Composition of fish skin collagen peptides and use thereof as a drug

Abstract

The invention relates to a composition of peptides having an aminogram in which: glycine, hydroxyproline and proline are in molar quantities such that the ratio of each quantity to the sum of the molar quantities of the amino acids in the composition is comprised between 20.0% and 24.5%, between 6.0% and 12.0% and between 10.6% and 14.6%, respectively; the peptide composition comprising a quantity of peptides with a molecular weight lower than 1400 Da such that the ratio of said quantity to the quantity of peptides in the composition is less than 40%; the molecular weight and the quantity of peptides in the composition being determined by exclusion chromatography. The invention likewise relates to such a composition to be used as a drug. The invention further relates to such a composition to be used as a food supplement.

Claims

1. A peptide composition having an aminogram in which: 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%; 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%; 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 peptide composition having, during analysis by exclusion chromatography during which each peptide of the peptide composition is eluted with a retention time that is representative of the apparent molecular weight of this peptide, an elution curve of the peptides having an area under the curve value corresponding to the peptides of apparent molecular weight of less than 1400 Da such that the ratio of this area value to the total area under the curve is less than 40%; said analysis being performed as described below: on a filtration column of dimensions 300×7.8 mm comprising a stationary phase formed of a silica gel with a porosity of 5 μm; the column being kept at a temperature of 40° C.; with, as mobile phase, a solution formed (A) of ultrapure water comprising 0.1% by volume of trifluoroacetic acid and (B) of acetonitrile, wherein the A/B volume ratio is 75/25; introducing, at the top of the gel filtration column, a volume of a solution comprising the peptide composition; the flow rate of the mobile phase in the column being 0.6 ml/min, and; the peptides of the composition being detected by absorbance at a wavelength of 214 nm.

2. The composition as claimed in claim 1, wherein each peptide of the composition has an apparent molecular weight of between 200 Da and 12000 Da.

3. The composition as claimed in claim 1, wherein the peptides have a mean apparent molecular weight of between 2500 Da and 3600 Da.

4. The composition as claimed in claim 1, wherein it has, by chromatographic analysis on an anion exchange column during which each peptide of the peptide composition is eluted from the column with a retention time that is representative of its charge: an area value under a peak corresponding to anionic peptides; an area value under a peak corresponding to neutral peptides, and; an area value under a peak corresponding to cationic peptides; such that 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, to the neutral peptides and to the cationic peptides of the composition is between 27.0% and 45%; 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 under the conditions described below: using a chromatographic column of dimensions 100×7.8 mm comprising, as stationary phase, a hydrophilic anion exchange resin functionalized with quaternary ammonium groups with a particle size of 10 μm; using, as first mobile phase for elution of the cationic peptides and neutral peptides, a 5 mM aqueous Tris buffer (C) at pH 8.35 for a duration of 7 minutes starting from the introduction of the composition to be analyzed at the top of the column, then a second mobile phase for elution of the anionic peptides, in which the ratio of the volume of a buffer (D) formed of 5 mM Tris, 5 M NaCl at pH 8.35 to the volume of buffer (C) increases linearly from 0 to 100% in 30 minutes; with a flow rate of the mobile phase of 1 ml/min in the column; the analysis being performed at a temperature of 25° C., and; with detection by absorbance at a wavelength of 214 nm at the column outlet.

5. The composition as claimed in claim 1, wherein the peptides have, during a reversed-phase liquid chromatography hydrophobicity analysis, a retention time of between 16 min and 36 min; said hydrophobicity analysis being performed under the conditions below: using a chromatography column of dimensions 250×4.6 mm having a stationary phase formed of silica grafted with butyl groups, of a particle size of 5 μm and of a porosity value of 300 Å; using, as first mobile phase for elution of the hydrophilic peptides, a solution (E) of trifluoroacetic acid at 0.1% in ultrapure water for a duration of 7 minutes starting from the introduction of the composition to be analyzed at the top of the column, then a second mobile phase for elution of the hydrophobic peptides in which the ratio of the volume of a solution (F) of trifluoroacetic acid at 0.1% in water comprising 40% acetonitrile to the volume of the solution (E) increases linearly from 0 to 40% in 30 minutes; with a flow rate of the mobile phase of 0.6 ml/min in the column; the analysis being performed at a temperature of 40° C., and; with detection by absorbance at a wavelength of 214 nm at the column outlet.

6. The composition as claimed in claim 1, wherein the composition is in a liquid state.

7. The composition as claimed in claim 1, wherein the composition is in a solid state.

8. The composition as claimed in claim 1, wherein the composition is devoid of carbohydrate.

9. The composition as claimed in claim 1, wherein the composition is devoid of fats.

10. The composition as claimed in claim 1, wherein the peptides of the composition are water-soluble.

11. The composition as claimed in claim 1, wherein the peptides of the composition result from a controlled enzymatic hydrolysis of collagen from skin of at least one fish selected from the group formed of fish from the family Pangasiidae and from the family Cichlidae.

12. The composition as claimed in claim 1, wherein the peptide composition is extracted from a skin of a fish previously pretreated with an acid treatment.

13. A medicament comprising the composition as claimed in claim 1.

14. A human food composition comprising the composition as claimed in claim 1.

Description

(1) Other aims, characteristics and advantages of the invention will become apparent on reading the following description, given non-limitingly and which refers to the appended drawings, in which:

(2) FIG. 1 is a chromatogram representing an analysis of a peptide composition according to the invention by gel filtration;

(3) FIG. 2 is a chromatogram representing an HPLC analysis of a peptide composition according to the invention on an anion exchange resin;

(4) FIG. 3 is a chromatogram representing an analysis of a peptide composition according to the invention by reversed-phase chromatography;

(5) FIG. 4 is a graphical depiction representing the change in the body mass of mice in which colonic inflammation has been induced by dextran sulfate sodium (DSS);

(6) FIG. 5 is a graphical depiction in a histogram of the result of an IL-1β assay in the colon of mice by the ELISA method;

(7) FIG. 6 is a graphical depiction in a histogram of the result of an IL-6 assay in the colon of mice by the ELISA method;

(8) FIG. 7 is a graphical depiction in a histogram of the result of a TNF-α assay in the colon of mice by the ELISA method;

(9) FIG. 8 is a graphical depiction in a histogram of the result of a TGF-β assay in the colon of mice by the ELISA method;

(10) FIG. 9 is a graphical depiction in a histogram of the result of an assay by PCR of the fungal flora in the colon of mice;

(11) FIG. 10 is a graphical depiction in a histogram of the result of an assay by PCR of Saccharomyces cerevisiae in the colon of mice;

(12) FIG. 11 is a graphical depiction in a histogram of the result of an assay by PCR of the Enterobacteriaceae in the colon of mice;

(13) FIG. 12 is a graphical depiction in a histogram of the result of an assay by PCR of the Firmicutes in the colon of mice;

(14) FIG. 13 is a graphical depiction in a histogram of the result of an assay by PCR of the Bacteroidetes in the colon of mice;

(15) FIG. 14 is a graphical depiction in a histogram of the result of an assay by PCR of Faecalibacterium prausnitzii in the colon of mice;

(16) FIG. 15 is a graphical depiction in a histogram of the result of an assay by PCR of Lactobacillus murinus in the colon of mice;

(17) FIG. 16 is a graphical depiction in a histogram of an analysis by quantitative RT-PCR of the messenger RNA of TGF-β in the colon of mice;

(18) FIG. 17 is a graphical depiction in a histogram of an analysis by quantitative RT-PCR of the messenger RNA of inducible NO synthase (iNOS) in the colon of mice;

(19) FIG. 18 is a graphical depiction in a histogram of an analysis by quantitative RT-PCR of the messenger RNA of Fizz1 in the colon of mice;

(20) FIG. 19 is a graphical depiction in a histogram of an analysis by quantitative RT-PCR of the messenger RNA of 5-LOX in the colon of mice;

(21) FIG. 20 is a graphical depiction in a histogram of an analysis by quantitative RT-PCR of the messenger RNA of 12/15-LOX in the colon of mice.

PROCESS FOR PREPARING A COMPOSITION ACCORDING TO THE INVENTION

(22) Skins of temperate-water fish are removed or purchased, especially from fish of the family Pangasiidae—especially Pangasius hypophtalmus (or Pangasianodon hypophtalmus), Pangasius pangasius, Pangasius bocourti—and/or from catfish or from the family Cichlidae—especially from the genus Oreochromis, in particular Oreochromis niloticus or from the genus Tilapia.

(23) A succession of steps of washing the skin, acid treatment of the washed skin and extraction and purification of the collagen are carried out. A step of enzymatic hydrolysis of the collagen from the fish skins thus obtained is then carried out so as to form the composition according to the invention. For this purpose, water is heated to a temperature of between 70° C. and 75° C. A weight of collagen from fish skins is gradually poured into the hot water, with stirring, such that the proportion by weight of collagen in the water is 45%, and the pH of the solution is adjusted to pH 6.0. An amount of cysteine protease of plant origin is then added to the solution of re-melted collagen. As cysteine protease, at least one protease of Carica papaïa is chosen, especially lypaine (Lypaine®, LYVEN, Collombelles, France) in the dry state. The weight ratio of cysteine protease to the weight of collagen is adapted depending on the desired hydrolysis conditions. For example, the weight ratio of cysteine protease to the weight of collagen is 0.2%. The temperature of the solution is kept at a value of between 65° C. and 70° C., adapted to promote the enzymatic activity of the cysteine protease and to maintain the optimum fluidity of the collagen hydrolyzate, taking into account the fact that the viscosity of the collagen hydrolyzate decreases with the hydrolysis time. The solution is kept at this temperature for a duration of the order of 45 minutes.

(24) The enzymatic hydrolysis reaction is stopped by heating the collagen hydrolyzate to a temperature greater than the denaturation temperature of the cysteine protease, for example to a temperature of between 85° C. and 90° C. for 20 minutes. The hydrolyzate of collagen from fish skins is optionally subjected to a step of filtration then to a step of pasteurization for a duration of at least 2 minutes at a pasteurization temperature of between 85° C. and 90° C. at least.

(25) The hydrolyzate of collagen or collagen peptides is then subjected to a step of drying under conditions suitable for forming a composition according to the invention, formed of a powder of predetermined particle size.

(26) Structural Characterizations of the Peptides of the Composition According to the Invention

(27) Amino Acid Composition

(28) A characterization of the composition of peptides resulting from the hydrolysis of collagen from skins of temperate-water fish according to the invention is carried out by assaying the free and total amino acids using an amino acid analyzer or using high performance liquid chromatography (HPLC) equipment in accordance with standard ISO 13903:2005. By way of comparison, the amino acid composition of a hydrolyzate of collagen from skins of cold-water fish (Alaska pollock), taken from deep water of the Bering Sea (Alaska) is determined. The comparative results are presented in table 6 below.

(29) TABLE-US-00001 TABLE 6 Collagen from temperate-water Collagen from fish according to cold-water fish, Amino acid, mol % the invention for comparison Glycine 20.0-24.5 25.1-34.7 Alanine  7.3-11.3  8.6-10.9 Proline 10.6-14.6 8.4-9.8 Glutamic acid  8.0-13.0 4.2-6.8 Serine 1.5-5.5 6.3-8.6 Arginine  6.9-10.9  6.2-10.3 Hydroxyproline  6.0-12.0 5.2-5.5 Aspartic acid 3.1-7.1 3.7-5.2 Threonine 0.7-4.7 3.3-3.7 Lysine 1.5-5.5 3.2-4.5 Leucine 0.6-4.6 1.8-3.6 Valine   0-0.40 1.6-2.9 Histidine .sup. 0-3.3 1.5-1.6 Phenylalanine 0.3-4.3 1.3-2.4 Methionine .sup. 0-2.5 1.2-2.9 Isoleucine .sup. 0-3.5 1.0-2.0 Hydroxylysine .sup. 0-3.5 .sup. 0-0.9 Tyrosine .sup. 0-1.5 0.3-1.0 Cysteine .sup. 0-2.0 0 Tryptophan 0 0

(30) A characterization of a peptide composition according to the invention by the distribution of the apparent molecular weights of the peptides, by the polarity of the peptides and by the hydrophobicity of the peptides is also carried out.

(31) Analysis of the Apparent Molecular Weights of the Peptides

(32) The distribution of apparent molecular weights of the peptides constituting 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 silica-based gel (BioSep-SEC-S2000, Phenomenex, Le Peck, France) with a porosity of 5 μm. The filtration column is kept at a temperature of 40° C. The mobile phase consists of a mixture comprising (A) ultrapure water with trifluoroacetic acid (0.1% by volume) added thereto and (B) acetonitrile (A/B; 75/25; v/v). The flow rate of the mobile phase is kept at 0.6 ml/min. The volume of the solution comprising the peptide composition to be analyzed is 25 μl. The detection is carried out at the outlet of the gel permeation column by measuring absorbance at a wavelength of 214 nm. In parallel, a calibration curve for determining an apparent molecular weight as a function of retention time is produced. In order to produce this calibration curve, known peptides of molecular weight of between 100 Da and 30 kDa are chosen. The known standards are proline, glutathione, ribonuclease A and trypsin, of respective apparent molecular weights of 115 Da, 307 Da, 13.7 kDa and 28.2 kDa.

(33) The apparent molecular weights and the retention times expressed in minutes of the standards are given in table 1 below:

(34) TABLE-US-00002 TABLE 1 Standard MW, Da Retention time, min Proline   115 17.193 Glutathione   307 16.938 Ribonuclease A 13 700 10.275 Trypsin 28 161 10.232

(35) The peptides are eluted from the column in succession as a function of their decreasing apparent molecular weight. The retention time values of each family of peptides of the composition to be analyzed, corresponding to a peak on the chromatogram, are read off at the top of each peak of the chromatogram and converted into an apparent molecular weight value by comparison with the calibration curve. The relative values of the amounts of each family of peptides correspond to the value of the area under the curve corresponding to each peak of the chromatogram. These values are expressed by the ratio of the value of the area under the curve corresponding to a family of peptides to the sum of the area values of each family of peptides.

(36) The values of retention time (min), corresponding apparent molecular weights (Da) and percentage of the area under the curve (expressed as percentage of the total area under the curve) corresponding to each family of peptides of the chromatogram shown in FIG. 1 are given in table 2 below. Each group of peptides corresponding to a peak on the chromatogram is identified by the apparent molecular weight value corresponding to the maximum of this peak on the chromatogram.

(37) TABLE-US-00003 TABLE 2 Retention time Apparent molecular Area under the (min) weight (Da) curve (%) 11.130 10 869   6.6 11.560 8096 7.0 12.072 5703 9.6 12.430 4461 13.0 12.962 3100 13.6 13.678 1897 19.0 14.638  983 21.6 16.217  333 9.6

(38) The proportion of peptides of the composition according to the invention—the chromatogram of which is shown in FIG. 1 and the values of which are given in table 2—for which the apparent molecular weight is less than 1400 Da is 31.2% (apparent molecular weights with values 983 Da and 333 Da) relative to all the peptides of the composition.

(39) The mean apparent molecular weight of the peptides of the composition according to the invention, the apparent molecular weight values of which are given in table 2, is 3442 Da. The mean apparent molecular weight of the peptides of the composition is defined as the mean of the weighted apparent molecular weight values corresponding to each group of peptides of the composition corresponding to the same peak on the chromatogram. The weighted apparent molecular weight value of a group of peptides of the same peak on the chromatogram corresponds to the apparent molecular weight value at the top (maximum) of the peak, weighted by the ratio of the value of the area under the curve of the corresponding peak to the (total) area under the curve of the chromatogram. “Area under the curve” or “area under the peak” is intended to mean the area of the space between the curve tracing 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 curve of the chromatogram, enclosing a top (or maximum) of the curve of the chromatogram.

(40) By way of generalization, table 3 below presents the mean values of the retention times (min), of the corresponding apparent molecular weights (Da) and of the percentage of the area under the curve for each family of peptides, corresponding to separate analyses of three peptide compositions according to the invention.

(41) TABLE-US-00004 TABLE 3 Retention time Molecular weight (min) (Da) Area (%) 11.13 ± 0.15 10 870 ± 830.sup.  4.6 ± 2 11.50 ± 0.20 8596 ± 700 5.6 ± 2 11.7 ± 0.1 7180 ± 100 4.5 ± 1 12.27 ± 0.3  5703 ± 400 9.6 ± 2 12.45 ± 0.20 4430 ± 100 13.0 ± 1  13.00 ± 0.06 3100 ± 100 13.5 ± 1  13.67 ± 0.06 1870 ± 40  20.0 ± 1  14.64 ± 0.02 983 ± 20 22.5 ± 2  16.18 ± 0.04 340 ± 10  12.0 ± 2.5 16.79 ± 0.01 224 ± 5    0.7 ± 0.15

(42) The mean proportion of peptides of compositions according to the invention—the values of which are given in table 3—and the molecular weight of which is less than 1400 Da is 35.2% (essentially corresponding to the apparent molecular weights of 983 Da and 340 Da).

(43) Analysis of the Polarity of the Constituent Peptides

(44) The proportion of anionic peptides in the composition according to the invention, the distribution of the apparent molecular weights of which is given in table 2, is analyzed, that is to say the proportion of peptides having 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, that is to say the proportion of peptides having an overall 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, Montlugon, France) with a particle size of 10 μm. The ion exchange HPLC chromatography column is of dimensions 100×7.8 mm.

(45) The HPLC chromatographic column is conditioned by ion exchange in a tris(hydroxymethyl)aminomethane (Tris) buffer at a concentration of 5 mM in water and the pH of which is adjusted to the value of 8.35.

(46) The temperature of the column is kept at a temperature of 25° C. The flow rate of the mobile phase in the column is 1 ml/min. A sample of the peptide composition to be analyzed is prepared such that the concentration thereof is 2 g/l, by dilution in ultrapure water. A volume of 90 μl of this solution to be analyzed is introduced at the top of the column. The detection is carried out by continuously measuring the absorbance at 214 nm.

(47) Starting from the introduction of the sample at the top of the column, the mobile phase consists of 5 mM Tris at pH 8.35 (solution A) for a duration of 7 minutes, then of a mobile phase in which a solution B, formed of 5 mM Tris, 5 M NaCl, pH 8.35, increases linearly from 0 to 100% in solution A in 30 minutes. The elution is then maintained by solution B for 2 minutes.

(48) The chromatogram obtained is shown in FIG. 2. The anionic peptides leave the column with a retention time of between 10 min and 17.5 min corresponding to an NaCl concentration of between 0.5 M and 1.7 M. The proportion of anionic peptides in the peptide composition according to the invention, the analysis of which is shown 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, the analysis of which is shown in FIG. 2, is 57.5%.

(49) By way of generalization, this analysis is reproduced on three peptide compositions according to the invention. The mean proportion of anionic peptides in these compositions according to the invention is between 27.9% and 42.5% and the mean proportion of neutral and cationic peptides in these compositions according to the invention is between 57.5% and 72.1%.

(50) Analysis of the Hydrophobicity of the Constituent Peptides

(51) The hydrophobicity of the constituent peptides of the composition according to the invention is analyzed by reversed-phase liquid chromatography on a column of silica grafted with butyl groups (Vydac 214TP™ C.sub.4, Grace, Epernon, France), of dimensions 250×4.6 mm. The particle size of the silica is 5 μm and the porosity thereof is 300 Å.

(52) The column is conditioned in ultrapure water acidified with 0.1% of trifluoroacetic acid. The temperature of the column is kept at a temperature of 40° C. The flow rate of the mobile phase in the column is 0.6 ml/min. A sample of the peptide composition to be analyzed is prepared such that the concentration thereof is 2 μg/l, by dilution in ultrapure water. A volume of 100 μl of this solution to be analyzed is introduced at the top of the column. Detection is carried out by continuously measuring the absorbance at 214 nm.

(53) Starting from the introduction of the sample at the top of the column, the mobile phase consists of acidified water (solution A) for a duration of 7 minutes, then of a mobile phase in which a solution B, formed of water acidified with 0.1% (by volume) of trifluoroacetic acid and comprising 40% of acetonitrile increases linearly from 0 to 100% in solution A in 30 minutes.

(54) The chromatogram obtained is shown 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.

(55) Biological Effects of a Composition According to the Invention

(56) Effect on Colonization by Candida albicans and on Digestive Candidiasis

(57) The effect of a composition according to the invention on digestive candidiasis induced in female C57BL/6 mice, aged 8 weeks and of a weight of between 20-25 g is analyzed. These mice are fed for a duration of 21 days with the composition according to the invention at an amount of 4 g/kg/day. On the 21.sup.st day, digestive candidiasis is induced by force-feeding each mouse with an amount of 5×10.sup.7 yeast cells. Between 2 days (D2) and 7 days (D7) after induction, the mice's stools are collected and the yeast load is evaluated (CFU/mg of stools) on a chromogenic medium. The results are given in table 4 below in comparison with the values measured on mice in which digestive candidiasis is induced but which are not treated with the composition according to the invention.

(58) TABLE-US-00005 TABLE 4 Composition Day after according to the induction invention CFU/mg stools D 3 Yes 125 D 3 No 263 D 4 Yes 125 D 4 No 275 D 5 Yes 150 D 5 No 250

(59) The composition according to the invention induces a reduction in the load of Candida albicans.

(60) Anti-Inflammatory Peptide Composition According to the Invention

(61) A comparative analysis is carried out of the stimulation of expression of inflammatory type 1 macrophage (M1) receptors and/or anti-inflammatory type 2 macrophage (M2) receptors by a composition of peptides according to the invention resulting from an enzymatic hydrolysis of collagen from skins of temperate-water fish and by a composition of peptides resulting from an enzymatic hydrolysis of collagen from skins of cold-water fish (outside the invention).

(62) Macrophages/monocytes from healthy human subjects were cultured for 24 hours in the presence of a pretreatment composition (control without collagen peptides, invention, outside of the invention, table 7) at a concentration of 100 μg/ml of culture medium. The effect of this pretreatment on the expression of characteristic receptors of type 1 macrophages (M1, inflammatory) and on the expression of characteristic receptors of type 2 macrophages (M2, anti-inflammatory) is evaluated by the level of production of oxygen free radicals (reactive oxygen species, ROS) by the receptors of type 1 macrophages specifically stimulated by a phorbol ester (“12-myristate-13-acetate-phorbol, TPA”) at a concentration of 100 μM or by the receptors of type 2 macrophages specifically stimulated by non-opsonized zymosan (NOZ) at a concentration of 100 μg/ml. The level of production of oxygen free radicals is measured by chemiluminescence in the presence of luminol at a concentration of 66 μM. The results presented in table 7 below represent the mean values obtained with three assays.

(63) TABLE-US-00006 TABLE 7 Luminescence, arbitrary units ×10.sup.−8 Pretreatment Without Outside collagen invention the invention Inducer Not 1.23 ± 0.16 1.64 ± 0.10 1.80 ± 0.18 induced NOZ 4.49 ± 0.43 5.06 ± 0.02 3.91 ± 0.38 TPA 9.96 ± 0.40 6.75 ± 0.11  1.4 ± 0.59

(64) It is observed that the macrophages/monocytes not treated (not induced) by NOZ and TPA have a level of production of oxygen free radicals that is substantially constant depending on the nature of the pretreatment (without collagen, according to the invention and outside the invention).

(65) The macrophages/monocytes pretreated with a composition of peptides according to the invention, that is to say resulting from an enzymatic hydrolysis of collagen from skins of temperate-water fish, have an increased expression of the receptors of anti-inflammatory type 2 macrophages (M2) revealed by the chemiluminescence intensity (5.06×10.sup.8 AU) induced by NOZ, compared to the macrophages/monocytes pretreated with a composition of peptides outside the invention, that is to say resulting from an enzymatic hydrolysis of collagen from skins of cold-water fish (3.91×10.sup.8 AU).

(66) The macrophages/monocytes pretreated with a composition of peptides according to the invention, that is to say resulting from an enzymatic hydrolysis of collagen from skins of temperate-water fish, have a decreased expression of the receptors of inflammatory type 1 macrophages (M1) revealed by the chemiluminescence intensity (6.75×10.sup.8 AU) induced by TPA, compared to the macrophages/monocytes pretreated with a composition of peptides outside the invention, that is to say resulting from an enzymatic hydrolysis of collagen from skins of cold-water fish (1.49×10.sup.9 AU) 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 resulting from an enzymatic hydrolysis of collagen from skins of cold-water fish, by increasing the level of expression of the receptors of anti-inflammatory type 2 macrophages (M2) and by decreasing the level of expression of the receptors of inflammatory type 1 macrophages (M1).

(67) The preparation of a composition of peptides according to the invention resulting from skins of temperate-water fish and having an aminogram in which: 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%; 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%; 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%; has such an anti-inflammatory phenotype, confirmed by colonic inflammation assays on a murine model, which is not found with a composition of peptides resulting from skins of cold-water fish and having an aminogram as described in table 6.
Colonic Inflammation in a Murine Model

(68) The anti-inflammatory effect of the peptide composition according to the invention is demonstrated on a murine model of pharmacological inflammation induced by dextran sulfate sodium (DSS, MP Biomedical LLC, Canada) characterized by weight loss and bloody diarrhea.

(69) The effect of the peptide composition according to the invention in limiting the weight loss of mice treated with DSS was studied. Female C57BL/6 laboratory mice aged from 10 to 11 weeks and of a weight of between 20 and 25 grams were treated for 7 days (D.sub.1 to D.sub.7) with DSS dissolved at an amount of 1.5% (weight/volume) in the mice's drinking water. These mice are also treated for 12 days (D.sub.1 to D.sub.12) with the composition according to the invention at an amount of 0.1 g/kg/day; 1 g/kg/day and 4 g/kg/day. This amount of composition according to the invention is dispensed in the drinking water of the mice. On D.sub.12, the mice are euthanized.

(70) 5 batches of mice are prepared, each batch containing 10 mice, in which: batch 1 is treated with DSS for 7 days; batch 2 is treated with DSS for 7 days and with the composition according to the invention at an amount 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 an amount 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 an amount of 4 g/kg/day for 12 days; batch 5 is treated with DSS for 7 days and with hydrolyzed casein not in accordance with the invention at an amount of 0.1 g/kg/day for 12 days.

(71) In parallel, a control is carried out on 5 mice which are not treated with DSS and for which inflammation is not induced, and which are not treated with a peptide composition according to the invention.

(72) 1. Study of Body Weight

(73) The weight of each mouse is measured each day and the weight loss undergone by each mouse from each batch is calculated. The results are presented in FIG. 4, in which the control is represented by empty circles (∘), batch 1 is represented by filled-in circles (□), batch 2 is represented by empty squares (□), batch 3 is represented by filled-in squares (.square-solid.), batch 4 is represented by empty triangles (Δ) and batch 5 is represented by filled-in triangles (.box-tangle-solidup.). It is observed that, from 0.1 g/kg/day (batch 2, □), the composition according to the invention limits the mice's weight loss. This limiting is also observed for 1 g/kg/day (batch 3, .square-solid.) and for 4 g/kg/day (batch 4, Δ). It is not observed for the treatment with hydrolyzed casein (batch 5, .box-tangle-solidup.), for which the weight loss is comparable to the weight loss of the mice from batch 1 (•).

(74) The composition according to the invention makes it possible to limit, or even to virtually entirely do away with, the weight loss caused by the inflammation induced by the dextran sulfate sodium (DSS) in mice. The composition according to the invention is capable of being used as medicament, especially for the treatment of colonic inflammation.

(75) 2. Histology

(76) The transversal histological sections of colons from mice treated with DSS alone exhibit, after bichromatic staining with hematoxylin and eosin, significant infiltrations of inflammatory cells at the mucosa and sub-mucosa. The thickness of the epithelium is reduced. The epithelium exhibits extensive ulceration. The transversal histological sections of colons from mice treated with DSS and with the composition according to the invention at an amount of 0.1 g/kg/day, 1 g/kg/day and 4 g/kg/day exhibit, after bichromatic staining with hematoxylin and eosin, tightly packed, straight tubular glands representative of a healthy and functional epithelium.

(77) 3. Macroscopic Score

(78) For each treatment condition, a macroscopic score is calculated from notation created according to the Wallace scale relating to the appearance of the stools, the damaged appearance of the colon, the weight of the colon and the length of the colon, according to the Wallace scale (E. S. Kimball, N. H. Wallace, C. R. Schneider, M. R. D'Andrea and P. J. Hornby; 2004; Neurogastroenterol Motil; 16, 811-818. Vanilloid receptor 1 antagonists attenuate disease severity in dextran sulfate sodium-induced colitis in mice). The more inflammatory the colon is, the higher the value of the macroscopic score, and the healthier the colon is, the lower the value of the macroscopic score.

(79) The mean values and the standard deviation of the macroscopic score of the mice from batches 1 to 5 are given in table 5 below.

(80) TABLE-US-00007 TABLE 5 Mean macroscopic Standard score deviation Batch 1 5.14 0.46 Batch 2 (0.1 g/kg/day) 1.30 0.33 p < 0.01 Batch 3 (1 g/kg/day) 1.90 0.5 p < 0.01 Batch 4 (4 g/kg/day) 3.00 0.33 p < 0.01 Batch 5 5.00 0.61

(81) A reduction in the macroscopic score is observed, induced by treatment with the composition according to the invention, that is to say an improvement in the inflammatory state of the colon induced by the composition according to the invention.

(82) 4. Inhibition of the Expression of Pro-Inflammatory Markers in Mice

(83) On D.sub.12, the mice are euthanized and the level of expression at the colon of pro-inflammatory cytokines is assayed by the ELISA technique: IL-1β: The level of IL-1β is analyzed by the ELISA technique and expressed in picograms (pg) of IL-1β per milligram (mg) of colon. The results are shown in FIG. 5, in which the first column (white column) corresponds to the analysis carried out on control mice. The second column (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. A statistically significant (p<0.05) reduction in the expression of IL-1β is observed in the colon of mice treated with the peptide composition according to the invention for doses of 0.1 g/kg/day, 1 g/kg/day and 4 g/kg/day compared to the colon of mice in which inflammation is induced by DSS and compared to mice in which inflammation is induced by DSS and treated with hydrolyzed casein. This effect was confirmed by analysis of IL-1β messenger RNA 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 picograms (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 carried out on control mice. The second column (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. A statistically significant reduction in the expression of IL6 is observed in the colon of mice treated with the peptide composition according to the invention 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 to the colon of mice in which inflammation is induced by DSS and compared to mice in which inflammation is induced by DSS and treated with hydrolyzed casein. This effect was confirmed by analysis of IL6 messenger RNA by quantitative RT-PCR; TNF-α: The level of TNF-α is analyzed by the ELISA technique and expressed in picograms (pg) of TNF-α per milligram (mg) of colon. The results are shown in FIG. 7, in which the first column (white column) corresponds to the analysis carried out on control mice. The second column (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. A statistically significant reduction in the expression of TNF-α is observed in the colon of mice treated with the peptide composition according to the invention for 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) compared to the colon of mice in which inflammation is induced by DSS and compared to mice in which inflammation is induced by DSS and treated with hydrolyzed casein.

(84) The analysis by quantitative RT-PCR (reverse transcriptase polymerase chain reaction) of the messenger RNAs of MCP1 shows a statistically significant reduction in these mRNAs induced by DSS, in particular for the doses of 0.1 g/kg/day (p<0.01) and 1 g/kg/day (p<0.05) of peptide composition according to the invention.

(85) The analysis by quantitative RT-PCR (reverse transcriptase polymerase chain reaction) of the messenger RNAs of 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 (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. A statistically significant reduction in the iNOS mRNAs induced by DSS is observed for doses of 0.1 g/kg/day (p<0.05) and 1 g/kg/day (p<0.05) of the peptide composition according to the invention.

(86) 5. Stimulation of the Expression of Anti-Inflammatory Markers in Mice

(87) On D.sub.12, the mice are euthanized and the level of expression at the colon of anti-inflammatory markers is assayed: TGF-β: The level of TGF-β is analyzed by the ELISA technique and expressed in picograms (pg) of TGF-β per milligram (mg) of colon. The results are shown in FIG. 8, in which the first column (white column) corresponds to the analysis carried out on control mice. The second column (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. Stimulation of the expression of TGF-β is observed in the colon of mice treated with the peptide composition according to the invention for the dose of 0.1 g/kg/day compared to the colon of mice in which inflammation is induced by DSS. This effect was confirmed by analysis of TGF-β messenger RNA 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 (FIG. 16); Fizz1: Fizz1 is a marker of anti-inflammatory M2 macrophages. The analysis by quantitative RT-PCR (reverse transcriptase polymerase chain reaction) of the messenger RNAs of Fizz1 is shown in FIG. 18, in which the first column (white column) corresponds to the analysis carried out on control mice. The second column (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. A statistically significant increase in the Fizz1 messenger RNAs, decreased by DSS, is 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) of the peptide composition according to the invention; a statistically significant increase in the Ym1 messenger RNAs, decreased by DSS, was also observed for doses of 0.1 g/kg/day (p<0.05) of the peptide composition according to the invention.
6. Effect of the Peptide Composition According to the Invention on the Colonic Flora—Colon Microbiota

(88) On D.sub.12, the mice are euthanized and the colonic flora of these mice is quantified by PCR (polymerase chain reaction). The values are standardized relative to the total amount of bacteria or fungi and relative to β-actin. β-actin constitutes the reference gene enabling standardization relative to the amount of colonic tissue analyzed. In particular, the following are quantified: the total fungal flora by quantitative amplification of the ITS1-2 fungal ribosomal DNA. The ratio R of the amount of ITS1-2 fungal ribosomal DNA to the amount of β-actin DNA is shown in FIG. 9, in which the first column (white column) corresponds to the assay carried out on control mice. The second column (column with diagonal hatching) corresponds to the assay carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the assay carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the assay carried out on the mice from batch 3. The fifth column (gray column) corresponds to the assay carried out on the mice from batch 2. The sixth column (black column) corresponds to the assay carried out on the mice from batch 5. The peptide composition according to the invention restores the fungal flora in particular at the dose of 4 g/kg/day; the yeast Saccharomyces cerevisiae by quantitative amplification of the 26S ribosomal DNA. The ratio R of the amount of 26S ribosomal DNA to the amount of β-actin DNA is shown in FIG. 10. The first column (white column) corresponds to the assay carried out on control mice. The second column (column with diagonal hatching) corresponds to the assay carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the assay carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the assay carried out on the mice from batch 3. The fifth column (gray column) corresponds to the assay carried out on the mice from batch 2. The sixth column (black column) corresponds to the assay carried out on the mice from batch 5. The peptide composition according to the invention statistically significantly restores the Saccharomyces cerevisiae flora at doses of 0.1 g/kg/day (p<0.05), 1 g/kg/day (p<0.05) and 4 μg/kg/day (p<0.05). In this instance, S. cerevisiae has anti-inflammatory potential; the Enterobacteriaceae flora by quantitative amplification of the 16S ribosomal DNA. The ratio R of the amount of 16S ribosomal DNA to the amount of β-actin DNA is shown in FIG. 11. The first column (white column) corresponds to the assay carried out on control mice. The second column (column with diagonal hatching) corresponds to the assay carried out on the mice from batch 1. The third column (gray column) corresponds to the assay carried out on the mice from batch 2. The fourth column (black column) corresponds to the assay carried out on the mice from batch 5. The peptide composition according to the invention enables a reduction in the Enterobacteriaceae flora, induced by DSS, at a concentration of 0.1 g/kg/day. Enterobacteriaceae are associated with a pro-inflammatory potential; the Firmicutes flora by quantitative amplification of the 16S ribosomal DNA on a fragment of the gene enabling analysis of diversity in the phylum. The ratio R of the amount of such a 16S ribosomal DNA to the amount of β-actin DNA is shown in FIG. 12. The first column (white column) corresponds to the assay carried out on control mice. The second column (column with diagonal hatching) corresponds to the assay carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the assay carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the assay carried out on the mice from batch 3. The fifth column (gray column) corresponds to the assay carried out on the mice from batch 2. The sixth column (black column) corresponds to the assay carried out on the mice from batch 5. The peptide composition according to the invention induces a statistically significant reduction in the Firmicutes flora, induced by DSS, at concentrations of 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 induces a reduction in the non-induced Firmicutes flora at these same concentrations. It should be noted that an increase in the Firmicutes flora is generally observed in a known manner during digestive inflammation (IBDs, inflammatory bowel diseases); the Bacteroidetes flora by quantitative amplification of the 16S ribosomal DNA on a fragment of the gene enabling analysis of diversity in the phylum. The ratio R of the amount of such a 16S ribosomal DNA to the amount of β-actin DNA is shown in FIG. 13. The first column (column with diagonal hatching) corresponds to the assay carried out on the mice from batch 1. The second column (column with vertical hatching) corresponds to the assay carried out on the mice from batch 4. The third column (column with horizontal hatching) corresponds to the assay carried out on the mice from batch 3. The fourth column (gray column) corresponds to the assay carried out on the mice from batch 2. The fifth column (black column) corresponds to the assay carried out on the mice from batch 5. The peptide composition according to the invention induces a statistically significant reduction in the Bacteroidetes flora, induced by DSS, at concentrations of 0.1 g/kg/day (p<0.05), 1 g/kg/day (p<0.05) and 4 g/kg/day (p<0.05). An increase in Bacteroidetes is generally observed in a known manner during digestive inflammation (IBD); the Faecalibacterium prausnitzii flora by quantitative amplification of specific DNA. The ratio R of the amount of such a specific DNA to the amount of β-actin DNA is shown in FIG. 14. The first column (white column) corresponds to the assay carried out on control mice. The second column (column with diagonal hatching) corresponds to the assay carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the assay carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the assay carried out on the mice from batch 3. The fifth column (gray column) corresponds to the assay carried out on the mice from batch 2. The sixth column (black column) corresponds to the assay carried out on the mice from batch 5. The peptide composition according to the invention induces a statistically significant increase in the Faecalibacterium prausnitzii flora, destroyed by DSS, at concentrations of 0.1 g/kg/day (p<0.01), 1 g/kg/day (p<0.01) and 4 g/kg/day (p<0.01). An increase in Faecalibacterium prausnitzii is generally observed in a known manner during digestive inflammation (IBD). Faecalibacterium prausnitzii also allegedly has anti-inflammatory properties; the Lactobacillus murinus flora by quantitative amplification of specific DNA. The ratio R of the amount of such a specific DNA to the amount of β-actin DNA is shown in FIG. 15, in which the first column (white column) corresponds to the assay carried out on control mice. The second column (column with diagonal hatching) corresponds to the assay carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the assay carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the assay carried out on the mice from batch 3. The fifth column (gray column) corresponds to the assay carried out on the mice from batch 2. The sixth column (black column) corresponds to the assay carried out on the mice from batch 5. The peptide composition according to the invention induces an increase in the 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 allegedly has anti-inflammatory properties.
7. Effect of the Peptide Composition According to the Invention on Enzymes of the Lipid Metabolism of Arachidonic Acid: 5-LOX: The analysis by quantitative RT-PCR (reverse transcriptase polymerase chain reaction) of the messenger RNAs of the enzyme 5-lipoxygenase (5-LOX) is shown in FIG. 19, in which the first column (white column) corresponds to the analysis carried out on control mice. The second column (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. A statistically significant reduction in the expression of 5-LOX was observed for doses of the peptide composition according to the invention of 0.1 g/kg/day (p<0.01) and 1 g/kg/day (p<0.01) relative to the control not induced by DSS. The peptide composition according to the invention has an inhibitory effect on the expression of 5-LOX, promoting the production of pro-inflammatory lipid mediators; 12/15-LOX: The analysis by quantitative RT-PCR (reverse transcriptase polymerase chain reaction) of the messenger RNAs of the enzyme 12/15-lipoxygenase (12/15-LOX) is shown in FIG. 20, in which the first column (white column) corresponds to the analysis carried out on control mice. The second column (column with diagonal hatching) corresponds to the analysis carried out on the mice from batch 1. The third column (column with vertical hatching) corresponds to the analysis carried out on the mice from batch 4. The fourth column (column with horizontal hatching) corresponds to the analysis carried out on the mice from batch 3. The fifth column (gray column) corresponds to the analysis carried out on the mice from batch 2. The sixth column (black column) corresponds to the analysis carried out on the mice from batch 5. A statistically significant increase in the expression of 12/15-LOX was observed for doses of the peptide composition according to the invention of 0.1 g/kg/day (p<0.01) and 1 g/kg/day (p<0.01) relative to the control not induced by DSS.

(89) It goes without saying that the invention may be subject to numerous variant embodiments and applications. In particular, different uses as medicament may vary without departing from the scope of protection of the invention.