EP4376865A2 - Pulse protein-based composition for activating the synthesis of fgf19 - Google Patents

Pulse protein-based composition for activating the synthesis of fgf19

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Publication number
EP4376865A2
EP4376865A2 EP22750660.7A EP22750660A EP4376865A2 EP 4376865 A2 EP4376865 A2 EP 4376865A2 EP 22750660 A EP22750660 A EP 22750660A EP 4376865 A2 EP4376865 A2 EP 4376865A2
Authority
EP
European Patent Office
Prior art keywords
leu
ser
ala
proteins
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22750660.7A
Other languages
German (de)
French (fr)
Inventor
Catherine Lefranc-Millot
Hubert Vidal
Clémence DEFOIS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roquette Freres SA
Original Assignee
Roquette Freres SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roquette Freres SA filed Critical Roquette Freres SA
Publication of EP4376865A2 publication Critical patent/EP4376865A2/en
Pending legal-status Critical Current

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Classifications

    • 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
    • A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
    • 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
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • 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
    • A23J3/14—Vegetable proteins
    • 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
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • 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
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • A23L33/185—Vegetable proteins

Definitions

  • the present invention consists of a composition of legume proteins, preferably pea or faba bean, the degree of hydrolysis of which is less than 10%, in particular for therapeutic use, preferably in the prevention and/or or the treatment of a disease capable of being treated by an activation of the synthesis of FGF19.
  • legume proteins preferably pea or faba bean
  • a fibroblast is a human cell present in the connective tissue.
  • the most important role of fibroblasts is to maintain the extracellular matrix of connective tissues, and to repair damage due to trauma. They also serve to regulate the organization and differentiation of cells in surrounding tissues.
  • These fibroblasts will, among other things, secrete the extracellular matrix, that is to say the proteins which form the fibers of the connective tissue and the glycoproteins of the ground substance. They are also involved in the metabolism of lipoproteins (LDL) and cholesterol.
  • LDL lipoproteins
  • Fibroblast growth factors (or FGF, English acronym for Fibroblast Growth Factor) form a family comprising 23 proteins identified to date in humans (FGF1, FGF2, etc. FGF23) which activate migration and multiplication fibroblasts. These factors are generally secreted by fibroblasts.
  • FGF19 (for the acronym for “Fibroblast growth factor 19”) is a protein belonging to this family of fibroblast growth factors. Its synthesis is activated by FXR (for the English acronym of "Farnesoid X receptor"), itself stimulated by bile salts, which has the effect of inhibiting gluconeogenesis and the synthesis of bile salts and activate protein and glycogen synthesis. It also plays on lipid and calcium metabolism.
  • a low serum FGF19 level is related to more serious forms of non-alcoholic fatty liver disease or NASH (Alisi A, Ceccarelli S, Panera N et al. Association between serum atypical fibroblast growth factors 21 and 19 and pediatric nonalcoholic fatty liver disease, PLoS One, 2013;8:e6716). Its use in the fight against NASH has therefore been studied. In an animal model, it unfortunately seems to promote the formation of hepatocellular carcinoma. An analogue of the molecule has been developed, without oncogenesis activity. It appears to show promise in the treatment of non-alcoholic fatty liver disease, decreasing the lipid content of liver cells. The use of an analog is however restrictive due to the need for the exogenous synthesis of said analog and its consumption as a drug. There is therefore an unmet need to naturally activate the in-vivo synthesis of FGF19 to prevent or treat NASH.
  • Sarcopenia is a geriatric syndrome characterized initially by a decrease in muscle capacity due to age and which, as it gets worse, will cause a deterioration in muscle strength and physical performance.
  • the sarcopenia observed in the elderly is attributable to the aging process but can be accelerated by pathological and behavioral factors such as malnutrition and a sedentary lifestyle.
  • FGF19 has recently emerged as an opportunity to fight the prevalence of sarcopenia (“Fibroblast growth factor 19 regulates skeletal muscle mass and improves muscle wasting in mice.” Benoit & al., Nat Med. 2017 Aug;23(8) :990-996).
  • sarcopenia Fibroblast growth factor 19 regulates skeletal muscle mass and improves muscle wasting in mice. Benoit & al., Nat Med. 2017 Aug;23(8) :990-996.
  • the present invention therefore consists first of all of a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10% for therapeutic use in a subject who needs them, preferably for use in the prevention and/or treatment of a disease capable of being treated by an activation of the synthesis of FGF19, in particular a disease associated with a dysregulation of the signaling pathway of FGF19.
  • the present invention also consists of a pharmaceutical composition intended to overexpress FGF19 comprising legume proteins, preferably pea or horse bean, the degree of hydrolysis of which is less than 10% and optionally a pharmaceutically acceptable excipient.
  • FIG. 1 shows the effects of different digestates on the expression of FGF19.
  • HT29 cells were exposed to different protein digests (PD) alone or in combination with an FXR agonist.
  • Fig. 2 shows the effects of different digestates on the expression of FGF19.
  • HT29 cells were exposed to different protein digests (PD) alone or in combination with an FXR agonist.
  • Fig. 2 shows the effects of different digestates on the expression of FGF19.
  • FIG. 2a shows the increase in the expression of FGF19 mRNA following treatment of HT29 cells with RQ3 and RQ7 digests.
  • HT29 cells were exposed to 0.1, 0.5, and 1 mM GW4064 and a natural FXR agonist, acid chenodeoxycholique (CDCA) at 50 mM alone or in combination for 6 hours with the RQ3 or RQ7 extracts.
  • a natural FXR agonist acid chenodeoxycholique
  • FIG. 2b shows the increase in the production and secretion of the FGF19 protein following the treatment of FIT29 cells with RQ3 and RQ7 digestates.
  • HT29 cells were exposed to GW4064 at 0, 1, 0.5 and 1 mM and a natural FXR agonist, chenodeoxycholic acid (CDCA) at 50 mM alone or in combination for 6h (left) and 24h (right) with extracts RQ3 or RQ7.
  • FIG. 3 shows the increase in the level of expression of FGF19 (mRNA) following co-culture treatment of Caco-2 and HT29-MTX cells with RQ3 and RQ7 digests.
  • composition of legume proteins according to the invention
  • the inventors have shown that a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10%, makes it possible to activate the expression of FGF19 in human cells.
  • the present invention therefore consists first of all of a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10% for therapeutic use in a subject who needs them, preferably for use in the prevention and/or treatment of a disease capable of being treated by an activation of the synthesis of FGF19, in particular a disease associated with a dysregulation of the signaling pathway of FGF19.
  • protein or “polypeptide” should be understood in the present application as the macromolecules formed from one or more polypeptide chains consisting of the sequence of amino acid residues linked together by peptide bonds.
  • legume will be understood in the present application to include the family of dicotyledonous plants of the order Fabales. It is one of the most important families of flowering plants, the third after Orchidaceae and Asteraceae by the number of species. It has about 765 genera comprising more than 19,500 species.
  • Several legumes are important cultivated plants including soybeans, beans, peas, chickpeas, field beans, groundnuts, cultivated lentils, cultivated alfalfa, various clovers, broad beans, carob, liquorice.
  • the present invention relates more particularly to globulins (approximately 50-60% of pea proteins) and albumins (20-25%).
  • Pea globulins are mainly subdivided into three subfamilies: legumes, vicilins and convicilins.
  • the proteins extracted from these legumes mainly belong to the globulin and albumin subgroups.
  • the legume protein mainly consists of globulins, in particular it contains more than 90% by weight of globulins relative to the total weight of the proteins.
  • Globulins can be distinguished from albumins by various methods well known to those skilled in the art, in particular by their solubility in water, albumins being soluble in pure water whereas globulins are only soluble in salt water.
  • the albumins and globulins present in a mixture can also be identified by electrophoresis or chromatography.
  • the legume protein according to the invention contains more than 90% by weight of globulins relative to the total weight of the proteins.
  • the term “pea” being considered here in its broadest sense and including in particular all varieties of “smooth pea”("smoothpea”) and “wrinkled pea”("wrinkledpea”), and all mutant varieties of “smooth pea” and “wrinkled pea” and this, regardless of the uses for which said varieties are generally intended (human food, animal nutrition and/or other uses).
  • the term “pea” in the present application includes the varieties of peas belonging to the genus Pisum and more particularly to the species sativum and aestivum.
  • Said mutant varieties are in particular those called “r mutants”, “rb mutants”, “rug 3 mutants”, “rug 4 mutants”, “rug 5 mutants” and “lam mutants” as described in the article by CL Heydley and para. entitled “Developing novel pea starches” Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87.
  • bean means the group of annual plants of the species Vicia faba, belonging to the group of legumes of the family Fabaceae, subfamily Faboideae, tribe Fabeae. A distinction is made between Minor and Major varieties. In the present invention, wild varieties and those obtained by genetic engineering or varietal selection are all excellent sources.
  • the legume proteins preferably pea or faba bean, having a degree of hydrolysis of less than 10% are the only protein source within the composition.
  • legume proteins are characterized by an amino acid profile deficient in certain amino acids (methionine and cysteine) when compared with the profiles required in human nutrition. .
  • This deficiency is often overcome by making a mixture with other protein sources such as animal proteins or cereal proteins such as rice or wheat.
  • the Applicant aims on the contrary to exploit to the maximum the effect of legume proteins, preferentially pea or faba bean, having a degree of hydrolysis of less than 10%, present in the composition in order to overexpress maximum FGF19. Any attempt to mix with other protein sources to restore the amino acid profile will result in a diminished effect on FGF19 overexpression.
  • the degree of hydrolysis can be determined by measuring the free amino nitrogen content relative to the total nitrogen according to the degree of hydrolysis test described below. After.
  • amino nitrogen groups of the free amino acids of the sample react with N-acetyl-L-cysteine and O-Phthaldialdehyde (OPA) to form isoindole derivatives.
  • OPA O-Phthaldialdehyde
  • the amount of isoindole derivative formed during this reaction is stoichiometric with the amount of free amino nitrogen. It is the isoindole derivative which is measured by the increase in absorbance at 340 nm.
  • test portion P* of the sample to be analyzed is introduced in a 100 mL beaker. This test portion will be 0.5 to 5.0 g depending on the amino nitrogen content of the sample.
  • Solution #1 is prepared by dissolving one tablet from vial 1 of the Megazyme kit in 3 mL of distilled water and shaking until completely dissolved. One tablet should be provided per test.
  • Solution No. 1 is prepared extemporaneously.
  • a blank, a standard and a sample are prepared directly in the tanks of the spectrophotometer under the following conditions: blank: introduce 3.00 ml of solution No. 1 and 50 pl of distilled water - standard: introduce 3, 00 ml of solution n°1 and 50 ⁇ l of vial 3 of the kit
  • Sample Megazyme Add 3.00 mL of solution #1 and 50 mL of sample preparation. The contents of each tank are mixed and the absorbance measurement (A1) of the solutions is read after approximately 2 minutes using a spectrophotometer at 340 nm (spectrophotometer equipped with tanks with a 1.0 cm optical path, capable of measuring at a wavelength of 340 nm, and checked according to the procedure described in the manufacturer's technical manual relating thereto).
  • the reactions are then initiated immediately by adding 100 ⁇ L of solution No. 2 which corresponds to the OPA solution from bottle 2 of the Megazyme kit in each spectrophotometer tank.
  • the absorbance A2 measurement of the blank, of the standard and of the sample is then read using a spectrophotometer at 340 nm.
  • the protein nitrogen content is determined according to the DUMAS method according to the ISO 16634 - 2016 standard. It is expressed as a percentage by weight relative to the weight of the product. - Calculation of the degree of hydrolysis
  • pea proteins having a degree of hydrolysis strictly between 5% and 10%. Indeed, if the degree of hydrolysis is higher or lower, it is found that the effect of activating the synthesis of FGF19 is less effective.
  • the legume proteins are pea proteins, the degree of hydrolysis of which is preferably between 5% and 10%.
  • pea proteins having a degree of hydrolysis strictly between 5% and 10%, are obtained by any means well known to those skilled in the art.
  • the starting base is a flour, preferably a concentrate (more than 50% protein on the DM), even more preferably an isolate (more than 80% protein on the DM). Any process well known to those skilled in the art for this will be used, whether by dry process (extraction without the use of aqueous solvent, e.g. via turboseparation) or by wet process (obtaining a flour by grinding, suspending flour in an aqueous solvent, extraction of insoluble compounds, in particular starch and internal fibers, isoelectric precipitation of proteins belonging to the globulin groups, recovery of the latter by centrifugation).
  • dry process extraction without the use of aqueous solvent, e.g. via turboseparation
  • wet process obtaining a flour by grinding, suspending flour in an aqueous solvent, extraction of insoluble compounds, in particular starch and internal fibers, isoelectric precipitation of proteins belonging to the globulin groups, recovery of the latter by centrifugation).
  • the pea proteins obtained by the wet process are particularly preferred because it leads to the obtaining of an isolate which concentrates to the maximum the capacities of the latter to activate the synthesis of FGF19.
  • the pea proteins (flour, preferably concentrate, even more preferably isolate) will then be hydrolysed.
  • Any method of protein hydrolysis whether chemical, physical, enzymatic or biological, well known to those skilled in the art can be used in order to obtain the legume proteins, preferably pea proteins, to be used for the invention.
  • Enzymatic hydrolysis will be preferred, in particular hydrolysis carried out with protease belonging to the aminopeptidase subgroup originating from Aspergillus Orizae.
  • the preparation of the pea protein hydrolyzate suitable for the invention therefore preferably comprises enzymatic or non-enzymatic hydrolysis, so that said pea protein isolate has a degree of hydrolysis (DH) between 5% and 10%, preferably between 6% and 8%, even more specifically from 6.5% to 7%.
  • DH degree of hydrolysis
  • the hydrolysis is carried out by an endopeptidase.
  • a non-specific endopeptidase is chosen, derived from a strain of Aspergillus, in particular a strain of Aspergillus spp or Aspergillus Oryzae. More particularly, an endopeptidase EC 3-4-11 is chosen.
  • the exact amount of enzyme added to the suspension to achieve the desired characteristics of pea protein isolates will vary depending on specific characteristics such as the enzyme or enzyme system used; the desired final degree of hydrolysis; and/or desired molecular/final weight distribution. Since these parameters are known, one skilled in the art can easily determine the appropriate conditions to obtain the desired characteristics of the pea protein isolate.
  • the initial pea proteins used to prepare the pea protein isolate according to the invention is a composition of pea proteins as described in application WO 2007/17572 or prepared by a method as described in application WO 2007/17572 (the teaching being incorporated by reference).
  • the initial pea protein composition is the composition marketed by ROQUETTE FRERES under the brand name NUTRALYS® S85F.
  • the pea protein suspension is brought to a value of 5 to 20% by weight of dry matter, in particular 15 to 20%.
  • the reaction temperature is adjusted to a value between 50 and 60°C, preferably around 55°C.
  • the enzyme system or an enzyme is added to the suspension in amounts in the range of about 0.3 to 1% w/v.
  • the hydrolysis reaction is typically carried out in a desired time in order to obtain the desired degree of hydrolysis and / or the desired desired molecular weight profile, in this case for a period of about 45 minutes to about 2 h 30 minutes, preferably about 1 hour.
  • the time required for the hydrolysis reaction depends on the characteristics as indicated above, but can be easily determined by those skilled in the art.
  • the suspension containing pea proteins can be hydrolyzed using non-enzymatic means, for example by mechanical (physical) and/or chemical hydrolysis. This technique is also well known in the state of the art.
  • the hydrolysis reaction is stopped, for example, by inactivation of the enzyme, or by other conventional means.
  • the inactivation of the enzyme is carried out by heat treatment.
  • the enzyme preparation may suitably be inactivated by raising the temperature of the incubation suspension to a temperature at which the enzymes become inactivated, for example at about 70°C for about 10 minutes.
  • the pea protein isolates thus obtained are then treated at high temperature for a short time (HTST), then pasteurized, optionally concentrated to a dry matter of 10 to 30%, before being dried by atomization.
  • the isolate can be pasteurized at a temperature between 130°C and 150°C for a period of about 1 second to about 30 seconds.
  • the legume proteins are faba bean proteins whose degree of hydrolysis is not modified by hydrolysis after its extraction, preferably with a degree of hydrolysis of between 0% and 5%.
  • faba bean proteins compared to pea proteins. Indeed, the synthesis activation effect is more important in particular in a context of cellular cooperation. This finding of greater efficacy was demonstrated using faba bean proteins whose degree of hydrolysis was not increased with enzymatic, physical or biological hydrolysis.
  • the fava bean protein is obtained by any means well known to those skilled in the art, whether by dry route (extraction without the use of aqueous solvent, p.e.
  • Faba bean proteins obtained by the wet process are particularly preferred because it leads to obtaining an isolate which thus concentrates as much as possible its capacities to activate the synthesis of FGF19.
  • a particularly preferred method for obtaining faba bean proteins is as follows:
  • the first step consists in the implementation of faba bean seeds. These still have their protective outer fibers, also called "hulls" in English.
  • the seeds can undergo a pre-treatment likely to include steps of cleaning, sieving (separation of the seeds from the pebbles for example), soaking, bleaching, toasting.
  • the heat treatment schedule will be 3 minutes at 80°C.
  • Non-limiting examples of varieties are for example the Tiffany, FFS or YYY varieties.
  • bean seed varieties will be used whose content of tannins and/or polyphenols is naturally low, such as the Organdi variety. Such varieties are known and likely to be obtained by varietal crossing and/or genetic modifications.
  • the second step aims for the most efficient separation possible of the outer fibers and the cotyledons. It is initiated for example by a first grinding of faba bean seeds using a stone mill.
  • a particular and particularly suitable example of such a stone mill is for example marketed by the company Alma®.
  • the seed will be introduced into a space made up of two stone discs, one of which is rotating.
  • this technique is of particular interest because it will cause a very effective separation of the external fibers and the cotyledons of the seeds.
  • the inter-disc space is adjusted between 0.4 and 0.6 mm.
  • the ground material obtained then undergoes the application of an upward flow of air, against the current.
  • the different solid particles will be classified according to their density.
  • two fractions are obtained: a light fraction mainly containing the external fibers or “hulls” and a “heavy” fraction mainly containing the cotyledons.
  • a particular and particularly suitable example of a suitable device is for example the MZMZ 1-40 marketed by the company Hosokawa-alpine®.
  • the heavy fraction, enriched in cotyledons, will then be ground using a knife mill.
  • a knife grinder is, for example, the SM300 marketed by the company Retsch®.
  • the succession of the three operations mentioned above within the second step aims to very finely separate the outer fibers and the cotyledons, avoiding degrading these two parts and mixing them.
  • the third step aims to reduce the particle size of the heavy fraction enriched in cotyledons by grinding them using a roller mill.
  • a particular and particularly suitable example of such a roller mill is for example the MLU 202, marketed by the company Bühler®. It is used here in order to reduce the particle size of the flour globally, in order to obtain a homogeneous and sufficiently fine powder to allow the following step 4 to be facilitated.
  • the preferred particle size is between 200 and 400 microns, preferably 300 microns.
  • a laser particle size measuring device is preferably used, although any method is possible, such as sieving.
  • the step of reducing the particle size of the heavy fraction enriched in cotyledons can be carried out in the presence of an aqueous solvent, preferably water.
  • an aqueous solvent preferably water.
  • the fourth step below is merged with the third step which are then carried out concomitantly.
  • the fourth step aims to suspend the powder obtained in the previous third step in an aqueous solvent, preferably in water.
  • the aim here is to carry out a selective extraction of certain compounds, mainly proteins as well as salts and sugars, by solubilizing them.
  • the pH of the solution is advantageously adjusted towards a neutral pH in order to limit as much as possible the solubilization of tannins and polyphenols. This pH rectification can be carried out before and/or after suspending the powder in the aqueous solvent.
  • the aqueous solvent is preferably water.
  • the latter can nevertheless be supplemented, for example with compounds making it possible to facilitate solubilization.
  • the pH of the aqueous solvent is adjusted between 6 and 8, preferably 7. Any acidic or basic reagent such as soda, lime, citric or hydrochloric acid is possible, but potash and ascorbic acid are preferred.
  • the temperature is adjusted between 2°C and 30°C, preferentially between 10°C and 30°C, preferentially between 15°C and 25°C, even more preferentially at 20°C. This temperature is regulated throughout the extraction reaction.
  • the powder obtained is mixed in order to obtain a suspension of between 5% and 25%, preferentially between 5% and 15%, preferentially between 7% and 13%, even more preferentially between 9% and 11%, the most preferred being 10%, the percentage being expressed as weight of powder per total weight of water/powder suspension.
  • the suspension is agitated using any apparatus well known to those skilled in the art, for example a tank equipped with an agitator, equipped with blades, marine propellers, or any equipment allowing effective agitation.
  • the extraction time preferably with stirring, is between 5 and 25 minutes, preferably between 10 and 20 minutes, even more preferably 15 minutes.
  • the fifth step aims to separate by centrifugation the soluble fraction and the solid fraction obtained during the fourth step.
  • the preferred industrial principle can be found in patent application EP1400537 which is incorporated herein by reference. The principle of this process is to start by using a hydrocyclone to extract a fraction enriched in starch, then to use a horizontal decanter to extract a fraction enriched in internal fibers. Nevertheless, it is possible to use an industrial centrifuge which will extract a fraction enriched in starch and internal fibres. In all cases, solid fractions and a liquid fraction are obtained which concentrate the majority of the proteins.
  • the sixth step aims to acidify the isoelectric pH of the bean proteins, around 4.5, then to subject the solution to heating in order to cause the so-called globulin proteins to coagulate, which will be separated by centrifugation.
  • the acidification is carried out at a pH between 4 and 5, preferably 4.5.
  • hydrochloric acid at around 7% by weight, but all types of acids, mineral or organic, can be used, such as citric acid. Even more preferably, the use of ascorbic acid pure or in combination with another mineral or organic acid is also possible. The use of ascorbic acid to acidify improves the final color.
  • Any means of heating is then possible, for example by means of a stirred tank equipped with a double jacket and/or coil or an in-line cooker by steam injection (“jet cooker”).
  • the heating temperature is advantageously between 45°C and 75°C, preferentially between 50°C and 70°C, even more preferentially between 55°C and 65°C, the most preferred being 60°C.
  • the heating time is advantageously between 5 minutes and 25 minutes, preferentially between 10 and 20 minutes, the most preferred being 10 minutes.
  • the protein composition mainly globulin, will coagulate and precipitate within the solution. It will be separated by any centrifugation technique, such as the Flottwegg® Sedicator.
  • the solution residual obtained concentrates sugars, salts and albumins, it is called faba bean solubles. It will be treated separately, preferably evaporated and/or dried.
  • the protein composition is then diluted to approximately 15-20% by weight of dry matter and neutralized to a pH of between 5.5 and 6.5, preferably 6.5, using any type of basic agent, preferably potash at 20% by weight.
  • the protein composition can then undergo a heat treatment, preferably at a temperature of 135° C. by direct injection of steam through a nozzle and cooling by vacuum flash effect at 65° C.
  • the protein composition obtained can be used directly, for example by being hydrolyzed by a protease or even textured by an extruder.
  • the protein composition useful for the invention is dried.
  • the preferred mode of drying is atomization, especially using a multiple-effect atomizer.
  • Typical parameters are an inlet temperature of 200°C and a vapor temperature of 85-90°C.
  • FGF19 is a gene coding for a protein belonging to the family of fibroblast growth factors which govern nutrient metabolism.
  • the FGF19 gene (Gene ID: 9965 updated on 8-07-2021) codes for a 1821 bp transcript (NCBI Reference Sequence: NM_005117.3, updated on June 29, 2021) which codes for an FGF19 protein (NCBI Reference Sequence: NP_0051108.1, updated June 29, 2021).
  • FGF19 also called FGF15 in rodents, is a member of a subfamily of fibroblast growth factors that regulate nutrient metabolism. FGF19 is expressed and secreted in the distal small intestine, by cells of the biliary and intestinal epithelium, where its synthesis is upregulated after postprandial absorption of bile acids.
  • activation of the synthesis or "overexpression of the gene” must be understood within the meaning of the invention as an exogenous activation (that is to say through a compound, a foreign molecule) mRNA or protein synthesis in the cells of a living organism, in particular the synthesis of FGF19 in the context of this invention.
  • Protein synthesis is the set of biochemical processes allowing cells to produce their proteins from their genes. It covers the steps of transcription of DNA into messenger RNA, aminoacylation of transfer RNA, translation of messenger RNA into polypeptide chains, post-translational modifications of the latter, and finally folding of the proteins thus produced. . It is tightly regulated at multiple levels, mainly during transcription and during translation. In the case of humans, the gene coding for the expression of FGF19 is located on human chromosome 11.
  • the activation of the synthesis of FGF19 can be determined by measuring the level of expression of the FGF19 gene, in particular by measuring either the level of expression of the mRNA of FGF19, or the level of expression of the FGF19 protein by any method known to those skilled in the art.
  • the expression of FGF19 is increased when the level of expression is at least 1.5 or 2, 3, 4, 5 times greater than in untreated cells.
  • FGF19 protein or "FGF19 polypeptide” should be understood within the meaning of the invention as a polypeptide, that is to say a chain of amino acids, having a homology with the particular sequence amino acid naturally secreted in mammalian organisms.
  • FGF19 polypeptide or “FGF19 polypeptide” should be understood within the meaning of the invention as a polypeptide, that is to say a chain of amino acids, having a homology with the particular sequence amino acid naturally secreted in mammalian organisms.
  • FGF19 polypeptide FGF19 polypeptide
  • FGF19 polypeptide and “FGF15/19” denote the native sequence of a natural form of an FGF19 polypeptide as expressed in any mammalian organism. This term includes any naturally occurring isoform, which encompasses variant forms such as alternatively spliced forms, allelic variant forms, and unprocessed and processed forms of FGF19, such as FGF19 polypeptide forms comprising a signal peptide
  • FGF19 polypeptide also includes fragments of a natural form of an FGF19 polypeptide, in particular recombinant fragments having the same biological activity as said natural form.
  • FGF19 includes all the FGF19 polypeptides exhibiting at least 50% identity with the human sequence represented in SEQ ID NO 1.
  • the expression "an FGF19 polypeptide exhibiting at least 50% identity with the human sequence represented in SEQ ID NO 1” denotes a polypeptide, member of the FGF19 family, having an amino acid sequence exhibiting at least 50% amino acid identity with the reference sequence . This requires that, after alignment, 50% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence
  • amino acid identity it is meant that the same amino acid is observed on both sequences. The identity does not take into account post-translational modifications that may occur on amino acids.
  • Identity is determined using computer analysis, such as the ClustalW computer alignment program, and the default settings suggested therein.
  • the ClustalW software is available from the website http://www.clust.org/clust2/. Using this program with its default settings, the part of a query and a "reference polypeptide" are aligned. The number of fully conserved residues is counted and divided by the length of the reference polypeptide.
  • the "reference polypeptide” has the sequence as represented in SEQ ID NO 1
  • the terms "at least 50% identity” indicate that the percentage identity between the two sequences, the query and the reference polypeptide of SEQ ID 1, is at least 50.55, 60.65 ,70, 75,80, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% with respect to the sequence SEQ ID NO: 1.
  • the FGF19 polypeptide is chosen from the polypeptide
  • FGF15 expressed in mice the FGF19 polypeptide expressed in humans, or the homologs of FGF15 and FGF19 expressed in other mammals such as rat, dog, cat, sheep, cattle, horse, pig, goat, rabbit.
  • the members of the FGF19 family include in particular: the human FGF19 polypeptide of 216 amino acids (including 24 amino acids constituting the signal peptide) whose sequence is represented in SEQ ID NO 1;
  • the present invention consists more particularly in the therapeutic use of a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10% in order to activate the synthesis of FGF19 in a subject and to prevent or treat diseases susceptible to be treated by an overexpression of FGF19, in particular diseases associated with the deregulation of FGF19.
  • Subjects suffering from diseases associated with the deregulation of FGF19 present a deregulation of the expression of the FGF19 gene, in particular a reduction in the synthesis of FGF19 or a reduction in the signaling pathway of FGF19, in particular a reduction in the activation of factors in the signaling pathway downstream of FGF19 such as ERK, PI3K, GSK3p.
  • the composition as described above is used for the treatment and/or prevention of a disease, preferably a disease capable of being treated by activating the synthesis of FGF19, in particular a disease associated with dysregulation of the FGF19 signaling pathway, preferably sarcopenia or a metabolic disease such as inflammatory bowel disease, primary bile acid malabsorption, obesity or non-alcoholic steatopathy ( NAFLD, from its English acronym “non alcoholic fatty liver disease”) such as non-alcoholic steatohepatitis (NASH, from its English acronym “non-alcoholic steatohepatitis”) in a subject.
  • a disease preferably a disease capable of being treated by activating the synthesis of FGF19, in particular a disease associated with dysregulation of the FGF19 signaling pathway, preferably sarcopenia or a metabolic disease such as inflammatory bowel disease, primary bile acid malabsorption, obesity or non-alcoholic steatopathy ( NAFLD, from its English acronym “non alcoholic fatty liver
  • subject or “patient” must be understood within the meaning of the invention as any living being belonging to the animal kingdom. In a non-exhaustive manner, we therefore mean dogs, cats, cows, pigs.
  • subject is meant a human being, even more preferably a human being having a disease or a syndrome capable of being treated by the activation of the synthesis of FGF19.
  • the method according to the invention is characterized in that the subject is a human being, in particular an elderly person.
  • the subject is a human being, in particular an elderly person.
  • elderly person we mean a human being over 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 years old.
  • the subject is an animal, in particular selected from the list of cats, dogs, cows, sheep, pigs, chickens and turkeys.
  • treatment includes a curative treatment resulting in a cure or a treatment alleviating, ameliorating, eliminating, reducing and/or stabilizing the symptoms of a disease or the suffering which it causes.
  • prevention corresponds to a prophylactic or preventive treatment, which includes both a treatment leading to the prevention of a disease and a treatment reducing and/or delaying the incidence of a disease or the risk that it occurs. .
  • the composition according to the invention aims to increase muscle fiber anabolism via the activation of the synthesis of FGF19 and to prevent or treat diseases associated with muscle loss, in particular sarcopenia.
  • Sarcopenia is a geriatric syndrome characterized by a decrease in muscle capacity due to age and which, as it gets worse, will cause a deterioration in muscle strength and physical performance.
  • the composition according to the invention preferably makes it possible to prevent or treat sarcopenia and to improve or eradicate the disease or the symptoms associated with the disease, such as the deterioration of muscular strength and physical performance.
  • the composition according to the invention makes it possible to prevent or treat a metabolic disease via the activation of the synthesis of FGF19, in particular non-alcoholic steatohepatitis (or NASH).
  • NASH is liver damage that results from an excessive accumulation of lipids in hepatocytes leading to lipotoxicity and inflammatory lesions of hepatocytes.
  • the pathophysiology involves fat accumulation (steatosis), inflammation and, variably, fibrosis.
  • the composition according to the invention makes it possible to prevent or treat NASH and to improve or eradicate the disease or the symptoms associated with NASH, such as a reduction in the accumulation of lipids in the hepatocytes and inflammatory lesions. hepatocytes in the patient.
  • composition according to the invention therefore makes it possible to envisage, via the stimulation of the synthesis of FGF19, a pathway in order to combat the prevalence of this NASH.
  • the present disclosure also consists of the use of a composition comprising a legume protein as described above for the preparation of a medicament for use in the treatment of a disease capable of being treated by activation of FGF19 synthesis as previously described, preferably sarcopenia or NASH.
  • the present disclosure is a method for treating a disease capable of being treated by the activation of the synthesis of FGF19 as described above, preferably sarcopenia or NASH in a subject in need comprising administering a therapeutically effective amount of a composition comprising a legume protein as previously described to a subject.
  • a therapeutically effective amount refers to a dose sufficient to reverse, alleviate or inhibit the progression of the disorder or condition to which this term applies, or reverse, alleviate or inhibit the progression of one or more symptoms of the disorder or condition to which this term applies.
  • the effective dose is determined and adjusted according to factors such as the composition used, the route of administration, the physical characteristics of the individual considered such as sex, age and weight, the concomitant medication, and other factors, which those skilled in the medical field will recognize.
  • the composition also comprises at least one FXR agonist (acronym for the English term “Farnesoid X receptor”).
  • FXR agonists are well known to those skilled in the art and can be chosen by way of non-limiting examples from: obeticholic acid (OCA), Chenodeoxycholic acid (CDCA), 6a-ethyl-chenodeoxycholic acid (6-ECDCA), Alisol B 23-acetate (AB23A), cafestol, Fexaramine, GW4064 (3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole) and Tropifexor, preferably GW4064 and Chenodeoxycholic acid (CDCA).
  • the composition according to the present disclosure may be included in a pharmaceutical composition, optionally in combination with a pharmaceutically acceptable excipient.
  • the present disclosure thus consists of a pharmaceutical composition intended to activate the synthesis of FGF19 comprising legume proteins, preferentially selected from peas or fava beans, the degree of hydrolysis of which is less than 10% as described above, optionally in combination with a pharmaceutically acceptable excipient.
  • the first essential constituents for the pharmaceutical composition intended to activate the synthesis of FGF19 are legume proteins, preferably pea or faba bean proteins, the degree of hydrolysis of which is less than 10% as described in the preceding paragraphs.
  • the two preferred alternative modes respectively use a pea protein, the degree of hydrolysis of which is preferably between 5% and 10%, or a faba bean protein, the degree of hydrolysis of which is not modified by hydrolysis after its extraction, preferentially with a degree of hydrolysis comprised between 0% and 10%, preferentially between 0% and 5%.
  • these proteins constitute the only protein sources of the composition according to the invention. Unlike the solutions well known in the prior art, the composition does not use a combination with other protein sources, for example rice or wheat proteins.
  • the composition in this preferential mode aims to maximize the ability of legume proteins to activate the synthesis of FGF19. It is possible to add amino acids to improve the profile of the composition, although this is not necessary.
  • pharmaceutical composition should be understood as any composition intended to treat a subject. Non-limiting examples of such pharmaceutical compositions are given below in this application.
  • a "pharmaceutically acceptable excipient” refers to a vehicle which does not produce an adverse, allergic or other untoward reaction when administered to a mammal, particularly a human, as the case may be.
  • a pharmaceutically acceptable carrier or excipient means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.
  • the pharmaceutical composition contains carriers, which are pharmaceutically acceptable for a formulation capable of being administered orally.
  • composition can be any composition well known to those skilled in the art, such as (and in a non-limiting manner) ready-to-drink drinks (RTD), powders to be rehydrated (powder-mix), compotes, gels, bread products, dairy products, .
  • RTD ready-to-drink drinks
  • powders to be rehydrated powders to be rehydrated
  • compotes compotes
  • gels bread products, dairy products, .
  • the invention relates to the application of these formulations in drinks, by means of mixtures of powders to be reconstituted, for dietetic nutrition (sport, slimming), and in ready-to-drink drinks for clinical (oral route or enteral bag) and dietary, where low viscosity of the drink and improved solubility of the legume protein are desired
  • the invention relates to the application of these nutritional formulations in dairy or vegetable drinks, in fermented milks of the yoghurt type (stirred, Greek style, to drink) and in dairy or vegetable creams, frozen desserts or sorbets.
  • the invention relates to the application of these nutritional formulations in biscuits, muffins, pancakes, nutritional bars (intended for specialized/slimming or sports nutrition), in gluten-free breads or breads enriched with proteins, in small cereals obtained by high-protein extrusion cooking ("crisps"), where high-protein solutions are more particularly sought without negative impact on the preparation process or the texture of the preparations or finished products.
  • crisps high-protein extrusion cooking
  • the term "powdered nutritional formulations” means powdered formulations comprising at least, preferably only, a legume protein, and in particular pea or faba bean protein, according to the invention, which are reconstitutable with an aqueous liquid, and which are suitable for oral administration to a human.
  • dry mix refers to the mixing of the components or ingredients to form a basic nutritional powder or, to the addition of a dry component, by powder or granule or powder-based ingredient to form a powdered nutritional formulation.
  • the oral powder formulations of the present invention are generally in the form of flowable or substantially fluid particulate compositions, or at least particulate compositions which can be easily molded and measured with a spoon. or other similar device, wherein the compositions can easily be reconstituted by the intended user with an aqueous solution, typically water, to form a liquid formulation for immediate oral or enteral use.
  • aqueous solution typically water
  • immediate oral or enteral use generally means for about 48 hours, more typically for about 24 hours, preferably immediately after reconstitution.
  • the powder formulations can be formulated with any type and quantity of nutrients sufficient to form a dietary supplement, or a specialized nutritional formulation intended for use in people following a particular diet intended for sports dietetics and of thinness.
  • the powdered nutritional formulation can be formulated for use: for feeding an overweight person following a slimming diet and wishing to limit muscle weight loss, to repair the muscles after an intense effort, for example in the sportsman, to ensure in the sportsman the maintenance or the construction of the muscular mass, or as a meal replacement for people wishing to lose weight via a satietogenic effect.
  • Powder formulations can have a caloric density to suit the nutritional needs of the end user, although in most cases reconstituted powders will comprise from about 350 to about 400 kcal/100 ml.
  • Powder formulations may have a protein level tailored to the nutritional needs of the end user, although in most cases reconstituted powders comprise from about 20 to about 91 g protein/100 g, including from about 40 to about 65 g of protein / 100g.
  • the formulation may comprise between 20 and 95% protein relative to the total weight of the formulation, for example between 20-90%, 30-80%, or 40-60%.
  • the legume protein isolate preferably pea or faba bean
  • the legume protein isolate can represent 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total protein of the formulation, or any combination of these percentage ranges. 100% representing the ultimate preferential mode to maximize the effect of FGF19 overexpression
  • powdered food formulations can have a fat content adapted to the nutritional needs of the end user, although in most cases, reconstituted powders comprise from about 0.5 to about 13 g /100 g, including about 3 to about 7 g /100 g.
  • the formulation may comprise between 0 and 20% of lipids relative to the total weight of the formulation, for example between 0.5-15%, 1-10%, or 3-7% (in particular % by weight).
  • the powdered nutritional formulations of the present invention may be packaged and sealed in single or multi-use containers and then stored at ambient conditions for up to about 36 months or more, more typically about 12 to about 24 months. month.
  • multi-use containers they can be opened and covered for repeated use by the end user, provided that the covered package is then stored under ambient conditions (e.g. avoiding extreme temperatures) and contents used in about a month or two.
  • ambient conditions e.g. avoiding extreme temperatures
  • the fields of application of the nutritional formulations according to the invention are in particular: - dietary nutrition (sport, slimming), clinical nutrition (in the form of a drink, dessert cream or enteral pouch), dairy products ( in the form of yoghurts, dairy drinks, dairy creams, frozen desserts or sorbets). - biscuit products, pastry products, bread products and high-protein cereal products.
  • Ready-to-drink protein or high-protein drinks then allow the body to benefit from a choice protein intake, with fewer calories.
  • These high-protein drinks must: be rich in protein, low in carbohydrates and in fats; to have good taste ; be designed to aid weight loss by stimulating fat loss and aiding muscle recovery; - be satietogenic; allow you to cope with cravings, without added sugars or fats; have a balanced content of essential amino acids, fibre, vitamins and minerals; be low calorie.
  • These ready-to-drink drinks can advantageously be prepared with legume protein isolates, preferably fava beans or peas, in accordance with the invention. They can also be used as the sole source of proteins, preferably because they maximize the effect of overexpression of FGF19.
  • vegetable drinks that are alternatives to cow's milk, contain on average from 4.5 to 11 g of protein per 100 ml of drink, preferably of the order of 7 g of protein per 100 ml, and are very low in fiber (about 0.5 to 1 g per 100 ml).
  • the beverage may comprise between 1 and 20% protein relative to the total weight of the beverage, for example between 3-15%, or 6-8%.
  • the pea protein isolate according to the present invention can represent 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total protein, or a any combination of these percentage ranges. Preferably, it represents at least 52%. In particular, the pea protein intake is between 52 and 100% of the total protein intake.
  • the pea protein intake can range from 0 to 100%, preferably from 0.01 or 0.1 to 100%.
  • the pea protein isolate according to the present invention may be 0.1-10%, 10-20%, 20-30%, 40-50%, 50-60%, 60-70%, 70- 80%, 80-90% or 90-100% of total protein, or any combination of these percentage ranges.
  • these protein or protein-enriched drinks are not only effective for rapid intake of muscles. This type of drink is also very beneficial as part of a slimming diet based on protein consumption.
  • slimming drinks are ideal for aiding weight loss. They allow more particularly to: provide a satiety effect protect the muscles and tone the body, preventing weight regain.
  • these slimming drinks have: a balanced content of essential amino acids, fibres, vitamins and minerals a reduced content of sugars, fats and calories.
  • enteral nutrition is a therapeutic nutrition solution by tube which is used when the digestive tract is functional and accessible but when the patient cannot eat normally or in cases of undernutrition. severe. This technique makes it possible to supply the nutrients directly to the digestive tract. It replaces, totally or partially, the traditional oral diet with “complete” nutritious formulas providing all the nutrients needed by the body.
  • These nutritional mixtures are composed of proteins, lipids, carbohydrates, vitamins and minerals with or without fibers.
  • polymeric mixtures standards
  • predigested semi-elementary
  • the semi-elementary mixtures are iso or high-calorie normal or high-protein mixtures, based on peptides and medium-chain triglycerides.
  • Pea protein isolates as a protein source, by their functional properties are particularly well suited for this use.
  • Example 1 Preparation of a composition comprising a hydrolyzed pea protein whose degree of hydrolysis (DH) is between 5% and 10%
  • Pea protein (marketed by the applicant company under the brand name NUTRALYS® S85F) is suspended at 15% w/w in 8500 liters of water preheated to 55°C.
  • Example 2 Preparation of a Food Composition Comprising a Non-Hydrolysed Bean Protein
  • Bean seeds of the Tiffany variety were first processed using a stone mill (Alma®). The ground material was then treated by turbo separation using a so-called "zig-zag" system (MZM 1-40, Hosokawa-alpine®) with an air speed of 4.0 m.s-1 (23 m3.h- 1). A light fraction containing the external fibers and a heavy fraction containing the cotyledons are thus obtained. The heavy fraction is then processed using a knife mill (SM300, Retsch®) whose rotation is 700 rpm and the outlet is equipped with a 6 mm grid. The heavy fraction is then ground using a roller mill (MLU 202, Bühler®). A flour is finally obtained whose particle size is less than 300 ⁇ m.
  • This flour is suspended at 10% by weight of dry matter in drinking water at 20°C.
  • the pH is adjusted to 7 by adding potash.
  • This supernatant is acidified to pH 4.5 by adding hydrochloric acid at approximately 7% by weight. It is heated to 60°C by injecting steam into a double jacket of the tank, where it is homogenized for 15 minutes.
  • the sediment is diluted to approximately 15-20% by weight of dry matter and neutralized to pH 6.5 by adding 20% potassium hydroxide. Heat treatment is carried out at 135°C using a nozzle and cooling is carried out by vacuum flash effect at 65°C. The product is finally atomized (inlet temperature of 200°C and vapor temperature of 85-90°C)
  • RQ4 Nutralys® W (wheat protein isolate)
  • RQ5 Nutralys® H85 (pea protein hydrolyzate whose DH measured according to the protocol described in the present application is equal to 22)
  • Tubermine GP potato protein isolate
  • the in vitro digestion model used simulates the digestion of food by mimicking three different phases of digestion: the oral phase, the gastric phase and the intestinal phase.
  • a hydration phase is first carried out. 6g of proteins are dissolved, in a buffer composed of calcium chloride (CaCl2) at a final concentration of 2.6 mM, for one hour at 37° C. with stirring.
  • the simulation of the gastric phase is done with the addition of a buffer solution containing hydrogen chloride (HCl) at 157.8 mM, and CaCl2 at 157.8 mM.
  • the reaction mixture is adjusted to a pH of 2 with a solution of 4M hydrochloric acid (HCl) or 4M sodium hydroxide (NaOH).
  • porcine pepsin (P7000, Sigma) are added to the rest of the reaction mixture in order to start the digestion of the proteins.
  • Digestion by the intestinal phase continues with the addition of a solution containing 210 mL of phosphate buffered saline (PBS) pH 7.4, CaCl2 at 558 pM and NaOH at 20.9 mM.
  • PBS phosphate buffered saline
  • CaCl2 calcium phosphate buffered saline
  • NaOH NaOH
  • the enzymes are subsequently inactivated by placing the mixture for 10 minutes in a water bath at 90°C.
  • the digestates obtained are frozen and then freeze-dried. All the proteins digested in vitro then underwent a post-treatment described below: - Dissolution in a sterile PBS solution at a concentration of 100 mg/mL Homogenization for 1 hour at 40°C with stirring (80 rpm)
  • FXR receptor agonist GW4064 chenodeoxycholic acid (CDCA), - dexamethasone (glucocorticoid receptor agonist)
  • the HT29 (ECACC 91072201), HT29-MTX and Caco-2 lines derived from a human colorectal adenocarcinoma, conventionally used as cell lines representative of human enterocytes, will be used.
  • the cells are systematically cultured in Dulbecco's modified Eagle medium (DMEM) 4.5 g/L of glucose supplemented with 1% sodium pyruvate, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). ) decomposed (Eurobio, Les Ulis, France).
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • the HT29 cells were seeded at a density of 7.5 x 10 4 cells/cm 2 with medium changed every 2 days and the cells were exposed to the different experimental conditions 2 days after confluence with exhausted complete medium in FBS.
  • the Caco-2 and HT29-MTX cells were seeded in a ratio of 1:1 at a density of 2.5 ⁇ 10 4 cells/cm 2 .
  • the cells were cultured for 14 days in complete medium to allow differentiation. Medium was changed in both compartments every other day until exposure to complete medium was depleted in the FBS.
  • TEER transepithelial electrical resistance
  • Millicell-ERS Millicell-ERS
  • Crop supply cell was from Dutscher (Issy-les-Moulineaux, France), unless otherwise indicated.
  • the protocol for exposing these cell cultures to the various samples and agonists is as follows: the cells were treated for 6 hours with (i) either DP at 1 mg/mL (this concentration did not show no toxic effect on HT29 cells) or nuclear receptor agonists, and (ii) the combination of DP and nuclear receptor agonists.
  • the different vehicle conditions like DP were dissolved in PBS and the agonists in DMSO. After exposure, cell supernatants were stored at -80°C for measurements of FGF19 protein secretion, and cell monolayers were maintained at -80°C until complete RNA extraction.
  • the effect of this exposure is measured by quantifying the FGF19 messenger RNA using the following protocol:
  • the expression of the target genes was evaluated by quantifying their mRNA level in the cells by RT -qPCR.
  • Total RNAs were isolated with TRI-Reagent solution (Sigma Aldrich).
  • First-order cDNAs were synthesized from 1 pg of total RNA with the TAKARA Prime ScriptTM RT Reagent Kit (TAKARA Bio, Saint-Germain-en-Laye, France).
  • qPCR assays were performed using the SYBR® Premix Ex-TaqTM Kit (TAKARA Bio) on Rotor-GeneTM 6000 (Corbett Research, Mortlake, Australia). .
  • Human TATA-binding protein (TBP) mRNA levels were used to normalize the data.
  • the toxicity of the DPs was evaluated on the HT29 cells in 96-well plates after 6 hours of exposure, at 1 and 2 mg/mL, in order to define the highest concentration that does not cause cell degradation or lysis. Under such conditions, no DP showed any toxic effect (as assessed by LDH release) at 1 or 2 mg/mL except DP #3 (RQ3) which induced the appearance of round-shaped cells predicting cell detachment at 2 mg/mL. It was therefore decided to choose the dose of 1 mg/mL to continue the tests on FGF19 expression.
  • the [fig. 1] summarizes the results obtained.
  • HT29 cells were exposed to the different DPs alone or in combination with an FXR agonist to determine their potential ability to increase FGF19 gene expression.
  • RQ3 and RQ7 has the maximum capacity for overexpression of the gene coding for FGF19 compared to other proteins and in particular compared to proteins from the animal world such as RQ1 and RQ2.
  • RQ3 is more effective than RQ5, which is also a pea protein isolate but with a degree of hydrolysis of 22, therefore greater than 10.
  • Example 6 Confirmation of the effect The capacity of the RQ3 and RQ7 digestates to increase the level of expression of the FGF19 mRNA as well as the production and the secretion of proteins was then evaluated.
  • HT29 cells were exposed to GW4064 at 0.1, 0.5, and 1 mM and a natural FXR agonist, chenodeoxycholic acid (CDCA) at 50 ⁇ M alone or in combination with RQ3 or RQ7 extracts.
  • the level of protein expression is similar to that of transcript expression, although co-incubation of RQ3 and RQ7 with FXR agonists showed less potentiation of FGF19 protein expression than mRNA.
  • RQ3 digest co-incubated with CDCA at 50 pM and GW4064 at 0.1 and 1 pM caused an increase in the level of FGF19 proteins of 3.1, 3.3 and 4.8 times respectively compared to the control condition ( vehicle) ([Fig. 2b], left).
  • the RQ7 digestate induced an increase of 2.5, 3 and 4.2 times, respectively.
  • RQ3 and RQ7 The capacity of RQ3 and RQ7 to induce the expression of FGF19 was evaluated in an insert co-culture model to approximate intestinal physiological conditions with apical and basolateral exchanges.
  • Caco-2 cells allow establishment of intercellular junctions providing a selectively permeable barrier and HT29-MTX cells have the ability to produce mucus at the top of the monolayer.
  • This type of co-culture of the two cell lines therefore provides a model constituting the two most represented cell types in a normal epithelium, enterocytes and goblet cells.
  • RQ3 and RQ7 showed potentiation of FGF19 expression when co-incubated with GW4064 at 0.2 mM [Fig. 3], although this effect is less potent than previously observed in plaque experiments containing only HT29 cells.

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Abstract

The present invention is a pulse protein composition, preferably of pea or faba bean, the degree of hydrolysis of which is less than 10%, in particular for therapeutic use, preferably in the prevention and/or treatment of a disease susceptible to treatment by activation of the synthesis of FGF19.

Description

Description Description
Titre: METHODE D’ACTIVATION DE LA SYNTHESE DUTitle: METHOD FOR ACTIVATING THE SYNTHESIS OF
FGF19 FGF19
Domaine technique [0001] La présente invention consiste en une composition de protéines de légumineuse, préférentiellement de pois ou de féverole, dont le degré d’hydrolyse est inférieur à 10%, en particulier pour une utilisation thérapeutique, de préférence dans la prévention et/ou le traitement d’une maladie susceptible d’être traitée par une activation de la synthèse du FGF19. Art antérieur TECHNICAL FIELD [0001] The present invention consists of a composition of legume proteins, preferably pea or faba bean, the degree of hydrolysis of which is less than 10%, in particular for therapeutic use, preferably in the prevention and/or or the treatment of a disease capable of being treated by an activation of the synthesis of FGF19. Prior art
[0002] Un fibroblaste est une cellule humaine présente dans le tissu conjonctif. Le rôle le plus important des fibroblastes est de maintenir la matrice extracellulaire des tissus conjonctifs, et de réparer les lésions dues à un traumatisme. Ils servent aussi à réguler l'organisation et la différenciation des cellules des tissus environnants. Ces fibroblastes vont entre-autre sécréter la matrice extracellulaire, c'est-à-dire les protéines qui forment les fibres du tissu conjonctif et les glycoprotéines de la substance fondamentale. Ils interviennent également dans le métabolisme des lipoprotéines (LDL) et du cholestérol. [0002] A fibroblast is a human cell present in the connective tissue. The most important role of fibroblasts is to maintain the extracellular matrix of connective tissues, and to repair damage due to trauma. They also serve to regulate the organization and differentiation of cells in surrounding tissues. These fibroblasts will, among other things, secrete the extracellular matrix, that is to say the proteins which form the fibers of the connective tissue and the glycoproteins of the ground substance. They are also involved in the metabolism of lipoproteins (LDL) and cholesterol.
[0003] Les facteurs de croissance des fibroblastes (ou FGF, acronyme anglais de Fibroblast Growth Factor) forment une famille comportant 23 protéines identifiées à ce jour chez l'homme (FGF1, FGF2... FGF23) qui activent la migration et la multiplication des fibroblastes. Ces facteurs sont généralement sécrétés par des fibroblastes. [0003] Fibroblast growth factors (or FGF, English acronym for Fibroblast Growth Factor) form a family comprising 23 proteins identified to date in humans (FGF1, FGF2, etc. FGF23) which activate migration and multiplication fibroblasts. These factors are generally secreted by fibroblasts.
[0004] Le FGF19 (pour l’acronyme anglais de « Fibroblast growth factor 19 ») est une protéine appartenant à cette famille des facteurs de croissances des fibroblastes. Sa synthèse est activée par le FXR (pour l’acronyme anglais de « Farnesoid X receptor »), lui-même stimulé par les sels biliaires, ce qui a pour conséquence d'inhiber la néoglucogenèse et la synthèse des sels biliaires et d'activer la synthèse protéique et du glycogène. Il joue également sur le métabolisme lipidique et du calcium. [0004] FGF19 (for the acronym for “Fibroblast growth factor 19”) is a protein belonging to this family of fibroblast growth factors. Its synthesis is activated by FXR (for the English acronym of "Farnesoid X receptor"), itself stimulated by bile salts, which has the effect of inhibiting gluconeogenesis and the synthesis of bile salts and activate protein and glycogen synthesis. It also plays on lipid and calcium metabolism.
[0005] Chez l’Homme, un taux sérique bas en FGF19 est en rapport avec des formes plus graves de stéatose hépatique non alcoolique ou NASH (Alisi A, Ceccarelli S, Panera N et al. Association between sérum atypical fibroblast growth factors 21 and 19 and pédiatrie nonalcoholic fatty liver disease, PLoS One, 2013;8:e6716). Son utilisation afin de lutter contre la NASH a donc été étudiée. Sur un modèle animal, il semble hélas favoriser la formation d'un carcinome hépatocellulaire. Un analogue de la molécule a été développé, sans activité d'oncogenèse. Il semble être prometteur dans le traitement de la stéatose hépatique non alcoolique, diminuant le contenu en lipides des cellules hépatiques. L’utilisation d’un analogue est cependant contraignante du fait de la nécessité de la synthèse exogène dudit analogue et de sa consommation en tant que médicament. Il existe donc un besoin insatisfait de naturellement activer la synthèse in-vivo de FGF19 pour prévenir ou traiter la NASH. [0005] In humans, a low serum FGF19 level is related to more serious forms of non-alcoholic fatty liver disease or NASH (Alisi A, Ceccarelli S, Panera N et al. Association between serum atypical fibroblast growth factors 21 and 19 and pediatric nonalcoholic fatty liver disease, PLoS One, 2013;8:e6716). Its use in the fight against NASH has therefore been studied. In an animal model, it unfortunately seems to promote the formation of hepatocellular carcinoma. An analogue of the molecule has been developed, without oncogenesis activity. It appears to show promise in the treatment of non-alcoholic fatty liver disease, decreasing the lipid content of liver cells. The use of an analog is however restrictive due to the need for the exogenous synthesis of said analog and its consumption as a drug. There is therefore an unmet need to naturally activate the in-vivo synthesis of FGF19 to prevent or treat NASH.
[0006] La sarcopénie est un syndrome gériatrique se caractérisant dans un premier temps par une diminution des capacités musculaires due à l'âge et qui en s’aggravant sera à l’origine d’une détérioration de la force musculaire et des performances physiques. La sarcopénie observée chez la personne âgée est imputable au processus de vieillissement mais peut être accélérée par des facteurs pathologiques et comportementaux tels que la dénutrition et la sédentarité. Le FGF19 est récemment apparu comme une opportunité afin de lutter contre la prévalence de la sarcopénie (« Fibroblast growth factor 19 régulâtes skeletal muscle mass and améliorâtes muscle wasting in mice. » Benoit & al., Nat Med. 2017 Aug;23(8):990-996). Tout comme le traitement de la NASH, Il existe donc un besoin insatisfait de naturellement activer la synthèse in-vivo de FGF19 afin de lutter contre la prévalence de ce syndrome. [0006] Sarcopenia is a geriatric syndrome characterized initially by a decrease in muscle capacity due to age and which, as it gets worse, will cause a deterioration in muscle strength and physical performance. The sarcopenia observed in the elderly is attributable to the aging process but can be accelerated by pathological and behavioral factors such as malnutrition and a sedentary lifestyle. FGF19 has recently emerged as an opportunity to fight the prevalence of sarcopenia (“Fibroblast growth factor 19 regulates skeletal muscle mass and improves muscle wasting in mice.” Benoit & al., Nat Med. 2017 Aug;23(8) :990-996). Like the treatment of NASH, there is therefore an unmet need to naturally activate the in-vivo synthesis of FGF19 in order to combat the prevalence of this syndrome.
Résumé Summary
[0007] Il est du mérite de la demanderesse d’avoir mis en évidence qu’une composition de protéines de légumineuse, particulièrement de protéines de pois légèrement hydrolysées ou de protéines de féverole non hydrolysées permettait d’activer la synthèse in-vitro de FGF19 dans des cultures cellulaires humaines, en particulier lorsque ces protéines représentent l’unique source protéique. Leurs ingestions quotidiennes dans la ration alimentaire permettent donc d’envisager de naturellement surexprimer la synthèse de FGF19 et donc lutter contre la prévalence ou le traitement de syndromes tels que la NASH ou la sarcopénie, citées ici de manière préférentielle et non exhaustives. [0007] It is to the credit of the applicant to have demonstrated that a composition of legume proteins, particularly slightly hydrolyzed pea proteins or non-hydrolyzed faba bean proteins, allowed to activate the in-vitro synthesis of FGF19 in human cell cultures, in particular when these proteins represent the sole protein source. Their daily ingestions in the food ration therefore make it possible to envisage naturally overexpressing the synthesis of FGF19 and therefore combating the prevalence or the treatment of syndromes such as NASH or sarcopenia, cited here in a preferential and non-exhaustive manner.
[0008] La présente invention consiste donc tout d’abord en une composition de protéines de légumineuse, préférentiellement de pois ou de féverole, dont le degré d’hydrolyse est inférieur à 10% pour une utilisation thérapeutique chez un sujet qui en a besoin, de préférence pour une utilisation dans la prévention et/ou le traitement d’une maladie susceptible d’être traitée par une activation de la synthèse du FGF19, notamment une maladie associée à une dérégulation de la voie de signalisation du FGF19. The present invention therefore consists first of all of a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10% for therapeutic use in a subject who needs them, preferably for use in the prevention and/or treatment of a disease capable of being treated by an activation of the synthesis of FGF19, in particular a disease associated with a dysregulation of the signaling pathway of FGF19.
[0009] La présente invention consiste également en une composition pharmaceutique destinée à surexprimer le FGF19 comportant des protéines de légumineuse, préférentiellement de pois ou de féverole, dont le degré d’hydrolyse est inférieur à 10% et optionnellement un excipient pharmaceutiquement acceptable. The present invention also consists of a pharmaceutical composition intended to overexpress FGF19 comprising legume proteins, preferably pea or horse bean, the degree of hydrolysis of which is less than 10% and optionally a pharmaceutically acceptable excipient.
[0010] L’invention sera mieux comprise à la lecture de la description détaillée ci- dessous Brève description des dessins Fig. 1 [0010] The invention will be better understood on reading the detailed description below Brief description of the drawings Fig. 1
[0011] [Fig. 1] montre les effets des différents digestats sur l’expression du FGF19. Les cellules HT29 ont été exposées aux différents digestats protéiques (DP) seuls ou en combinaison avec un agoniste FXR. Fig. 2 [0011] [Fig. 1] shows the effects of different digestates on the expression of FGF19. HT29 cells were exposed to different protein digests (PD) alone or in combination with an FXR agonist. Fig. 2
[0012] [Fig. 2a] montre l’augmentation de l’expression de l’ARNm FGF19 suite au traitement de cellules HT29 aux digestats RQ3 et RQ7. Les cellules HT29 ont été exposées à GW4064 à 0,1, 0,5 et 1 mM et à un agoniste FXR naturel, l’acide chenodeoxycholique (CDCA) à 50 mM seul ou en combinaison pendant 6h avec les extraits RQ3 ou RQ7. [0012] [Fig. 2a] shows the increase in the expression of FGF19 mRNA following treatment of HT29 cells with RQ3 and RQ7 digests. HT29 cells were exposed to 0.1, 0.5, and 1 mM GW4064 and a natural FXR agonist, acid chenodeoxycholique (CDCA) at 50 mM alone or in combination for 6 hours with the RQ3 or RQ7 extracts.
[0013] [Fig. 2b] montre l’augmentation de la production et la sécrétion de la protéine FGF19 suite au traitement de cellules FIT29 aux digestats RQ3 et RQ7. Les cellules HT29 ont été exposées à GW4064 à 0, 1 , 0,5 et 1 mM et à un agoniste FXR naturel, l’acide chenodeoxycholique (CDCA) à 50 mM seul ou en combinaison pendant 6h (gauche) et 24h (droite) avec les extraits RQ3 ou RQ7. [0013] [Fig. 2b] shows the increase in the production and secretion of the FGF19 protein following the treatment of FIT29 cells with RQ3 and RQ7 digestates. HT29 cells were exposed to GW4064 at 0, 1, 0.5 and 1 mM and a natural FXR agonist, chenodeoxycholic acid (CDCA) at 50 mM alone or in combination for 6h (left) and 24h (right) with extracts RQ3 or RQ7.
Fig. 3 Fig. 3
[0014] [Fig. 3] montre l’augmentation du niveau d’expression du FGF19 (ARNm) suite au traitement de co-culture de cellules Caco-2 et HT29-MTX avec des digestats RQ3 et RQ7. [0014] [Fig. 3] shows the increase in the level of expression of FGF19 (mRNA) following co-culture treatment of Caco-2 and HT29-MTX cells with RQ3 and RQ7 digests.
Description détaillée detailed description
Composition de protéines de légumineuse selon l’inventionComposition of legume proteins according to the invention
[0015] Les inventeurs ont montré qu’une composition de protéines de légumineuse, de préférence de pois ou de féverole dont le degré d’hydrolyse est inférieur à 10% permettait d’activer l’expression du FGF19 dans des cellules humaines. [0015] The inventors have shown that a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10%, makes it possible to activate the expression of FGF19 in human cells.
[0016] La présente invention consiste donc tout d’abord en une composition de protéines de légumineuse de préférence de pois ou de féverole dont le degré d’hydrolyse est inférieur à 10% pour une utilisation thérapeutique chez un sujet qui en a besoin, de préférence pour une utilisation dans la prévention et/ou le traitement d’une maladie susceptible d’être traitée par une activation de la synthèse du FGF19, notamment une maladie associée à une dérégulation de la voie de signalisation du FGF19. The present invention therefore consists first of all of a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10% for therapeutic use in a subject who needs them, preferably for use in the prevention and/or treatment of a disease capable of being treated by an activation of the synthesis of FGF19, in particular a disease associated with a dysregulation of the signaling pathway of FGF19.
[0017] Le terme « protéine » ou « polypeptide » doit se comprendre dans la présente demande comme les macromolécules formées d'une ou de plusieurs chaînes polypeptidiques constituées de l'enchaînement de résidus d'acides aminés liés entre eux par des liaisons peptidiques. [0017] The term “protein” or “polypeptide” should be understood in the present application as the macromolecules formed from one or more polypeptide chains consisting of the sequence of amino acid residues linked together by peptide bonds.
[0018] Par « légumineuse », on comprendra dans la présente demande la famille de plantes dicotylédones de l'ordre des Fabales. C'est l'une des plus importantes familles de plantes à fleurs, la troisième après les Orchidaceae et les Asteraceae par le nombre d'espèces. Elle compte environ 765 genres regroupant plus de 19 500 espèces. Plusieurs légumineuses sont d'importantes plantes cultivées parmi lesquelles le soja, les haricots, les pois, le pois chiche, la féverole, l'arachide, la lentille cultivée, la luzerne cultivée, différents trèfles, les fèves, le caroubier, la réglisse. [0018] The term “legume” will be understood in the present application to include the family of dicotyledonous plants of the order Fabales. It is one of the most important families of flowering plants, the third after Orchidaceae and Asteraceae by the number of species. It has about 765 genera comprising more than 19,500 species. Several legumes are important cultivated plants including soybeans, beans, peas, chickpeas, field beans, groundnuts, cultivated lentils, cultivated alfalfa, various clovers, broad beans, carob, liquorice.
[0019] Dans le cadre particulier des protéines de pois, la présente invention concerne plus particulièrement les globulines (environ 50-60% des protéines du pois) et les albumines (20-25%). Les globulines de pois se subdivisent principalement en trois sous-familles : les légumines, les vicilines et les convicilines. In the particular context of pea proteins, the present invention relates more particularly to globulins (approximately 50-60% of pea proteins) and albumins (20-25%). Pea globulins are mainly subdivided into three subfamilies: legumes, vicilins and convicilins.
[0020] Les protéines extraites de ces légumineuses appartiennent majoritairement aux sous-groupes des globulines et des albumines. Dans la présente invention, la protéine de légumineuse est majoritairement constituée de globulines, en particulier elle contient plus de 90% en poids de globulines par rapport au poids total des protéines. Les globulines peuvent être distinguées des albumines par diverses méthodes bien connues de l’homme du métier, notamment par leur solubilité dans l’eau, les albumines étant solubles dans l’eau pure alors que les globulines sont uniquement solubles dans l’eau salée. On peut également identifier les albumines et globulines présentes dans un mélange par électrophorèse ou chromatographie. Une méthode préférée est décrite dans l’article « Peptide and protein molecular weight détermination by electrophoresis using a high-molarity tris buffer System without urea. » Fling SP, Gregerson DS, Anal. Biochem. 1986;155:83-88. [0020] The proteins extracted from these legumes mainly belong to the globulin and albumin subgroups. In the present invention, the legume protein mainly consists of globulins, in particular it contains more than 90% by weight of globulins relative to the total weight of the proteins. Globulins can be distinguished from albumins by various methods well known to those skilled in the art, in particular by their solubility in water, albumins being soluble in pure water whereas globulins are only soluble in salt water. The albumins and globulins present in a mixture can also be identified by electrophoresis or chromatography. A preferred method is described in the article “Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer System without urea. Fling SP, Gregerson DS, Anal. Biochem. 1986;155:83-88.
[0021] La protéine de légumineuse selon l’invention contient plus de 90% en poids de globulines par rapport au poids total des protéines. [0022] Le terme « pois » étant ici considéré dans son acception la plus large et incluant en particulier toutes les variétés de « pois lisse » (« smooth pea ») et « de pois ridés » (« wrinkled pea »), et toutes les variétés mutantes de « pois lisse » et de « pois ridé » et ce, quelles que soient les utilisations auxquelles on destine généralement lesdites variétés (alimentation humaine, nutrition animale et/ou autres utilisations). [0023] Le terme « pois » dans la présente demande inclut les variétés de pois appartenant au genre Pisum et plus particulièrement aux espèces sativum et aestivum. Lesdites variétés mutantes sont notamment celles dénommées « mutants r », « mutants rb », « mutants rug 3 », « mutants rug 4 », « mutants rug 5 » et « mutants lam » tels que décrits dans l’article de C-L Heydley et al. intitulé « Developing novel pea starches » Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87. The legume protein according to the invention contains more than 90% by weight of globulins relative to the total weight of the proteins. The term "pea" being considered here in its broadest sense and including in particular all varieties of "smooth pea"("smoothpea") and "wrinkled pea"("wrinkledpea"), and all mutant varieties of “smooth pea” and “wrinkled pea” and this, regardless of the uses for which said varieties are generally intended (human food, animal nutrition and/or other uses). [0023] The term "pea" in the present application includes the varieties of peas belonging to the genus Pisum and more particularly to the species sativum and aestivum. Said mutant varieties are in particular those called “r mutants”, “rb mutants”, “rug 3 mutants”, “rug 4 mutants”, “rug 5 mutants” and “lam mutants” as described in the article by CL Heydley and para. entitled “Developing novel pea starches” Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87.
[0024] Par « féverole », on entend le groupe des plantes annuelles de l'espèce Vicia faba, appartenant au groupe des légumineuses de la famille des Fabaceae, sous-famille des Faboideae, tribu des Fabeae. On distingue les variétés Minor et Major. Dans la présente invention, les variétés sauvages et celles obtenues par génie génétique ou sélection variétales sont toutes d’excellentes sources. [0024] The term “bean bean” means the group of annual plants of the species Vicia faba, belonging to the group of legumes of the family Fabaceae, subfamily Faboideae, tribe Fabeae. A distinction is made between Minor and Major varieties. In the present invention, wild varieties and those obtained by genetic engineering or varietal selection are all excellent sources.
[0025] De manière préférée, les protéines de légumineuse, préférentiellement de pois ou de féverole, possédant un degré d’hydrolyse inférieur à 10% sont la seule source protéique au sein de la composition. En effet, il est bien connu dans le domaine de l’invention que les protéines de légumineuses, sont caractérisées par un profil d’acides aminés déficient en certains acides aminés (méthionine et cystéine) quand on le compare avec les profils requis en nutrition humaine. Cette déficience est souvent palliée en réalisant un mélange avec d’autres sources protéiques telles que les protéines animales ou les protéines de céréales comme le riz ou le blé. On peut citer par exemple « Protein content and amino acid composition of commercially available plant-based protein isolâtes” (Gorissen & al., Amino Acids 50, 1685-1695, 2018). [0025] Preferably, the legume proteins, preferably pea or faba bean, having a degree of hydrolysis of less than 10% are the only protein source within the composition. Indeed, it is well known in the field of the invention that legume proteins are characterized by an amino acid profile deficient in certain amino acids (methionine and cysteine) when compared with the profiles required in human nutrition. . This deficiency is often overcome by making a mixture with other protein sources such as animal proteins or cereal proteins such as rice or wheat. We can cite for example “Protein content and amino acid composition of commercially available plant-based protein isolates” (Gorissen & al., Amino Acids 50, 1685-1695, 2018).
[0026] Dans ce mode préférentiel, la Demanderesse vise au contraire à exploiter au maximum l’effet des protéines de légumineuse, préférentiellement de pois ou de féverole, possédant un degré d’hydrolyse inférieur à 10%, présente dans la composition afin de surexprimer au maximum le FGF19. Toute tentative de mélange avec d’autres sources protéiques afin de rétablir le profil en acides aminés aura pour conséquence un abaissement de l’effet sur la surexpression du FGF19. [0027] Le degré d’hydrolyse peut être déterminé en mesurant la teneur en azote aminé libre par rapport à l’azote total selon le Test de degré d’hydrolyse décrit ci- après. Son principe consiste tout d’abord en la détermination de la teneur en azote aminé (NH2 libre) sur l’échantillon de protéines avec le kit MEGAZYME (référence K-PANOPA), suivi de la détermination de la teneur en azote protéique (azote total) de l’échantillon, enfin du calcul du degré d’hydrolyse à l’aide de ces deux teneurs. - Détermination de la teneur en azote aminé : [0026] In this preferential mode, the Applicant aims on the contrary to exploit to the maximum the effect of legume proteins, preferentially pea or faba bean, having a degree of hydrolysis of less than 10%, present in the composition in order to overexpress maximum FGF19. Any attempt to mix with other protein sources to restore the amino acid profile will result in a diminished effect on FGF19 overexpression. The degree of hydrolysis can be determined by measuring the free amino nitrogen content relative to the total nitrogen according to the degree of hydrolysis test described below. After. Its principle consists first of all in the determination of the amino nitrogen content (free NH2) on the protein sample with the MEGAZYME kit (reference K-PANOPA), followed by the determination of the protein nitrogen content (total nitrogen ) of the sample, finally the calculation of the degree of hydrolysis using these two contents. - Determination of amino nitrogen content:
[0028] Les groupes « azote aminé » des acides aminés libres de l’échantillon réagissent avec le N-acétyl-L-cystéine et l’OPhthaldialdéhyde (OPA) pour former des dérivés d’isoindole. [0028] The “amino nitrogen” groups of the free amino acids of the sample react with N-acetyl-L-cysteine and O-Phthaldialdehyde (OPA) to form isoindole derivatives.
[0029] La quantité de dérivé d’isoindole formée au cours de cette réaction est stoechiométrique avec la quantité d’azote aminé libre. C’est le dérivé d’isoindole qui est mesuré par l’augmentation de l’absorbance à 340 nm. The amount of isoindole derivative formed during this reaction is stoichiometric with the amount of free amino nitrogen. It is the isoindole derivative which is measured by the increase in absorbance at 340 nm.
[0030] Dans un bêcher de 100 mL, on introduit une prise d’essai P* exactement pesée, de l’échantillon à analyser. Cette prise d’essai sera de 0,5 à 5,0 g en fonction de la teneur en azote aminé de l’échantillon. On ajoute environ 50 mL d’eau distillée, on homogénéise et on transvase dans une fiole jaugée de 100 mL. On ajoute 5 mL de dodécyle sulfate de sodium (SDS) à 20% et on complète avec de l’eau distillée pour atteindre un volume de 100 mL. On agite pendant 15 minutes avec un agitateur magnétique à 1000 rpm. On prépare une solution n°1 en dissolvant un comprimé du flacon 1 du kit Megazyme dans 3 mL d’eau distillée et on agite jusqu'à dissolution complète. Il faut prévoir un comprimé par essai. La solution n°1 est préparée extemporanément. In a 100 mL beaker, an exactly weighed test portion P* of the sample to be analyzed is introduced. This test portion will be 0.5 to 5.0 g depending on the amino nitrogen content of the sample. Add approximately 50 mL of distilled water, homogenize and transfer to a 100 mL volumetric flask. Add 5 mL of 20% sodium dodecyl sulfate (SDS) and make up with distilled water to reach a volume of 100 mL. Stirred for 15 minutes with a magnetic stirrer at 1000 rpm. Solution #1 is prepared by dissolving one tablet from vial 1 of the Megazyme kit in 3 mL of distilled water and shaking until completely dissolved. One tablet should be provided per test. Solution No. 1 is prepared extemporaneously.
[0031] On prépare un blanc, un standard et un échantillon directement dans les cuves du spectrophotomètre dans les conditions suivantes : blanc : introduire 3,00 ml de la solution n°1 et 50 pl d’eau distillée - standard : introduire 3,00 ml de la solution n°1 et 50 pl du flacon 3 du kitA blank, a standard and a sample are prepared directly in the tanks of the spectrophotometer under the following conditions: blank: introduce 3.00 ml of solution No. 1 and 50 pl of distilled water - standard: introduce 3, 00 ml of solution n°1 and 50 µl of vial 3 of the kit
Megazyme échantillon : introduire 3,00 ml de la solution n°1 et 50 mI de la préparation de l’échantillon. [0032] On mélange le contenu de chaque cuve et on lit la mesure d’absorbance (A1) des solutions après 2 mn environ au spectrophotomètre à 340 nm (spectrophotomètre équipé de cuves de 1,0 cm de trajet optique, pouvant mesurer à une longueur d’onde de 340 nm, et vérifié selon le mode opératoire décrit dans le manuel technique du constructeur qui s’y rapporte). Sample Megazyme: Add 3.00 mL of solution #1 and 50 mL of sample preparation. The contents of each tank are mixed and the absorbance measurement (A1) of the solutions is read after approximately 2 minutes using a spectrophotometer at 340 nm (spectrophotometer equipped with tanks with a 1.0 cm optical path, capable of measuring at a wavelength of 340 nm, and checked according to the procedure described in the manufacturer's technical manual relating thereto).
[0033] On amorce ensuite les réactions immédiatement en ajoutant 100 pL de la solution n°2 qui correspond à la solution d’OPA du flacon 2 du kit Megazyme dans chaque cuve de spectrophotomètre. The reactions are then initiated immediately by adding 100 μL of solution No. 2 which corresponds to the OPA solution from bottle 2 of the Megazyme kit in each spectrophotometer tank.
[0034] On mélange le contenu de chaque cuve et on les place environ 20 minutes dans l’obscurité. [0034] The contents of each tank are mixed and placed for about 20 minutes in the dark.
[0035] On lit ensuite la mesure d’absorbance A2 du blanc, du standard et de l’échantillon au spectrophotomètre à 340 nm. The absorbance A2 measurement of the blank, of the standard and of the sample is then read using a spectrophotometer at 340 nm.
[0036] La teneur en azote aminé libre, exprimée en pourcentage en poids par rapport au poids du produit, est donnée par la formule suivante : [Math 1] ou : The free amino nitrogen content, expressed as a percentage by weight relative to the weight of the product, is given by the following formula: [Math 1] or:
ΔAech =Aech2 - Aechl ΔAblc =Ablc2 - Ablc1 Aech2 = absorbance de l’échantillon après ajout de la solution n°2 Aechl = absorbance de l’échantillon après ajout de la solution n°1 Ablc2 = absorbance du blanc après ajout de la solution n°2 Ablc1 = absorbance du blanc après ajout de la solution n°1 V = volume de la fiole m = masse de la prise d’essai en g ΔAech =Aech2 - Aechl ΔAblc =Ablc2 - Ablc1 Aech2 = absorbance of the sample after addition of solution n°2 Aechl = absorbance of the sample after addition of solution n°1 Ablc2 = absorbance of the blank after addition of the solution n°2 Ablc1 = absorbance of the blank after addition of solution n°1 V = volume of the flask m = mass of the test sample in g
6803 = coefficient d’extinction du dérivé d’isoindole à 340 nm (en L. mol'1. cm-1). 14,01 = masse molaire de l’azote (en g. moM) 6803 = extinction coefficient of the isoindole derivative at 340 nm (in L. mol −1 . cm −1 ). 14.01 = molar mass of nitrogen (in g. moM)
3,15 = volume final dans la cuve (en mL) 3.15 = final volume in the tank (in mL)
0,05 = prise d’essai dans la cuve (en mL) - Détermination de la teneur en azote protéique : 0.05 = test portion in tank (in mL) - Determination of protein nitrogen content:
La teneur d’azote protéique est déterminée selon la méthode de DUMAS selon la norme ISO 16634 - 2016. Elle est exprimée en pourcentage en poids par rapport au poids du produit. - Calcul du degré d’hydrolyse The protein nitrogen content is determined according to the DUMAS method according to the ISO 16634 - 2016 standard. It is expressed as a percentage by weight relative to the weight of the product. - Calculation of the degree of hydrolysis
Le degré d’hydrolyse (DH) est calculé avec la formule suivante : [Math 2] The degree of hydrolysis (DH) is calculated with the following formula: [Math 2]
[0037] Comme il sera démontré plus loin dans la partie relative aux exemples de cette demande de brevet, il est important au sens de l’invention d’utiliser des protéines de pois, possédant un degré d’hydrolyse strictement compris entre 5% et 10%. En effet, si le degré d’hydrolyse est supérieur ou inférieur on constate que l’effet d’activation de la synthèse du FGF19 est moins performant. As will be demonstrated later in the part relating to the examples of this patent application, it is important within the meaning of the invention to use pea proteins, having a degree of hydrolysis strictly between 5% and 10%. Indeed, if the degree of hydrolysis is higher or lower, it is found that the effect of activating the synthesis of FGF19 is less effective.
Procédé de préparation des protéines de légumineuse selon l’inventionProcess for the preparation of legume proteins according to the invention
[0038] Dans un mode premier préférentiel de l’invention, les protéines de légumineuse sont des protéines de pois dont le degré d’hydrolyse est préférentiellement compris entre 5% et 10%. In a preferred first mode of the invention, the legume proteins are pea proteins, the degree of hydrolysis of which is preferably between 5% and 10%.
[0039] Les protéines de pois, possédant un degré d’hydrolyse strictement compris entre 5% et 10% sont obtenues par tout moyen bien connu de l’homme de l’art. The pea proteins, having a degree of hydrolysis strictly between 5% and 10%, are obtained by any means well known to those skilled in the art.
[0040] La base de départ est une farine, préférentiellement un concentrât (plus de 50% de protéines sur la MS), encore plus préférentiellement un isolat (plus de 80% de protéines sur la MS). Tout procédé bien connu de l’homme du métier pour ce faire sera utilisé, que ce soit par voie sèche (extraction sans utilisation de solvant aqueux p.e. via turboséparation) ou par voie humide (obtention d’une farine par broyage, mise en suspension de la farine dans un solvant aqueux, extraction des composés insoluble en particulier l’amidon et les fibres internes, précipitation isoélectrique des protéines appartenant aux groupes des globulines, récupération de celle-ci par centrifugation). Les protéines de pois obtenues par voie humide sont particulièrement préférées car elle mène à l’obtention d’un isolat qui concentre au maximum les capacités de celui-ci à aller activer la synthèse du FGF19. [0041] Les protéines de pois (farine, préférentiellement concentrât, encore plus préférentiellement isolat) seront ensuite hydrolysées. Toute méthode d’hydrolyse protéique qu’elle soit chimique, physique, enzymatique ou biologique bien connue de l’homme du métier est utilisable afin d’obtenir les protéines de légumineuse, préférentiellement de pois, à utiliser pour l’invention. L’hydrolyse enzymatique sera préférée, en particulier une hydrolyse réalisée avec protéase appartenant au sous- groupe des aminopeptidase provenant d ’Aspergillus Orizae. The starting base is a flour, preferably a concentrate (more than 50% protein on the DM), even more preferably an isolate (more than 80% protein on the DM). Any process well known to those skilled in the art for this will be used, whether by dry process (extraction without the use of aqueous solvent, e.g. via turboseparation) or by wet process (obtaining a flour by grinding, suspending flour in an aqueous solvent, extraction of insoluble compounds, in particular starch and internal fibers, isoelectric precipitation of proteins belonging to the globulin groups, recovery of the latter by centrifugation). The pea proteins obtained by the wet process are particularly preferred because it leads to the obtaining of an isolate which concentrates to the maximum the capacities of the latter to activate the synthesis of FGF19. The pea proteins (flour, preferably concentrate, even more preferably isolate) will then be hydrolysed. Any method of protein hydrolysis, whether chemical, physical, enzymatic or biological, well known to those skilled in the art can be used in order to obtain the legume proteins, preferably pea proteins, to be used for the invention. Enzymatic hydrolysis will be preferred, in particular hydrolysis carried out with protease belonging to the aminopeptidase subgroup originating from Aspergillus Orizae.
[0042] La préparation de l’hydrolysat de protéines de pois adapté à l’invention comporte donc préférentiellement une hydrolyse par voie enzymatique ou non enzymatique, de manière à ce que ledit isolat de protéines de pois présente un degré d'hydrolyse (DH) compris entre 5 % et 10 %, de préférence entre 6 % et 8 %, de manière encore plus spécifique de 6,5 % à 7 %. The preparation of the pea protein hydrolyzate suitable for the invention therefore preferably comprises enzymatic or non-enzymatic hydrolysis, so that said pea protein isolate has a degree of hydrolysis (DH) between 5% and 10%, preferably between 6% and 8%, even more specifically from 6.5% to 7%.
[0043] Dans un premier mode de réalisation, l’hydrolyse est réalisée par une endopeptidase. Il est choisi une endopeptidase non spécifique, dérivée d’une souche d ’Aspergillus en particulier une souche de Aspergillus spp ou Aspergillus Oryzae. Il est choisi plus particulièrement une endopeptidase EC 3-4-11. In a first embodiment, the hydrolysis is carried out by an endopeptidase. A non-specific endopeptidase is chosen, derived from a strain of Aspergillus, in particular a strain of Aspergillus spp or Aspergillus Oryzae. More particularly, an endopeptidase EC 3-4-11 is chosen.
[0044] La quantité exacte d'enzyme ajoutée à la suspension pour obtenir les caractéristiques souhaitées des isolats de protéines de pois variera en fonction de caractéristiques spécifiques telles que l'enzyme ou le système enzymatique utilisés ; le degré final désiré d’hydrolyse ; et/ou la distribution de poids moléculaire / final souhaité. Étant donné que ces paramètres sont connus, l'homme de l'art peut facilement déterminer les conditions appropriées pour obtenir les caractéristiques désirées de l’isolat de protéines de pois. The exact amount of enzyme added to the suspension to achieve the desired characteristics of pea protein isolates will vary depending on specific characteristics such as the enzyme or enzyme system used; the desired final degree of hydrolysis; and/or desired molecular/final weight distribution. Since these parameters are known, one skilled in the art can easily determine the appropriate conditions to obtain the desired characteristics of the pea protein isolate.
[0045] Dans un mode de réalisation particulier, les protéines de pois initiale utilisées pour préparer l’isolat de protéines de pois selon l’invention est une composition de protéines de pois telle que décrites dans la demande WO 2007/17572 ou préparée par un procédé tel que décrit dans la demande WO 2007/17572 (l’enseignement étant incorporé par référence). Dans un mode de réalisation particulier, la composition de protéines de pois initiale est la composition commercialisée par ROQUETTE FRERES sous le nom de marque NUTRALYS® S85F. [0046] Dans un mode de réalisation préféré de l’invention, la suspension de protéines de pois est amenée à une valeur de 5 à 20 % en poids de matière sèche, en particulier de 15 à 20 %. La température de réaction est ajustée à une valeur comprise entre 50 et 60°C, de préférence de l’ordre de 55°C. En règle générale, le système d'enzyme ou une enzyme est ajouté à la suspension en des quantités dans la gamme d'environ 0,3 à 1 % poids/volume. La réaction d'hydrolyse est typiquement effectuée dans une durée souhaitée afin d'obtenir le degré d'hydrolyse et/ou le profil désiré de poids moléculaire désiré, en l’occurrence ici pendant une période d'environ 45 minutes à environ 2 h 30 minutes, de préférence d'environ 1 heure. Une fois encore, le temps nécessaire à la réaction d'hydrolyse dépend des caractéristiques comme indiqué ci-dessus, mais peut être facilement déterminée par l'homme du métier. In a particular embodiment, the initial pea proteins used to prepare the pea protein isolate according to the invention is a composition of pea proteins as described in application WO 2007/17572 or prepared by a method as described in application WO 2007/17572 (the teaching being incorporated by reference). In a particular embodiment, the initial pea protein composition is the composition marketed by ROQUETTE FRERES under the brand name NUTRALYS® S85F. In a preferred embodiment of the invention, the pea protein suspension is brought to a value of 5 to 20% by weight of dry matter, in particular 15 to 20%. The reaction temperature is adjusted to a value between 50 and 60°C, preferably around 55°C. Generally, the enzyme system or an enzyme is added to the suspension in amounts in the range of about 0.3 to 1% w/v. The hydrolysis reaction is typically carried out in a desired time in order to obtain the desired degree of hydrolysis and / or the desired desired molecular weight profile, in this case for a period of about 45 minutes to about 2 h 30 minutes, preferably about 1 hour. Once again, the time required for the hydrolysis reaction depends on the characteristics as indicated above, but can be easily determined by those skilled in the art.
[0047] Dans d'autres réalisations, la suspension contenant des protéines de pois peut être hydrolysée en utilisant des moyens non enzymatiques, par exemple par hydrolyse mécanique (physique) et/ou chimique. Cette technique est également bien connue dans l'état de l’art. [0047] In other embodiments, the suspension containing pea proteins can be hydrolyzed using non-enzymatic means, for example by mechanical (physical) and/or chemical hydrolysis. This technique is also well known in the state of the art.
[0048] Une fois que les protéines de pois ont été hydrolysées au degré souhaité, la réaction d'hydrolyse est arrêtée, par exemple, par inactivation de l'enzyme, ou par d'autres moyens classiques. Dans un mode de réalisation, l'inactivation de l'enzyme est effectuée par traitement thermique. Conformément à la pratique établie, la préparation d'enzyme peut convenablement être inactivée en augmentant la température de la suspension d'incubation à une température à laquelle les enzymes deviennent inactivées, par exemple à environ 70°C pendant environ 10 minutes. Les isolats de protéines de pois ainsi obtenus sont ensuite traités à haute température courte durée (HTST), puis pasteurisés, éventuellement concentrés à une matière sèche de 10 à 30 %, avant d'être séchés par atomisation. Par exemple, l’isolat peut être pasteurisé à une température comprise entre 130°C et 150°C pendant une période d’environ 1 seconde à environ 30 secondes. [0048] Once the pea proteins have been hydrolyzed to the desired degree, the hydrolysis reaction is stopped, for example, by inactivation of the enzyme, or by other conventional means. In one embodiment, the inactivation of the enzyme is carried out by heat treatment. In accordance with established practice, the enzyme preparation may suitably be inactivated by raising the temperature of the incubation suspension to a temperature at which the enzymes become inactivated, for example at about 70°C for about 10 minutes. The pea protein isolates thus obtained are then treated at high temperature for a short time (HTST), then pasteurized, optionally concentrated to a dry matter of 10 to 30%, before being dried by atomization. For example, the isolate can be pasteurized at a temperature between 130°C and 150°C for a period of about 1 second to about 30 seconds.
[0049] Dans un second mode préférentiel alternatif de l’invention, les protéines de légumineuse sont des protéines de féverole dont le degré d’hydrolyse n’est pas modifié par hydrolyse après son extraction, préférentiellement avec un degré d’hydrolyse compris entre 0% et 5%. In a second alternative preferential mode of the invention, the legume proteins are faba bean proteins whose degree of hydrolysis is not modified by hydrolysis after its extraction, preferably with a degree of hydrolysis of between 0% and 5%.
[0050] Comme il sera démontré plus loin dans la partie relative aux exemples de cette demande de brevet, il est préférable au sens de l’invention d’utiliser des protéines de féverole par rapport aux protéines de pois. En effet, l’effet d’activation de synthèse est plus important en particulier dans un contexte de coopération cellulaire. Cette constatation d’une efficacité plus importante a été démontrée à l’aide de protéines de féverole dont le degré d’hydrolyse n’a pas été augmenté avec une hydrolyse enzymatique, physique ou biologique. [0051] La protéine de féverole est obtenue par tout moyen bien connu de l’homme de l’art que ce soit par voie sèche (extraction sans utilisation de solvant aqueux p.e. via turboséparation) ou par voie humide (obtention d’une farine par broyage, mise en suspension de la farine dans un solvant aqueux, extraction des composés insoluble en particulier l’amidon et les fibres internes, précipitation isoélectrique des protéines appartenant aux groupe des globulines, récupération de celle-ci par centrifugation). Les protéines de féveroles obtenues par voie humide sont particulièrement préférées car elle mène à l’obtention d’un isolat qui concentre ainsi au maximum les capacités de celui-ci à aller activer la synthèse du FGF19. As will be demonstrated later in the part relating to the examples of this patent application, it is preferable within the meaning of the invention to use faba bean proteins compared to pea proteins. Indeed, the synthesis activation effect is more important in particular in a context of cellular cooperation. This finding of greater efficacy was demonstrated using faba bean proteins whose degree of hydrolysis was not increased with enzymatic, physical or biological hydrolysis. The fava bean protein is obtained by any means well known to those skilled in the art, whether by dry route (extraction without the use of aqueous solvent, p.e. via turboseparation) or by wet route (obtaining a flour by grinding, suspending the flour in an aqueous solvent, extraction of insoluble compounds, in particular starch and internal fibers, isoelectric precipitation of proteins belonging to the globulin group, recovery of the latter by centrifugation). Faba bean proteins obtained by the wet process are particularly preferred because it leads to obtaining an isolate which thus concentrates as much as possible its capacities to activate the synthesis of FGF19.
[0052] Un procédé particulièrement préféré pour l’obtention des protéines de féverole est le suivant : A particularly preferred method for obtaining faba bean proteins is as follows:
[0053] La première étape consiste en la mise en oeuvre de graines de féverole. Celles-ci comportent encore leurs fibres externes protectrices, appelées également « hulls » en anglais. Les graines peuvent subir un pré-traitement susceptible de comporter des étapes de nettoyage, de tamisage (séparation des graines des cailloux par exemple), de trempage, de blanchiment, de toastage. De manière préférée, si un blanchiment est effectué, le barème du traitement thermique sera de 3 minutes à 80°C. Des exemples non-limitatifs de variétés sont par exemple les variétés Tiffany, FFS ou YYY. De manière préférentielle, on utilisera des variétés de graines de féveroles dont la teneur en tanins et/ou polyphénols est naturellement basse telles que la variété Organdi. De telles variétés sont connues et susceptibles d’être obtenues par croisement variétal et/ou modifications génétiques. [0054] La seconde étape vise la séparation la plus efficace possible des fibres externes et des cotylédons. Elle est initiée par exemple par un premier broyage des graines de féverole à l’aide d’un moulin à pierre. Un exemple particulier et particulièrement approprié d’un tel moulin à pierre est par exemple commercialisé par la société Alma®. Comme précédemment décrit, la graine va être introduite dans un espace constitué de deux disques en pierre, dont l’un est en rotation. La demanderesse s’est aperçue que cette technique est particulièrement d’intérêt car elle va provoquer une séparation très efficace des fibres externes et des cotylédons des graines. De manière préférée, l’espace inter-disque est ajustée entre 0,4 et 0,6 mm. The first step consists in the implementation of faba bean seeds. These still have their protective outer fibers, also called "hulls" in English. The seeds can undergo a pre-treatment likely to include steps of cleaning, sieving (separation of the seeds from the pebbles for example), soaking, bleaching, toasting. Preferably, if bleaching is carried out, the heat treatment schedule will be 3 minutes at 80°C. Non-limiting examples of varieties are for example the Tiffany, FFS or YYY varieties. Preferably, bean seed varieties will be used whose content of tannins and/or polyphenols is naturally low, such as the Organdi variety. Such varieties are known and likely to be obtained by varietal crossing and/or genetic modifications. The second step aims for the most efficient separation possible of the outer fibers and the cotyledons. It is initiated for example by a first grinding of faba bean seeds using a stone mill. A particular and particularly suitable example of such a stone mill is for example marketed by the company Alma®. As previously described, the seed will be introduced into a space made up of two stone discs, one of which is rotating. The applicant has noticed that this technique is of particular interest because it will cause a very effective separation of the external fibers and the cotyledons of the seeds. Preferably, the inter-disc space is adjusted between 0.4 and 0.6 mm.
[0055] Le broyât obtenu subit ensuite l’application d’un flux d’air ascendant, à contre-courant. Les différentes particules solides vont être classifiées selon leur densité. Typiquement, après équilibre, on obtient deux fractions : une fraction légère contenant majoritairement les fibres externes ou « hulls » et une fraction « lourde » contenant majoritairement les cotylédons. Un exemple particulier et particulièrement approprié d’un appareillage adéquat est par exemple le MZMZ 1- 40 commercialisé par la société Hosokawa-alpine®. [0055] The ground material obtained then undergoes the application of an upward flow of air, against the current. The different solid particles will be classified according to their density. Typically, after equilibrium, two fractions are obtained: a light fraction mainly containing the external fibers or “hulls” and a “heavy” fraction mainly containing the cotyledons. A particular and particularly suitable example of a suitable device is for example the MZMZ 1-40 marketed by the company Hosokawa-alpine®.
[0056] La fraction lourde, enrichie en cotylédons, va ensuite être broyée à l’aide d’un moulin à couteaux. Un exemple particulier et particulièrement approprié d’un tel moulin à couteaux est par exemple le SM300 commercialisé par la société Retsch®. The heavy fraction, enriched in cotyledons, will then be ground using a knife mill. A particular and particularly suitable example of such a knife grinder is, for example, the SM300 marketed by the company Retsch®.
[0057] La succession des trois opérations ci-dessus citées au sein de la seconde étape vise à séparer de manière très fine les fibres externes et les cotylédons, en évitant de dégrader ces deux parties et mélanger celle-ci. [0058] La troisième étape vise à réduire la granulométrie de la fraction lourde enrichie en cotylédons par leur broyage à l’aide d’un broyeur à rouleaux. Un exemple particulier et particulièrement approprié d’un tel broyeur à rouleaux est par exemple le MLU 202, commercialisé par la société Bühler®. Il est ici utilisé afin de réduire la granulométrie de la farine de manière globale, afin d’obtenir une poudre homogène et suffisamment fine afin de permettre à l’étape 4 suivante d’être facilitée. La granulométrie préférée est comprise entre 200 et 400 microns, préférentiellement 300 microns. Afin de mesurer cette granulométrie, on utilise préférentiellement un appareil de granulométrie laser, bien que toute méthode soit possible telle que le tamisage. The succession of the three operations mentioned above within the second step aims to very finely separate the outer fibers and the cotyledons, avoiding degrading these two parts and mixing them. The third step aims to reduce the particle size of the heavy fraction enriched in cotyledons by grinding them using a roller mill. A particular and particularly suitable example of such a roller mill is for example the MLU 202, marketed by the company Bühler®. It is used here in order to reduce the particle size of the flour globally, in order to obtain a homogeneous and sufficiently fine powder to allow the following step 4 to be facilitated. The preferred particle size is between 200 and 400 microns, preferably 300 microns. In order to measure this particle size, a laser particle size measuring device is preferably used, although any method is possible, such as sieving.
[0059] De manière alternative, l’étape de réduction de la granulométrie de la fraction lourde enrichie en cotylédons peut être réalisée en présence de solvant aqueux, préférentiellement de l’eau. Dans ce cas, la quatrième étape ci-dessous est fusionnée avec la troisième étape qui sont alors réalisées de manière concomitante. Alternatively, the step of reducing the particle size of the heavy fraction enriched in cotyledons can be carried out in the presence of an aqueous solvent, preferably water. In this case, the fourth step below is merged with the third step which are then carried out concomitantly.
[0060] La quatrième étape vise à mettre en suspension la poudre obtenue dans la troisième étape précédente dans un solvant aqueux, préférentiellement dans de l’eau. Le but est ici de réaliser une extraction sélective de certains composés, majoritairement les protéines ainsi que les sels et les sucres, en les solubilisant. Le pH de la solution est avantageusement rectifié vers un pH neutre afin de limiter au maximum la solubilisation des tanins et polyphénols. Cette rectification de pH peut être effectuée avant et/ou après suspension de la poudre dans le solvant aqueux. The fourth step aims to suspend the powder obtained in the previous third step in an aqueous solvent, preferably in water. The aim here is to carry out a selective extraction of certain compounds, mainly proteins as well as salts and sugars, by solubilizing them. The pH of the solution is advantageously adjusted towards a neutral pH in order to limit as much as possible the solubilization of tannins and polyphenols. This pH rectification can be carried out before and/or after suspending the powder in the aqueous solvent.
[0061] Le solvant aqueux est préférentiellement de l’eau. Celle-ci peut néanmoins être additivée, par exemple avec des composés permettant de faciliter la solubilisation. Le pH du solvant aqueux est ajusté entre 6 et 8, préférentiellement 7. Tout réactif acide ou basique tel que la soude, la chaux, l’acide citrique ou chlorhydrique est envisageable, mais la potasse et l’acide ascorbique sont préférés. La température est ajustée entre 2°c et 30°c, préférentiellement entre 10°C et 30°C, préférentiellement entre 15°C et 25°C, encore plus préférentiellement à 20°C. Cette température est régulée tout au long de la réaction d’extraction. The aqueous solvent is preferably water. The latter can nevertheless be supplemented, for example with compounds making it possible to facilitate solubilization. The pH of the aqueous solvent is adjusted between 6 and 8, preferably 7. Any acidic or basic reagent such as soda, lime, citric or hydrochloric acid is possible, but potash and ascorbic acid are preferred. The temperature is adjusted between 2°C and 30°C, preferentially between 10°C and 30°C, preferentially between 15°C and 25°C, even more preferentially at 20°C. This temperature is regulated throughout the extraction reaction.
[0062] La poudre obtenue est délayée afin d’obtenir une suspension comprise entre 5% et 25%, préférentiellement entre 5% et 15%, préférentiellement entre 7% et 13%, encore plus préférentiellement entre 9% et 11%, le plus préféré étant 10%, le pourcentage étant exprimé en poids de poudre par poids total de suspension eau/poudre. La suspension est agitée à l’aide de tout appareillage bien connu de l’Homme du métier, par exemple une cuve équipée d’un agitateur, équipé de pales, d’hélices marines, ou de tout équipement permettant une agitation efficace. Le temps d’extraction, préférentiellement sous agitation, est compris entre 5 et 25 minutes, préférentiellement entre 10 et 20 minutes, encore plus préférentiellement 15 minutes. The powder obtained is mixed in order to obtain a suspension of between 5% and 25%, preferentially between 5% and 15%, preferentially between 7% and 13%, even more preferentially between 9% and 11%, the most preferred being 10%, the percentage being expressed as weight of powder per total weight of water/powder suspension. The suspension is agitated using any apparatus well known to those skilled in the art, for example a tank equipped with an agitator, equipped with blades, marine propellers, or any equipment allowing effective agitation. The extraction time, preferably with stirring, is between 5 and 25 minutes, preferably between 10 and 20 minutes, even more preferably 15 minutes.
[0063] La cinquième étape vise à séparer par centrifugation la fraction soluble et la fraction solide obtenues lors de la quatrième étape. On peut retrouver le principe industriel préféré dans la demande de brevet EP1400537 qui est incorporée ici par référence. Le principe de ce procédé est de commencer en utilisant un hydrocyclone afin d’extraire une fraction enrichie en amidon, puis d’utiliser une décanteuse horizontale afin d’extraire une fraction enrichie en fibres interne. Néanmoins, il est possible d’utiliser une centrifugeuse industrielle qui va extraire une fraction enrichie en amidon et en fibres internes. Dans tous les cas, on obtient des fractions solides et une fraction liquide qui concentre la majorité des protéines. The fifth step aims to separate by centrifugation the soluble fraction and the solid fraction obtained during the fourth step. The preferred industrial principle can be found in patent application EP1400537 which is incorporated herein by reference. The principle of this process is to start by using a hydrocyclone to extract a fraction enriched in starch, then to use a horizontal decanter to extract a fraction enriched in internal fibers. Nevertheless, it is possible to use an industrial centrifuge which will extract a fraction enriched in starch and internal fibres. In all cases, solid fractions and a liquid fraction are obtained which concentrate the majority of the proteins.
[0064] La sixième étape vise à acidifier au pH isoélectrique des protéines de féverole, autour de 4,5, puis de faire subir à la solution un chauffage afin de faire coaguler les protéines dites globulines, qui seront séparées par centrifugation. [0065] L’acidification est réalisée à un pH entre 4 et 5, préférentiellement 4,5.The sixth step aims to acidify the isoelectric pH of the bean proteins, around 4.5, then to subject the solution to heating in order to cause the so-called globulin proteins to coagulate, which will be separated by centrifugation. The acidification is carried out at a pH between 4 and 5, preferably 4.5.
Celle-ci est réalisée préférentiellement avec de l’acide chlorhydrique à 7% massique environ, mais tous types d’acides, minéraux ou organiques, sont utilisables tels que l’acide citrique. De manière encore plus préférentielle, l’utilisation d’acide ascorbique pur ou en combinaison avec un autre acide minéral ou organique, est également possible. L’utilisation d’acide ascorbique pour acidifier permet une amélioration de la coloration finale. Tout moyen de chauffage est ensuite possible, par exemple au moyen d’une cuve agitée équipée d’une double enveloppe et/ou serpentin ou un cuiseur en ligne par injection de vapeur (« jet cooker » en anglais). La température de chauffage est avantageusement comprise entre 45°C et 75°C, préférentiellement entre 50°C et 70°C, encore plus préférentiellement entre 55°C et 65°C, le plus préféré étant 60°C. Le temps de chauffage est avantageusement compris entre 5 minutes et 25 minutes, préférentiellement entre 10 et 20 minutes, le plus préféré étant 10 minutes. This is preferably carried out with hydrochloric acid at around 7% by weight, but all types of acids, mineral or organic, can be used, such as citric acid. Even more preferably, the use of ascorbic acid pure or in combination with another mineral or organic acid is also possible. The use of ascorbic acid to acidify improves the final color. Any means of heating is then possible, for example by means of a stirred tank equipped with a double jacket and/or coil or an in-line cooker by steam injection (“jet cooker”). The heating temperature is advantageously between 45°C and 75°C, preferentially between 50°C and 70°C, even more preferentially between 55°C and 65°C, the most preferred being 60°C. The heating time is advantageously between 5 minutes and 25 minutes, preferentially between 10 and 20 minutes, the most preferred being 10 minutes.
[0066] La composition protéique, majoritairement de la globuline, va coaguler et précipiter au sein de la solution. Elle sera séparée par toute technique de centrifugation, comme par exemple le Sédicanteur Flottwegg®. La solution résiduelle obtenue concentre sucres, sels et albumines, elle est appelée solubles de féverole. Elle sera traitée à part, préférentiellement évaporée et/ou séchée. The protein composition, mainly globulin, will coagulate and precipitate within the solution. It will be separated by any centrifugation technique, such as the Flottwegg® Sedicator. The solution residual obtained concentrates sugars, salts and albumins, it is called faba bean solubles. It will be treated separately, preferably evaporated and/or dried.
[0067] La combinaison de la précipitation isoélectrique avec un chauffage contrôlé proposé par l’invention permet l’obtention : - d’un floc de protéines coagulées, donnant après traitement requis le produit revendiqué dans la présente demande, et The combination of isoelectric precipitation with controlled heating proposed by the invention makes it possible to obtain: - a floc of coagulated proteins, giving after required treatment the product claimed in the present application, and
- de solubles résiduels contenant entre autres des protéines solubles (albumines), sels et sucres - residual solubles containing, among other things, soluble proteins (albumins), salts and sugars
[0068] Dans une septième étape, la composition protéique est ensuite diluée à environ 15-20% en poids de matière sèche et neutralisée à pH compris entre 5,5 et 6,5, préférentiellement 6,5, à l’aide de tout type d’agent basique, préférentiellement de la potasse à 20% massique. In a seventh step, the protein composition is then diluted to approximately 15-20% by weight of dry matter and neutralized to a pH of between 5.5 and 6.5, preferably 6.5, using any type of basic agent, preferably potash at 20% by weight.
[0069] La composition protéique peut ensuite subir un traitement thermique, préférentiellement à une température de 135°C par injection directe de vapeur par tuyère et refroidissement par effet flash sous-vide à 65°C. The protein composition can then undergo a heat treatment, preferably at a temperature of 135° C. by direct injection of steam through a nozzle and cooling by vacuum flash effect at 65° C.
[0070] La composition protéique obtenue peut être utilisée directement par exemple en étant hydrolysée par une protéase ou bien texturée par une extrudeuse. The protein composition obtained can be used directly, for example by being hydrolyzed by a protease or even textured by an extruder.
[0071] Dans une huitième étape, la composition protéique utile pour l’invention est séchée. Le mode préféré de séchage est l’atomisation, en particulier à l’aide d’un atomiseur à multiple effets. Les paramètres typiques sont une température d’entrée de 200°C et une température des buées à 85-90°C. In an eighth step, the protein composition useful for the invention is dried. The preferred mode of drying is atomization, especially using a multiple-effect atomizer. Typical parameters are an inlet temperature of 200°C and a vapor temperature of 85-90°C.
[0072] Utilisation thérapeutique [0072] Therapeutic use
[0073] La composition selon la présente divulgation permet d’activer la synthèse du FGF19 dans les cellules humaines. [0074] Le FGF19 est un gène codant pour une protéine appartenant à la famille des facteurs de croissances des fibroblastes qui régissent le métabolisme des nutriments. Chez l’homme, Le gène FGF19 (Gene ID : 9965 mis à jour le 8-07-2021) code pour un transcrit de 1821 pb (NCBI Reference Sequence: NM_005117.3, mis à jour le 29 juin 2021) qui code pour une protéine FGF19 (NCBI Reference Sequence : NP_0051108.1 , mis à jour le 29 juin 2021). Le FGF19, également appelé FGF15 chez les rongeurs, est un membre d'une sous-famille de facteurs de croissance des fibroblastes qui régissent le métabolisme des nutriments. Le FGF19 est exprimé et sécrété dans l'intestin grêle distal, par les cellules de l'épithélium biliaire et intestinal, où sa synthèse est régulée positivement après l'absorption postprandiale des acides biliaires. The composition according to the present disclosure makes it possible to activate the synthesis of FGF19 in human cells. FGF19 is a gene coding for a protein belonging to the family of fibroblast growth factors which govern nutrient metabolism. In humans, the FGF19 gene (Gene ID: 9965 updated on 8-07-2021) codes for a 1821 bp transcript (NCBI Reference Sequence: NM_005117.3, updated on June 29, 2021) which codes for an FGF19 protein (NCBI Reference Sequence: NP_0051108.1, updated June 29, 2021). FGF19, also called FGF15 in rodents, is a member of a subfamily of fibroblast growth factors that regulate nutrient metabolism. FGF19 is expressed and secreted in the distal small intestine, by cells of the biliary and intestinal epithelium, where its synthesis is upregulated after postprandial absorption of bile acids.
[0075] Le terme « activation de la synthèse » ou « surexpression du gène » doit se comprendre au sens de l’invention comme une activation exogène (c’est à dire par le biais d’un composé, d’une molécule étrangère) de la synthèse d’ARNm ou de protéine dans les cellules d’un organisme vivant, en particulier la synthèse du FGF19 dans le cadre de cette invention. The term "activation of the synthesis" or "overexpression of the gene" must be understood within the meaning of the invention as an exogenous activation (that is to say through a compound, a foreign molecule) mRNA or protein synthesis in the cells of a living organism, in particular the synthesis of FGF19 in the context of this invention.
[0076] La synthèse des protéines est l'ensemble des processus biochimiques permettant aux cellules de produire leurs protéines à partir de leurs gènes. Elle recouvre les étapes de transcription de l'ADN en ARN messager, d'aminoacylation des ARN de transfert, de traduction de l'ARN messager en chaînes polypeptidiques, de modifications post-traductionnelles de ces dernières, et enfin de repliement des protéines ainsi produites. Elle est étroitement régulée à de multiples niveaux, principalement lors de la transcription et lors de la traduction. Pour le cas de l’être humain, le gène codant pour l’expression du FGF19 est situé sur le chromosome 11 humain. [0076] Protein synthesis is the set of biochemical processes allowing cells to produce their proteins from their genes. It covers the steps of transcription of DNA into messenger RNA, aminoacylation of transfer RNA, translation of messenger RNA into polypeptide chains, post-translational modifications of the latter, and finally folding of the proteins thus produced. . It is tightly regulated at multiple levels, mainly during transcription and during translation. In the case of humans, the gene coding for the expression of FGF19 is located on human chromosome 11.
[0077] L’activation de la synthèse du FGF19 peut être déterminée en mesurant le niveau d’expression du gène FGF19, en particulier en mesurant soit le niveau de l’expression de l’ARNm du FGF19, soit le niveau d’expression de la protéine de FGF19 par toutes méthodes connues de l’homme du métier. En particulier, l’expression du FGF19 est augmentée lorsque le niveau d’expression est au moins 1.5 ou 2, 3, 4, 5 fois plus important que dans les cellules non traitées. The activation of the synthesis of FGF19 can be determined by measuring the level of expression of the FGF19 gene, in particular by measuring either the level of expression of the mRNA of FGF19, or the level of expression of the FGF19 protein by any method known to those skilled in the art. In particular, the expression of FGF19 is increased when the level of expression is at least 1.5 or 2, 3, 4, 5 times greater than in untreated cells.
[0078] Le terme « protéine de FGF19 » ou « polypeptide de FGF19 » doit se comprendre au sens de l’invention comme un polypeptide, c'est-à-dire une chaîne d'acides aminés, ayant une homologie avec la séquence particulière d’acide aminé naturellement secrétée chez les organismes mammifères. [0079] Selon l'invention, les termes " polypeptide FGF19 ", "FGF19" et "FGF15/19" désignent la séquence native d'une forme naturelle d'un polypeptide FGF19 tel qu'exprimé dans n'importe quel organisme mammifère. Ce terme comprend n'importe quelle isoforme d'origine naturelle, qui englobe les formes variantes telles que des formes épissées alternativement, les formes de variants alléliques, et les formes non traitées et traitées de FGF19, telles que les formes de polypeptide FGF19 comprenant un peptide signal The term "FGF19 protein" or "FGF19 polypeptide" should be understood within the meaning of the invention as a polypeptide, that is to say a chain of amino acids, having a homology with the particular sequence amino acid naturally secreted in mammalian organisms. According to the invention, the terms “FGF19 polypeptide”, “FGF19” and “FGF15/19” denote the native sequence of a natural form of an FGF19 polypeptide as expressed in any mammalian organism. This term includes any naturally occurring isoform, which encompasses variant forms such as alternatively spliced forms, allelic variant forms, and unprocessed and processed forms of FGF19, such as FGF19 polypeptide forms comprising a signal peptide
[0080] Ce terme "polypeptide FGF19" comprend également des fragments d'une forme naturelle d'un polypeptide FGF19, en particulier des fragments recombinants ayant la même activité biologique que ladite forme naturelle This term "FGF19 polypeptide" also includes fragments of a natural form of an FGF19 polypeptide, in particular recombinant fragments having the same biological activity as said natural form.
[0081] Dans le sens de l'invention, le terme "FGF19" comprend tous les polypeptides FGF19 présentant au moins 50 % d'identité avec la séquence humaine représentée dans SEQ ID NO 1. L'expression "un polypeptide FGF19 présentant au moins 50 % d'identité avec la séquence humaine représentée dans SEQ ID NO 1" désigne un polypeptide, membre de la famille FGF19, ayant une séquence d'acides aminés présentant au moins 50 % d'identité d'acide aminé avec la séquence de référence. Ceci nécessite que, après l'alignement, 50 % des acides aminés dans la séquence candidate sont identiques aux acides aminés correspondants dans la séquence de référence [0082] Par « identité de l'acide aminé », on entend que le même acide aminé est observé sur les deux séquences. L'identité ne prend pas en compte les modifications post-traduction qui peuvent se produire sur les acides aminés. L'identité selon la présente invention est déterminée à l'aide d'une analyse informatique, telle que le programme d'alignement d'ordinateur ClustalW, et les paramètres par défaut suggérés dans celui-ci. Le logiciel ClustalW est disponible à partir du site Web http://www.clust.org/clust2/. En utilisant ce programme avec ses réglages par défaut, la partie d'une requête et d'un "polypeptide de référence" sont alignées. Le nombre de résidus totalement conservés est compté et divisé par la longueur du polypeptide de référence. Selon la présente invention, le "polypeptide de référence" présente la séquence telle que représentée dans SEQ ID NO 1 [0083] Les termes "au moins 50 % d'identité" indiquent que le pourcentage d'identité entre les deux séquences, la requête et le polypeptide de référence de SEQ ID 1, est d'au moins 50,55, 60,65,70, 75,80, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 ou 100 % par rapport à la séquence SEQ ID NO : 1. [0084] Par conséquent, le polypeptide FGF19 est choisi parmi le polypeptide[0081] Within the meaning of the invention, the term "FGF19" includes all the FGF19 polypeptides exhibiting at least 50% identity with the human sequence represented in SEQ ID NO 1. The expression "an FGF19 polypeptide exhibiting at least 50% identity with the human sequence represented in SEQ ID NO 1" denotes a polypeptide, member of the FGF19 family, having an amino acid sequence exhibiting at least 50% amino acid identity with the reference sequence . This requires that, after alignment, 50% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence [0082] By "amino acid identity" it is meant that the same amino acid is observed on both sequences. The identity does not take into account post-translational modifications that may occur on amino acids. Identity according to the present invention is determined using computer analysis, such as the ClustalW computer alignment program, and the default settings suggested therein. The ClustalW software is available from the website http://www.clust.org/clust2/. Using this program with its default settings, the part of a query and a "reference polypeptide" are aligned. The number of fully conserved residues is counted and divided by the length of the reference polypeptide. According to the present invention, the "reference polypeptide" has the sequence as represented in SEQ ID NO 1 The terms "at least 50% identity" indicate that the percentage identity between the two sequences, the query and the reference polypeptide of SEQ ID 1, is at least 50.55, 60.65 ,70, 75,80, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% with respect to the sequence SEQ ID NO: 1. [0084] Consequently, the FGF19 polypeptide is chosen from the polypeptide
FGF15 exprimé chez les souris, le polypeptide FGF19 exprimé chez l'homme, ou les homologues de FGF15 et FGF19 exprimés chez d'autres mammifères tels que rat, chien, chat, mouton, bétail, cheval, porc, chèvre, lapin. FGF15 expressed in mice, the FGF19 polypeptide expressed in humans, or the homologs of FGF15 and FGF19 expressed in other mammals such as rat, dog, cat, sheep, cattle, horse, pig, goat, rabbit.
[0085] Les membres de la famille FGF19 comprennent notamment : - le polypeptide FGF19 humain de 216 acides aminés (y compris 24 acides aminés constituant le peptide signal) dont la séquence est représentée dans SEQ ID NO 1 ;The members of the FGF19 family include in particular: the human FGF19 polypeptide of 216 amino acids (including 24 amino acids constituting the signal peptide) whose sequence is represented in SEQ ID NO 1;
- le polypeptide de mus musculus FGF15 de 218 acides aminés (y compris 25 acides aminés constituant le peptide signal) dont la séquence est représentée dans SEQ ID NO 10. - l'une quelconque des autres séquences telles que représentées dans SEQ ID 2,- the mus musculus FGF15 polypeptide of 218 amino acids (including 25 amino acids constituting the signal peptide) whose sequence is represented in SEQ ID NO 10. - any of the other sequences as represented in SEQ ID 2,
3, 4, 5, 6, 7, 8, ou 9, telle que présentées dans la table 1 ci-dessous : [tableau 1] : 3, 4, 5, 6, 7, 8, or 9, as presented in Table 1 below: [Table 1]:
[0086] La présente invention consiste plus particulièrement en l’utilisation thérapeutique d’une composition de protéines de légumineuse, préférentiellement de pois ou de féverole dont le degré d’hydrolyse est inférieur à 10% afin d’activer la synthèse du FGF19 chez un sujet et de prévenir ou traiter les maladies susceptibles d’être traitées par une surexpression du FGF19, notamment les maladies associées à la dérégulation du FGF19. The present invention consists more particularly in the therapeutic use of a composition of legume proteins, preferably peas or fava beans, the degree of hydrolysis of which is less than 10% in order to activate the synthesis of FGF19 in a subject and to prevent or treat diseases susceptible to be treated by an overexpression of FGF19, in particular diseases associated with the deregulation of FGF19.
[0087] Les sujets atteints de maladies associées à la dérégulation du FGF19 présentent une dérégulation de l’expression du gène FGF19, en particulier une réduction de la synthèse de FGF19 ou une diminution de la voie de signalisation du FGF19, notamment une diminution de l’activation des facteurs de la voie de signalisation en aval de FGF19 telle que ERK, PI3K, GSK3p. Subjects suffering from diseases associated with the deregulation of FGF19 present a deregulation of the expression of the FGF19 gene, in particular a reduction in the synthesis of FGF19 or a reduction in the signaling pathway of FGF19, in particular a reduction in the activation of factors in the signaling pathway downstream of FGF19 such as ERK, PI3K, GSK3p.
[0088] Dans un mode de réalisation particulier, la composition telle que décrite précédemment est utilisée pour le traitement et/ou la prévention d’une maladie, de préférence une maladie susceptible d’être traitée par une activation de la synthèse du FGF19, notamment une maladie associée à une dérégulation de la voie de signalisation du FGF19, de préférence la sarcopénie ou une maladie métabolique telle qu’une maladie inflammatoire chronique de l’intestin, la malabsorption des acides biliaires primaire, l’obésité ou la stéatopathie non alcoolique (NAFLD, de son acronyme anglais « non alcoholic fatty liver disease ») telle que la stéatohépatite non alcoolique (NASH, de son acronyme anglophone « non-alcoholic steatohepatitis ») chez un sujet. In one particular embodiment, the composition as described above is used for the treatment and/or prevention of a disease, preferably a disease capable of being treated by activating the synthesis of FGF19, in particular a disease associated with dysregulation of the FGF19 signaling pathway, preferably sarcopenia or a metabolic disease such as inflammatory bowel disease, primary bile acid malabsorption, obesity or non-alcoholic steatopathy ( NAFLD, from its English acronym “non alcoholic fatty liver disease”) such as non-alcoholic steatohepatitis (NASH, from its English acronym “non-alcoholic steatohepatitis”) in a subject.
[0089] Le terme « sujet » ou « patient » doit se comprendre au sens de l’invention comme tout être vivant appartenant au règne animal. De manière non exhaustive, on entend donc les chiens, les chats, les vaches, les porcs. De manière préférée, par « sujet » on entend un être humain, de manière encore plus préférée un être humain ayant une maladie ou un syndrome susceptible d’être traité par l’activation de la synthèse de FGF19. The term “subject” or “patient” must be understood within the meaning of the invention as any living being belonging to the animal kingdom. In a non-exhaustive manner, we therefore mean dogs, cats, cows, pigs. Preferably, by “subject” is meant a human being, even more preferably a human being having a disease or a syndrome capable of being treated by the activation of the synthesis of FGF19.
[0090] De manière préférée, la méthode selon l’invention est caractérisée en ce que le sujet est un être humain, en particulier une personne âgée. Par « personne âgée » on entend un être humain ayant plus de 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 ans. Preferably, the method according to the invention is characterized in that the subject is a human being, in particular an elderly person. By “elderly person” we mean a human being over 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 years old.
[0091] De manière préférée, le sujet est un animal, en particulier sélectionné dans la liste des chats, chiens, vaches, des moutons, des porcs, des poulets et des dindes [0092] Le terme « traitement » inclut un traitement curatif aboutissant à une guérison ou un traitement allégeant, améliorant, éliminant, réduisant et/ou stabilisant les symptômes d'une maladie ou la souffrance qu'elle provoque. Le terme « prévention » correspond à un traitement prophylactique ou préventif, qui comprend aussi bien un traitement aboutissant à la prévention d'une maladie qu'un traitement réduisant et/ou retardant l'incidence d'une maladie ou le risque qu'elle survienne. Preferably, the subject is an animal, in particular selected from the list of cats, dogs, cows, sheep, pigs, chickens and turkeys. [0092] The term "treatment" includes a curative treatment resulting in a cure or a treatment alleviating, ameliorating, eliminating, reducing and/or stabilizing the symptoms of a disease or the suffering which it causes. The term "prevention" corresponds to a prophylactic or preventive treatment, which includes both a treatment leading to the prevention of a disease and a treatment reducing and/or delaying the incidence of a disease or the risk that it occurs. .
[0093] Dans un premier mode de réalisation préféré, la composition selon l’invention vise à augmenter l’anabolisme de fibre musculaire via l’activation de la synthèse du FGF19 et à prévenir ou traiter des maladies associées à une perte musculaire, en particulier la sarcopénie. La sarcopénie est un syndrome gériatrique se caractérisant par une diminution des capacités musculaires due à l’âge et qui en s’aggravant sera à l’origine d’une détérioration de la force musculaire et des performances physiques. [0094] La composition selon l’invention permet de préférence de prévenir ou traiter la sarcopénie et d’améliorer ou éradiquer la maladie ou les symptômes associée à la maladie, telle que la détérioration de la force musculaire et des performances physiques. In a first preferred embodiment, the composition according to the invention aims to increase muscle fiber anabolism via the activation of the synthesis of FGF19 and to prevent or treat diseases associated with muscle loss, in particular sarcopenia. Sarcopenia is a geriatric syndrome characterized by a decrease in muscle capacity due to age and which, as it gets worse, will cause a deterioration in muscle strength and physical performance. The composition according to the invention preferably makes it possible to prevent or treat sarcopenia and to improve or eradicate the disease or the symptoms associated with the disease, such as the deterioration of muscular strength and physical performance.
[0095] La consommation de protéine de légumineuse est déjà bien connue mais dans ce but il est clairement recommandé de combiner avec d’autres sources de protéines afin d’améliorer le profil des acides aminés. La stratégie préférentielle innovante découverte par la demanderesse simplifie la chose en se concentrant sur l’activité propre sur la surexpression du FGF19. Cette surexpression est maximale pour les protéines de légumineuses, préférentiellement de pois et ou de féverole, quand le degré d’hydrolyse est très précisément sélectionné. Cette approche est donc en complète opposition avec les stratégies couramment utilisées dans l’art antérieur. The consumption of legume protein is already well known but for this purpose it is clearly recommended to combine with other protein sources in order to improve the amino acid profile. The innovative preferential strategy discovered by the applicant simplifies the matter by focusing on the specific activity on the overexpression of FGF19. This overexpression is maximal for legume proteins, preferably peas and/or fava beans, when the degree of hydrolysis is very precisely selected. This approach is therefore in complete opposition to the strategies commonly used in the prior art.
[0096] Dans un autre mode de réalisation préféré, la composition selon l’invention permet de prévenir ou traiter une maladie métabolique via l’activation de la synthèse du FGF19, en particulier la stéatohépatite non alcoolique (ou NASH). [0097] La NASH est une atteinte hépatique qui résulte d’une accumulation excessive de lipides dans les hépatocytes entraînant une lipotoxicité et des lésions inflammatoires des hépatocytes. La physiopathologie implique l'accumulation de graisse (stéatose), une inflammation et, de manière variable, une fibrose. [0098] La composition selon l’invention permet de prévenir ou traiter la NASH et d’améliorer ou éradiquer la maladie ou les symptômes associée à la NASH, telle qu’une diminution de l’accumulation des lipides dans les hépatocytes et des lésions inflammatoires des hépatocytes chez le patient. In another preferred embodiment, the composition according to the invention makes it possible to prevent or treat a metabolic disease via the activation of the synthesis of FGF19, in particular non-alcoholic steatohepatitis (or NASH). [0097] NASH is liver damage that results from an excessive accumulation of lipids in hepatocytes leading to lipotoxicity and inflammatory lesions of hepatocytes. The pathophysiology involves fat accumulation (steatosis), inflammation and, variably, fibrosis. The composition according to the invention makes it possible to prevent or treat NASH and to improve or eradicate the disease or the symptoms associated with NASH, such as a reduction in the accumulation of lipids in the hepatocytes and inflammatory lesions. hepatocytes in the patient.
[0099] A ce jour, peu voire aucun traitement n’existe. Une alimentation réduite et équilibrée, de l’exercice, la consommation de café ou de vitamine E sont pour le moment les seules traitement conseillés par G « American Liver Foundation » (https://liverfoundation.org/for-patients/about-the-liver/diseases-of-the- liver/nonalcoholic-steatohepatitis-information-center/nash-treatment/). Ces recommandations sont hélas bien maigres et parfois difficilement applicables (exercice physique impossible, consommation de caféine). La composition selon l’invention permet donc d’envisager via la stimulation de synthèse du FGF19 une voie afin de lutter contre la prévalence de cette NASH. To date, little or no treatment exists. A reduced and balanced diet, exercise, consumption of coffee or vitamin E are currently the only treatments recommended by the G "American Liver Foundation" (https://liverfoundation.org/for-patients/about-the -liver/diseases-of-the-liver/nonalcoholic-steatohepatitis-information-center/nash-treatment/). These recommendations are unfortunately very meager and sometimes difficult to apply (physical exercise impossible, caffeine consumption). The composition according to the invention therefore makes it possible to envisage, via the stimulation of the synthesis of FGF19, a pathway in order to combat the prevalence of this NASH.
[0100] La présente divulgation consiste également en l’utilisation d’une composition comprenant une protéine de légumineuse telle que décrite précédemment pour la préparation d’un médicament pour l’utilisation dans le traitement d’une maladie susceptible d’être traitée par l’activation de la synthèse du FGF19 telle que décrite précédemment, de préférence la sarcopénie ou la NASH. [0100] The present disclosure also consists of the use of a composition comprising a legume protein as described above for the preparation of a medicament for use in the treatment of a disease capable of being treated by activation of FGF19 synthesis as previously described, preferably sarcopenia or NASH.
[0101] Dans un autre mode de réalisation particulier, la présente divulgation est une méthode de traitement d’une maladie susceptible d’être traitée par l’activation de la synthèse du FGF19 telle que décrite précédemment, de préférence la sarcopénie ou la NASH chez un sujet dans le besoin comprenant l’administration d’une quantité thérapeutiquement efficace d’une composition comprenant une protéine de légumineuse telle que décrite précédemment chez un sujet. In another particular embodiment, the present disclosure is a method for treating a disease capable of being treated by the activation of the synthesis of FGF19 as described above, preferably sarcopenia or NASH in a subject in need comprising administering a therapeutically effective amount of a composition comprising a legume protein as previously described to a subject.
[0102] Dans le contexte de la divulgation, une quantité thérapeutiquement efficace fait référence à une dose suffisante pour inverser, soulager ou inhiber la progression du trouble ou de l'état auquel ce terme s'applique, ou inverser, soulager ou inhiber la progression d'un ou plusieurs symptômes du trouble ou de l'état auquel ce terme s'applique. [0102] In the context of the disclosure, a therapeutically effective amount refers to a dose sufficient to reverse, alleviate or inhibit the progression of the disorder or condition to which this term applies, or reverse, alleviate or inhibit the progression of one or more symptoms of the disorder or condition to which this term applies.
[0103] La dose efficace est déterminée et ajustée en fonction de facteurs tels que la composition utilisée, la voie d'administration, les caractéristiques physiques de l'individu considéré telles que le sexe, l'âge et le poids, la médication concomitante, et d'autres facteurs, que les personnes compétentes dans le domaine médical reconnaîtront. The effective dose is determined and adjusted according to factors such as the composition used, the route of administration, the physical characteristics of the individual considered such as sex, age and weight, the concomitant medication, and other factors, which those skilled in the medical field will recognize.
[0104] Selon un mode de réalisation particulier de l’invention, la composition comprend en outre au moins un agoniste de FXR (acronyme pour le terme anglais « Farnesoid X receptor »). Les agonistes de FXR sont bien connus par l’homme du métier et peuvent être choisis à titre d’exemples non limitatifs parmi : obeticholic acid (OCA), Chenodeoxycholic acid (CDCA), 6a-ethyl-chenodeoxycholic acid (6- ECDCA), Alisol B 23-acetate (AB23A), Cafestol, Fexaramine, GW4064 (3-(2,6- Dichlorophenyl)-4-(3’-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole) et Tropifexor, de préférence GW4064 et Chenodeoxycholic acid (CDCA). According to a particular embodiment of the invention, the composition also comprises at least one FXR agonist (acronym for the English term “Farnesoid X receptor”). FXR agonists are well known to those skilled in the art and can be chosen by way of non-limiting examples from: obeticholic acid (OCA), Chenodeoxycholic acid (CDCA), 6a-ethyl-chenodeoxycholic acid (6-ECDCA), Alisol B 23-acetate (AB23A), Cafestol, Fexaramine, GW4064 (3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole) and Tropifexor, preferably GW4064 and Chenodeoxycholic acid (CDCA).
[0105] La composition selon la présente divulgation peut être inclus dans une composition pharmaceutique, optionnellement en combinaison avec un excipient pharmaceutiquement acceptable. [0106] La présente divulgation consiste ainsi en une composition pharmaceutique destinée à activer la synthèse du FGF19 comprenant des protéines de légumineuse, préférentiellement sélectionnée parmi le pois ou la féverole, dont le degré d’hydrolyse est inférieur à 10% tel que décrit précédemment, optionnellement en combinaison avec un excipient pharmaceutiquement acceptable. [0107] Les premiers constituants primordiaux pour la composition pharmaceutique destinée à activer la synthèse du FGF19 sont les protéines de légumineuse, préférentiellement de pois ou de féverole, dont le degré d’hydrolyse est inférieur à 10% telles que décrites dans les paragraphes précédents. En particulier, les deux modes alternatifs préférentiels utilisent respectivement une protéine de pois dont le degré d’hydrolyse est préférentiellement compris entre 5% et 10% ou une protéine de féverole dont le degré d’hydrolyse n’est pas modifié par hydrolyse après son extraction, préférentiellement avec un degré d’hydrolyse compris entre 0% et 10%, préférentiellement entre 0% et 5%. [0105] The composition according to the present disclosure may be included in a pharmaceutical composition, optionally in combination with a pharmaceutically acceptable excipient. The present disclosure thus consists of a pharmaceutical composition intended to activate the synthesis of FGF19 comprising legume proteins, preferentially selected from peas or fava beans, the degree of hydrolysis of which is less than 10% as described above, optionally in combination with a pharmaceutically acceptable excipient. The first essential constituents for the pharmaceutical composition intended to activate the synthesis of FGF19 are legume proteins, preferably pea or faba bean proteins, the degree of hydrolysis of which is less than 10% as described in the preceding paragraphs. In particular, the two preferred alternative modes respectively use a pea protein, the degree of hydrolysis of which is preferably between 5% and 10%, or a faba bean protein, the degree of hydrolysis of which is not modified by hydrolysis after its extraction, preferentially with a degree of hydrolysis comprised between 0% and 10%, preferentially between 0% and 5%.
[0108] Dans un mode préférentiel, ces protéines constituent les seules sources de protéines de la composition selon l’invention. A l’inverse des solutions bien connues dans l’art antérieur la composition n’utilise pas de combinaison avec d’autres sources protéiques par exemple des protéines de riz ou de blé. La composition dans ce mode préférentiel vise à maximiser la capacité des protéines de légumineuse afin d’activer la synthèse du FGF19. Il est possible d’ajouter des acides aminés afin d’améliorer le profil de la composition, bien que cela ne soit pas nécessaire. [0109] Le terme « composition pharmaceutique » doit se comprendre comme toute composition destinée à soigner un sujet. Des exemples non limitatifs de telles compositions pharmaceutiques sont données dans la suite de cette présente demande. In a preferential mode, these proteins constitute the only protein sources of the composition according to the invention. Unlike the solutions well known in the prior art, the composition does not use a combination with other protein sources, for example rice or wheat proteins. The composition in this preferential mode aims to maximize the ability of legume proteins to activate the synthesis of FGF19. It is possible to add amino acids to improve the profile of the composition, although this is not necessary. The term “pharmaceutical composition” should be understood as any composition intended to treat a subject. Non-limiting examples of such pharmaceutical compositions are given below in this application.
[0110] Un "excipient pharmaceutiquement acceptable" se réfère à un véhicule qui ne produit pas de réaction indésirable, allergique ou autre réaction fâcheuse lorsqu'il est administré à un mammifère, en particulier un humain, selon le cas. Un support ou excipient pharmaceutiquement acceptable désigne une charge solide, semi- solide ou liquide non toxique, un diluant, un matériau d'encapsulation ou un auxiliaire de formulation de tout type. [0111] De préférence, la composition pharmaceutique contient des véhicules, qui sont pharmaceutiquement acceptables pour une formulation susceptible d'être administré oralement. [0110] A "pharmaceutically acceptable excipient" refers to a vehicle which does not produce an adverse, allergic or other untoward reaction when administered to a mammal, particularly a human, as the case may be. A pharmaceutically acceptable carrier or excipient means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type. [0111] Preferably, the pharmaceutical composition contains carriers, which are pharmaceutically acceptable for a formulation capable of being administered orally.
La composition peut être toute composition bien connue de l’homme du métier comme par exemple (et de manière non limitative) des boissons prête à être consommées (RTD), des poudres à réhydrater (powder-mix), des compotes, des gels, des produits de panification, des produits laitiers, . The composition can be any composition well known to those skilled in the art, such as (and in a non-limiting manner) ready-to-drink drinks (RTD), powders to be rehydrated (powder-mix), compotes, gels, bread products, dairy products, .
[0112] Plus particulièrement, l’invention concerne l’application de ces formulations en boissons, par le biais de mélanges de poudres à reconstituer, pour la nutrition diététique (sport, minceur), et en boissons prêtes-à-boire pour la nutrition clinique (voie orale ou poche entérale) et diététique, où l’on recherche une faible viscosité de la boisson et une amélioration de la solubilité de la protéine de légumineuseMore particularly, the invention relates to the application of these formulations in drinks, by means of mixtures of powders to be reconstituted, for dietetic nutrition (sport, slimming), and in ready-to-drink drinks for clinical (oral route or enteral bag) and dietary, where low viscosity of the drink and improved solubility of the legume protein are desired
[0113] Également, l’invention concerne l’application de ces formulations nutritionnelles en boissons laitières ou végétales, en laits fermentés de type yaourts (brassés, à la grecque, à boire) et en crèmes laitières ou végétales, desserts glacés ou sorbets. Also, the invention relates to the application of these nutritional formulations in dairy or vegetable drinks, in fermented milks of the yoghurt type (stirred, Greek style, to drink) and in dairy or vegetable creams, frozen desserts or sorbets.
[0114] Enfin, l’invention concerne l’application de ces formulations nutritionnelles en biscuits, muffins, pancakes, barres nutritionnelles (destinés à la nutrition spécialisée / minceur ou sportif), en pains ou pains sans gluten enrichis en protéines, en petites céréales obtenues par cuisson extrusion (« crisps ») hyperprotéinés, où l’on recherche plus particulièrement des solutions hyperprotéinées sans impact négatif sur le procédé de préparation ou la texture des préparations ou produits finis. Finally, the invention relates to the application of these nutritional formulations in biscuits, muffins, pancakes, nutritional bars (intended for specialized/slimming or sports nutrition), in gluten-free breads or breads enriched with proteins, in small cereals obtained by high-protein extrusion cooking ("crisps"), where high-protein solutions are more particularly sought without negative impact on the preparation process or the texture of the preparations or finished products.
[0115] Au sens de l’invention, on entend par « formulations nutritionnelles en poudre », des formulations en poudre comprenant au moins, préférentiellement uniquement, une protéine de légumineuse, et en particulier de pois ou de féverole, selon l’invention, qui sont reconstituables avec un liquide aqueux, et qui conviennent pour l'administration orale à un être humain. [0115] Within the meaning of the invention, the term "powdered nutritional formulations" means powdered formulations comprising at least, preferably only, a legume protein, and in particular pea or faba bean protein, according to the invention, which are reconstitutable with an aqueous liquid, and which are suitable for oral administration to a human.
[0116] L'expression « mélange à sec » tel qu'utilisé ici, sauf indication contraire, se réfère au mélange des composants ou des ingrédients pour former une poudre nutritionnelle de base ou, à l'addition d'un composant sec, en poudre ou granulé ou d'un ingrédient à base poudre pour former une formulation nutritionnelle en poudre. [0116] The term "dry mix" as used herein, unless otherwise specified, refers to the mixing of the components or ingredients to form a basic nutritional powder or, to the addition of a dry component, by powder or granule or powder-based ingredient to form a powdered nutritional formulation.
[0117] Tous les pourcentages, parties et rapports, tel qu'utilisé ici, se rapportent au poids de la formulation totale, sauf indication contraire. [0117] All percentages, parts and ratios, as used herein, refer to the weight of the total formulation, unless otherwise indicated.
[0118] Les formulations orales en poudre de la présente invention sont généralement sous la forme de compositions particulaires aptes à l'écoulement ou sensiblement fluides, ou au moins des compositions particulaires qui peuvent être facilement moulées et mesurées à l'aide d'une cuillère ou d'un autre dispositif similaire, dans lequel les compositions peuvent facilement être reconstitués par l'utilisateur prévu avec une solution aqueuse, typiquement de l'eau, pour former une formulation liquide pour utilisation orale ou entérale immédiate. Dans ce contexte, l'utilisation « immédiatement » signifie généralement pendant environ 48 heures, plus typiquement pendant environ 24 heures, de préférence juste après la reconstitution. [0118] The oral powder formulations of the present invention are generally in the form of flowable or substantially fluid particulate compositions, or at least particulate compositions which can be easily molded and measured with a spoon. or other similar device, wherein the compositions can easily be reconstituted by the intended user with an aqueous solution, typically water, to form a liquid formulation for immediate oral or enteral use. In this context, "immediately" use generally means for about 48 hours, more typically for about 24 hours, preferably immediately after reconstitution.
[0119] Les formulations en poudre peuvent être formulées avec tous types et quantités de nutriments suffisants de manière à former un complément alimentaire, ou une formulation nutritionnelle spécialisée destinée à être utilisée chez les personnes suivant un régime alimentaire particulier destiné à la diététique du sport et de la minceur. [0119] The powder formulations can be formulated with any type and quantity of nutrients sufficient to form a dietary supplement, or a specialized nutritional formulation intended for use in people following a particular diet intended for sports dietetics and of thinness.
[0120] Dans des exemples de réalisations, la formulation nutritionnelle en poudre peut être formulée pour une utilisation : pour l’alimentation d’une personne en surcharge pondérale suivant un régime amaigrissant et souhaitant limiter la perte de poids musculaire, pour réparer les muscles après un effort intense, par exemple chez le sportif, pour assurer chez le sportif le maintien ou la construction de la masse musculaire, ou comme substitut de repas pour des personnes désirant perdre du poids via un effet satiétogène. [0120] In exemplary embodiments, the powdered nutritional formulation can be formulated for use: for feeding an overweight person following a slimming diet and wishing to limit muscle weight loss, to repair the muscles after an intense effort, for example in the sportsman, to ensure in the sportsman the maintenance or the construction of the muscular mass, or as a meal replacement for people wishing to lose weight via a satietogenic effect.
[0121] Les formulations en poudre peuvent avoir une densité calorique adaptée aux besoins nutritionnels de l'utilisateur final, bien que dans la plupart des cas, les poudres reconstituées comprennent d'environ 350 à environ 400 kcal / 100 ml. [0121] Powder formulations can have a caloric density to suit the nutritional needs of the end user, although in most cases reconstituted powders will comprise from about 350 to about 400 kcal/100 ml.
[0122] Les formulations en poudre peuvent présenter un taux protéique adapté aux besoins nutritionnels de l'utilisateur final, bien que dans la plupart des cas, les poudres reconstituées comprennent d'environ 20 à environ 91 g de protéines / 100 g, y compris d'environ 40 à environ 65 g de protéines / 100g. [0123] Ainsi, la formulation peut comprendre entre 20 et 95 % de protéines par rapport au poids totale de la formulation, par exemple entre 20-90%, 30-80%, ou 40-60%. [0122] Powder formulations may have a protein level tailored to the nutritional needs of the end user, although in most cases reconstituted powders comprise from about 20 to about 91 g protein/100 g, including from about 40 to about 65 g of protein / 100g. [0123] Thus, the formulation may comprise between 20 and 95% protein relative to the total weight of the formulation, for example between 20-90%, 30-80%, or 40-60%.
[0124] Par exemple, l’isolat de protéines de légumineuses, préférentiellement de pois ou de féverole, selon la présente invention peut représenter 40-50%, 50-60%, 60-70%, 70-80%, 80-90% ou 90-100% de la protéine totale de la formulation, ou une quelconque combinaison de ces gammes de pourcentages. 100% représentant le mode préférentiel ultime afin de maximiser l’effet de surexpression du FGF19 For example, the legume protein isolate, preferably pea or faba bean, according to the present invention can represent 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total protein of the formulation, or any combination of these percentage ranges. 100% representing the ultimate preferential mode to maximize the effect of FGF19 overexpression
[0125] Par ailleurs, les formulations alimentaires en poudre peuvent présenter un taux de matière grasse adapté aux besoins nutritionnels de l'utilisateur final, bien que dans la plupart des cas, les poudres reconstituées comprennent d'environ 0,5 à environ 13 g /100 g, y compris d'environ 3 à environ 7 g /100 g. Ainsi, la formulation peut comprendre entre 0 et 20 % de lipides par rapport au poids totale de la formulation, par exemple entre 0,5-15%, 1 -10%, ou 3-7% (en particulier % en poids). [0126] Les formulations nutritionnelles en poudre de la présente invention peuvent être emballées et scellées dans des conteneurs simples ou multi-usage, puis stockées dans des conditions ambiantes jusqu'à environ 36 mois ou plus, plus typiquement d'environ 12 à environ 24 mois. [0125] Furthermore, powdered food formulations can have a fat content adapted to the nutritional needs of the end user, although in most cases, reconstituted powders comprise from about 0.5 to about 13 g /100 g, including about 3 to about 7 g /100 g. Thus, the formulation may comprise between 0 and 20% of lipids relative to the total weight of the formulation, for example between 0.5-15%, 1-10%, or 3-7% (in particular % by weight). [0126] The powdered nutritional formulations of the present invention may be packaged and sealed in single or multi-use containers and then stored at ambient conditions for up to about 36 months or more, more typically about 12 to about 24 months. month.
[0127] Pour multi-utilisation des conteneurs, ils peuvent être ouverts et couverts pour une utilisation répétée par l'utilisateur final, à condition que le paquet recouvert soit ensuite stocké dans des conditions ambiantes (par exemple, éviter les températures extrêmes) et les contenus utilisés dans environ un mois ou deux. [0127] For multi-use containers, they can be opened and covered for repeated use by the end user, provided that the covered package is then stored under ambient conditions (e.g. avoiding extreme temperatures) and contents used in about a month or two.
[0128] Les domaines d’applications des formulations nutritionnelles selon l’invention sont notamment : - la nutrition diététique (sport, minceur), la nutrition clinique (sous forme de boisson, de crème dessert ou de poche entérale), les produits laitiers (sous forme de yaourts, boissons laitières, crèmes laitières, desserts glacés ou sorbets). - les produits de biscuiterie, produits de pâtisserie, produits de panification et produits céréaliers hyperprotéinés. The fields of application of the nutritional formulations according to the invention are in particular: - dietary nutrition (sport, slimming), clinical nutrition (in the form of a drink, dessert cream or enteral pouch), dairy products ( in the form of yoghurts, dairy drinks, dairy creams, frozen desserts or sorbets). - biscuit products, pastry products, bread products and high-protein cereal products.
[0129] Dans le domaine du sport, il est connu que les protéines participent à l'entretien et à la croissance du muscle. L’apport en protéines est également important pour les athlètes qui pratiquent la musculation ou le renforcement musculaire. [0129] In the field of sport, it is known that proteins participate in the maintenance and growth of muscle. Protein intake is also important for athletes who practice bodybuilding or muscle strengthening.
[0130] Les boissons protéinées ou hyperprotéinées prêtes à boire permettent alors de faire bénéficier l’organisme d'un apport protéique de choix, les calories en moins. [0131] Ces boissons hyperprotéinées doivent : être riches en protéines, pauvres en glucides et en graisses ; avoir bon goût ; être conçues pour aider à perdre du poids, en stimulant la perte de graisse et en aidant à la récupération musculaire ; - être satiétogènes ; permettre de faire face aux fringales, sans sucres ni graisses ajoutées ; présenter un contenu équilibré en acides aminés essentiels, en fibres, en vitamines et minéraux ; être hypocaloriques. [0132] Ces boissons prêtes-à-boire peuvent être avantageusement préparées avec les isolats de protéines de légumineuses, préférentiellement de féveroles ou de pois, conformément à l’invention. Elles peuvent être d’ailleurs utilisées comme seule source de protéines, de manière préférée car maximisant l’effet de surexpression du FGF19. [0133] Par exemple, les boissons végétales alternatives au lait de vache, contiennent en moyenne de 4,5 à 11 g de protéines pour 100 ml de boisson, de préférence de l’ordre de 7 g de protéines pour 100 ml, et sont très pauvres en fibres (de l’ordre de 0,5 à 1 g pour 100 ml). [0130] Ready-to-drink protein or high-protein drinks then allow the body to benefit from a choice protein intake, with fewer calories. These high-protein drinks must: be rich in protein, low in carbohydrates and in fats; to have good taste ; be designed to aid weight loss by stimulating fat loss and aiding muscle recovery; - be satietogenic; allow you to cope with cravings, without added sugars or fats; have a balanced content of essential amino acids, fibre, vitamins and minerals; be low calorie. These ready-to-drink drinks can advantageously be prepared with legume protein isolates, preferably fava beans or peas, in accordance with the invention. They can also be used as the sole source of proteins, preferably because they maximize the effect of overexpression of FGF19. For example, vegetable drinks that are alternatives to cow's milk, contain on average from 4.5 to 11 g of protein per 100 ml of drink, preferably of the order of 7 g of protein per 100 ml, and are very low in fiber (about 0.5 to 1 g per 100 ml).
[0134] Ainsi, la boisson peut comprendre entre 1 et 20 % de protéines par rapport au poids totale de la boisson, par exemple entre 3-15%, ou 6-8%. Thus, the beverage may comprise between 1 and 20% protein relative to the total weight of the beverage, for example between 3-15%, or 6-8%.
[0135] Par exemple, l’isolat de protéines de pois selon la présente invention peut représenter 50-60%, 60-70%, 70-80%, 80-90% ou 90-100% de la protéine totale, ou une quelconque combinaison de ces gammes de pourcentages. De préférence, il représente au moins 52 %. Notamment, l’apport en protéines de pois est compris entre 52 et 100 % de l’apport en protéines totales. For example, the pea protein isolate according to the present invention can represent 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total protein, or a any combination of these percentage ranges. Preferably, it represents at least 52%. In particular, the pea protein intake is between 52 and 100% of the total protein intake.
[0136] Pour les boissons prêtes à boire, l’apport en protéines de pois peut aller de 0 à 100%, de préférence de 0,01 ou 0,1 à 100 %. Par exemple, l’isolat de protéines de pois selon la présente invention peut représenter 0, 1 -10%, 10-20%, 20-30%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90% ou 90-100% de la protéine totale, ou une quelconque combinaison de ces gammes de pourcentages. For ready-to-drink beverages, the pea protein intake can range from 0 to 100%, preferably from 0.01 or 0.1 to 100%. For example, the pea protein isolate according to the present invention may be 0.1-10%, 10-20%, 20-30%, 40-50%, 50-60%, 60-70%, 70- 80%, 80-90% or 90-100% of total protein, or any combination of these percentage ranges.
[0137] Dans le domaine des boissons « minceur », i.e. destinées à être utilisées dans des régimes hypocaloriques ou destinées à la perte de poids, comme mentionné précédemment, ces boissons protéinées ou enrichies en protéines ne sont pas uniquement efficaces pour une prise rapide de muscles. Ce type de boisson est également très avantageux dans le cadre d'un régime minceur basé sur la consommation de protéines. In the field of "slimming" drinks, i.e. intended for use in low-calorie diets or intended for weight loss, as mentioned previously, these protein or protein-enriched drinks are not only effective for rapid intake of muscles. This type of drink is also very beneficial as part of a slimming diet based on protein consumption.
[0138] Il est connu que les boissons minceurs sont idéales pour aider à la perte de poids. Elles permettent plus particulièrement de : procurer un effet de satiété protéger les muscles et de tonifier le corps, évitant la reprise de poids. It is known that slimming drinks are ideal for aiding weight loss. They allow more particularly to: provide a satiety effect protect the muscles and tone the body, preventing weight regain.
[0139] Comme pour les boissons « sport », ces boissons minceurs présentent : un contenu équilibré en acides aminés essentiels, en fibres, en vitamines et minéraux un contenu réduit en sucres, en matières grasses et en calories. [0160] As with “sport” drinks, these slimming drinks have: a balanced content of essential amino acids, fibres, vitamins and minerals a reduced content of sugars, fats and calories.
[0140] C’est ainsi que les boissons protéinées sont en effet d'une grande efficacité pour perdre rapidement quelques kilos. Ces préparations riches en protéines réduisent ou stoppent simplement la sensation de faim chez la personne qui en consomme. En prenant par exemple une telle boisson, l'utilisateur peut réduire de manière considérable la quantité de nourriture à consommer, et permettre une perte de poids rapide (dans le cadre d’un processus de substitut de repas pour contrôle du poids, ou substitut de la ration journalière totale pour contrôle du poids). [0141] En nutrition clinique, il est connu que la nutrition entérale est une solution thérapeutique de nutrition par sonde qui est utilisée lorsque le tube digestif est fonctionnel et accessible mais lorsque le patient ne peut s’alimenter normalement ou encore dans les cas de dénutrition sévère. [0142] Cette technique permet d’apporter directement les nutriments dans le tube digestif. Elle remplace, de manière totale ou partielle, l’alimentation orale traditionnelle par des formules nutritives « complètes » apportant l’ensemble des nutriments nécessaires à l’organisme. [0140] This is how protein drinks are indeed highly effective for rapidly losing a few pounds. These protein-rich preparations simply reduce or stop the feeling of hunger in the person who consumes them. By taking such a drink, for example, the user can considerably reduce the amount of food to be consumed, and allow rapid weight loss (as part of a process of meal replacement for weight control, or the total daily ration for weight control). [0141] In clinical nutrition, it is known that enteral nutrition is a therapeutic nutrition solution by tube which is used when the digestive tract is functional and accessible but when the patient cannot eat normally or in cases of undernutrition. severe. This technique makes it possible to supply the nutrients directly to the digestive tract. It replaces, totally or partially, the traditional oral diet with “complete” nutritious formulas providing all the nutrients needed by the body.
[0143] Ces formules sont généralement conditionnées dans des poches souples (en PVC) et administrées au moyen de sondes naso-gastriques ou, gastrostomies, naso-jéjunale, naso-duodénale, jéjunostomie. These formulas are generally packaged in flexible pouches (made of PVC) and administered by means of naso-gastric or gastrostomy, naso-jejunal, naso-duodenal or jejunostomy tubes.
[0144] Ces mélanges nutritionnels sont composés de protéines, lipides, glucides, vitamines et minéraux avec ou sans fibres. These nutritional mixtures are composed of proteins, lipids, carbohydrates, vitamins and minerals with or without fibers.
[0145] On distingue plusieurs catégories : les mélanges polymériques (standards) et les mélanges semi-élémentaires (« prédigérés »), ces derniers étant indiqués dans des cas bien spécifiques (syndrome du grêle court, insuffisance pancréatique exocrine, etc.) : There are several categories: polymeric mixtures (standards) and semi-elementary ("predigested") mixtures, the latter being indicated in very specific cases (short bowel syndrome, exocrine pancreatic insufficiency, etc.):
* Mélanges polymériques : * Polymer blends:
- hypocaloriques (0,5 - 0,75 kcal /ml), normo ou hyperprotéinés, avec ou sans fibres - isocaloriques (1 kcal / ml), normo ou hyperprotéinés, avec ou sans fibres - hypocaloric (0.5 - 0.75 kcal / ml), normal or high protein, with or without fiber - isocaloric (1 kcal / ml), normal or high protein, with or without fiber
- hypercaloriques (1 ,25 -1 ,5 kcal / ml) normo ou hyperprotéinés, avec ou sans fibres- hypercaloric (1.25 -1.5 kcal / ml) normal or high protein, with or without fiber
* formules spécifiques (troubles du métabolisme glycémique, insuffisance respiratoire). * specific formulas (glycemic metabolism disorders, respiratory failure).
[0146] Les semi-élémentaires sont des mélanges iso ou hypercaloriques normo ou hyperprotéinés, à base de peptides et de triglycérides à chaînes moyennes. The semi-elementary mixtures are iso or high-calorie normal or high-protein mixtures, based on peptides and medium-chain triglycerides.
[0147] Les isolats de protéines de pois, comme source de protéines, par leurs propriétés fonctionnelles sont particulièrement bien adaptés à cet usage. Pea protein isolates, as a protein source, by their functional properties are particularly well suited for this use.
[0148] Par ailleurs, ils permettent de préserver les mêmes propriétés que les protéines de lait, et ce pour un coût moindre. [0149] L’invention sera mieux comprise avec les exemples suivants qui n’ont pour but que de mieux faire comprendre celle-ci. Ceux-ci n’ont aucune portée limitative. [0148] Furthermore, they make it possible to preserve the same properties as milk proteins, and this at a lower cost. The invention will be better understood with the following examples which are intended only to better understand it. These have no limiting scope.
Exemples Examples
[0150] Exemple 1 : Préparation d’une composition comportant une protéine de pois hydrolysée dont le degré d’hydrolyse (DH) est compris entre 5% et 10% Example 1: Preparation of a composition comprising a hydrolyzed pea protein whose degree of hydrolysis (DH) is between 5% and 10%
[0151] De la protéine de pois (commercialisée par la société demanderesse sous le nom de marque NUTRALYS® S85F) est mise en suspension à 15% p/p dans 8500 litres d'eau préchauffée à 55°C. Pea protein (marketed by the applicant company under the brand name NUTRALYS® S85F) is suspended at 15% w/w in 8500 liters of water preheated to 55°C.
[0152] Après agitation 3h à 55°C, on ajoute 0,5 % (poids/poids) de d'endoprotéase FLAVORPRO 750 MDP (de la société BIOCATALYST) et on laisse s’établir la réaction enzymatique durant 1 h à 55°C. On inhibe ensuite la réaction par chauffage du milieu à 70°C et on maintient à cette température pendant 10 minutes minimum. After stirring for 3 hours at 55° C., 0.5% (weight/weight) of FLAVORPRO 750 MDP endoprotease (from the company BIOCATALYST) is added and the enzymatic reaction is allowed to establish for 1 hour at 55° C. vs. The reaction is then inhibited by heating the medium to 70° C. and this temperature is maintained for a minimum of 10 minutes.
[0153] On réalise enfin un traitement UHT (barème 140°C - 10 s) et un séchage par atomisation. La poudre obtenue est caractérisée par un degré hydrolyse de 7 et une matière sèche de 95% Finally, a UHT treatment (scale 140° C. - 10 s) and spray drying are carried out. The powder obtained is characterized by a degree of hydrolysis of 7 and a dry matter of 95%.
[0154] Exemple 2 : Préparation d’une composition alimentaire comportant une protéine de féverole non hydrolysée Example 2: Preparation of a Food Composition Comprising a Non-Hydrolysed Bean Protein
[0155] Des graines de féverole de la variété Tiffany ont d’abord été traitées à l’aide d’un moulin à pierre (Alma®). Le broyât a ensuite été traité par turboséparation à l’aide d’un système dit « zig-zag » (MZM 1-40, Hosokawa-alpine®) avec une vitesse d’air de 4.0 m.s-1 (23 m3.h-1). On obtient ainsi une fraction légère contenant les fibres externes et une fraction lourde contenant les cotylédons. La fraction lourde est ensuite traitée à l’aide d’un moulin à couteaux (SM300, Retsch®) dont la rotation est de 700 tr/min et la sortie équipée d’une grille de 6 mm. La fraction lourde est ensuite broyée à l’aide d’un broyeur à rouleaux (MLU 202, Bühler®). On obtient finalement une farine dont la taille de particules est inférieure à 300 pm. [0155] Bean seeds of the Tiffany variety were first processed using a stone mill (Alma®). The ground material was then treated by turbo separation using a so-called "zig-zag" system (MZM 1-40, Hosokawa-alpine®) with an air speed of 4.0 m.s-1 (23 m3.h- 1). A light fraction containing the external fibers and a heavy fraction containing the cotyledons are thus obtained. The heavy fraction is then processed using a knife mill (SM300, Retsch®) whose rotation is 700 rpm and the outlet is equipped with a 6 mm grid. The heavy fraction is then ground using a roller mill (MLU 202, Bühler®). A flour is finally obtained whose particle size is less than 300 μm.
[0156] Cette farine est mise en suspension à 10% en poids de matière sèche dans de l’eau potable à 20°C. Le pH est ajusté à 7 par ajout de potasse. On pratique une homogénéisation pendant 15 minutes toujours à 20°C. La solution est ensuite envoyée sur un décanteur Sedicanter de la société Flottweg (Vitesse du bol : 60% (environ 3500g), Vitesse de la vis à 60% pour une Vr =18.8, Pipette pour le surnageant (overflow) à 140 mm, Alimentation à 1m3/h) et on récupère le surnageant liquide contenant les protéines. This flour is suspended at 10% by weight of dry matter in drinking water at 20°C. The pH is adjusted to 7 by adding potash. We practice a homogenization for 15 minutes still at 20°C. The solution is then sent to a Sedicanter decanter from Flottweg (Bowl speed: 60% (about 3500g), Screw speed at 60% for a Vr =18.8, Pipette for the supernatant (overflow) at 140 mm, Feeding at 1m 3 /h) and the liquid supernatant containing the proteins is recovered.
[0157] Ce surnageant est acidifié à pH 4.5 par ajout d’acide chlorhydrique à 7% massique environ. On chauffe à 60°C par injection de vapeur dans une double enveloppe de la cuve, où l’on pratique une homogénéisation pendant 15 minutes. On utilise une seconde fois le Sedicanter de Flottweg (Vitesse du bol à 60%, (environ 3500g) Vitesse de la vis à 10% pour une Vr =3.5 jusqu’à 40% (Vr = 12.6) Pipette pour l’overflow à 140 mm au départ jusqu’à 137 Alimentation à 700 l/h) mais cette fois ci pour récupérer le sédiment où se trouvent les protéines coagulées. This supernatant is acidified to pH 4.5 by adding hydrochloric acid at approximately 7% by weight. It is heated to 60°C by injecting steam into a double jacket of the tank, where it is homogenized for 15 minutes. The Flottweg Sedicanter is used a second time (Bowl speed at 60%, (about 3500g) Screw speed at 10% for a Vr =3.5 up to 40% (Vr = 12.6) Pipette for the overflow at 140 mm initially up to 137 Feeding at 700 l/h) but this time to recover the sediment where the coagulated proteins are found.
[0158] Le sédiment est dilué à environ 15-20% en poids de matière sèche et neutralisé à pH 6,5 par ajout de potasse à 20%. On pratique un traitement thermique à 135°C à l’aide d’une tuyère et on réalise un refroidissement par effet flash sous- vide à 65°C. Le produit est enfin atomisé (température d’entrée de 200°C et température des buées à 85-90°C) The sediment is diluted to approximately 15-20% by weight of dry matter and neutralized to pH 6.5 by adding 20% potassium hydroxide. Heat treatment is carried out at 135°C using a nozzle and cooling is carried out by vacuum flash effect at 65°C. The product is finally atomized (inlet temperature of 200°C and vapor temperature of 85-90°C)
[0159] Exemple 3 : Matériels et Méthodes [0159] Example 3: Materials and Methods
[0160] En plus des deux échantillons décrits dans les exemples 1 et 2 ci-dessus et dénommé respectivement RQ3 et RQ7, les protéines listées ci-dessous ont été utilisées à titre comparatif : In addition to the two samples described in examples 1 and 2 above and called RQ3 and RQ7 respectively, the proteins listed below were used for comparison:
- RQ1 : protéine de lactosérum PRODIETF90 (INGREDIA) - RQ1: whey protein PRODIETF90 (INGREDIA)
- RQ2 : Albumine d’œuf standard - RQ2: Standard egg albumin
- RQ4 : Nutralys® W (isolat de protéine de blé) - RQ5 : Nutralys® H85 (hydrolysat de protéine de pois dont le DH mesuré selon le protocole décrit dans la présente demande est égale à 22) - RQ4: Nutralys® W (wheat protein isolate) - RQ5: Nutralys® H85 (pea protein hydrolyzate whose DH measured according to the protocol described in the present application is equal to 22)
- RQ6 : PROATEIN® (concentrât de protéine d’avoine) - RQ6: PROATEIN® (oat protein concentrate)
- RQ8 : Tubermine GP (isolat de protéine de pomme de terre) - RQ8: Tubermine GP (potato protein isolate)
- RQ9 : Solulys® 095E (corn steep) - RQ10 : albumine de pois obtenue selon l’exemple 2 du brevet WO 2018/197822- RQ9: Solulus® 095 E (corn steep) - RQ10: pea albumin obtained according to example 2 of patent WO 2018/197822
- RQ11 : protéines totales de pois (albumines + globulines) obtenues selon les exemples 1 et 2 du brevet WO2019/068998, dont le degré d’hydrolyse n’a pas été modifié [0161] Les échantillons sont tout d’abord hydrolysés in vitro afin de simuler leur digestion consécutive à leur absorption par un sujet. Le protocole utilisé est le suivant : - RQ11: total pea proteins (albumins + globulins) obtained according to examples 1 and 2 of patent WO2019/068998, the degree of hydrolysis of which has not been modified [0161] The samples are first of all hydrolyzed in vitro in order to simulate their digestion following their absorption by a subject. The protocol used is as follows:
[0162] Le modèle de digestion in vitro utilisé simule la digestion des aliments en mimant trois différentes phases de la digestion: la phase orale, la phase gastrique et la phase intestinale. Afin de simuler la mastication et l’humidification par la salive, une phase d’hydratation est d’abord réalisée. 6g de protéines sont solubilisées, dans un tampon composé de chlorure de calcium (CaCI2) à une concentration finale de 2.6 mM, pendant une heure à 37°C sous agitation. La simulation de la phase gastrique se fait avec l’ajout d’une solution tampon contenant du chlorure d’hydrogène (HCl) à 157.8 mM, et du CaCI2 à 157.8 mM. Le mélange réactionnel est ajusté à un pH 2 avec une solution d’acide chlorhydrique (HCl) 4M ou d’hydroxyde de sodium (NaOH) 4M. Puis 0.6g de pepsine porcine (P7000, Sigma) sont ajoutés dans le reste du mélange réactionnel afin d’entamer la digestion des protéines. Les réactifs sont placés 2 heures sous agitation à 37°C avec des ajustements de pH réguliers au cours du temps (pH = 2 ± 0.5). La digestion par la phase intestinale se poursuit avec l’ajout d’une solution contenant 210 mL de tampon phosphate buffered saline (PBS) pH 7.4, du CaCI2 à 558 pM et du NaOH à 20.9 mM. Le pH du mélange réactionnel est ensuite ajusté à 6.8. Puis 1.2 g de pancréatine d’origine porcine (P7545, Sigma) est ajouté pour terminer l’hydrolyse des protéines en petits peptides. Le mélange est incubé 4 heures sous agitation à 37°C avec des ajustements de pH au cours du temps (pH = 6.8 ± 0.5). Les enzymes sont par la suite inactivées en plaçant le mélange 10 minutes au bain-marie à 90°C. Les digestats obtenus sont congelés puis lyophilisés. L’ensemble des protéines digérées in vitro ont ensuite subi un post-traitement décrit ci-après : - Dissolution dans une solution de PBS stérile à une concentration de 100 mg/mL Homogénéisation pendant 1 heure à 40°c sous agitation (80 tr/min)The in vitro digestion model used simulates the digestion of food by mimicking three different phases of digestion: the oral phase, the gastric phase and the intestinal phase. In order to simulate chewing and humidification by saliva, a hydration phase is first carried out. 6g of proteins are dissolved, in a buffer composed of calcium chloride (CaCl2) at a final concentration of 2.6 mM, for one hour at 37° C. with stirring. The simulation of the gastric phase is done with the addition of a buffer solution containing hydrogen chloride (HCl) at 157.8 mM, and CaCl2 at 157.8 mM. The reaction mixture is adjusted to a pH of 2 with a solution of 4M hydrochloric acid (HCl) or 4M sodium hydroxide (NaOH). Then 0.6 g of porcine pepsin (P7000, Sigma) are added to the rest of the reaction mixture in order to start the digestion of the proteins. The reagents are stirred for 2 hours at 37° C. with regular pH adjustments over time (pH=2±0.5). Digestion by the intestinal phase continues with the addition of a solution containing 210 mL of phosphate buffered saline (PBS) pH 7.4, CaCl2 at 558 pM and NaOH at 20.9 mM. The pH of the reaction mixture is then adjusted to 6.8. Then 1.2 g of pancreatin of porcine origin (P7545, Sigma) is added to complete the hydrolysis of the proteins into small peptides. The mixture is incubated for 4 hours with stirring at 37° C. with pH adjustments over time (pH=6.8±0.5). The enzymes are subsequently inactivated by placing the mixture for 10 minutes in a water bath at 90°C. The digestates obtained are frozen and then freeze-dried. All the proteins digested in vitro then underwent a post-treatment described below: - Dissolution in a sterile PBS solution at a concentration of 100 mg/mL Homogenization for 1 hour at 40°C with stirring (80 rpm)
Centrifugation 15 min à 5000g Centrifugation 15 min at 5000g
Filtration sur filtres de porosité 0,45 microns Filtration on 0.45 micron porosity filters
[0163] Des aliquots baptisés digestats protéiques (ou DP dans la suite de cette demande) d’1 ml_ sont préparés et stockés à -20°c [0163] Aliquots called protein digestates (or DP in the remainder of this application) of 1 ml_ are prepared and stored at -20°C
[0164] On utilisera également les réactifs suivants dissous dans du DMSO Hybri- MaxTM filtré stérile: The following reagents dissolved in sterile filtered Hybri-MaxTM DMSO will also be used:
FXR receptor agonist GW4064, chenodeoxycholic acid (CDCA), - dexamethasone (glucocorticoid receptor agonist) FXR receptor agonist GW4064, chenodeoxycholic acid (CDCA), - dexamethasone (glucocorticoid receptor agonist)
[0165] Pour les cultures cellulaires, on utilisera les lignées HT29 (ECACC 91072201), HT29-MTX and Caco-2 dérivées d’un adenocarcinoma humain colorectal, classiquement utilisés comme lignées cellulaires représentatives d'entérocytes humains. Les cellules sont systématiquement cultivées dans le milieu Eagle modifié de Dulbecco (DMEM) 4,5 g/L de glucose additionné de 1 % de pyruvate de sodium, de 1 % de pénicilline-streptomycine et de 10 % de sérum fœtal-bovin (FBS) décomposé (Eurobio, Les Ulis, France). Pour les expériences sur plaques de 12 et 96 puits, les cellules HT29 ont été ensemencées à une densité de 7,5 x 104 cellules/cm2 avec un milieu changé tous les 2 jours et les cellules ont été exposées aux différentes conditions expérimentales 2 jours après la confluence avec un milieu complet épuisé dans FBS. Pour les plaques 12 puits (Costar®, Cambridge, MA), les cellules Caco-2 et HT29-MTX ont été ensemencées dans un rapport de 1:1 à une densité de 2,5 x 104 cellules/cm2. Les cellules ont été cultivées pendant 14 jours en milieu complet pour permettre la différenciation. Le milieu a été changé dans les deux compartiments tous les deux jours jusqu'à ce que l'exposition au milieu complet soit épuisée dans le FBS. L'intégrité de la monocouche cellulaire a été évaluée en mesurant la résistance électrique transépithéliale (TEER) à l'aide d'un Millicell-ERS (Millipore Corp., Billerica, MA). L'approvisionnement en culture cellulaire provenait de Dutscher (Issy-les-Moulineaux, France), sauf indication contraire. For the cell cultures, the HT29 (ECACC 91072201), HT29-MTX and Caco-2 lines derived from a human colorectal adenocarcinoma, conventionally used as cell lines representative of human enterocytes, will be used. The cells are systematically cultured in Dulbecco's modified Eagle medium (DMEM) 4.5 g/L of glucose supplemented with 1% sodium pyruvate, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). ) decomposed (Eurobio, Les Ulis, France). For the 12- and 96-well plate experiments, the HT29 cells were seeded at a density of 7.5 x 10 4 cells/cm 2 with medium changed every 2 days and the cells were exposed to the different experimental conditions 2 days after confluence with exhausted complete medium in FBS. For the 12-well plates (Costar®, Cambridge, MA), the Caco-2 and HT29-MTX cells were seeded in a ratio of 1:1 at a density of 2.5×10 4 cells/cm 2 . The cells were cultured for 14 days in complete medium to allow differentiation. Medium was changed in both compartments every other day until exposure to complete medium was depleted in the FBS. Cell monolayer integrity was assessed by measuring transepithelial electrical resistance (TEER) using a Millicell-ERS (Millipore Corp., Billerica, MA). Crop supply cell was from Dutscher (Issy-les-Moulineaux, France), unless otherwise indicated.
[0166] Le protocole d’exposition de ces cultures cellulaires aux différents échantillons et agonistes est le suivant : les cellules ont été traitées pendant 6 h avec (i) soit de l'DP à 1 mg/mL (cette concentration n'a montré aucun effet toxique sur les cellules HT29) ou des agonistes des récepteurs nucléaires, et (ii) la combinaison du DP et des agonistes des récepteurs nucléaires. Les différentes conditions du véhicule comme DP ont été dissous dans du PBS et les agonistes dans du DMSO. Après l'exposition, les surnageants cellulaires ont été stockés à -80 °C pour les mesures de la sécrétion de protéines FGF19, et les monocouches cellulaires ont été maintenues à -80 °C jusqu'à l'extraction totale de l'ARN. The protocol for exposing these cell cultures to the various samples and agonists is as follows: the cells were treated for 6 hours with (i) either DP at 1 mg/mL (this concentration did not show no toxic effect on HT29 cells) or nuclear receptor agonists, and (ii) the combination of DP and nuclear receptor agonists. The different vehicle conditions like DP were dissolved in PBS and the agonists in DMSO. After exposure, cell supernatants were stored at -80°C for measurements of FGF19 protein secretion, and cell monolayers were maintained at -80°C until complete RNA extraction.
[0167] On mesure l’effet de cette exposition en quantifiant l’ARN messager du FGF19 à l’aide du protocole suivant : L’expression des gènes cibles a été évaluée par la quantification de leur niveau d’ARNm dans les cellules par RT-qPCR. Les ARN totaux ont été isolés avec la solution TRI-Reagent (Sigma Aldrich). Des ADN complémentaires de premier rang ont été synthétisés à partir de 1 pg d’ARN totaux avec le kit de réactifs TAKARA Prime ScriptMC RT (TAKARA Bio, Saint-Germain- en-Laye, France). Les essais qPCR ont été effectués à l’aide du kit SYBR® Premix Ex-Taq™ (TAKARA Bio) sur Rotor-GeneTM 6000 (Corbett Research, Mortlake, Australie). . Les niveaux d’ARNm de la protéine de liaison TATA humaine (TBP) ont été utilisés pour normaliser les données. The effect of this exposure is measured by quantifying the FGF19 messenger RNA using the following protocol: The expression of the target genes was evaluated by quantifying their mRNA level in the cells by RT -qPCR. Total RNAs were isolated with TRI-Reagent solution (Sigma Aldrich). First-order cDNAs were synthesized from 1 pg of total RNA with the TAKARA Prime Script™ RT Reagent Kit (TAKARA Bio, Saint-Germain-en-Laye, France). qPCR assays were performed using the SYBR® Premix Ex-Taq™ Kit (TAKARA Bio) on Rotor-GeneTM 6000 (Corbett Research, Mortlake, Australia). . Human TATA-binding protein (TBP) mRNA levels were used to normalize the data.
[0168] [0168]
[0169] Exemple 4 : Evaluation de la viabilité cellulaire Example 4: Evaluation of cell viability
[0170] La toxicité des DP, préalablement préchauffés pendant 10 min à 90 °C, a été évaluée sur les cellules HT29 dans des plaques de 96 puits après 6 heures d'exposition, à 1 et 2 mg/mL, afin de définir la concentration la plus élevée qui n'entraîne ni dégradation ni lyse des cellules. Dans de telles conditions, aucun DP n'a montré d'effet toxique (tel qu'évalué par la libération de la LDH) à 1 ou 2 mg/mL sauf le DP n°3 (RQ3) qui a induit l'apparition de cellules de forme ronde prédisant le détachement cellulaire à 2 mg/mL. Il a donc été décidé de choisir la dose de 1 mg/mL pour poursuivre les tests sur l'expression FGF19. The toxicity of the DPs, previously preheated for 10 min at 90° C., was evaluated on the HT29 cells in 96-well plates after 6 hours of exposure, at 1 and 2 mg/mL, in order to define the highest concentration that does not cause cell degradation or lysis. Under such conditions, no DP showed any toxic effect (as assessed by LDH release) at 1 or 2 mg/mL except DP #3 (RQ3) which induced the appearance of round-shaped cells predicting cell detachment at 2 mg/mL. It was therefore decided to choose the dose of 1 mg/mL to continue the tests on FGF19 expression.
[0171] Exemple 5 : Effets des échantillons sur l’expression du FGF19 Example 5: Effects of the samples on the expression of FGF19
[0172] La [fig. 1] résume les résultats obtenus. Les cellules HT29 ont été exposées aux différents DP seuls ou en combinaison avec un agoniste FXR pour déterminer leur capacité potentielle à augmenter l’expression du gène FGF19. En tant qu’agoniste FXR, GW4064 aux doses de 0,1 et 1 mM, a augmenté l’expression du gène FGF19 de façon dose-dépendante de 2 et 6,2 fois, respectivement, par comparaison à la condition contrôle (véhicule=DMSO) L’exposition au DP seul n’a révélé aucun changement dans l’expression FGF19 par comparaison à la condition contrôle (véhicule=PBS). Cependant, le DP co-incubé avec GW4064 à 0,1 mM a potentialisé l’expression du FGF19 induite par l’agoniste FXR, avec une augmentation de 2,5 (RQ1) à 4 fois (RQ3 et RQ7) par rapport à leur condition contrôle (PBS/DMSO) et une augmentation de 1 ,3 à 2 fois par rapport à la condition 0,1 pM GW4064. [0172] The [fig. 1] summarizes the results obtained. HT29 cells were exposed to the different DPs alone or in combination with an FXR agonist to determine their potential ability to increase FGF19 gene expression. As an FXR agonist, GW4064 at doses of 0.1 and 1 mM increased FGF19 gene expression in a dose-dependent manner by 2 and 6.2 times, respectively, compared to the control condition (vehicle= DMSO) Exposure to DP alone revealed no change in FGF19 expression compared to the control condition (vehicle=PBS). However, DP co-incubated with 0.1 mM GW4064 potentiated FXR agonist-induced FGF19 expression, with a 2.5 (RQ1) to 4-fold (RQ3 and RQ7) increase over their control condition (PBS/DMSO) and an increase of 1.3 to 2 times compared to the 0.1 pM GW4064 condition.
[0173] On peut voir que RQ3 et RQ7 possède la capacité maximale de surexpression du gène codant pour le FGF19 par rapport aux autres protéines et en particulier par rapport aux protéines issues du monde animal telle que RQ1 et RQ2. [0174] On peut également noter que : It can be seen that RQ3 and RQ7 has the maximum capacity for overexpression of the gene coding for FGF19 compared to other proteins and in particular compared to proteins from the animal world such as RQ1 and RQ2. [0174] It can also be noted that:
- RQ3 est plus efficace que RQ5, qui est également un isolat de protéine de pois mais avec un degré d’hydrolyse de 22 donc supérieur à 10. - RQ3 is more effective than RQ5, which is also a pea protein isolate but with a degree of hydrolysis of 22, therefore greater than 10.
- RQ3 est également plus efficace que RQ 11 (protéine de pois totales non hydrolysées) Ces résultats démontrent bien que pour l’isolat de protéine de pois selon l’invention, la sélection d’un degré d’hydrolyse compris entre 5% et 10% est donc importante afin de maximiser l’effet - RQ3 is also more effective than RQ 11 (unhydrolyzed total pea protein) These results clearly demonstrate that for the pea protein isolate according to the invention, the selection of a degree of hydrolysis of between 5% and 10 % is therefore important in order to maximize the effect
[0175] Exemple 6 : Confirmation de l’effet [0176] La capacité des digestats RQ3 et RQ7 à augmenter le niveau d'expression de l'ARNm FGF19 ainsi que la production et la sécrétion de protéines a ensuite été évalué . Les cellules HT29 ont été exposées à GW4064 à 0,1, 0,5 et 1 mM et à un agoniste FXR naturel, l'acide chenodeoxycholique (CDCA) à 50 pM seul ou en combinaison avec les extraits RQ3 ou RQ7. Confirmant la première série d'expériences, GW4064 de 0,1 à 1 pM a augmenté l'expression du gène FGF19 de façon dose-dépendante de 2,3 à 6,6 fois, respectivement, par comparaison à la condition contrôle (véhicule=DMSO) [Fig. 2a], L’exposition au CDCA à 50 pM a montré une augmentation similaire de l’expression du gène GW4064 à 0,1 pM (le GW4064 étant donc plus puissant que le CDCA, en accord avec les données de la littérature). Les RQ3 et RQ7 co-incubés avec le CDCA à 50 pM ont tous les deux montré une augmentation de 5,3 fois par rapport à la condition contrôle (PBS/DMSO). Le digestat RQ3 co-incubé avec GW4064 à 0,1 , 0,5 et 1 pM a induit une augmentation de 4,5 ; 7 et 8,5 fois de l’expression du gène FGF19, respectivement. Le digestat RQ7 a induit une augmentation de 5,6 ; 7 et 7,3 fois, respectivement. Les deux digestats RQ3 et RQ7 co-incubés avec des agonistes FXR potentialisent l’effet observé avec l’agoniste seul sur l’expression de FGF19, avec un effet plus important pour les faibles doses d’agonistes. [0175] Example 6: Confirmation of the effect The capacity of the RQ3 and RQ7 digestates to increase the level of expression of the FGF19 mRNA as well as the production and the secretion of proteins was then evaluated. HT29 cells were exposed to GW4064 at 0.1, 0.5, and 1 mM and a natural FXR agonist, chenodeoxycholic acid (CDCA) at 50 µM alone or in combination with RQ3 or RQ7 extracts. Confirming the first series of experiments, GW4064 from 0.1 to 1 pM increased the expression of the FGF19 gene in a dose-dependent manner by 2.3 to 6.6 times, respectively, compared to the control condition (vehicle= DMSO) [Fig. 2a], Exposure to CDCA at 50 pM showed a similar increase in GW4064 gene expression at 0.1 pM (with GW4064 therefore being more potent than CDCA, in agreement with literature data). RQ3 and RQ7 co-incubated with 50 pM CDCA both showed a 5.3-fold increase over the control condition (PBS/DMSO). RQ3 digest co-incubated with GW4064 at 0.1, 0.5 and 1 pM induced an increase of 4.5; 7 and 8.5 times of FGF19 gene expression, respectively. The RQ7 digestate induced an increase of 5.6; 7 and 7.3 times, respectively. The two digests RQ3 and RQ7 co-incubated with FXR agonists potentiate the effect observed with the agonist alone on the expression of FGF19, with a greater effect for low doses of agonists.
[0177] Le niveau d’expression de la protéine est semblable à celui de l’expression du transcrit, bien que la co-incubation de RQ3 et RQ7 avec des agonistes FXR ait montré moins de potentialisation de l’expression de la protéine FGF19 que l’ARNm. Le digestat RQ3 co-incubé avec CDCA à 50 pM et GW4064 à 0,1 et 1 pM a provoqué une augmentation respective du niveau de protéines FGF19 de 3,1, 3,3 et 4,8 fois par rapport à la condition contrôle (véhicule) ([Fig. 2b], gauche). Le digestat RQ7 a quant à lui induit une augmentation de 2,5, 3 et 4,2 fois, respectivement. Comme on l’a observé au niveau de l’expression des gènes, une plus grande potentialisation de la synthèse de protéines FGF19 se produit à la dose la plus faible d’agonistes, avec presque aucune potentialisation induite par le DP à 1 pM de GW4064 par rapport à l’agoniste seul, ce qui suggère que l’effet maximal a été atteint. [0178] Les mêmes tendances ont été observées lorsque les cellules ont été incubées pendant 24 h avec les DP ([Fig. 2b], droite). RQ3 et RQ7 ont montré une potentialisation de la production de protéines FGF19, avec un effet maximal avec le GW4064 à 0,1 mM. [0179] Exemple 7 : Effet sur une co-culture The level of protein expression is similar to that of transcript expression, although co-incubation of RQ3 and RQ7 with FXR agonists showed less potentiation of FGF19 protein expression than mRNA. RQ3 digest co-incubated with CDCA at 50 pM and GW4064 at 0.1 and 1 pM caused an increase in the level of FGF19 proteins of 3.1, 3.3 and 4.8 times respectively compared to the control condition ( vehicle) ([Fig. 2b], left). The RQ7 digestate induced an increase of 2.5, 3 and 4.2 times, respectively. As observed at the gene expression level, greater potentiation of FGF19 protein synthesis occurs at the lowest dose of agonists, with almost no DP-induced potentiation at 1 µM GW4064 compared to agonist alone, suggesting that the maximum effect has been achieved. The same trends were observed when the cells were incubated for 24 h with the DPs ([Fig. 2b], right). RQ3 and RQ7 showed potentiation of FGF19 protein production, with maximum effect with GW4064 at 0.1 mM. [0179] Example 7: Effect on a co-culture
[0180] La capacité de RQ3 et RQ7 à induire l’expression de FGF19 a été évaluée dans un modèle de co-culture en insert pour se rapprocher des conditions physiologiques intestinales avec des échanges apicaux et basolatéraux. Les cellules Caco-2 permettent l’établissement de jonctions intercellulaires fournissant une barrière sélectivement perméable et les cellules HT29-MTX ont la capacité de produire du mucus au sommet de la monocouche. Ce type de co-culture des deux lignées cellulaires fournit donc un modèle constituant les deux types cellulaires les plus représentés dans un épithélium normal, des entérocytes et des cellules caliciformes. RQ3 et RQ7 ont montré une potentialisation de l’expression FGF19 lorsqu’ils ont été co-incubés avec GW4064 à 0,2 mM [Fig. 3], bien que cet effet soit moins puissant que celui observé précédemment dans des expériences sur plaques contenant uniquement des cellules HT29. The capacity of RQ3 and RQ7 to induce the expression of FGF19 was evaluated in an insert co-culture model to approximate intestinal physiological conditions with apical and basolateral exchanges. Caco-2 cells allow establishment of intercellular junctions providing a selectively permeable barrier and HT29-MTX cells have the ability to produce mucus at the top of the monolayer. This type of co-culture of the two cell lines therefore provides a model constituting the two most represented cell types in a normal epithelium, enterocytes and goblet cells. RQ3 and RQ7 showed potentiation of FGF19 expression when co-incubated with GW4064 at 0.2 mM [Fig. 3], although this effect is less potent than previously observed in plaque experiments containing only HT29 cells.
SEQUENCE LISTING SEQUENCE LISTING
<110> ROQUETTE FRERES <110> ROQUETTE BROTHERS
<120> METHODE D'ACTIVATION DE LA SYNTHESE DU FGF19 <130> B21101298 <120> FGF19 SYNTHESIS ACTIVATION METHOD <130> B21101298
<160> 10 <160> 10
<170> Patentln version 3.5 <170> Patentln version 3.5
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<211> 216 <211> 216
<212> PRT <212> PRT
<213> homo sapiens <213> homo sapiens
<400> 1 <400> 1
Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15
Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30
His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45 His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45
Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala 50 55 60 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala 50 55 60
Asp Gly Val Val Asp Cys Ala Arg Gly Gin Ser Ala His Ser Leu Leu 65 70 75 80 Asp Gly Val Val Asp Cys Ala Arg Gly Gin Ser Ala His Ser Leu Leu 65 70 75 80
Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His 85 90 95 Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His 85 90 95
Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gin Gly Leu 100 105 110 Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gin Gly Leu 100 105 110
Leu Gin Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 Leu Gin Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125
Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140
Leu Ser Ser Ala Lys Gin Arg Gin Leu Tyr Lys Asn Arg Gly Phe Leu Leu Ser Ser Ala Lys Gin Arg Gin Leu Tyr Lys Asn Arg Gly Phe Leu
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) 145 150 155 160 SHEET INCORPORATED BY REFERENCE (RULE 20.6) 145 150 155 160
Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro 165 170 175 Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro 165 170 175
Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu 180 185 190 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu 180 185 190
Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala 195 200 205 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala 195 200 205
Val Arg Ser Pro Ser Phe Glu Lys 210 215 Val Arg Ser Pro Ser Phe Glu Lys 210 215
<210> 2 <211> 220 <212> PRT <213> Sus scrofa <210> 2 <211> 220 <212> PRT <213> Sus scrofa
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Ala Met Arg Ser Ala Pro Ser Arg Cys Ala Val Val Arg Ala Leu Val 1 5 10 15 Ala Met Arg Ser Ala Pro Ser Arg Cys Ala Val Val Arg Ala Leu Val 1 5 10 15
Leu Ala Gly Leu Trp Leu Ala Ala Ala Gly Arg Pro Leu Ala Phe Ser 20 25 30 Leu Ala Gly Leu Trp Leu Ala Ala Ala Gly Arg Pro Leu Ala Phe Ser 20 25 30
Asp Ala Gly Pro His Val His Tyr Gly Trp Gly Glu Ser Val Arg Leu 35 40 45 Asp Ala Gly Pro His Val His Tyr Gly Trp Gly Glu Ser Val Arg Leu 35 40 45
Arg His Leu Tyr Thr Ala Ser Pro His Gly Val Ser Ser Cys Phe Leu 50 55 60 Arg His Leu Tyr Thr Ala Ser Pro His Gly Val Ser Ser Cys Phe Leu 50 55 60
Arg Ile His Ser Asp Gly Pro Val Asp Cys Ala Pro Gly Gin Ser Ala 65 70 75 80 Arg Ile His Ser Asp Gly Pro Val Asp Cys Ala Pro Gly Gin Ser Ala 65 70 75 80
His Ser Leu Met Glu Ile Arg Ala Val Ala Leu Ser Thr Val Ala Ile 85 90 95 His Ser Leu Met Glu Ile Arg Ala Val Ala Leu Ser Thr Val Ala Ile 85 90 95
Lys Gly Glu Arg Ser Gly Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys 100 105 110 Lys Gly Glu Arg Ser Gly Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys 100 105 110
Met Gin Gly Gin Thr Gin Tyr Ser Asp Glu Asp Cys Ala Phe Glu Glu 115 120 125 Met Gin Gly Gin Thr Gin Tyr Ser Asp Glu Asp Cys Ala Phe Glu Glu 115 120 125
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Trp Ser Lys Lys His His 130 135 140 SHEET INCORPORATED BY REFERENCE (RULE 20.6) Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Trp Ser Lys Lys His His 130 135 140
Leu Pro Val Ser Leu Ser Ser Ala Arg Gin Arg Gin Leu Tyr Lys Gly 145 150 155 160 Leu Pro Val Ser Leu Ser Ser Ala Arg Gin Arg Gin Leu Tyr Lys Gly 145 150 155 160
Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Ser Thr Leu 165 170 175 Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Ser Thr Leu 165 170 175
Pro Ala Glu Pro Glu Asp Leu Gin Asp Pro Phe Lys Ser Asp Leu Phe 180 185 190 Pro Ala Glu Pro Glu Asp Leu Gin Asp Pro Phe Lys Ser Asp Leu Phe 180 185 190
Ser Leu Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Arg Ile Ala Ala 195 200 205 Ser Leu Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Arg Ile Ala Ala 195 200 205
Lys Leu Gly Ala Val Lys Ser Pro Ser Phe Tyr Lys 210 215 220 Lys Leu Gly Ala Val Lys Ser Pro Ser Phe Tyr Lys 210 215 220
<210> 3 <210> 3
<211> 218 <212> PRT <213> Bos taurus <211> 218 <212> PRT <213> Bos taurus
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Met Arg Ser Ala Pro Ser Arg Cys Ala Val Ala Arg Ala Leu Val Leu 1 5 10 15 Met Arg Ser Ala Pro Ser Arg Cys Ala Val Ala Arg Ala Leu Val Leu 1 5 10 15
Ala Gly Leu Trp Leu Ala Ala Ala Gly Arg Pro Leu Ala Phe Ser Asp 20 25 30 Ala Gly Leu Trp Leu Ala Ala Ala Gly Arg Pro Leu Ala Phe Ser Asp 20 25 30
Ala Gly Pro His Val His Tyr Gly Trp Gly Glu Ser Val Arg Leu Arg 35 40 45 Ala Gly Pro His Val His Tyr Gly Trp Gly Glu Ser Val Arg Leu Arg 35 40 45
His Leu Tyr Thr Ala Gly Pro Gin Gly Leu Tyr Ser Cys Phe Leu Arg 50 55 60 His Leu Tyr Thr Ala Gly Pro Gin Gly Leu Tyr Ser Cys Phe Leu Arg 50 55 60
Ile His Ser Asp Gly Ala Val Asp Cys Ala Gin Val Gin Ser Ala His 65 70 75 80 Ile His Ser Asp Gly Ala Val Asp Cys Ala Gin Val Gin Ser Ala His 65 70 75 80
Ser Leu Met Glu Ile Arg Ala Val Ala Leu Ser Thr Val Ala Ile Lys 85 90 95 Ser Leu Met Glu Ile Arg Ala Val Ala Leu Ser Thr Val Ala Ile Lys 85 90 95
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) Gly Glu Ang Sen Val Leu Tyn Leu Cys Met Asp Ala Asp Gly Lys Met 100 105 110 SHEET INCORPORATED BY REFERENCE (RULE 20.6) Gly Glu Ang Sen Val Leu Tyn Leu Cys Met Asp Ala Asp Gly Lys Met 100 105 110
Gin Gly Leu Thr Gin Tyn Ser Ala Glu Asp Cys Ala Phe Glu Glu Glu 115 120 125 Gin Gly Leu Thr Gin Tyn Ser Ala Glu Asp Cys Ala Phe Glu Glu Glu 115 120 125
Ile Arg Pro Asp Gly Tyr Asn Val Tyr Trp Ser Arg Lys His His Leu 130 135 140 Ile Arg Pro Asp Gly Tyr Asn Val Tyr Trp Ser Arg Lys His His Leu 130 135 140
Pro Val Ser Leu Ser Ser Ser Arg Gin Arg Gin Leu Phe Lys Ser Arg 145 150 155 160 Pro Val Ser Leu Ser Ser Ser Arg Gin Arg Gin Leu Phe Lys Ser Arg 145 150 155 160
Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Ser Thr Ile Pro 165 170 175 Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Ser Thr Ile Pro 165 170 175
Ala Glu Pro Glu Asp Leu Gin Glu Pro Leu Lys Pro Asp Phe Phe Leu 180 185 190 Ala Glu Pro Glu Asp Leu Gin Glu Pro Leu Lys Pro Asp Phe Phe Leu 180 185 190
Pro Leu Lys Thr Asp Ser Met Asp Pro Phe Gly Leu Ala Thr Lys Leu 195 200 205 Pro Leu Lys Thr Asp Ser Met Asp Pro Phe Gly Leu Ala Thr Lys Leu 195 200 205
Gly Ser Val Lys Ser Pro Ser Phe Tyr Asn 210 215 Gly Ser Val Lys Ser Pro Ser Phe Tyr Asn 210 215
<210> 4 <210> 4
<211> 94 <211> 94
<212> PRT <212> PRT
<213> Equus Caballus <400> 4 <213> Equus Caballus <400> 4
Ala Ala Gly Arg Pro Leu Ala Leu Ser Asp Ala Gly Pro His Val His 1 5 10 15 Ala Ala Gly Arg Pro Leu Ala Leu Ser Asp Ala Gly Pro His Val His 1 5 10 15
Tyr Gly Trp Gly Glu Pro Ile Arg Leu Arg His Leu Tyr Thr Ala Gly 20 25 30 Tyr Gly Trp Gly Glu Pro Ile Arg Leu Arg His Leu Tyr Thr Ala Gly 20 25 30
Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Ala 35 40 45 Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Ala 35 40 45
Val Asp Cys Ala Arg Gly Gin Ser Ala His Ser Leu Val Glu Ile Arg 50 55 60 Val Asp Cys Ala Arg Gly Gin Ser Ala His Ser Leu Val Glu Ile Arg 50 55 60
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) Ala Val Ala Leu Ang Thn Val Ala Ile Lys Gly Val His Sen Val Ang 65 70 75 80 SHEET INCORPORATED BY REFERENCE (RULE 20.6) Ala Val Ala Leu Ang Thn Val Ala Ile Lys Gly Val His Sen Val Ang 65 70 75 80
Tyn Leu Cys Met Gly Ala Asp Gly Ang Met Gin Gly Leu Val 85 90 Tyn Leu Cys Met Gly Ala Asp Gly Ang Met Gin Gly Leu Val 85 90
<210> 5 <210> 5
<211> 137 <211> 137
<212> PRT <213> Ovis anies <212> PRT <213> Ovis anies
<400> 5 <400> 5
Leu Met Glu Ile Ang Ala Val Ala Leu Ser Thr Val Ala Ile Lys Gly 1 5 10 15 Leu Met Glu Ile Ang Ala Val Ala Leu Ser Thr Val Ala Ile Lys Gly 1 5 10 15
Glu Arg Ser Val Leu Phe Leu Cys Met Asp Ala Asp Gly Lys Met Gin 20 25 30 Glu Arg Ser Val Leu Phe Leu Cys Met Asp Ala Asp Gly Lys Met Gin 20 25 30
Gly Leu Thr Gin Tyr Ser Ala Glu Asp Cys Ala Phe Glu Glu Glu Ile 35 40 45 Gly Leu Thr Gin Tyr Ser Ala Glu Asp Cys Ala Phe Glu Glu Glu Ile 35 40 45
Arg Pro Asp Gly Tyr Asn Val Tyr Trp Ser Arg Lys His His Leu Pro 50 55 60 Arg Pro Asp Gly Tyr Asn Val Tyr Trp Ser Arg Lys His His Leu Pro 50 55 60
Val Ser Leu Ser Ser Ser Arg Gin Arg Gin Leu Phe Lys Ser Arg Gly 65 70 75 80 Val Ser Leu Ser Ser Ser Arg Gin Arg Gin Leu Phe Lys Ser Arg Gly 65 70 75 80
Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Ser Thr Ile Pro Ala 85 90 95 Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Ser Thr Ile Pro Ala 85 90 95
Glu Pro Glu Asp Leu Gin Glu Pro Leu Lys Pro Asp Phe Phe Leu Pro 100 105 110 Glu Pro Glu Asp Leu Gin Glu Pro Leu Lys Pro Asp Phe Phe Leu Pro 100 105 110
Leu Lys Thr Asp Ser Met Asp Pro Phe Gly Leu Ala Thr Lys Leu Gly 115 120 125 Leu Lys Thr Asp Ser Met Asp Pro Phe Gly Leu Ala Thr Lys Leu Gly 115 120 125
Ser Val Lys Ser Pro Ser Phe Tyr Thr 130 135 Ser Val Lys Ser Pro Ser Phe Tyr Thr 130 135
<210> 6 <211> 193 <210> 6 <211> 193
<212> PRT <212> PRT
<213> Canis familiairis <213> Canis familiaris
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) <400> 6 SHEET INCORPORATED BY REFERENCE (RULE 20.6) <400> 6
Pno Leu Ala Phe Sen Asp Ala Gly Pno His Val His Tyn Gly Tnp Gly 1 5 10 15 Pno Leu Ala Phe Sen Asp Ala Gly Pno His Val His Tyn Gly Tnp Gly 1 5 10 15
Glu Pno Ile Ang Leu Ang His Leu Tyn Thn Ala Gly Pro His Gly Leu 20 25 30 Glu Pno Ile Ang Leu Ang His Leu Tyn Thn Ala Gly Pro His Gly Leu 20 25 30
Sen Sen Cys Phe Leu Ang Ile Ang Ala Asp Gly Gly Val Asp Cys Ala 35 40 45 Sen Sen Cys Phe Leu Ang Island Ang Ala Asp Gly Gly Val Asp Cys Ala 35 40 45
Arg Gly Gin Ser Ala His Ser Leu Met Glu Met Arg Ala Val Ala Leu 50 55 60 Arg Gly Gin Ser Ala His Ser Leu Met Glu Met Arg Ala Val Ala Leu 50 55 60
Arg Thr Val Ala Ile Lys Gly Val His Ser Gly Arg Tyr Leu Cys Met 65 70 75 80 Arg Thr Val Ala Ile Lys Gly Val His Ser Gly Arg Tyr Leu Cys Met 65 70 75 80
Gly Ala Asp Gly Arg Met Gin Gly Leu Pro Gin Tyr Ser Ala Gly Asp 85 90 95 Gly Ala Asp Gly Arg Met Gin Gly Leu Pro Gin Tyr Ser Ala Gly Asp 85 90 95
Cys Thr Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Trp 100 105 110 Cys Thr Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Trp 100 105 110
Ser Lys Lys His His Leu Pro Ile Ser Leu Ser Ser Ala Lys Gin Arg 115 120 125 Ser Lys Lys His His Leu Pro Ile Ser Leu Ser Ser Ala Lys Gin Arg 115 120 125
Gin Leu Tyr Lys Gly Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro 130 135 140 Gin Leu Tyr Lys Gly Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro 130 135 140
Ile Leu Pro Gly Ser Pro Thr Glu Pro Arg Asp Leu Glu Asp His Val 145 150 155 160 Ile Leu Pro Gly Ser Pro Thr Glu Pro Arg Asp Leu Glu Asp His Val 145 150 155 160
Glu Ser Asp Gly Phe Ser Ala Ser Leu Glu Thr Asp Ser Met Asp Pro 165 170 175 Glu Ser Asp Gly Phe Ser Ala Ser Leu Glu Thr Asp Ser Met Asp Pro 165 170 175
Phe Gly Ile Ala Thr Lys Ile Gly Leu Val Lys Ser Pro Ser Phe Gin 180 185 190 Phe Gly Ile Ala Thr Lys Ile Gly Leu Val Lys Ser Pro Ser Phe Gin 180 185 190
Lys lily
<210> 7 <210> 7
<211> 219 <211> 219
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) <212> PRT <213> Felis catus SHEET INCORPORATED BY REFERENCE (RULE 20.6) <212> PRT <213> Felis catus
<400> 7 <400> 7
Met Ang Sen Ala Pno Sen Gin Cys Ala Val Thn Ang Ala Leu Val Leu 1 5 10 15 Met Ang Sen Ala Pno Sen Gin Cys Ala Val Thn Ang Ala Leu Val Leu 1 5 10 15
Ala Gly Leu Tnp Leu Ala Ala Ala Gly Ang Pno Leu Ala Phe Sen Asp 20 25 30 Ala Gly Leu Tnp Leu Ala Ala Ala Gly Ang Pno Leu Ala Phe Sen Asp 20 25 30
Ala Gly Pno His Val His Tyn Gly Tnp Gly Glu Pno Ile Ang Leu Arg 35 40 45 Ala Gly Pno His Val His Tyn Gly Tnp Gly Glu Pno Ile Ang Leu Arg 35 40 45
His Leu Tyr Thr Ala Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg 50 55 60 His Leu Tyr Thr Ala Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg 50 55 60
Ile Arg Ala Asp Gly Gly Val Asp Cys Ala Arg Ser Gin Ser Ala His 65 70 75 80 Island Arg Ala Asp Gly Gly Val Asp Cys Ala Arg Ser Gin Ser Ala His 65 70 75 80
Ser Leu Val Glu Ile Arg Ala Val Ala Leu Arg Thr Val Ala Ile Lys 85 90 95 Ser Leu Val Glu Ile Arg Ala Val Ala Leu Arg Thr Val Ala Ile Lys 85 90 95
Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Arg Met 100 105 110 Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Arg Met 100 105 110
Gin Gly Leu Leu Gin Tyr Ser Ala Gly Asp Cys Ala Phe Gin Glu Glu 115 120 125 Gin Gly Leu Leu Gin Tyr Ser Ala Gly Asp Cys Ala Phe Gin Glu Glu 115 120 125
Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu 130 135 140 Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu 130 135 140
Pro Val Ser Leu Ser Ser Ala Ile Gin Arg Gin Leu Tyr Lys Gly Arg 145 150 155 160 Pro Val Ser Leu Ser Ser Ala Ile Gin Arg Gin Leu Tyr Lys Gly Arg 145 150 155 160
Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Gly Ser Pro 165 170 175 Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met Leu Pro Gly Ser Pro 165 170 175
Ala Glu Pro Arg Asp Leu Gin Asp His Val Glu Ser Glu Arg Phe Ser 180 185 190 Ala Glu Pro Arg Asp Leu Gin Asp His Val Glu Ser Glu Arg Phe Ser 180 185 190
Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Ile Ala Thr Lys 195 200 205 Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe Gly Ile Ala Thr Lys 195 200 205
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) Met Gly Leu Val Lys Ser Pro Ser Phe Gin Lys 210 215 SHEET INCORPORATED BY REFERENCE (RULE 20.6) Met Gly Leu Val Lys Ser Pro Ser Phe Gin Lys 210 215
<210> 8 <211> 218 <212> PRT <210> 8 <211> 218 <212> PRT
<213> Onyctolagus cuniculus <400> 8 <213> Onyctolagus cuniculus <400> 8
Met Ang Ang Ala Pno Sen Gly Gly Ala Ala Ala Ang Ala Leu Val Leu 1 5 10 15 Met Ang Ang Ala Pno Sen Gly Gly Ala Ala Ala Ang Ala Leu Val Leu 1 5 10 15
Ala Gly Leu Tnp Leu Ala Ala Ala Ala Ang Pno Leu Ala Leu Sen Asp 20 25 30 Ala Gly Leu Tnp Leu Ala Ala Ala Ala Ang Pno Leu Ala Leu Sen Asp 20 25 30
Ala Gly Pno His Leu His Tyn Gly Tnp Gly Glu Pno Val Ang Leu Ang 35 40 45 Ala Gly Pno His Leu His Tyn Gly Tnp Gly Glu Pno Val Ang Leu Ang 35 40 45
His Leu Tyn Ala Thn Sen Ala His Gly Val Sen His Cys Phe Leu Ang 50 55 60 His Leu Tyn Ala Thn Sen Ala His Gly Val Sen His Cys Phe Leu Ang 50 55 60
Ile Ang Ala Asp Gly Ala Val Asp Cys Glu Arg Ser Gin Ser Ala His 65 70 75 80 Island Ang Ala Asp Gly Ala Val Asp Cys Glu Arg Ser Gin Ser Ala His 65 70 75 80
Ser Leu Leu Glu Ile Ang Ala Val Ala Leu Ang Thr Val Ala Phe Lys 85 90 95 Ser Leu Leu Glu Ile Ang Ala Val Ala Leu Ang Thr Val Ala Phe Lys 85 90 95
Gly Val His Ser Ser Arg Tyr Leu Cys Met Gly Ala Asp Gly Arg Met 100 105 110 Gly Val His Ser Ser Arg Tyr Leu Cys Met Gly Ala Asp Gly Arg Met 100 105 110
Arg Gly Gin Leu Gin Tyr Ser Glu Glu Asp Cys Ala Phe Gin Glu Glu 115 120 125 Arg Gly Gin Leu Gin Tyr Ser Glu Glu Asp Cys Ala Phe Gin Glu Glu 115 120 125
Ile Ser Ser Gly Tyr Asn Val Tyr Arg Ser Thr Thr His His Leu Pro 130 135 140 Ile Ser Ser Gly Tyr Asn Val Tyr Arg Ser Thr Thr His His Leu Pro 130 135 140
Val Ser Leu Ser Ser Ala Lys Gin Arg His Leu Tyr Lys Thr Arg Gly 145 150 155 160 Val Ser Leu Ser Ser Ala Lys Gin Arg His Leu Tyr Lys Thr Arg Gly 145 150 155 160
Phe Leu Pro Leu Ser His Phe Leu Pro Val Leu Pro Leu Ala Ser GluPhe Leu Pro Leu Ser His Phe Leu Pro Val Leu Pro Leu Ala Ser Glu
165 170 175 165 170 175
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) Glu Thn Ala Ala Leu Gly Asp His Pno Glu Ala Asp Leu Phe Sen Pno 180 185 190 SHEET INCORPORATED BY REFERENCE (RULE 20.6) Glu Thn Ala Ala Leu Gly Asp His Pno Glu Ala Asp Leu Phe Sen Pno 180 185 190
Pno Leu Glu Thn Asp Sen Met Asp Pno Phe Gly Met Ala Thr Lys Leu 195 200 205 Pno Leu Glu Thn Asp Sen Met Asp Pno Phe Gly Met Ala Thr Lys Leu 195 200 205
Gly Pro Val Lys Ser Pro Ser Phe Gin Lys 210 215 Gly Pro Val Lys Ser Pro Ser Phe Gin Lys 210 215
<210> 9 <210> 9
<211> 218 <212> PRT <211> 218 <212> PRT
<213> Rattus norvegicus <400> 9 <213> Rattus norvegicus <400> 9
Met Ala Arg Lys Trp Ser Gly Arg Ile Val Ala Arg Ala Leu Val Leu 1 5 10 15 Met Ala Arg Lys Trp Ser Gly Arg Ile Val Ala Arg Ala Leu Val Leu 1 5 10 15
Ala Thr Leu Trp Leu Ala Val Ser Gly Arg Pro Leu Val Gin Gin Ser 20 25 30 Ala Thr Leu Trp Leu Ala Val Ser Gly Arg Pro Leu Val Gin Gin Ser 20 25 30
Gin Ser Val Ser Asp Glu Gly Pro Leu Phe Leu Tyr Gly Trp Gly Lys 35 40 45 Gin Ser Val Ser Asp Glu Gly Pro Leu Phe Leu Tyr Gly Trp Gly Lys 35 40 45
Ile Thr Arg Leu Gin Tyr Leu Tyr Ser Ala Gly Pro Tyr Val Ser Asn 50 55 60 Island Thr Arg Leu Gin Tyr Leu Tyr Ser Ala Gly Pro Tyr Val Ser Asn 50 55 60
Cys Phe Leu Arg Ile Arg Ser Asp Gly Ser Val Asp Cys Glu Glu Asp 65 70 75 80 Cys Phe Leu Arg Ile Arg Ser Asp Gly Ser Val Asp Cys Glu Glu Asp 65 70 75 80
Gin Asn Glu Arg Asn Leu Leu Glu Phe Arg Ala Val Ala Leu Lys Thr 85 90 95 Gin Asn Glu Arg Asn Leu Leu Glu Phe Arg Ala Val Ala Leu Lys Thr 85 90 95
Ile Ala Ile Lys Asp Val Ser Ser Val Arg Tyr Leu Cys Met Ser Ala 100 105 110 Ile Ala Ile Lys Asp Val Ser Ser Val Arg Tyr Leu Cys Met Ser Ala 100 105 110
Asp Gly Lys Ile Tyr Gly Leu Ile Arg Tyr Ser Glu Glu Asp Cys Thr 115 120 125 Asp Gly Lys Ile Tyr Gly Leu Ile Arg Tyr Ser Glu Glu Asp Cys Thr 115 120 125
Phe Arg Glu Glu Met Asp Cys Leu Gly Tyr Asn Gin Tyr Arg Ser Met 130 135 140 Phe Arg Glu Glu Met Asp Cys Leu Gly Tyr Asn Gin Tyr Arg Ser Met 130 135 140
Lys His His Leu His Ile Ile Phe Ile Lys Ala Lys Pro Arg Glu Gin Lys His His Leu His Ile Ile Phe Ile Lys Ala Lys Pro Arg Glu Gin
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) 145 150 155 160 SHEET INCORPORATED BY REFERENCE (RULE 20.6) 145 150 155 160
Leu Gin Gly Gin Lys Pro Ser Asn Phe Ile Pro Ile Phe His Arg Ser 165 170 175 Leu Gin Gly Gin Lys Pro Ser Asn Phe Ile Pro Ile Phe His Arg Ser 165 170 175
Phe Phe Glu Ser Thr Asp Gin Leu Arg Ser Lys Met Phe Ser Leu Pro 180 185 190 Phe Phe Glu Ser Thr Asp Gin Leu Arg Ser Lys Met Phe Ser Leu Pro 180 185 190
Leu Glu Ser Asp Ser Met Asp Pro Phe Arg Met Val Glu Asp Val Asp 195 200 205 Leu Glu Ser Asp Ser Met Asp Pro Phe Arg Met Val Glu Asp Val Asp 195 200 205
His Leu Val Lys Ser Pro Ser Phe Gin Lys 210 215 His Leu Val Lys Ser Pro Ser Phe Gin Lys 210 215
<210> 10 <210> 10
<211> 218 <211> 218
<212> PRT <212> PRT
<213> mus musculus <213> mus musculus
<400> 10 <400> 10
Met Ala Arg Lys Trp Asn Gly Arg Ala Val Ala Arg Ala Leu Val Leu 1 5 10 15 Met Ala Arg Lys Trp Asn Gly Arg Ala Val Ala Arg Ala Leu Val Leu 1 5 10 15
Ala Thr Leu Trp Leu Ala Val Ser Gly Arg Pro Leu Ala Gin Gin Ser 20 25 30 Ala Thr Leu Trp Leu Ala Val Ser Gly Arg Pro Leu Ala Gin Gin Ser 20 25 30
Gin Ser Val Ser Asp Glu Asp Pro Leu Phe Leu Tyr Gly Trp Gly Lys 35 40 45 Gin Ser Val Ser Asp Glu Asp Pro Leu Phe Leu Tyr Gly Trp Gly Lys 35 40 45
Ile Thr Arg Leu Gin Tyr Leu Tyr Ser Ala Gly Pro Tyr Val Ser Asn 50 55 60 Island Thr Arg Leu Gin Tyr Leu Tyr Ser Ala Gly Pro Tyr Val Ser Asn 50 55 60
Cys Phe Leu Arg Ile Arg Ser Asp Gly Ser Val Asp Cys Glu Glu Asp 65 70 75 80 Cys Phe Leu Arg Ile Arg Ser Asp Gly Ser Val Asp Cys Glu Glu Asp 65 70 75 80
Gin Asn Glu Arg Asn Leu Leu Glu Phe Arg Ala Val Ala Leu Lys Thr 85 90 95 Gin Asn Glu Arg Asn Leu Leu Glu Phe Arg Ala Val Ala Leu Lys Thr 85 90 95
Ile Ala Ile Lys Asp Val Ser Ser Val Arg Tyr Leu Cys Met Ser Ala 100 105 110 Ile Ala Ile Lys Asp Val Ser Ser Val Arg Tyr Leu Cys Met Ser Ala 100 105 110
Asp Gly Lys Ile Tyr Gly Leu Ile Arg Tyr Ser Glu Glu Asp Cys Thr 115 120 125 Asp Gly Lys Ile Tyr Gly Leu Ile Arg Tyr Ser Glu Glu Asp Cys Thr 115 120 125
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) Phe Ang Glu Glu Met Asp Cys Leu Gly Tyr Asn Gin Tyr Arg Ser Met 130 135 140 SHEET INCORPORATED BY REFERENCE (RULE 20.6) Phe Ang Glu Glu Met Asp Cys Leu Gly Tyr Asn Gin Tyr Arg Ser Met 130 135 140
Lys His His Leu His Ile Ile Phe Ile Gin Ala Lys Pro Arg Glu Gin 145 150 155 160 Lys His His Leu His Ile Ile Phe Ile Gin Ala Lys Pro Arg Glu Gin 145 150 155 160
Leu Gin Asp Gin Lys Pro Ser Asn Phe Ile Pro Val Phe His Arg Ser 165 170 175 Leu Gin Asp Gin Lys Pro Ser Asn Phe Ile Pro Val Phe His Arg Ser 165 170 175
Phe Phe Glu Thr Gly Asp Gin Leu Arg Ser Lys Met Phe Ser Leu Pro 180 185 190 Phe Phe Glu Thr Gly Asp Gin Leu Arg Ser Lys Met Phe Ser Leu Pro 180 185 190
Leu Glu Ser Asp Ser Met Asp Pro Phe Arg Met Val Glu Asp Val Asp 195 200 205 Leu Glu Ser Asp Ser Met Asp Pro Phe Arg Met Val Glu Asp Val Asp 195 200 205
His Leu Val Lys Ser Pro Ser Phe Gin Lys 210 215 His Leu Val Lys Ser Pro Ser Phe Gin Lys 210 215
FEUILLE INCORPOREE PAR RENVOI (REGLE 20.6) SHEET INCORPORATED BY REFERENCE (RULE 20.6)

Claims

Revendications Claims
[Revendication 1] Composition de protéines de légumineuse dont le degré d’hydrolyse est inférieur à 10%, contenant de préférence plus de 90% en poids de globulines par rapport au poids total des protéines, pour une utilisation dans la prévention et/ou le traitement d’une maladie susceptible d’être traitée par une activation de la synthèse du FGF19 chez un sujet, caractérisée en ce que ladite maladie est la sarcopénie ou la stéatohépatite non alcoolique. [Claim 1] Composition of legume proteins with a degree of hydrolysis of less than 10%, preferably containing more than 90% by weight of globulins based on the total weight of the proteins, for use in the prevention and/or treatment of a disease capable of being treated by an activation of the synthesis of FGF19 in a subject, characterized in that the said disease is sarcopenia or non-alcoholic steatohepatitis.
[Revendication 2] Composition pour l’utilisation selon la revendication 1 caractérisée en ce que lesdites protéines de légumineuse sont la seule source protéique au sein de la composition. [Claim 2] A composition for use according to claim 1 characterized in that said legume protein is the sole protein source within the composition.
[Revendication 3] Composition pour l’utilisation selon la revendication 1 ou 2 caractérisée en ce que les protéines de légumineuse sont des protéines de pois dont le degré d’hydrolyse est préférentiellement compris entre 6% et 8%. [Claim 3] Composition for use according to Claim 1 or 2, characterized in that the legume proteins are pea proteins, the degree of hydrolysis of which is preferably between 6% and 8%.
[Revendication 4] Composition pour l’utilisation selon la revendication 1 ou 2 caractérisée en ce que les protéines de légumineuse sont des protéines de féverole dont le degré d’hydrolyse est compris entre 0% et 5%. [Claim 4] Composition for use according to Claim 1 or 2, characterized in that the legume proteins are faba bean proteins, the degree of hydrolysis of which is between 0% and 5%.
[Revendication 5] Composition pour l’utilisation selon l’une quelconque des revendications 1 à 4 caractérisée en ce que le sujet est un mammifère, de préférence un être humain, préférentiellement une personne ayant plus de 40 ans. [Claim 5] Composition for use according to any one of Claims 1 to 4, characterized in that the subject is a mammal, preferably a human being, preferably a person over 40 years old.
[Revendication 6] Composition pour l’utilisation selon l’une quelconque des revendications 1 à 5 comprenant en outre un agoniste de FXR choisis parmi : obeticholicacid (OCA), Chenodeoxycholicacid (CDCA), 6a-ethyl-chenodeoxycholic acid (6-ECDCA), Alisol B 23-acetate (AB23A), Cafestol, Fexaramine, GW4064 (3- (2,6-Dichlorophenyl)-4-(3’-carboxy-2-chlorostilben-4-yl)oxymethyl-5- isopropylisoxazole) et Tropifexor, de préférence GW4064 et Chenodeoxycholic acid (CDCA). [Claim 6] A composition for use according to any one of claims 1 to 5 further comprising an FXR agonist selected from: obeticholicacid (OCA), Chenodeoxycholicacid (CDCA), 6a-ethyl-chenodeoxycholic acid (6-ECDCA) , Alisol B 23-acetate (AB23A), Cafestol, Fexaramine, GW4064 (3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole) and Tropifexor , preferably GW4064 and Chenodeoxycholic acid (CDCA).
[Revendication 7] Composition pharmaceutique apte à surexprimer la synthèse du FGF19 comportant des protéines de légumineuse dont le degré d’hydrolyse est inférieur à 10% et optionnellement un excipient pharmaceutique acceptable, de préférence caractérisée en ce que lesdites protéines de légumineuse sont la seule source protéique au sein de ladite composition. [Claim 7] Pharmaceutical composition capable of overexpressing the synthesis of FGF19 comprising legume proteins whose degree of hydrolysis is less than 10% and optionally an acceptable pharmaceutical excipient, of preferably characterized in that said legume proteins are the only protein source within said composition.
[Revendication 8] Composition pharmaceutique selon la revendication 7 caractérisée en ce que les protéines de légumineuse sont : - des protéines de pois dont le degré d’hydrolyse est préférentiellement compris entre 6% et 8%, ou [Claim 8] Pharmaceutical composition according to Claim 7, characterized in that the legume proteins are: - pea proteins, the degree of hydrolysis of which is preferably between 6% and 8%, or
- des protéines de féverole dont le degré d’hydrolyse est compris entre entre 0% et 5%. - faba bean proteins whose degree of hydrolysis is between 0% and 5%.
[Revendication 9] Composition pharmaceutique selon la revendication 7 ou 8 comprenant en outre un agoniste de FXR choisis parmi : obeticholic acid (OCA), Chenodeoxycholic acid (CDCA), 6a-ethyl-chenodeoxycholic acid (6-ECDCA), Alisol B 23-acetate (AB23A), Cafestol, Fexaramine, GW4064 (3-(2,6-Dichlorophenyl)-4- (3’-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole) et Tropifexor, de préférence GW4064 et Chenodeoxycholic acid (CDCA). [Claim 9] Pharmaceutical composition according to claim 7 or 8 further comprising an FXR agonist chosen from: obeticholic acid (OCA), Chenodeoxycholic acid (CDCA), 6a-ethyl-chenodeoxycholic acid (6-ECDCA), Alisol B 23- acetate (AB23A), Cafestol, Fexaramine, GW4064 (3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole) and Tropifexor, preferably GW4064 and Chenodeoxycholic acid (CDCA).
EP22750660.7A 2021-07-26 2022-07-26 Pulse protein-based composition for activating the synthesis of fgf19 Pending EP4376865A2 (en)

Applications Claiming Priority (2)

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FR2108059A FR3125410B1 (en) 2021-07-26 2021-07-26 METHOD FOR ACTIVATING THE SYNTHESIS OF FGF19
PCT/EP2022/025353 WO2023006248A2 (en) 2021-07-26 2022-07-26 Method for activating the synthesis of fgf19

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EP4376865A2 true EP4376865A2 (en) 2024-06-05

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Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2844515B1 (en) 2002-09-18 2004-11-26 Roquette Freres PROCESS FOR EXTRACTING COMPONENTS OF PEA FLOUR
FR2882896B1 (en) * 2005-03-14 2007-05-04 Larena Sa FOOD COMPOSITION FOR PREVENTING FRAGILITY SYNDROME IN OLDER PEOPLE
FR2889416B1 (en) 2005-08-05 2007-10-26 Roquette Freres COMPOSITION OF PEAS PROTEINS
FR3065623B1 (en) * 2017-04-28 2021-07-09 Roquette Freres IMPROVED PEA ALBUMINS, PROCESS FOR OBTAINING THEIR APPLICATIONS
WO2019023245A1 (en) * 2017-07-25 2019-01-31 Cedars-Sinai Medical Center Methods for treating liver diseases
EP3691472B1 (en) 2017-10-04 2022-11-16 Roquette Frères Pea protein composition with improved nutritional quality
US20220053792A1 (en) * 2018-09-25 2022-02-24 Roquette Freres Food composition containing a mixture of leguminous proteins and casein
FR3089094B1 (en) * 2018-11-30 2022-04-15 Roquette Freres Soluble legume protein
AU2020281621A1 (en) * 2019-05-24 2021-12-16 Cosucra Groupe Warcoing S.A. Composition comprising inulin and protein

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FR3125410B1 (en) 2023-12-29
WO2023006248A3 (en) 2023-03-23
FR3125410A1 (en) 2023-01-27

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