a representative elution curve (i.e. a chromatogram) of the peptides having an area value under this curve (i.e., an area value representative of the mass quantity of peptides) corresponding to the peptides of apparent molecular weight between 1800 Da and 10 000 Da such that the ratio of this area value to the total area under the curve (corresponding to all the peptides of the composition) is greater than 35% -including between 35% and 70%, in particular between 45% and 65%. According to certain particular embodiments, this ratio is between 49% and 55%.
• the peptide composition according to the invention exhibits during its analysis by exclusion chromatography in which each peptide of the peptide composition is eluted with a retention time representative of the apparent molecular weight of this peptide, a representative elution curve (i.e. a chromato gram) of the peptides having an area value under this curve (i.e., an area value representative of the mass quantity of peptides ) corresponding to the peptides of apparent molecular weight between 600 Da and 1800 Da such that the ratio of this area value over the total area under the curve (corresponding to all the peptides of the composition) is between 15 % and 45% -in particular between 20% and 40%, especially between 25% and 35%. According to certain particular embodiments, this ratio is between 27% and 32%.
• the peptide composition according to the invention exhibits during its analysis by exclusion chromatography in which each peptide of the peptide composition is eluted with a retention time representative of the apparent molecular weight of this peptide, a representative elution curve (i.e. a chromato gram) of the peptides having an area value under this curve (i.e., an area value representative of the mass quantity of peptides ) corresponding to the peptides of apparent molecular weight less than 600 Da such that the ratio of this area value to the total area under the curve (corresponding to all the peptides of the composition) is less than 10%. According to certain particular embodiments, this ratio is between 8.5% and 14.5%.
• composition according to the invention present by chromatographic analysis on an anion exchange column in which each peptide of the peptide composition is eluted from the column with a retention time representative of its charge:
• the ratio of this area value under the peak corresponding to the anionic peptides to the sum of the area values under the peaks corresponding to the anionic peptides, the neutral peptides and the cationic peptides of the composition is between 27.0% and 45%, especially between 30% and 45%, in particular between 35% and 43%, preferably between 35% and 40%;
• the value of the area under the peak corresponding to the anionic peptides, the value of the area under the peak corresponding to the cationic peptides and the value of the area under the peak corresponding to the neutral peptides being determined by chromatographic analysis in conditions described below:
• chromatographic column of dimensions 100 x 7.8 mm comprising as stationary phase a hydrophilic anion exchange resin functionalized with quaternary ammonium groups, and with a particle size of 10 ⁇ ;
• the peptides of the composition according to the invention exhibit, during a hydrophobicity analysis by reverse phase liquid chromatography, a retention time of between 16 min and 36 min;
• hydrophobicity analysis being carried out under the following conditions: using a chromatography column of dimensions 250 ⁇ 4.6 mm having a stationary phase formed of silica grafted with butyl groups, with a particle size of 5 ⁇ and a porosity value of 300 A;
• a composition according to the invention comprises hydrophobic peptides by nature which are eluted from the above-mentioned column and under the conditions specified above with a retention time corresponding to a percentage of acetonitrile between 12% and 38%.
• the median retention time of the peptides of the composition according to the invention is 26 min corresponding to a percentage of acetonitrile of 25% in the eluent.
• the peptide composition is in the liquid state. It may be a solution of the peptide composition according to the invention in a liquid solvent, especially in an aqueous solvent.
• the peptide composition is in the solid state.
• the peptide composition may be in the form of a split state solid. It may be in particular a solid at least partially dehydrated state.
• the peptide composition according to the invention can be in the form of a powder.
• the peptide composition is free of carbohydrate.
• the peptide composition is free of fat.
• the dry matter content of the peptide composition comprises a mass proportion of collagen peptides greater than 95%, especially greater than 99%.
• the peptide composition has an amount of collagen peptides such that the ratio of the collagen peptide mass of the dry matter of the peptide composition to the dry matter mass of the peptide composition is greater than 95%, especially greater than 99%.
• the peptides of the composition are water-soluble.
• the peptides of the peptide composition are 100% soluble in water.
• the peptide composition is hydro-compatible.
• the peptides of the peptide composition are derived from a controlled enzymatic hydrolysis of skin collagen of at least one fish selected from the group consisting of fish of the family Pangasiidae - in particular Pangasius hypophtalmus (or Pangasianodon hypophtalmus), Pagasius pangasius, Pangasius bocourti and the family Cichlidae - especially the genus Oreochromis, especially Oreochromis niloticus or the genus Tilapia -.
• the peptides of the peptide composition are derived from a controlled enzymatic hydrolysis of skin collagen of at least one sinful fish in temperate water from a temperate region.
• the invention also extends to the use of such a peptide composition in a therapeutic treatment of the human or animal body.
• the invention therefore also extends to such a peptide composition for use as a medicament.
• the invention therefore extends to such a peptide composition for its use as a medicament in the preventive or curative treatment of at least one pathology of the human or animal body.
• the invention also extends in particular to a peptide composition for use as a medicament in at least one of the following treatments:
• the invention also extends to any use of a peptide composition according to the invention in human nutrition. According to some embodiments, the invention also extends to any use of a peptide composition according to the invention in human nutrition to the exclusion of any use as a medicament. In particular, advantageously the composition according to the invention is used as a dietary supplement.
• the invention also extends to a peptide composition obtained by a process in which:
• - skins of temperate water fish are selected, in particular from the family Pangasiidae and / or the family of
• At least one step of acid or alkali treatment of the skin adapted to allow extraction of at least a portion of the collagen of the skins, then;
• At least one liquid / solid extraction of the collagen is carried out in water brought to a temperature of between 60 ° C. and 98 ° C. vs.
• a separation step is then carried out -particularly a step of separation by decantation- of a fraction comprising solids (and fats) and of a solution comprising the collagen extracted, then the solution comprising the collagen extracted is subjected to a purification step, for example by filtration on land and / or demineralization on an ion exchange resin, adapted to form a purified solution comprising collagen, the dry matter of the purified solution comprising a mass proportion of collagen of at least 99%, especially at least 99.5%, in particular at least 99.8%.
• a purified collagen solution comprising substantially pure collagen is formed.
• such a purified solution of substantially colorless collagen is formed.
• such a purified collagen solution is formed substantially substantially completely elastin-free.
• the purified solution is concentrated to form a purified collagen gel and the purified collagen gel is then subjected to the collagen hydrolysis step.
• Such a method makes it possible to obtain a peptide composition according to the invention that is substantially colorless. Such a method makes it possible to obtain a peptide composition according to the invention substantially free of elastin. In particular, it makes it possible to obtain such a peptide composition according to the invention without any chromatographic step of purification of the peptide composition.
• a subsequent step of filtering the peptide composition is carried out.
• a step of pasteurization of the peptide composition is also carried out for a period of at least 2 minutes at a time.
• Pasteurization temperature between 85 ° C and 90 ° C minimum.
• a step of drying the peptide composition is carried out.
• This spray drying step is carried out so as to form a composition according to the invention which is substantially dehydrated and in the form of a powder.
• the invention therefore extends to a peptide composition obtained by a process in which:
• At least one acid treatment step of the skins adapted to allow extraction of at least a portion of the collagen from the skins, then;
• said peptide composition having an overall amino acid analysis wherein:
• the glycine is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 20.0% and 24.5%;
• the hydroxyproline is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 6.0% and 12.0%;
• the proline is in molar quantity such that the ratio of this quantity to the sum of the molar amounts of the amino acids in the composition is between 10.6% and 14.6%;
• the peptide composition exhibiting, in an exclusion chromatography analysis, in which each peptide of the peptide composition is eluted with a retention time representative of the apparent molecular weight of that peptide, an elution curve (c ') that is, a curve or a chromatogram elution profile) of peptides having a value of area under the curve (i.e., a value of area representative of the mass quantity of peptides) corresponding to peptides of apparent molecular weight less than 1400 Da such that the ratio of this area value to the total area under the curve (corresponding to all the peptides of the composition) is less than 40%;
• a filtration column of dimensions 300 x 7.8 mm comprising a stationary phase formed of a silica gel with a porosity of 5 ⁇ ;
• the column being maintained at a temperature of 40 ° C .;
• the invention also relates to a peptide composition, such a peptide composition for its use as a medicament, a process for obtaining such a peptide composition and such a peptide composition obtained by such a method of obtaining characterized in combination. by all or some of the characteristics mentioned above or below.
• FIG. 1 is a representative chromatogram of an analysis of a peptide composition according to the invention by gel filtration
• FIG. 2 is a representative chromatogram of an HPLC analysis of a peptide composition according to the invention on an anion exchange resin
• FIG. 3 is a representative chromatogram of an analysis of a peptide composition according to the invention by reverse phase chromatography
• FIG. 4 is a graphical representation representative of the evolution of the body mass of mice whose colonic inflammation is induced by sodium dextran sulfate (DSS);
• FIG. 5 is a graphical representation in histogram of the result of an IL1 assay in the mouse colon by the ELIS A method
• FIG. 6 is a graphical representation in histogram of the result of an IL6 assay in the mouse colon by the ELISA method
• FIG. 7 is a graphical representation in histogram of the result of a TNF-assay in the mouse colon by the ELISA method
• FIG. 8 is a graphical representation in histogram of the result of a TGF- ⁇ assay in the mouse colon by the ELISA method
• FIG. 9 is a graphical graphical representation of the result of a PCR assay of the fungal flora in the mouse colon.
• FIG. 10 is a graphical representation in histogram of the result of a Saccharomyces cerevisiae PCR assay in the mouse colon
• FIG. 11 is a graphical representation in histogram of the result of a PCR assay of enterobacteria in the mouse colon
• FIG. 12 is a graphical representation in histogram of the result of a PCR assay of firmicutes in the mouse colon
• FIG. 13 is a graphical representation in histogram of the result of a PCR assay for bacteroidetes in the mouse colon;
• FIG. 14 is a graphical representation in histogram of the result of a PCR assay of Faecalibacterium prausnitzii in the mouse colon;
• FIG. 15 is a graphical representation in histogram of the result of a Lactobacille murinus PCR assay in the mouse colon;
• FIG. 16 is a graphical representation in histogram of a quantitative RT-PCR analysis of messenger RNAs of TGF- ⁇ in the mouse colon;
• FIG. 17 is a graphical representation in histogram of a quantitative RT-PCR analysis of the messenger RNAs of the
• iNOS Inducible NO-synthase
• FIG. 18 is a graphical representation in histogram of a quantitative RT-PCR analysis of messenger RNAs of Fizz1 in the mouse colon;
• FIG. 19 is a graphical representation in histogram of a quantitative RT-PCR analysis of messenger RNAs of 5-LOX in the mouse colon;
• FIG. 20 is a graphical representation in histogram of a quantitative RT-PCR analysis of the messenger RNAs of 12/15-LOX in the mouse colon.
• PROCESS FOR PREPARING A COMPOSITION ACCORDING TO THE INVENTION Temperate water fish skins are harvested or purchased, in particular fish of the family Pangasiidae - in particular Pangasius hypophtalmus (or Pangasianodon hypophtalmus), Pagasius pangasius, of Pangasius bocourti and / or catfish or of the Cichlidae family - in particular of the genus Oreochromis, in particular Oreochromis niloticus or the genus Tilapia-.
• a succession of steps of washing the skin, acid treatment of the washed skin and extraction and purification of collagen are carried out.
• An enzymatic hydrolysis step of the fish skin collagen thus obtained is then carried out so as to form the composition according to the invention.
• water is heated to a temperature between 70 ° C and 75 ° C.
• a mass of fish skin collagen is gradually poured into hot water and with stirring, such that the proportion of mass of collagen in the water is 45%, and the pH of the solution is adjusted to pH 6.0.
• a quantity of cysteine protease of vegetable origin is then added to the solution of collapsed collagen.
• At least one protease of Carica papa ⁇ a in particular lypaine (Lypaine®, LYVEN, Collombelles, France) in the dry state, is chosen as cysteine protease.
• the ratio of the cysteine protease mass to the collagen mass is adapted according to the desired hydrolysis conditions. For example, the ratio of the cysteine protease mass to the collagen mass is 0.2%.
• the temperature of the solution is maintained between 65 ° C. and 70 ° C., which is suitable for promoting the enzymatic activity of the cysteine protease and for maintaining the optimum fluidity of the collagen hydrolyzate, taking into account that the Viscosity of the collagen hydrolyzate decreases with the hydrolysis time.
• the solution is maintained at this temperature for a duration of the order of 45 minutes.
• the enzymatic hydrolysis reaction is stopped by heating the collagen hydrolyzate to a temperature above the denaturation temperature of the cysteine protease, for example at a temperature between 85 ° C and 90 ° C for 20 minutes.
• the hydrolyzate of Fish skin collagen is optionally subjected to a filtration step and then to a pasteurization step for a period of at least 2 minutes at a pasteurization temperature between 85 ° C and 90 ° C minimum.
• the hydrolyzate of collagen or collagen peptides is then subjected to a drying step under conditions suitable for forming a composition according to the invention formed of a powder of predetermined particle size.
• a characterization of a peptide composition according to the invention is also carried out by the distribution of the apparent molecular weights of the peptides, by the polarity of the peptides and not the hydrophobicity of the peptides.
• the apparent molecular weight distribution of the constituent peptides of a composition according to the invention is analyzed by gel permeation on a liquid chromatography column of dimensions 300 ⁇ 7.8 mm in which the stationary phase consists of a gel based on silica (BioSep-SEC-S2000, Phenomenex, Peck, France) with a porosity of 5 ⁇ .
• the filtration column being maintained at a temperature of 40 ° C.
• the mobile phase consists of a mixture comprising (A) ultrapure water supplemented with trifluoroacetic acid (0.1% by volume) and (B) acetonitrile (A / B, 75/25, v / v).
• the flow rate of the mobile phase is maintained at 0.6 ml / min.
• the volume of the solution comprising the peptide composition to be analyzed is 25 ⁇ L ⁇ .
• the detection is carried out at the outlet of the gel permeation column by the measurement of absorbance at the wavelength of 214 nm.
• a calibration curve is obtained for determining an apparent molecular weight as a function of the retention time.
• known peptides of molecular weight between 100 Da and 30 kDa are chosen.
• the known standards are proline, glutathione, ribonuclease A and trypsin, with apparent molecular weights of 115 Da, 307 Da, 13.7 kDa and 28.2 kDa, respectively.
• the apparent molecular weights and the retention times expressed in minutes of the standards are given in Table 1 below:
• the peptides are eluted from the column successively according to their decreasing apparent molecular weight.
• the retention time values of each peptide family of the analyte corresponding to a peak of the chromatogram are recorded at the apex of each peak of the chromatogram and translated into an apparent molecular weight value as compared to the calibration curve.
• the relative values of the amounts of each peptide family correspond to the value of the air under the curve corresponding to each peak of the chromatograph. These values are expressed as the ratio of the area under the curve corresponding to a family of peptides to the sum of the area values of each peptide family.
• the proportion of peptides of the composition according to the invention - whose chromatogram is shown in FIG. 1 and whose values are given in Table 2 - whose apparent molecular weight is less than 1400 Da is 31.2% (apparent molecular weight of values 983 Da and 333 Da) with respect to all the peptides of the composition.
• the average apparent molecular weight of the peptides of the composition according to the invention whose apparent molecular weight values are given in Table 2 is 3442 Da.
• the average apparent molecular weight of the peptides of the composition is defined as the average of the weighted apparent molecular weight values corresponding to each group of peptides of the composition corresponding to the same peak of the chromatogram.
• the value of the weighted apparent molecular weight of a group of peptides from the same peak of the chromatogram corresponds to the value of the apparent molecular weight at the peak (maximum) of the peak weighted by the ratio of the value of the area under the curve of the peak corresponding to the (total) area under the curve of the chromatogram.
• Area under the curve or “area under the peak” means the area of the surface extending between the curve describing the peak of the chromatogram and the baseline of the chromatogram. In particular, the area under one of the peaks of the chromatogram extends between two minima of the chromatogram curve framing a peak (or maximum) of the chromatogram curve.
• compositions according to the invention - whose values are given in Table 3 - and whose molecular weight is less than 1400 Da is 35.2% (corresponding essentially to the apparent molecular weights of 983 Da and 340 da).
• the proportion of anionic peptides in the composition according to the invention is analyzed, the distribution of apparent molecular weights is given in Table 2, that is to say the proportion of peptides with a negative charge at pH 8.35.
• the proportion of neutral and / or cationic peptides in the composition according to the invention is also analyzed, ie the proportion of peptides presenting a generally neutral charge at pH 8.35 or having a positive charge at pH 8. 35.
• Such an analysis is carried out by ion exchange high performance liquid chromatography (HPLC) in which the stationary phase is an anion exchange resin (Hydrophase HP-SAX, Interchim, Montluzzo, France) with a particle size of 10 ⁇ .
• HPLC ion exchange chromatography column is 100 x 7.8 mm in size.
• the ion exchange HPLC column is conditioned in tris (hydroxymethyl) amino methane (Tris) buffer at a concentration of 5 mM in water and the pH is adjusted to 8.35.
• the temperature of the column is maintained at a temperature of 25 ° C.
• the flow rate of the mobile phase in the column and 1 mL / min.
• a sample of the peptide composition to be analyzed is prepared so that its concentration is 2 g / L by dilution in ultrapure water.
• a volume of 90 ⁇ l ⁇ of this solution to be analyzed is introduced at the top of the column.
• the detection is carried out by measuring the absorbance continuously at 214 nm.
• the mobile phase consists of 5 mM Tris pH 8.35 (solution A) for a period of 7 minutes, then a mobile phase in which a solution B formed of 5mM Tris, 5M NaC, pH 8.35 linearly increases from 0% to 100% in solution A in 30 minutes. Elution is then maintained with solution B for 2 minutes.
• the chromatogram obtained is represented in FIG. 2.
• the anionic peptides leave the column with a retention time of between 10 min and 17.5 min corresponding to a NaCt concentration of between 0.5 M and 1.7 M.
• the proportion of anionic peptides in the peptide composition according to the invention for which the analysis represented in FIG. 2 is 36.9%.
• the neutral and cationic peptides leave the column with a retention time of between 1 minute and 8 minutes.
• the proportion of neutral and cationic peptides in the peptide composition according to the invention in the analysis shown in FIG. 2 is 57.5%.
• compositions according to the invention As a generalization, this analysis is reproduced on three peptide compositions according to the invention.
• the average proportion of anionic peptides in these compositions according to the invention is between 27.9% and 42.5% and the average proportion of neutral and cationic peptides in these compositions according to the invention is between 57.5% and 72%. , 1%.
• the hydrophobicity of the peptides constituting the composition according to the invention is analyzed by reverse phase liquid chromatography on a silica column grafted with butyl groups (Vydac 214TP TM C 4 , Grace, Epernon, France) dimensions 250 x 4.6 mm.
• the granulometry of the silica is 5 ⁇ and its porosity is 300 A.
• the column is conditioned in ultrapure acidified water with 0.1% trifluoroacetic acid.
• the temperature of the column is maintained at a temperature of 40 ° C.
• the flow rate of the mobile phase in the column and 0.6 mL / min.
• a sample of the peptide composition to be analyzed is prepared so that its concentration is 2 g / L by dilution in ultrapure water.
• a volume of 100 ⁇ L ⁇ of this solution to be analyzed is introduced at the top of the column.
• the detection is performed by measuring the absorbance continuously at the wavelength of 214 nm.
• the mobile phase consists of acidified water (solution A) for a period of 7 minutes, then a mobile phase in which a solution B formed of Acidified water with 0.1% (by volume) trifluoroacetic acid and comprising 40% acetonitrile increases linearly from 0% to 100% in solution A in 30 minutes.
• the chromatogram obtained is represented in FIG. 3.
• the peptides of the composition according to the invention leave the column with a retention time of between 16 min and 36 min corresponding to a percentage of acetonitrile of between 12% and 38% in the eluent.
• the median retention time of the peptides of the composition according to the invention is 26 min corresponding to a percentage of acetonitrile of 25% in the eluent.
• mice The effect of a composition according to the invention on the induced gastric candidiasis in female C57BL / 6 mice, aged 8 weeks and weighing between 20-25 g, is analyzed. These mice are fed for a period of 21 days with the composition according to the invention at a rate of 4 g / kg / day. 21 th day, is induced gastrointestinal candidiasis by gavage to each mouse with a quantity of 5.10 7 yeast cells. Between 2 days (D2) and 7 days (D7) after induction, the feces of the mice are collected and the yeast load (CFU / mg of stool) is evaluated on a chromogenic medium. The results are given in Table 4 below in comparison with the values measured in mice whose digestive candidiasis is induced but which are not treated with the composition according to the invention.
• composition according to the invention induces a decrease in the load of Candida albicans.
• Macrophages / monocytes of healthy human subjects are cultured for 24 hours in the presence of a pretreatment composition (control without collagen peptides, invention, excluding the invention, Table 7) at a concentration of 100 ⁇ g / ml of culture medium.
• a pretreatment composition control without collagen peptides, invention, excluding the invention, Table 7.
• ROS Reactive Oxygen Species
• type 1 macrophage receptors specifically stimulated by a phorbol ester ("12-myristate-13-acetate-phorbol, TPA") at a concentration of 100 ⁇
• type 2 macrophage receptors specifically stimulated with non-opsonized Zymosan (ZNO) at a concentration of 100 ⁇ g / mL.
• ZNO non-opsonized Zymosan
• the level of free radical production of oxygen is measured by chemiluminescence in the presence of luminol at a concentration of 66 ⁇ .
• Table 7 represent the average values obtained with three tests.
• Non-induced by ZNO and TPA present a level of production of oxygen free radicals substantially constant according to the nature of the pretreatment (without collagen, according to the invention and outside the invention).
• the macrophages / monocytes pretreated with a peptide composition according to the invention exhibit an increased expression of type 2 macrophage receptors.
• M2 anti-inflammatory revealed by the intensity of chemiluminescence (5.06 10 8 ua) induced by ZNO, compared to macrophages / monocytes pretreated with a composition of peptides outside the invention, that is to say from hydrolysis enzymatic collagen cold water fish skin (3.91 10 8 ua).
• the macrophages / monocytes pretreated with a peptide composition according to the invention that is to say derived from an enzymatic hydrolysis of collagen from skins of temperate water fish, have a decreased expression of type 1 macrophage receptors.
• Inflammatory (Ml) levels revealed by TPA-induced chemiluminescence intensity (6.75 10 8 ua), by compared to macrophages / monocytes pretreated with a composition of peptides outside the invention, that is to say from an enzymatic hydrolysis of collagen cold water fish skins (1.40 10 9 ua).
• the peptide composition according to the invention has an anti-inflammatory phenotype compared to a composition of peptides outside the invention, that is to say from an enzymatic hydrolysis of collagen cold water fish skins by increasing the level of expression of macrophage type 2 (M2) anti-inflammatory receptors and by decreasing the level of expression of inflammatory macrophage type 1 (M1) receptors.
• M2 macrophage type 2
• M1 inflammatory macrophage type 1
• composition of peptides according to the invention derived from skins of temperate water fish and having an aminogram in which:
• the glycine is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 20.0% and 24.5%;
• the hydroxyproline is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 6.0% and 12.0%;
• the proline is in a molar amount such that the ratio of this amount to the sum of the molar amounts of the amino acids in the composition is between 10.6% and 14.6%;
• the anti-inflammatory effect of the peptide composition according to the invention is demonstrated on a murine model of pharmacological inflammation induced by sodium dextran sulfate (DSS, MP Biomedical LLC, Canada) characterized by weight loss and diarrhea bloody.
• the effect of the peptide composition according to the invention is studied to limit the weight loss of DSS-treated mice. 7 to 10 days old female C57BL / 6 laboratory mice weighing between 20 and 25 grams are treated for 7 days (D 1 to 7 ) with DSS dissolved at a rate of 1.5% (w / v). ) in the drinking water of the mice.
• mice are also treated for 12 days (July to 12 ) with the composition according to the invention at a rate of 0.1 g / kg / day; 1 g / kg / day and 4 g / kg / day.
• This quantity of composition according to the invention is delivered in the drinking water of the mice.
• the mice are euthanized.
• mice Five batches of mice are prepared, each batch comprising 10 mice, of which:
• batch 2 is treated with DSS for 7 days and with the composition according to the invention at the rate of 0.1 g / kg / day for 12 days;
• batch 3 is treated with DSS for 7 days and with the composition according to the invention at a rate of 1 g / kg / day for 12 days;
• batch 4 is treated with DSS for 7 days and with the composition according to the invention at the rate of 4 g / kg / day for 12 days;
• mice which are not treated with DSS and for which the inflammation is not induced, and which are not treated with a peptide composition according to the invention.
• composition according to the invention makes it possible to limit, or even to cancel almost completely, the weight loss caused by the inflammation induced by sodium dextran sulfate (DSS) in mice.
• the composition according to the invention is capable of being used as a medicament, especially for the treatment of colonic inflammation.
• Transverse histological sections of mouse colon treated with DSS alone show, after bichromatic staining with hematoxylin and eosin, important infiltrations of inflammatory cells in the mucosa and the submucosa.
• the thickness of the epithelium is reduced.
• the epithelium shows extensive ulcerations.
• the transverse histological sections of mouse colon treated with DSS and with the composition according to the invention at the rate of 0.1 g / kg / day, 1 g / kg / day and 4 g / kg / day show, after dichromatic staining by the hematoxylin and eosin of the tight and rectilinear tubular glands representative of a healthy and functional epithelium.
• a macroscopic score is calculated from Wallace scale scores for stool appearance, damaged colon, colon weight, and colon length.
• Wallace scale (ES Kimball, NH Wallace, CR Scnneider, MR D'Andrea and PJ Hornby, 2004, Neurogastroenterol Motil, 16, 811-818 Vanilloid receptor 1 antagonists attenuate disease severity in sodium dextran sulphate-induced colitis in mice) .
• the value of the macroscopic score is all the higher as the colon is inflammatory and is weaker as the colon is healthy.
• the mean values and the standard deviation of the macroscopic score of the mice of batches 1 to 5 are given in Table 5 below.
• a decrease in the macroscopic score induced by the treatment with the composition according to the invention is observed, that is to say an improvement in the inflammatory state of the colon induced by the composition according to the invention.
• mice On day 12 , the mice were euthanized and the level of expression in the colon of pro-inflammatory cytokines by the ELISA technique was assayed:
• IL- ⁇ The level of IL- ⁇ is analyzed by the ELISA technique and expressed in picogram ( ⁇ g) of IL-1 per milligram (mg) of colon. The results are shown in FIG. 5 in which the first column (white column) corresponds to the analysis performed on the control mice.
• the second column (oblique hatched column) corresponds to the analysis carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the analysis carried out on the mice in lot 4.
• the fourth column (column horizontal hatching) corresponds to the analysis carried out on the mice in lot 3.
• the fifth column (grayed-out column) corresponds to the analysis carried out on the mice in batch 2.
• the sixth column black solid column) corresponds to the analysis carried out in the mice of batch 5.
• This effect was confirmed by IL-1 messenger RNA analysis by quantitative RT-PCR in particular (p ⁇ 0.01) for the doses of 0.1 g / kg / day and 1 g / kg / day;
• IL6 The level of IL6 is analyzed by the ELISA technique and expressed in picogram (pg) of IL6 per milligram (mg) of colon. The results are shown in FIG. 6 in which the first column (white column) corresponds to the analysis performed on the control mice.
• the second column (oblique hatched column) corresponds to the analysis carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the analysis carried out on the mice in lot 4.
• the fourth column (column horizontal hatching) corresponds to the analysis carried out on the mice in lot 3.
• the fifth column (grayed-out column) corresponds to the analysis carried out on the mice in batch 2.
• the sixth column black solid column) corresponds to the analysis carried out in mice of batch 5.
• a statistically significant decrease in the expression of IL6 in the colon of mice treated with the peptide composition according to the invention is observed for doses of 0.1 g / kg / day (p ⁇ 0). , 01), 1 g / kg / day (p ⁇ 0.01) and 4 g / kg / day (p ⁇ 0.05) compared with the colon of mice whose inflammation is induced by the DSS and with respect to the mice of which inflammation is induced by DSS and treated with hydrolysed casein.
• This effect was confirmed by IL6 messenger RNA analysis by quantitative RT-PCR;
• TNF- ⁇ The level of TNF- ⁇ is analyzed by the ELISA technique and expressed in picogram ( ⁇ g) of TNF- ⁇ per milligram (mg) of colon. The results are shown in FIG. 7 in which the first column (white column) corresponds to the analysis performed on the control mice.
• the second column (oblique hatched column) corresponds to the analysis carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the analysis carried out on the mice in lot 4.
• the fourth column (column horizontal hatching) corresponds to the analysis carried out on the mice in lot 3.
• fifth column shade column
• the sixth column black solid column corresponds to the analysis carried out on the mice in batch 5.
• RT-PCR Quantitative Reverse Transcriptase Polymerase Chain Reaction
• the quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis of the messenger RNAs of the inducible NO-synthase (iNOS) is shown in FIG. 17 in which the first column (white column) corresponds to the analysis carried out on control mice.
• the second column (oblique hatched column) corresponds to the analysis carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the analysis carried out on the mice in lot 4.
• the fourth column (column horizontal hatching) corresponds to the analysis carried out on the mice in lot 3.
• the fifth column (grayed-out column) corresponds to the analysis carried out on the mice in batch 2.
• the sixth column black solid column corresponds to the analysis carried out in lot 5 mice.
• mice On day 12 , the mice were euthanized and the level of expression in the colon of anti-inflammatory markers was analyzed: - TGF- ⁇ .
• the TGF- ⁇ level is analyzed by the ELISA technique and expressed in picogram (pg) of TGF- ⁇ per milligram (mg) of colon.
• the results are shown in FIG. 8 in which the first column (white column) corresponds to the analysis performed on the control mice.
• the second column (oblique hatched column) corresponds to the analysis carried out on the mice of batch 1.
• the third column vertical hatch column
• mice The fourth column (column horizontal hatching) corresponds to the analysis carried out on the mice in lot 3.
• the fifth column corresponds to the analysis carried out on the mice in batch 2.
• the sixth column (black solid column) corresponds to the analysis carried out in mice of batch 5.
• a stimulation of the expression of TGF- ⁇ in the colon of mice treated with the peptide composition according to the invention is observed for the dose of 0.1 ⁇ g / kg / day relative to the colon of mice whose inflammation is induced by DSS. This effect was confirmed by analysis of messenger RNAs of TGF- ⁇ by quantitative RT-PCR, in particular for the doses of 0.1 g / kg / day and 1 g / kg / day of peptide composition according to the invention ( figure 16);
• Fizzl is a marker of anti-inflammatory M2 macrophages.
• the quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis of the messenger RNAs of Fizzl is shown in FIG. 18 in which the first column (white column) corresponds to the analysis performed on the control mice.
• the second column (oblique hatched column) corresponds to the analysis carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the analysis carried out on the mice in lot 4.
• the fourth column column (column horizontal hatching) corresponds to the analysis carried out on the mice in lot 3.
• the fifth column (grayed-out column) corresponds to the analysis carried out on the mice in batch 2.
• the sixth column corresponds to the analysis carried out on mice in batch 5.
• a statistically significant increase in Fizzl messenger RNA reduced by DSS was observed, for doses of 0.1 g / kg / day (p ⁇ 0.05), 1 g / kg / day (p ⁇ 0.01) and 4 g / kg / day (p ⁇ 0.05) composition of peptides according to the invention;
• mice were euthanized and the colic flora of these mice was quantified by PCR ("Polymerase Chain Reaction"). The values are normalized with respect to the total amount of bacteria or fungi and relative to ⁇ -actin. ⁇ -actin is the reference gene allowing normalization with respect to the amount of colonic tissue analyzed. In particular, we quantify:
• the ratio R of the amount of fungal ribosomal DNA ITS1-2 on the amount of DNA of ⁇ -actin is shown in FIG. 9 in which the first column (white column) corresponds to the assay performed on the control mice.
• the second column (oblique hatched column) corresponds to the assay carried out on the mice of batch 1.
• the third column corresponds to the assay performed on the mice of batch 4.
• the fourth column horizontal hatched column
• the fifth column corresponds to the assay performed on the mice in batch 2.
• the sixth column solid black column corresponds to the assay performed on the mice in batch 5.
• the composition of peptides according to the invention restore the fungal flora especially at a dose of 4 g / kg / day;
• the ratio R of the quantity of 26S ribosomal DNA on the amount of DNA of ⁇ -actin is shown in FIG. 10.
• the first column (white column) corresponds to the assay performed on the control mice.
• the second column (oblique hatched column) corresponds to the assay carried out on the mice of the batch 1.
• the third column (vertical hatched column) corresponds to the assay carried out on the mice of the lot 4.
• the fourth column horizontal hatched column
• the fifth column corresponds to the assay performed on the mice of batch 2.
• the sixth column corresponds to the assay performed on the mice of batch 5.
• the peptide composition according to the invention restores in a statistically significant manner the Saccharomyces cerevisiae flora at doses of 0.1 g / kg / day (p ⁇ 0.05), 1 g / kg / day (p ⁇ 0.05) and 4 g / kg / day (p ⁇ 0.05).
• S. cerevisiae has an anti-inflammatory potential
• the ratio R of the quantity of 16S ribosomal DNA to the amount of ⁇ -actin DNA is shown in FIG. 11.
• the first column (white column) corresponds to the assay performed on the control mice.
• the second column (oblique hatched column) corresponds to the assay performed on the batch 1 mice.
• the third column (grayed column) corresponds to the assay performed on the batch 2 mice.
• the fourth column (solid black column) corresponds to the assay performed. in the mice of batch 5.
• the peptide composition according to the invention makes it possible to reduce the DSS-induced enterobacteria flora at a concentration of 0.1 g / kg / day. Enterobacteria are associated with pro-inflammatory potential;
• the ratio R of the amount of such 16S ribosomal DNA on the amount of ⁇ -actin DNA is shown in FIG. 12.
• the first column (white column) corresponds to the assay performed on the control mice.
• the second column (oblique hatched column) corresponds to the assay carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the assay performed on the mice of batch 4.
• the fourth column (horizontal hatched column) corresponds to the assay carried out on the mice in batch 3.
• the fifth column corresponds to the assay performed on the mice of batch 2.
• the sixth column corresponds to the assay carried out on the mice of batch 5.
• the peptide composition according to the invention induces the statistically significant reduction of the firmicutes flora induced by DSS at 0.1 g / kg / day (p ⁇ 0.05), 1 g / kg / day (p ⁇ 0.05) and 4 g / kg / day (p ⁇ 0.05), but also decreased flora of firmicutes not induced at these same concentrations. It should be noted that the increase of firmicutes flora is generally observed in a known manner during digestive inflammation ("IBD, Inflammatory Bowel Diseases");
• the ratio R of the amount of such a 16S ribosomal DNA on the amount of ⁇ -actin DNA is shown in FIG. 13.
• the first column corresponds to the assay performed on the mice of batch 1.
• the second column corresponds to the assay carried out on the mice of batch 4.
• the third column corresponds to the assay carried out on the mice of batch 3.
• the fourth column corresponds to the assay
• the fifth column (black solid column) corresponds to the assay carried out on the mice of batch 5.
• the peptide composition according to the invention induces a statistically significant decrease of the bacteroidetes flora induced by DSS at concentrations 0.1 g / kg / day (p ⁇ 0.05), 1 g / kg / day (p ⁇ 0.05) and 4 g / kg / day (p ⁇ 0.05).
• the increase of bacteroidetes is generally observed in a known manner during digestive inflammation ("IBD");
• the ratio R of the amount of such specific DNA on the amount of DNA of ⁇ -actin is shown in FIG. 14.
• the first column (white column) corresponds to the assay performed on the control mice.
• the second column (oblique hatched column) corresponds to the assay carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the assay carried out on the mice of batch 4.
• the fourth column (horizontal hatched column) corresponds to the assay carried out on the mice of lot 3.
• the fifth column (grayed column) corresponds to the assay carried out on the mice of batch 2.
• the sixth column black solid column corresponds to the assay carried out on the mouse of lot 5.
• the peptide composition according to the invention induces the statistically significant increase of Faecalibacterium praustnizii flora destroyed by DSS at a concentration of 0.1 g / kg / day (p ⁇ 0.01), 1 g / kg / day (p ⁇ 0.01) and 4 g / kg / day (p ⁇ 0.01).
• the increase of Faecalibacterium praustnizii is generally observed in a known manner during digestive inflammation ("IBD"). Faecalibacterium praustnizii would also have anti-inflammatory properties;
• the ratio R of the amount of such specific DNA to the amount of ⁇ -actin DNA is shown in FIG. 15 in which the first column (white column) corresponds to the assay performed on the control mice.
• the second column (oblique hatched column) corresponds to the assay carried out on the mice of batch 1.
• the third column vertical hatch column
• the fourth column (horizontal hatched column) corresponds to
• the fifth column (gray column) corresponds to the assay performed on the mice in batch 2.
• the sixth column solid black column corresponds to the assay performed on the mice in batch 5.
• composition of Peptides according to the invention induced the increase of Lactobacillus murinus flora at concentrations of 0.1 g / kg / day, 1 g / kg / day and 4 g / kg / day (p ⁇ 0.05). Lactobacillus murinus also has anti-inflammatory properties.
• 5-LOX The quantitative reverse transcriptase polymerase reaction (RT-PCR) analysis of the messenger RNAs of the enzyme 5-lipoxygenase (5-LOX) is represented in FIG. 19 in which the first column (white column ) is the analysis performed on the control mice.
• the second column (oblique hatched column) corresponds to the analysis carried out on the mouse of lot 1.
• the third column (vertical hatch column) corresponds to the analysis carried out on the mice in lot 4.
• the fourth column horizontal hatched column) corresponds to the analysis carried out on the mice in batch 3.
• the fifth column shade column
• the sixth column black solid column corresponds to the analysis carried out on the mice in batch 5.
• the peptide composition according to the invention has an inhibitory effect on the expression of 5-LOX favoring the production of pro-inflammatory lipid mediators;
• 12/15-LOX The quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis of the messenger RNAs of the 12/15-lipoxygenase enzyme (12/15-LOX) is represented in FIG.
• the first column white column
• the second column (oblique hatched column) corresponds to the analysis carried out on the mice of batch 1.
• the third column (vertical hatch column) corresponds to the analysis carried out on the mice in lot 4.
• the fourth column (column horizontal hatching) corresponds to the analysis carried out on the mice in lot 3.
• the fifth column (grayed-out column) corresponds to the analysis carried out on the mice in batch 2.
• the sixth column black solid column corresponds to the analysis carried out in mice of batch 5.

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