Bioassimilable eggshell membrane

Abstract

The present invention relates to a composition comprising a peptide fraction, wherein: the peptide fraction comprises cysteine and methionine; the mass content of methionine of the peptide fraction is greater than 1% relative to the total amino acids of the peptide fraction; the cysteine of the peptide fraction is at least partially sulfonated, the degree of sulfonation of the cysteine being greater than 50%, preferably greater than 70%, and even more preferably greater than 80%.

Description

BIOASSIMILABLE SHELL MEMBRANE Scope of the invention

[0001] The present invention relates to a composition comprising a peptide fraction with a high mass content of cysteine ​​and a high degree of cysteine ​​sulfonation. The present invention also relates to a composition comprising a peptide fraction containing cysteine ​​and methionine, with a high mass content of methionine and a high degree of cysteine ​​sulfonation. The present invention further relates to a method for preparing a composition comprising an oxidative sulfitolysis step of shell membrane. The present invention also relates to a composition obtained by this method. The present invention further proposes these compositions for use as a drug, food supplement, medical device, cosmetic product, or biomaterial. Technical background

[0002] The agri-food industry produces a considerable amount of eggshells each year, considered a production waste in egg-breaking plants around the world, the majority of which is not properly treated for circular use (spreading, feeding livestock, etc.) and is destroyed.

[0003] The industrial-scale valorization of eggshells offers numerous opportunities, particularly due to their composition. Eggshells are composed of two usable raw materials: the shell itself, mainly composed of calcium carbonate (the inorganic part of the shell), and the shell membrane, containing over 500 proteins and other non-protein compounds, primarily glycosaminoglycans (the organic part of the eggshell).

[0004] Calcium carbonate from the shell can, for example, be used as: dietary supplements for bone health; in dairy products, drinks and cereals; medicines in the pharmaceutical industry; building materials as filler in cements, concretes and construction products; raw material for the production of various chemicals such as sodium bicarbonate; agricultural product to improve soil quality, increase the pH of acidic soils and provide nutrients to plants; fillers in the manufacture of plastics, paints and coatings; acidity neutralizing agent in water filtration systems.

[0005] The shell membrane is very rich in metabolites of biological interest, which may include more than 500 proteins and other non-protein compounds involved in the process of mineralization of the eggshell as well as in the protection of the embryo.

[0006] These proteins and other non-protein compounds of the shell membrane can, for example, be used as nutritional supplements, cosmetic products due to their moisturizing properties, biomaterials to promote healing or tissue regeneration, and agronomic products for organic fertilizers.

[0007] The shell membrane is neither naturally digestible nor bioavailable. This characteristic is linked to the chemical structure of the membrane.

[0008] The proteins in the shell membrane are not naturally water-soluble.

[0009] There are several experimental methods for extracting these proteins from the shell membrane starting from the raw eggshell: acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis.

[0010] Document EP3653721 discloses a process for hydrolyzing shell membrane using a solution comprising a denaturing agent, a reducing agent, a buffer, and an enzyme. This process allows for the efficient solubilization of the shell membrane.

[0011] The document CN117230141 relates to a process for extracting oligopeptide from shell membrane comprising an enzymatic hydrolysis step.

[0012] The CN116240255 document describes a process for preparing eggshell membrane extract comprising chondroitin sulfate and oligopeptides.

[0013] Acid hydrolysis and alkaline hydrolysis have the disadvantage of destroying certain valuable components and reducing the nutritional or therapeutic value of some proteins, particularly due to the operating conditions. Conversely, enzymatic hydrolysis can be less aggressive; however, because the shell membrane is highly resistant to enzymes, its effectiveness is very limited or even nonexistent.

[0014] Non-denaturing preparatory methods exist for extracting proteins from other biological sources, notably oxidative sulfitolysis, which can be used for extracting proteins from wool. This method involves grafting a sulfite group onto the thiol group of an amino acid, particularly cysteine ​​in disulfide bridges, within a protein chain. Oxidative sulfitolysis thus generates bioactive, non-denatured protein chains with sulfonated groups, and more specifically, containing sulfonated cysteines.

[0015] Cysteines play a leading role in cell biology and in cell biology for maintaining cell integrity, regulating metabolic pathways and protecting cells against oxidative stress.

[0016] The supply of a peptide fraction with a high proportion of cysteine ​​is therefore particularly sought after.

[0017] US patent 7148327 describes a process for preparing high molecular weight soluble proteins with reduced damage to the protein structure. Specifically, the process includes an oxidative sulfitolysis step and a step for extracting soluble proteins from a wool source. S-sulfonated keratin proteins are formed as a result of this process.

[0018] Document WO2010114938 relates to a process for the production of S-sulfonated keratin peptides from wool or wool fibers comprising an oxidative sulfitolysis step of the keratin source followed by an enzymatic hydrolysis reaction.

[0019] The document WO2016174367 describes a proteomelanic complex comprising a soluble or partially soluble protein extract rich in S-sulfonated residues and melanin, and the process for preparing this complex from wool, including an oxidative sulfitolysis step.

[0020] Document NZ 530229 describes a process for preparing keratin derivatives comprising an oxidative sulfitolysis step, implemented from wool or other keratin-rich materials.

[0021] However, the state of the art indicates oxidative sulfitolysis of wool or other keratin-rich materials, resulting in peptide fractions with a low proportion of sulfonated cysteine ​​and a negligible or zero proportion of methionine. Peptide fractions with a high proportion of sulfonated cysteine, however, yield more soluble fractions in physiological environments, leading to improved bioavailability. Furthermore, methionine is an essential amino acid.

[0022] It is therefore desirable to provide higher-performing compositions comprising bio-assimilable and bioactive peptide fractions with high nutritional or therapeutic value, as well as efficient and non-denaturing processes for manufacturing these compositions from readily available and abundant material, or even material in overproduction. Summary of the invention

[0023] The present invention relates to the following objects.

[0024] Item 1. Composition comprising a peptide fraction, in which: • the peptide fraction comprises cysteine ​​in a mass content greater than 8%, preferably greater than 9%, even more preferably greater than 10%, relative to the total amino acids of the peptide fraction; • the cysteine ​​of the peptide fraction is at least partially sulfonated, the degree of sulfonation of the cysteine ​​being greater than 50%, preferably greater than 70%, even more preferably greater than 80%.

[0025] Item 2. Composition according to item 1, wherein the peptide fraction comprises methionine in a mass content greater than 1%, preferably greater than 2%, even more preferably greater than 3%; and / or the peptide fraction comprises cysteine ​​and methionine in a total mass content greater than 9%, preferably greater than 10%, even more preferably greater than 12%.

[0026] Item 3. Composition according to item 1 or 2, comprising: • a polysaccharide fraction, preferably comprising galactomannans, chitin and / or glycosaminoglycans, the glycosaminoglycans preferably comprising chondroitin sulfate, dermatan sulfate, keratan sulfate and / or hyaluronic acid; in which the polysaccharide fraction includes, more particularly preferred, hyaluronic acid.

[0027] Item 4. Process for preparing a composition, comprising an oxidative sulfitolysis step of a starting material, said starting material comprising shell membranes.

[0028] Item 5. A process according to item 4, in which the oxidative sulfitolysis step is carried out in the presence of a sulfite salt, preferably sodium sulfite.

[0029] Item 6. A process according to item 4 or 5, wherein the oxidative sulfitolysis step is carried out in the presence of a metal as a catalyst, preferably a divalent metal, even more preferably copper.

[0030] Item 7. A process according to any one of items 4 to 6, wherein the oxidative sulfitolysis step is carried out in the presence of an oxidizing agent, preferably dioxygen.

[0031] Item 8. A process according to any one of items 4 to 7, comprising an enzymatic hydrolysis step of the product from the oxidative sulfitolysis step, preferably by a serine protease.

[0032] Item 9. A process according to any one of items 4 to 8, wherein the starting material comprises eggshells.

[0033] Item 10. A process according to item 9, comprising a solid / liquid separation step enabling the collection of a solid fraction comprising a mineral part of eggshells and a liquid fraction comprising a peptide fraction.

[0034] Item 11. A process according to any one of items 4 to 10, comprising a step of precipitating an insoluble peptide fraction and removing it.

[0035] Item 12. Composition which can be obtained by the process according to any one of items 4 to 11.

[0036] Item 13. Composition according to any one of items 1 to 4 or 12, for use as a medicinal product, preferably for the treatment and / or prevention of a dermatological condition or joint pathology.

[0037] Item 14. Use of the composition according to any one of items 1 to 4 or 12, as a food supplement, or in a medical device or cosmetic product.

[0038] Item 15. Composition comprising: • a peptide fraction in which the cysteine ​​is at least partially sulfonated; • a polysaccharide fraction, including galactomannans, chitin and / or glycosaminoglycans, the glycosaminoglycans preferably including chondroitin sulfate, dermatan sulfate, keratan sulfate and / or hyaluronic acid.

[0039] Item 16. Composition according to item 15, wherein the polysaccharide fraction comprises hyaluronic acid.

[0040] Item 17. Composition according to item 15 or 16, wherein the peptide fraction comprises cysteine ​​in a mass content exceeding 6%, or in a mass content exceeding 7%, or in a mass content exceeding 8%, preferably exceeding 9%, even more preferably exceeding 10%, relative to the total amino acids of the peptide fraction.

[0041] Item 18. Composition according to any one of items 15 to 17, wherein the peptide fraction comprises methionine in a mass content exceeding 1%, preferably exceeding 2%, even more preferably exceeding 3%; and / or the peptide fraction comprises cysteine ​​and methionine in a total mass content exceeding 9%, preferably exceeding 10%, even more preferably exceeding 12%.

[0042] Item 19. Composition according to any one of items 15 to 18, wherein the degree of sulfonation of cysteine ​​in the peptide fraction is greater than 50%, preferably greater than 70%, even more preferably greater than 80%.

[0043] Item 20. Composition according to any one of items 15 to 19, for use as a medicinal product, preferably for the treatment and / or prevention of a dermatological condition or joint pathology.

[0044] Item 21. Use of the composition according to any one of items 15 to 19, as a food supplement, or in a medical device or cosmetic product.

[0045] Item 22. Composition comprising a peptide fraction, in which: • the peptide fraction includes cysteine ​​and methionine; • methionine from the peptide fraction is present in a mass content greater than 1% relative to the total amino acids of the peptide fraction; • the cysteine ​​of the peptide fraction is at least partially sulfonated, the degree of sulfonation of the cysteine ​​being greater than 50%, preferably greater than 70%, even more preferably greater than 80%.

[0046] Item 23. Composition according to item 22, wherein the peptide fraction comprises cysteine ​​in a mass content exceeding 6% relative to the total amino acids of the peptide fraction, preferably exceeding 7%, preferably exceeding 8%, more preferably exceeding 9%, even more preferably exceeding 10%.

[0047] Item 24. Composition according to item 22 or 23, wherein the peptide fraction comprises methionine in a mass content exceeding 2% relative to the total amino acids of the peptide fraction, preferably exceeding 3%; and / or the peptide fraction comprises cysteine ​​and methionine in a total mass content exceeding 7% relative to the total amino acids of the peptide fraction, preferably exceeding 8%, preferably exceeding 9%, more preferably exceeding 10%, even more preferably exceeding 12%.

[0048] Item 25. Composition according to any one of items 22 to 24, wherein the peptide fraction has a mass average molecular weight Mw of 500 to 10000 g / mol, preferably of 700 to 5000 g / mol, preferably again of 1000 to 2000 g / mol.

[0049] Item 26. Composition according to one of items 22 to 25, further comprising: • a polysaccharide fraction, preferably comprising galactomannans, chitin and / or glycosaminoglycans, the glycosaminoglycans preferably comprising chondroitin sulfate, dermatan sulfate, keratan sulfate and / or hyaluronic acid; in which the polysaccharide fraction includes, more particularly preferred, hyaluronic acid.

[0050] Item 27. Composition according to any one of items 22 to 26, for use as a medicinal product, preferably for the treatment and / or prevention of a dermatological condition or joint pathology.

[0051] Item 28. Use of the composition according to any one of items 22 to 27, as a food supplement, or in a medical device or cosmetic product.

[0052] Item 29. Composition which can be obtained by the process according to any one of items 4 to 11, which also has the characteristics of any one of items 1 to 3, or of any one of items 15 to 19, or of any one of items 22 to 26.

[0053] The present invention addresses a need expressed in the prior art. More specifically, it provides a bioavailable and bioactive peptide composition with high nutritional or therapeutic value, as well as an efficient and non-denaturing process for manufacturing this composition. Preferably, the composition comprises a glycoprotein complex. The process used to obtain the peptide fraction of interest involves the oxidative sulfitolysis of a shell membrane, preferably followed by enzymatic hydrolysis. The process of the invention thus allows for the efficient valorization of a waste product from the agri-food industry. The process preserves the extracted peptides because it does not involve aggressive chemical treatment, high temperatures, or, more generally, denaturing conditions. Detailed description of the invention

[0054] Other features, aspects, objects and advantages of the present invention will become even clearer upon reading the description that follows.

[0055] The entire description below applies to items 1 to 29 shown above.

[0056] In this application, "peptide fraction" means all amino acids, peptides, polypeptides and proteins present in the composition.

[0057] In this application, "total amino acids" means all the amino acids contained in the peptide fraction. These amino acids may include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gin), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). These amino acids may be in their native form or with a modified side chain (e.g., sulfocysteine).

[0058] In this application, amino acid residues within a peptide, polypeptide, or protein are considered equivalent to amino acids. For example, the cysteine ​​content corresponds to the total of free cysteine ​​(if present) and cysteine ​​residues within peptides, polypeptides, or proteins, in the peptide fraction.

[0059] By "sulfonation rate" of cysteine, it is understood that the mass ratio of the amount of sulfonate cysteines to the total amount of cysteine ​​in the peptide fraction is the mass ratio of the amount of sulfonate cysteines to the total amount of cysteine ​​in the peptide fraction.

[0060] In the present invention, "bio-assimilable" means the ability of a molecule or composition to pass through biological barriers, such as the skin including the scalp, or the gastrointestinal barrier, and to become available in the circulation, advantageously without being degraded, partially or totally eliminated.

[0061] In the present invention, "oxidative sulfitolysis" refers to the covalent grafting of a sulfite group onto a thiol group in the presence of an oxidizing agent. The presence of this oxidizing agent allows for a sulfonation rate of cysteines exceeding 50%, as will be explained in more detail below.

[0062] In the present invention, the term "shell membrane" refers to a membrane derived from an eggshell. The eggshell comprises a solid, essentially mineral structure, composed mainly of calcium salts, and the shell membrane. The shell membrane includes proteins and other biological molecules such as polysaccharides and glycosaminoglycans.

[0063] The invention relates to a composition comprising a peptide fraction, in which: • the peptide fraction includes cysteine • the cysteine ​​of the peptide fraction is at least partially sulfonated, the degree of sulfonation of the cysteine ​​may be greater than 50%, preferably greater than 70%, even more preferably greater than 80%.

[0064] The peptide fraction may comprise cysteine ​​in a mass content greater than 6% relative to the total amino acids of the peptide fraction, preferably greater than 7%, preferably greater than 8%, more preferably greater than 9%, even more preferably greater than 10%.

[0065] The peptide fraction advantageously comprises methionine. The mass content of methionine in the peptide fraction is preferably greater than 1%, preferably even greater than 2%, and even more preferably greater than 3%, relative to the total amino acids in the peptide fraction.

[0066] Methionine is an essential amino acid obtained through diet because it is not metabolized by the human body. In comparison, it is absent or present in negligible amounts in keratin products.

[0067] The total mass content of sulfur-containing amino acids, namely cysteine ​​and methionine, is preferably greater than 7% relative to the total amino acids of the peptide fraction, preferably even greater than 8%, preferably greater than 9%, more preferably greater than 10%, and even more preferably greater than 12%.

[0068] The peptide fraction may have, in particular, a mass average molecular weight (Mw) of 500 to 10,000 g / mol, preferably 700 to 5,000 g / mol, and more preferably 1,000 to 2,000 g / mol, for example, 1,200 to 1,800 g / mol. It may also have a number average molecular weight (Mn) of 100 to 5,000 g / mol, preferably 150 to 2,000 g / mol, and more preferably 200 to 1,000 g / mol, for example, 300 to 400 g / mol. The molecular weight distribution can be determined by HPLC-UV. The peptide fraction may have a polydispersity index (Mw / Mn) of 1.5 to 10, preferably 2 to 7, and more preferably 3 to 5, for example, 4 to 4.5.

[0069] In some embodiments, the peptide fraction may comprise a mass proportion of molecules with a molecular weight greater than 6000 g / mol of less than 1%, preferably less than 0.5%. The peptide fraction may comprise a mass proportion of molecules with a molecular weight between 200 and 800 g / mol representing 40 to 80%, preferably 50 to 70%.

[0070] Preferably, the peptide fraction can be essentially devoid of free amino acids (mass proportion less than 5%, or 2%, or 1%).

[0071] The sulfone group of cysteine ​​is a polar group. This group helps improve the solubility of the peptide fraction in a physiological environment, allowing for better bio-assimilation by the human body.

[0072] The peptide fraction may preferably be completely soluble, for example in an aqueous solution at pH 4. Alternatively, in other embodiments, the peptide fraction may comprise a soluble part and an insoluble part.

[0073] The mass content of cysteine ​​(or any other amino acid) relative to the total amino acids of the peptic fraction can be evaluated using a chemical analysis by aminogram.

[0074] Preferably, the chemical analysis by aminogram includes an acid hydrolysis step of the peptide fraction; a separation step of free amino acids by chromatography, preferably liquid chromatography; a detection step of amino acids, preferably by mass spectrometry or by UV detection.

[0075] In the peptide fraction, the mass contents of amino acids, relative to the total amino acids in the peptide fraction, can be as follows: • Asp: 4 to 15%, preferably 6 to 13%, preferably 8 to 11%; • Thr: 2 to 10%, preferably 3 to 8%, preferably 4 to 7%; • Ser: from 2 to 10%, preferably from 3 to 8%, preferably even from 4 to 7%; • Glu: from 8 to 19%, preferably from 10 to 17%, preferably even from 12 to 15%; • Pro and HPro: from 4 to 15%, preferably from 6 to 13%, preferably even from 8 to 11%; • Gly: from 2 to 10%, preferably from 3 to 8%, preferably even more from 4 to 7%; • Ala: from 0.5 to 7%, preferably from 1 to 6%, preferably even more from 2 to 5%; • Cys: from 8 to 20%, preferably from 10 to 16%, preferably even from 11 to 14%; • Val: from 2 to 12%, preferably from 3 to 10%, preferably even more from 5 to 8%; • Met: from 1 to 9%, preferably from 2 to 7%, preferably even more from 3 to 6%; • Island: from 0.5 to 6%, preferably from 1 to 5%, preferably even from 2 to 4%; • Leu: from 1 to 9%, preferably from 2 to 7%, preferably even from 3 to 6%; • Tyr: 0.2 to 5%, preferably 0.5 to 4%, preferably 1 to 3%; • Phe: 0.2 to 5%, preferably 0.5 to 4%, preferably 1 to 3%; • His: 0.5 to 6%, preferably 1 to 5%, preferably 2 to 4%; • Lilies: from 2 to 10%, preferably from 3 to 8%, preferably even more from 4 to 7%; • Arg: from 2 to 10%, preferably from 3 to 8%, preferably even from 4 to 7%; • Trp: from 0.5 to 6%, preferably from 1 to 5%, preferably still from 1.5 to 3.5%.

[0076] The presence of sulfonated cysteine ​​is a marker of the completion of the oxidative sulfitolysis process.

[0077] The degree of cysteine ​​sulfonation can be assessed by a separation or non-separation method, preferably by the "Biotin Switch Technique" described in the article by O. Rudyk, P. Eaton, Biochemical methods for monitoring protein thiol redox states in biological systems, Redox Biology, 2014, 2, 803. Even more preferably, characterization can be carried out by HPLC, as described in the article by B. Garcia-Alonso, MJ Pena-Egido, G. Garcia-Moreno, S-sulfonate determination and formation in meat products, J. Agric. Food Chem., 2001, 49, 423. Characterization can also be carried out even more preferably by Fourier transform cyclotron resonance mass spectrometry as described in the article by KF Medzihradszky, S. Guan, DAMaltby, AL Burlingame, Sulfopeptide fragmentation in electron-capture and electrontransferdissociation, J. Am. Soc. Mass. Spectrom., 2007, 18, 1617-1624.

[0078] The degree of sulfonation of cysteine ​​can also be assessed by other separation methods: • Matrix-assisted desorption / ionization coupled with standard mass spectrometry or time-of-flight mass spectrometry (MALDI-MS or MALDI-TOF) as described for example in the article by J. Alcock, MV Perkins, JM Chalker, Chemical methods for mapping cysteine ​​oxidation, Chem. Soc. Rev., 2018, 47, 31. • Tandem mass spectrometry (MS / MS) with dynamic simulations (computational models to simulate the chemical reactions observed in MS / MS) as described for example in the article by V. Macaluso, D. Scuderi, ME Crestoni, S. Fornarini, D. Corinti, E. Dalloz, E. Martinez-Nunez, WL Hase, R. Spezia, L-cysteine ​​modified by S-Sulfation: Consequence on fragmentation processes elucidated by tandem mass spectrometry and chemical dynamics simulations, The Journal of Physical Chemistry A, 2019, 123, 17. • Collision-induced dissociation coupled with tandem mass spectrometry (CID MS / MS) as described in the article by L. Sleno, DA Volmer, Ion activation methods for tandem mass spectrometry, Journal of Mass Spectrometry, 2004, 39, 1091. • Electrospray-induced mass spectrometry (ESI-MS) as described in the article by A. Lim, T. Prokaeva, ME McComb, LH Connors, M. Skinner, CE Costello, Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis, Protein Science, 2003, 12, 1775.

[0079] The degree of sulfonation of cysteine ​​in the peptide fraction can, for example, be 50 to 60%, or 60 to 70%, or 70 to 80%, or 80 to 90%, or 90 to 95%, or 95 to 100%.

[0080] In one embodiment, the composition may comprise from 0.01 to 100 ppm of metals, preferably from 0.05 to 50 ppm, even more preferably from 0.1 to 30 ppm, by weight.

[0081] Preferably, the metals are chosen from divalent metals, even more preferably from residues of copper, calcium or zinc.

[0082] The composition according to the invention may also include polysaccharides (referred to herein as polysaccharide fraction).

[0083] The said polysaccharide fraction may include galactomannans, chitin and / or glycosaminoglycans.

[0084] Preferably, the glycosaminoglycans are chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid, preferably hyaluronic acid.

[0085] The composition may include from 36 to 99.9%, preferably from 40 to 85%, even more preferably from 45 to 68% peptide fraction (by mass of dry matter).

[0086] The composition may include from 0 to 10%, preferably from 0.5 to 8%, even more preferably from 2 to 6% of polysaccharide fraction (by mass of dry matter).

[0087] Preferably, hyaluronic acid represents, in the composition, a mass content of 0 to 10%, preferably of 0.5 to 8%, even more preferably of 2 to 6% (in dry matter).

[0088] The composition can be formulated in dry (solid) form, for example as a powder (preferably water-soluble), in semi-solid form, or in liquid form (especially as an aqueous solution or emulsion). Preparation process Oxidative sulfitolysis

[0089] The invention also relates to a method for preparing a composition, comprising an oxidative sulfitolysis step of a starting material, said starting material comprising shell membranes.

[0090] Shell membranes come from eggshells.

[0091] In one embodiment, the starting material comprises shell membranes without the mineral portion of the eggshells. Thus, the process may include a preliminary step of separating the shell membranes from the eggshells to provide the starting material. This separation is preferably mechanical, including, for example, grinding, ultrasonic treatment, and sieving; high-pressure heating; and / or sorting by cyclonic airflow.

[0092] However, preferably the starting material may include eggshells (with shell membrane and mineral part).

[0093] Eggshells can come from any oviparous or ovoviviparous animal, vertebrate or invertebrate, for example, from the eggs of birds, fish, insects or reptiles, preferably from birds, and even more preferably from birds of the Galliformes group. They can notably come from the eggs of chickens, quail, ducks, geese, ostriches, preferably from chicken eggs.

[0094] The shell membrane is generally composed of two submembranes, an inner shell membrane and an outer shell membrane. In the context of this invention, the shell membrane may refer to either the inner shell membrane, the outer shell membrane, or preferably both.

[0095] The shell membrane includes proteins and polysaccharides.

[0096] The polysaccharides of the shell membrane may include galactomannans, chitin, and glycosaminoglycans, notably chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. Preferably, the shell membrane comprises a hyaluronic acid content of 0.1 to 30% by weight, preferably 0.1 to 20% by weight, preferably 0.5 to 10% by weight, and even more preferably 1 to 8% by weight.

[0097] Shell membrane proteins may include cysteine-rich shell membrane proteins (CREMPs) and glycoproteins.

[0098] These glycoproteins may include ovoglobulins, ovomucin, ovotransferrin, ovomucoid, and collagen.

[0099] This collagen can notably be of type I, IV and / or V.

[0100] In one embodiment, the process includes a crushing step and optionally a washing step of eggshells, used as starting material.

[0101] In another embodiment, the starting material consists of eggshells (optionally washed) obtained without prior crushing.

[0102] For the purposes of oxidative sulfitolysis, the starting material can be placed in a tank, preferably including an agitation system (e.g., an agitation shaft) and an aeration system, e.g., bubbling, i.e., by injecting air or dioxygen (e.g., essentially pure) preferably at the lowest point of the reaction medium through a bubbling wand or a sinter.

[0103] The starting material can thus be suspended by stirring in an aqueous solution to form a reaction medium. The reaction medium can then be bubbling to introduce a gaseous oxidizing agent, as described below.

[0104] The oxidative sulfitolysis step may include: a) the reduction of cysteines by grafting of sulfite groups; b) the oxidation of the thiol functions of cysteine ​​in the presence of a catalyst; c) the reoxidation of the catalyst.

[0105] Cysteine ​​reduction involves the reduction of disulfide bridges (RSS-R') between two cysteine ​​residues, which can be carried out using a sulfite salt. More specifically, the sulfite ion SO₄²⁻ 2 ' gives up two electrons causing the disulfide bridge to break so as to form a residue with a thiol group (R-SH) and a residue with a thiosulfonate group (RS-SOs'), which is called sulfonated cysteine.

[0106] The residue with a thiol group thus formed can be oxidized to form a new disulfide bridge with another thiol group, in the presence of the catalyst, via electron exchange. Indeed, the catalyst, for example in the form of a divalent metal ion, undergoes reduction, changing from an oxidation state (+II) to an oxidation state (+1), allowing the formation of this disulfide bridge.

[0107] The catalyst can be reoxidized using an oxidizing agent, which can be, for example, the oxygen supplied to the solution.

[0108] Substeps a), b), and c) of the oxidative sulfitolysis step are repeated so that the sulfonation rate is preferably greater than 50%, preferably greater than 70%, and even more preferably greater than 80%. Non-oxidative sulfitolysis theoretically allows a maximum sulfonation rate of 50% to be achieved.

[0109] The solution used to form the reaction medium may include a sulfite salt.

[0110] The said sulfite salt may be sodium sulfite, calcium sulfite, potassium sulfite, magnesium sulfite or ammonium sulfite, preferably sodium sulfite.

[0111] The solution used to form the reaction medium may include a catalyst, in particular an organic catalyst or a metal, or a Lewis acid such as borane.

[0112] The organic catalyst can be chosen from among tertiary amines.

[0113] Preferably the catalyst is a metal, in particular a transition metal.

[0114] The transition metal can be chosen from copper, manganese, iron, and is preferably copper. The catalyst can be, for example, in the form of a cuprammonium complex. The copper can be added to the solution as a metallic salt, for example, copper sulfate.

[0115] Preferably, the solution used to form the reaction medium includes the catalyst, the sulfite salt, a base and an acid.

[0116] The base can be chosen from ammonia, oxalate, glycine, histidine, ethylenediamine, acetylacetone, diethyleneamine, triethylenetetramine, and preferably is ammonia.

[0117] Preferably, the mass concentration of the base in the solution is 0.05 to 20%, preferably 0.1 to 5%, even more preferably 0.25 to 1%.

[0118] The acid can be chosen from sulfuric acid, hydrochloric acid, chloric acid, permanganic acid, acetic acid, oxalic acid, methanesulfonic acid, phosphoric acid, trifluoroacetic acid, thioglycolic acid, fumaric acid, tartaric acid, propionic acid, formic acid, itaconic acid, trichloroacetic acid, citric acid, preferably sulfuric acid.

[0119] Preferably, the molar concentration of the acid in the solution is 0.1 to 10 mol. 1 , preferably from 0.5 to 5 mol.L 1 , preferably in concentrations of 1 to 3 mol. 1 .

[0120] The concentration of sulfite salt in the solution can be from 5 to 10g / L, preferably from 10 to 50 g / L, even more preferably from 20 to 30 g / L.

[0121] The reaction medium can be prepared, for example, by preparing a solution, which can be obtained by mixing: • an aqueous solution comprising the catalyst, the base and the acid; • an aqueous solution comprising the sulfite salt; then by adding this solution to the starting material.

[0122] The mass ratio of the starting material to the solution, within the reaction medium, can be, for example, from 1 to 20%, preferably from 5 to 15%.

[0123] The oxidative sulfitolysis step can be implemented in the presence of an oxidizing agent.

[0124] The oxidizing agent can be chosen from a gas, a liquid or a solid, preferably a gas.

[0125] The gaseous oxidizing agent can be dioxygen, carbon dioxide, ozone, nitrogen dioxide, preferably dioxygen.

[0126] The liquid oxidizing agent can be hydrogen peroxide, sodium perchlorate, sodium hypochlorite, peracetic acid, bromine, nitric acid, chloric acid, manganese.

[0127] The oxidizing agent, preferably dioxygen, can be introduced into the reaction medium by ventilation and / or bubbling, at a flow rate between 0.1 and 100 Nl / minute of air per 100 liters of reaction medium, preferably between 1 and 50 Nl / minute of air per 100 liters of solution, very preferably between 5 and 20 Nl / minute of air per 100 liters of reaction medium.

[0128] The oxidizing agent, preferably hydrogen peroxide, can be introduced into the mixture by adding it in solution.

[0129] The addition of an oxidizing agent can be initiated before the reactants are brought into contact and carried out throughout the oxidative sulfitolysis step; alternatively, it can be carried out periodically or alternately after the reactants have been brought into contact and the reaction has begun. Preferably, the oxidative sulfitolysis step comprises a first substep carried out without external addition of an oxidizing agent (in particular, without the injection of air or oxygen), followed by a second substep carried out with external addition of an oxidizing agent (for example, with the injection of air or oxygen). This configuration helps to limit the conversion of sulfite to sulfate, which could deplete the medium of sulfite.

[0130] The temperature of the oxidative sulfitolysis step can be from 10 to 60°C, preferably from 20 to 40°C.

[0131] The pH of the reaction medium in the oxidative sulfitolysis step can be a basic pH, preferably from 8 to 10.

[0132] The duration of the oxidative sulfitolysis stage can be from 6 hours to 5 days, preferably from 12 hours to 4 days, preferably from 18 hours to 3 days, and even more preferably from 1 to 2 days. Enzymatic hydrolysis

[0133] The process according to the invention may include an enzymatic hydrolysis step of the product from the oxidative sulfitolysis step.

[0134] This enzymatic hydrolysis step aims to break amino acid bonds and also release polysaccharides bound to certain proteins. The release of these polysaccharides improves bioavailability.

[0135] The enzymatic hydrolysis step can be carried out using an endopeptidase, in particular a non-specific endopeptidase.

[0136] The enzyme may include trypsin, chymotrypsin, pepsin, papain, bromelain, protease, preferably a protease.

[0137] The protease can notably be a serine protease or a prolyl endopeptidase, preferably a serine protease.

[0138] Preferably, the enzyme is added directly to the reaction medium after the oxidative sulfitolysis step.

[0139] The pH of the reaction mixture in the enzymatic hydrolysis step can be basic, preferably between 8 and 10. The pH can be adjusted by adding base or acid during the enzymatic hydrolysis to maintain it within a desired range. For example, a basic solution, such as sodium hydroxide, with a mass concentration of 0.01 N to 10 N, preferably 1 N to 3 N, can be added to the reaction mixture during the enzymatic hydrolysis step.

[0140] It is possible to stop the pH adjustment (for example, by stopping the addition of basic solution) before the enzymatic hydrolysis reaction is complete. The pH then tends to decrease until the enzyme is no longer active. Alternatively, or cumulatively, it is possible to include an enzyme inactivation step as described below.

[0141] The temperature of the enzymatic hydrolysis step can be from 20 to 80°C, preferably from 30 to 70°C, even more preferably from 40 to 60°C.

[0142] Preferably, the enzymatic hydrolysis step is carried out under agitation.

[0143] The duration of the enzymatic hydrolysis step can be from 1 hour to 3 days, preferably from 6 hours to 2 days, preferably from 12 hours to 2 days, and even more preferably from 18 hours to 36 hours. Solid / liquid separation

[0144] When the starting material includes eggshells, the process according to the invention may include a solid / liquid separation step at the end of enzymatic hydrolysis, allowing the collection of a solid fraction comprising a mineral part of the eggshells, and a liquid fraction including in particular a peptide fraction.

[0145] Preferably, the solid fraction comprising a mineral part of the eggshells thus collected is dissolved by acid hydrolysis.

[0146] This acid hydrolysis can be carried out using a strong acid, preferably hydrochloric acid or sulfuric acid.

[0147] This acid hydrolysis can also be carried out using a weak acid, preferably acetic acid, oxalic acid, formic acid, ascorbic acid, citric acid.

[0148] Preferably, the mass (or molar) concentration of the acid can be from 0.01 N to 10 N, preferably from 1 to 8 N.

[0149] Acid hydrolysis of the shells allows for the dissolution of calcium salts present in eggshells, particularly calcium carbonate.

[0150] When the starting material includes shell membranes separated from the mineral part of eggshells, this solid / liquid separation may be unnecessary, and a liquid fraction can be obtained directly from enzymatic hydrolysis.

[0151] In one embodiment, the enzyme present in the liquid fraction is inactivated, for example by acidifying the medium. The exact conditions for enzyme deactivation vary depending on the enzyme used.

[0152] For example, the medium can be acidified using a solution of sulfuric acid or hydrochloric acid.

[0153] For example, the pH of the liquid fraction of the enzyme inactivation step can be adjusted to a value of 2 to 6, preferably 3 to 5.

[0154] For example, the temperature of the liquid fraction of the enzyme inactivation step can be from 20 to 80°C, preferably from 30 to 70°C, even more preferably from 40 to 60°C.

[0155] The liquid fraction can then be cooled to a temperature of 0 to 40°C, preferably from 10 to 30°C.

[0156] In one embodiment, decreasing the pH of the liquid fraction and / or decreasing the temperature leads to the precipitation of insoluble constituents. These precipitated insoluble constituents can be separated from the liquid fraction, for example, by passing the liquid fraction through a filter, such as a cellulose pre-coating filter or diatomaceous earth, for example, under vacuum. Alternatively, other separation techniques can be used to remove the precipitate, such as filter presses or centrifugation.

[0157] Insoluble constituents may include insoluble peptides (or polypeptides or proteins).

[0158] In one embodiment, the separated insoluble components are eliminated.

[0159] In another embodiment, these constituents are used, for example, in biomaterials or other textile applications.

[0160] Preferably, the liquid fraction (if necessary after separation of insoluble constituents) is subjected to one or more purification treatments, including, for example, passage through an ion-exchange column to remove most of the metallic species from the fraction. Preferably, the pH of the liquid fraction during the purification treatment(s) is between 2 and 4.

[0161] Optionally, the peptide fraction can also be separated from the polysaccharide fraction to obtain the peptide fraction on the one hand, and the polysaccharide fraction on the other. The process of the invention can therefore be used to produce a composition comprising the peptide fraction and / or a composition comprising the polysaccharide fraction. The composition comprising the polysaccharide fraction can be used as such. Alternatively, the peptide fraction and the polysaccharide fraction can be retained in the liquid fraction.

[0162] The purified liquid fraction can be concentrated, for example, by spray drying, vacuum evaporation, falling-float evaporation, rotary vacuum drying, or freeze-drying. Pre-concentration on a membrane can be performed. The pH of the fraction as it undergoes the concentration step is preferably between 4 and 6.

[0163] The invention also relates to a composition that can be obtained by the process according to the invention.

[0164] According to one embodiment, the composition that can be obtained by this process has the characteristics of the composition described above. Uses

[0165] In one embodiment, the composition according to the invention or the composition that can be obtained by the process according to the invention is used as a medicinal product, food supplement, or in a medical device or cosmetic product.

[0166] Preferably, the product is suitable for the prevention and / or treatment of dermatological conditions such as those involving disruption of the skin barrier, or joint disorders, particularly for the reconstruction of connective tissues. Preferably, the product is suitable as an anti-inflammatory, particularly in the prevention and / or treatment of joint disorders. The anti-inflammatory activity of the composition according to the invention has been demonstrated in vitro by quantifying the IL-6 and IL-10 markers in the supernatants of macrophage cultures stimulated by lipopolysaccharides.

[0167] The drug may be presented in particular as a formulation for topical use, such as a cream or lotion; or as a formulation for oral administration.

[0168] Preferably, the food supplement is suitable for the sectors of joint well-being, beauty, skin and hair health.

[0169] Preferably, the medical device can be a dressing, prosthesis or orthosis, the composition being used as a biomaterial in such a device, for example in the form of a coating.

[0170] Preferably, the cosmetic product may contain a cosmetically acceptable composition and vehicle. It may be administered orally, topically, by injection, or intraocularly and may be presented in solid, semi-solid, or emulsion form, particularly as a cream, or in liquid form, particularly as a lotion. For this purpose, it may be used for the care of hair, nails, and / or skin, and may be formulated, in particular, as a cream or lotion. Examples Example 1: Preparation of a peptide fraction Preparing the eggshells

[0171] A mass of 4000 kg of eggshells is isolated, then these are crushed, washed and drained.

[0172] Crushed eggshells are added to a production tank comprising a shaft for agitation and an oxygenation system with air supply by bubbling.

[0173] Eggshells are suspended by stirring in softened water with oxygenation of the solution. Preparation of the copper-ammonium solution and the sulfite solution for the oxidative sulfitolysis reaction

[0174] In a separate tank, 40 liters of a 25% aqueous ammonia solution (mass concentration) are mixed with 15 kg of copper sulfate (Cu(SC)4)2.5 H2O) in 1500 L of water, followed by sulfuric acid (2 mol / L concentration). -1 ) is added until a pH of 8.5 is reached.

[0175] A mass of 60 kg of sodium sulfite Na2SOs is dissolved in a volume of 500 liters of water. The resulting reducing solution is mixed and then added to the basic copper sulfate solution. The process of oxidative sulfitolysis

[0176] The copper-sulfite solution is added to the tank containing the eggshells to allow the separation of proteins corresponding to the breaking of disulfide bonds in the protein chains by oxidative sulfitolysis.

[0177] Initially, the temperature is maintained between 20 and 40°C without the addition of oxygen by bubbling.

[0178] The reaction mixture is kept under stirring with a basic pH between 8 and 10.

[0179] In the second stage, the temperature is maintained between 20 and 40°C with oxygen added by bubbling. The reaction mixture is continuously stirred at a basic pH between 8 and 10.

[0180] This operation is conducted over a period of 1 day. Enzymatic hydrolysis / enzymatic inactivation

[0181] A mass of 350 g of enzyme (alkaline serine protease) with an enzymatic activity of 580000 U / g in the medium is added to the tank with the reaction medium.

[0182] The pH in the medium is maintained between 8 and 10 and the temperature in the medium is fixed at 50°C.

[0183] The reaction mixture is placed under agitation for 24 hours.

[0184] A sodium hydroxide solution at a mass concentration of 80 g / L (2N) is added as the enzymatic hydrolysis progresses in order to stabilize the pH of the medium between 8 and 10. When the pH no longer changes, it means that the reaction is complete and therefore the addition of sodium hydroxide is no longer necessary.

[0185] The solid fraction (mineral fraction including shells) and the liquid fraction (organic matter including soluble organic substances of interest and in particular the peptide fraction) are separated.

[0186] The solid mineral fraction, including the shells, is dissolved by acid hydrolysis to extract the calcium salts. Sulfuric acid is used at a molar concentration of 98.1 g / L.

[0187] The liquid fraction containing the enzyme used is inactivated by acidifying the medium with a sulfuric acid solution at a mass concentration of 98.1 gL -1 leading to enzymatic inactivation and initial precipitation of peptides insoluble under these conditions. The temperature of the medium is 50°C with a pH of the solution fixed at 4.

[0188] After enzymatic inactivation, the temperature is then lowered to 20°C, leading to the precipitation of certain peptides and maintaining the copper in an optimal solubility and oxidation state for subsequent isolation from the soluble fraction. Isolation of the soluble peptide fraction

[0189] The precipitate is removed by passing the liquid phase through a cellulose filter.

[0190] The liquid phase (or permeate) is subjected to a passage through an ion exchange column in order to remove excess copper from the fraction.

[0191] The purified peptide fraction is concentrated by vacuum evaporation-concentration with pH adjustment. Example 2: Evaluation of the cysteine ​​content of the peptic fraction from eggshell membrane

[0192] The amino acid composition of the peptide fraction obtained from the extraction described above from eggshell membranes is evaluated by aminogram.

[0193] Sample preparation includes a hydrolysis phase using a hydrochloric acid solution (concentration of 6 mol / L). 1) for 24 hours at a temperature of 110°C in order to break the peptide bonds and thus obtain free amino acids, followed by performic oxidation of the sample.

[0194] Table 1 below summarizes the mass contents (expressed as a percentage) of each amino acid present in the peptide fraction from the shell membrane relative to the total amino acids of said peptide fraction. [Table 1] &

[0195] Table 2 below summarizes the molecular weight distribution of the peptide fraction from the shell membrane. [Table 2] < < < < < < < < < < < < < < Example 3: Evaluation of the cysteine ​​content of the peptic fraction from sheep's wool (comparative)

[0196] For comparison, the amino acid composition of the peptide fraction obtained from an extraction from sheep's wool (subjected to oxidative sulfitolysis and enzymatic hydrolysis as described above) is also evaluated by aminogram, as in Example 2.

[0197] Table 3 below summarizes the mass contents (expressed as a percentage) of each amino acid present in the peptide fraction from sheep's wool relative to the total amino acids in said peptide fraction. [Table 3] &

[0198] Thus, the peptide fraction derived from the shell membrane has a cysteine ​​mass content of 11.6% relative to the total amino acids, while the peptide fraction derived from sheep's wool has a cysteine ​​mass content of 7.6% relative to the total amino acids. The cysteine ​​mass content of the peptide fraction derived from the shell membrane is therefore significantly higher than that derived from sheep's wool.

[0199] Similarly, the peptide fraction from the shell membrane has a methionine mass content of 3.4% relative to total amino acids, while the peptide fraction from sheep's wool has a methionine mass content of 0.6% relative to total amino acids. The methionine mass content of the peptide fraction from the shell membrane is therefore significantly higher than that from sheep's wool.

Claims

Demands 1. Composition comprising a peptide fraction, in which: • the peptide fraction includes cysteine ​​and methionine; • methionine from the peptide fraction is present in a mass content greater than 1% relative to the total amino acids of the peptide fraction; • the cysteine ​​of the peptide fraction is at least partially sulfonated, the degree of sulfonation of the cysteine ​​being greater than 50%, preferably greater than 70%, even more preferably greater than 80%.

2. Composition according to claim 1, wherein the peptide fraction comprises cysteine ​​in a mass content greater than 6% relative to the total amino acids of the peptide fraction, preferably greater than 7%, more preferably greater than 8%, more preferably greater than 9%, even more preferably greater than 10%.

3. Composition according to any one of claims 1 to 2, wherein the peptide fraction comprises methionine in a mass content greater than 2% relative to the total amino acids of the peptide fraction, preferably greater than 3%; and / or the peptide fraction comprises cysteine ​​and methionine in a total mass content greater than 7% relative to the total amino acids of the peptide fraction, preferably greater than 8%, more preferably greater than 9%, more preferably greater than 10%, even more preferably greater than 12%.

4. Composition according to any one of claims 1 to 3, wherein the peptide fraction has a mass average molecular weight Mw of 500 to 10000 g / mol, preferably of 700 to 5000 g / mol, preferably again of 1000 to 2000 g / mol.

5. Composition according to any one of claims 1 to 4, further comprising: • a polysaccharide fraction, preferably comprising galactomannans, chitin and / or glycosaminoglycans, the glycosaminoglycans preferably comprising chondroitin sulfate, dermatan sulfate, keratan sulfate and / or hyaluronic acid; wherein the polysaccharide fraction more particularly preferably comprises hyaluronic acid.

6. A process for preparing a composition, comprising an oxidative sulfitolysis step of a starting material, said starting material comprising shell membranes.

7. A process according to claim 6, wherein the oxidative sulfitolysis step is carried out in the presence of a sulfite salt, preferably sodium sulfite.

8. A process according to any one of claims 6 to 7, wherein the oxidative sulfitolysis step is carried out in the presence of a metal as a catalyst, preferably a divalent metal, even more preferably copper.

9. A process according to any one of claims 6 to 8, wherein the oxidative sulfitolysis step is carried out in the presence of an oxidizing agent, preferably dioxygen.

10. A process according to any one of claims 6 to 9, comprising an enzymatic hydrolysis step of the product from the oxidative sulfitolysis step, preferably by a serine protease.

11. A method according to any one of claims 6 to 9, wherein the starting material comprises eggshells.

12. A process according to claim 11, comprising a solid / liquid separation step enabling the collection of a solid fraction comprising a mineral part of the eggshells and a liquid fraction comprising a peptide fraction.

13. A process according to any one of claims 6 to 12, comprising a step of precipitating an insoluble peptide fraction and removing it.

14. Composition obtainable by the process according to any one of claims 6 to 13.

15. Composition according to any one of claims 1 to 6 or according to claim 14, for use as a medicinal product, preferably for the treatment and / or prevention of a dermatological condition, cosmetic dermatology, or joint pathology.

16. Use of the composition according to any one of claims 1 to 6 or according to claim 14, for the manufacture of a food supplement, or a medical device or a cosmetic product.

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