Heparin and a mixture of native proteins and peptides from waste tissues of slaughtered animals

Abstract

The present invention relates to a method for obtaining heparin, native proteins and peptides, and other compounds of interest, by fractionation of mammalian intestinal mucosa. Certain new fractions or extracts of the mucosa containing said compounds are also disclosed.

Description

[Technical field]

[0001] This application claims the benefit of European Patent Application No. 22382214.9, filed March 8, 2022.

[0002] The present invention relates to the field of obtaining heparin, proteins and peptides in native form and other compounds of interest from animals slaughtered for meat consumption. [Background technology]

[0003] In the processing of some parts of animals slaughtered mainly for food consumption, the remaining extracted viscera, which are generally not offered to meat consumers other than the parts used as food, are further processed to obtain products of several purposes in other fields. For example, it is widely known how to obtain heparin of high purity grade, useful in medicine, from mucosal tissues, especially from the intestinal mucosa. After recovery of the mucosa, the intestinal tubular structure is reused for many different purposes, including even medical applications, mainly as casings or grafts in sausage processing.

[0004] These processes for extracting heparin involve, for example, aggressive mechanical homogenization of mucosal tissue (i.e. intestine) in the presence of proteolytic enzymes and at high temperatures (60°C-80°C or even higher) and / or by salting out the homogenate. This is followed by a precipitation step, an optional extraction step and chromatography. These methods yield, on the one hand, heparin in several purity grades depending on the steps of the process, and, on the other hand, protein hydrolysates, which are considered by-products and are often used in feed as peptide supplements. Examples of literature that disclose methods for the extraction of heparin and other by-products from the small intestinal mucosa include Chinese Patent Application No. 107236059 (Shenqing) and Chinese Patent No. 106035980B (Jiangnan University).

[0005] Proteins in their natural state, with their intact structure and function, that have not been denatured by heat, chemicals, enzymatic reactions or other denaturing agents, are called "native proteins". Up to now, these heparin extraction methods do not obtain proteins or peptides in native form, because high temperatures, enzymatic treatments and mechanical stress reduce them to peptide hydrolysates, which are mainly composed of very small peptides and free amino acids that are not of high value. On the other hand, the requirements imposed by health authorities for regulatory and medical use to obtain heparin with appropriate purity grades do not allow many variations on the methods for obtaining said compound.

[0006] Active enzymes and mixtures of active enzymes have also been obtained from mucosal tissues of slaughtered animals. For example, China Patent No. 102839162B (Tibet Liyang technology Co Ltd) discloses a method for obtaining alkaline phosphatase from animal viscera (including porcine intestinal mucosa), in which the mucosa is first cut, homogenized at 4°C, then precipitated with acid, extracted with butanol, and dialyzed. It is further salted out with ammonium sulfate, and finally the salted out fraction is subjected to several chromatography columns for further purification. An alkaline phosphatase product with a specific activity of 415U / mg is thus obtained. However, this method does not allow the option of obtaining heparin or other enzymes in accordance with established regulations.

[0007] Proteolytic enzymes are obtained from fish digestive tracts, for example, according to the method disclosed in Russian Patent No. 2610669, in which the tissue is homogenized, filtered and the proteins are salted out with ammonium sulfate, with a prior step of removing nucleic acids and lipids, the precipitate is then dissolved in a buffer and subjected to chromatography.

[0008] All these methods for recovering one or more enzymes in active form from animal mucosal tissues include a series of steps designed to preserve the enzyme integrity, they are carried out at low and moderate temperatures (4°C to 37°C) and they are carried out under physiological conditions (pH and ionic forces).

[0009] In all the methods disclosed above, either for the extraction of heparin or obtaining active enzyme, the main objective is to recover the desired product in a balanced condition of high yield, high activity and high purity.

[0010] Besides these methods of extraction from the tissues of slaughtered animals, many enzymes are currently obtained using biotechnological methods, including the expression of enzymes in bioreactors. In these biotechnological methods, host cells (yeast, bacteria, etc.) are modified using recombinant gene techniques to express the enzyme of interest when cultivated. However, these methods are usually expensive and require many reagents and energy suppliers to control the growth conditions of the cells. Therefore, they are usually considered to be methods with a certain degree of complexity.

[0011] Enzymes are used in several industrial and medical fields: cellulases and ligninases in the biofuel industry, nucleases in molecular biology, amylases and proteases in food processing, and xylanases in the paper industry. Many enzymes are also used in clinical diagnostics. In the field of feed supplements and additives, phytases have been used to release phosphorus from phytic acid, which is commonly found in feed compositions.

[0012] Regardless of existing methods, there remains a need for alternative methods for valorizing the products of slaughtered animals, which are obtained during a reproducible, uncomplicated and inexpensive process, which allows obtaining a high yield of useful by-products as reliably as possible, and which comply with regulations, if any. In the case of certain enzymes, there remains a need in the field of food additives for active enzymes which can be obtained by a reliable and economical process. Summary of the Invention

[0013] With the aim of valuing as many products as possible from discarded tissues of slaughtered animals, the inventors propose a new method that allows to simultaneously obtain mammalian intestinal mucosa heparin and proteins and peptides in native form in an inexpensive and sustainable manner. Heparin is obtained in a grade useful for pharmaceutical applications (i.e., pharmaceutical grade compliant with regulations). Furthermore, proteins and peptides are obtained in their native state, so that they are in their properly folded and assembled form with operative structure and function (e.g., enzymatic, structural, hormonal, etc.).

[0014] The present invention is therefore embedded in the context of slaughterhouse tissue processing where value-added products are obtained after tissue homogenization such as heparin.

[0015] This new method involves homogenization of mammalian tissues under mild conditions (low temperature and physiological pH) that help preserve the stability and activity of isolated proteins and peptides. Several additional steps are also performed, including fractionation and chromatography of the homogenate material.

[0016] Accordingly, a first aspect of the present invention comprises the steps of: (i) adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa to obtain preserved mucosa, or storing the aqueous extract at a temperature that allows the proteins, peptides and heparin to be preserved in a native state; (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa; (iii) subjecting the diluted mucosa of step (ii) or the preserved mucosa of (i) to homogenization at a temperature below 40° C. to lyse the cell membranes of the mucosal cells and obtain a stable homogenate of the mucosa containing heparin and mucosal proteins and peptides; (iv) separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iii) by one or more physical and / or chemical means selected from the group consisting of centrifugation, filtration and / or ultrafiltration, optionally using a detergent, and combinations thereof, to obtain a pellet (P) containing heparin and a supernatant fraction (SN) containing proteins and mucosal proteins and peptides; (va) subjecting the pellet fraction (P) of (iv) containing heparin to an alkaline proteolysis process with a proteolytic enzyme to obtain a mixture of heparin and protein hydrolysates, and subjecting the heparin-containing mixture to one or more precipitation steps, and optionally one or more extraction steps, and one or more chromatography steps to purify said heparin from the protein hydrolysates; and (vb) subjecting the supernatant (SN) of (iv) containing the mixture of solubilized and / or suspended mucosal proteins and peptides to one or more of the following successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and / or ion exchange chromatography steps in order to separate the proteins and peptides according to either their solubility, isoelectric point and / or molecular weight (the order or sequence of these steps may be interchangeable). The present invention relates to a method for simultaneously obtaining heparin and proteins and peptides in the native state by fractionation of mammalian intestinal mucosa, comprising:

[0017] Therefore, the following steps: (i) adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa to obtain preserved mucosa, or storing the aqueous extract at a temperature that allows the proteins, peptides and heparin to be preserved in a native state; (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa; (iii) subjecting the diluted mucosa of step (ii) or the preserved mucosa of (i) to homogenization at a temperature below 25° C. to lyse the cell membranes of the mucosal cells and obtain a stable homogenate of the mucosa containing heparin and mucosal proteins and peptides; and (iv) separating the heparin and mucosal proteins and peptides, typically contained in the stable homogenate obtained in step (iii), by one or more physical and / or chemical means selected from the group consisting of centrifugation, filtration and / or ultrafiltration, optionally using a detergent, and combinations thereof, to obtain a pellet (P) containing the heparin and a supernatant fraction (SN) containing the proteins and mucosal proteins and peptides. A method is disclosed for the simultaneous access of heparin and native proteins and peptides by fractionation of mammalian intestinal mucosa, comprising:

[0018] Another aspect of the present invention is a method for producing a method for manufacturing a semiconductor device comprising the steps of: (i) adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa to obtain preserved mucosa, or storing the extract at a temperature that allows the proteins, peptides and heparin to be preserved in a native state; (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa; (iiia) subjecting the diluted mucosa of step (ii) or the preserved mucosa of (i) to homogenization at a temperature below 40° C. and optionally in the presence of detergents and / or hydrolytic enzymes such as phospholipases to lyse the cell membranes of the mucosal cells to obtain a stable homogenate of the mucosa containing heparin and mucosal proteins and peptides; (iva) separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iiia) by one or more physical and / or chemical means selected from the group consisting of centrifugation and filtration (microfiltration and / or ultrafiltration), optionally using a detergent, and combinations thereof, to obtain a pellet (P) containing heparin and a supernatant fraction (SN) containing proteins and mucosal proteins and peptides, (v.a') subjecting the pellet fraction (P) of (iva) containing heparin to an alkaline proteolysis process with proteases to obtain a mixture of heparin and protein hydrolysates, and subjecting the heparin-containing mixture to one or more precipitation steps, and optionally one or more extraction steps, and one or more chromatography steps to purify said heparin from the protein hydrolysates; (v.b') subjecting the supernatant (SN) of (iva) containing the mixture of solubilized and / or suspended mucosal proteins and peptides to one or more of the following successive filtration, ultrafiltration steps, solvent extraction, protein precipitation, enzymatic hydrolysis, gel permeation, ion exchange chromatography, size exclusion chromatography or affinity chromatography steps in order to separate the proteins and peptides according to either their solubility, isoelectric point and / or molecular weight (the order or sequence of these steps may be interchangeable). The present invention relates to a method for simultaneously obtaining heparin and proteins and peptides in the native state by fractionation of mammalian intestinal mucosa, comprising:

[0019] A further aspect of the present invention is a method for obtaining heparin by fractionation of mammalian intestinal mucosa comprising steps (i), (ii), (iiia), (iva) and (v.a') as defined herein.

[0020] A further aspect of the present invention is a method for obtaining proteins and peptides in the native state by fractionation of mammalian intestinal mucosa comprising steps (i), (ii), (iiia), (iva) and (v.b') as defined herein.

[0021] Several compositions containing the compounds of interest are obtained by this method, and the compositions, particularly the fractions from mammalian intestinal mucosa containing purified acidic or basic proteins (i.e., enzymes), are then useful for the general applications for which they are known to be useful or for any new applications.

[0022] Accordingly, another aspect of the present invention is a composition comprising an acidic fraction of mammalian intestinal mucosa acidic proteins and peptides, comprising: (vb1') subjecting the supernatant (SN) of step (v.b') containing the mixture of solubilized and / or suspended mucosal proteins and peptides to filtration through a filter with a maximum cutoff of 100 μm to obtain a filtered supernatant containing the majority of mucosal proteins and peptides, in order to retain small particles and lipid residues that were not pelleted in step (iva); (vb4') subjecting the supernatant of step (vb1') to an anion exchange chromatography column to obtain a bound fraction (SNbf') and an unbound fraction (SNubf') and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cut-off of 1-5 KDa to obtain an acidic fraction of the supernatant containing acidic proteins and peptides. and wherein the process comprises steps (i), (ii), (iiia) and (iva) as defined herein, Or alternatively, (vb1') subjecting the supernatant (SN) of step (v.b') containing the mixture of solubilized and / or suspended mucosal proteins and peptides to filtration through a filter with a maximum cutoff of 100 μm in order to retain small particles and lipid residues that were not pelleted in step (iva) to obtain a filtered supernatant containing the majority of the mucosal proteins and peptides; (vb4a) subjecting the supernatant of step (vb1') to a cation exchange chromatography column to obtain a bound fraction (SNbf) and an unbound fraction (SNubf), and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa to obtain a basic fraction of the supernatant containing basic proteins and peptides; (vb5a) subjecting the unbound fraction (SNubf) of step (vb4a) to an anion exchange chromatography column to obtain a bound fraction (SNubfbf) and an unbound fraction (SNubub), and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa to obtain a supernatant acidic fraction containing acidic proteins and peptides. The present invention relates to a composition obtainable by a process comprising steps (i), (ii), (iiia) and (iva) as defined herein, further comprising:

[0023] Another aspect of the present invention is a method for producing a (vb5') subjecting the unbound fraction (SNubf') to a cation exchange chromatography column to obtain a bound fraction (SNubfbf') and an unbound fraction (SNubub'), and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1 to 5 KDa to obtain a supernatant basic fraction containing basic proteins and peptides. and further comprising steps (i), (ii), (iiia), (iva), (vb1') and (vb4') as defined herein for obtaining an acidic fraction of the supernatant comprising acidic proteins and peptides, or alternatively, obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1′) and (vb4a) as defined herein, The basic fraction of basic proteins and peptides of the mammalian intestinal mucosa.

[0024] Another aspect of the present invention relates to a composition S Nubub' comprising mammalian intestinal mucosa proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1'), (vb4') and (vb5') as defined herein.

[0025] Another aspect of the present invention relates to a composition S Nubub comprising mammalian intestinal mucosa proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1'), (vb4a) and (vb5a) as defined herein.

[0026] The present invention also provides fractions containing pharmaceutical grade heparin, as illustrated in the examples, and therefore heparin that complies with all regulatory aspects by health authorities.

[0027] In the context of this method, a mixture of native proteins of mammalian intestinal mucosa, including those with high enzymatic activity, is obtained in high yields. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] All terms used herein in this application are to be understood in their ordinary meanings known in the art unless otherwise specified. Other more specific definitions for certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims unless a specifically and explicitly set forth definition provides a broader definition.

[0029] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise specified, definite articles used herein, such as "the," also include plural nouns.

[0030] As used herein, the term "about" or "around" refers to a range of values ​​of ±10% of the specified value. For example, the expression "about 10" or "around 10" includes ±10% of 10, i.e., 9 to 11.

[0031] When enzyme activity is stated, it is defined as moles of substrate converted per unit time = rate x reaction volume. Enzyme activity is a measure of the amount of active enzyme and therefore depends on the conditions (mainly pH and temperature). The SI unit is the katal (1 katal = 1 mol / sec), which is too large. A more practical and commonly used value is the enzyme unit (U) = 1 μmol / min. 1 U corresponds to 16.67 nanokatals.

[0032] The specific activity of an enzyme is another common unit. It is the activity of the enzyme per milligram of total protein (expressed in μmol / min / mg). Specific activity gives a measure of the enzyme purity in a mixture. It is the micromoles of product formed by the enzyme under given conditions in a given amount of time (min) per milligram of total protein. Specific activity is equal to the reaction rate multiplied by the reaction volume divided by the mass of the total protein. The SI unit is katal / kg, but a more practical unit is μmol / mg / min. Specific activity is a measure of the enzyme processivity (the ability of the enzyme to process) at a specific (usually saturating) substrate concentration and is usually constant for pure enzymes.

[0033] The enzymatic activity of the enzyme obtained by the described method can also be expressed in units of activity per kilogram of mucosa (expressed in μmol / min / kg). This parameter gives a measure of the amount of enzyme extracted from the initial raw material.

[0034] The term "mammalian intestinal mucosa" according to the present invention is a term used in the technical terminology of processing animal by-products in slaughterhouses, such as visceral material from animals. It includes all the material obtained after scraping of the intestine, i.e. the small intestine. This scraping results in a clean tube / membrane, and the scraped material is the so-called mammalian intestinal mucosa, a complex mixture including the intestinal epithelium, the lamina propria (mucosa) and the muscularis mucosa (mucosa), the microvilli including the brush border, and the lumen. This complex mixture is in the present application subjected to homogenization and further processing in some examples and embodiments to obtain the enzymes and heparin of interest.

[0035] As mentioned above, the first aspect of the present invention comprises the steps of: (i) adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa to obtain preserved mucosa, or storing the extract at a temperature that allows the proteins, peptides and heparin to be preserved in a native state; (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa; (iii) subjecting the diluted mucosa of step (ii) or the preserved mucosa of (i) to homogenization at a temperature below 40° C. to lyse the cell membranes of the mucosal cells and obtain a stable homogenate of the mucosa containing heparin and mucosal proteins and peptides; (iv) separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iii) by one or more physical and / or chemical means selected from the group consisting of centrifugation, filtration and / or ultrafiltration, optionally using a detergent, and combinations thereof, to obtain a pellet (P) containing heparin and a supernatant fraction (SN) containing proteins and mucosal proteins and peptides; (va) subjecting the pellet fraction (P) of (iv) containing heparin to an alkaline proteolysis process with a proteolytic enzyme to obtain a mixture of heparin and protein hydrolysates, and subjecting the heparin-containing mixture to one or more precipitation steps, and optionally one or more extraction steps, and one or more chromatography steps to purify said heparin from the protein hydrolysates; and (vb) subjecting the supernatant (SN) containing the mixture of solubilized and / or suspended mucosal proteins and peptides to one or more of successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and / or ion exchange chromatography steps in order to separate the proteins and peptides according to either their solubility, isoelectric point and / or molecular weight (the order or sequence of these steps may be interchangeable). The present invention relates to a method for simultaneously obtaining heparin and proteins and peptides in the native state by fractionation of mammalian intestinal mucosa, comprising:

[0036] Steps (va) and (vb) are also referred to herein as (vP) and (v.SN), respectively, to refer to the management or further processing of the (P) and (SN) fractions of (iv) that allow simultaneous access to mammalian intestinal mucosa heparin and proteins and peptides in the native state.

[0037] "Simultaneous" should be understood to include not only simultaneous processing with the necessary adapted equipment, but also processing in which two compositions of interest are obtained from the same intestinal mucosa, different in time or place. In other words, heparin is obtained, while other proteins and peptides of interest are largely preserved and also purified from other fractions obtained from the same mucosa. This is true of the method of the present invention, since up to now the proteolytic steps applied in processes for obtaining heparin in high yields from mammalian intestinal mucosa only allowed to obtain mixtures of peptides other than heparin, and thus non-functional, i.e. native proteins and peptides.

[0038] The inventors have surprisingly discovered a combination of steps and pretreatments which, when combined, allow for obtaining both heparin and native proteins and peptides (including functional enzymes) of mammalian intestinal mucosa with high yields and purity grades.

[0039] The resulting fractions contain several purified grades of heparin or proteins and peptides depending on the type, number and sequence of the physical and / or chemical means applied in steps (iv) and subsequently (va) or (vb). Naturally, the content of each of the fractions in each case is a valuable product or composition resulting from the method of the invention, which allows the simultaneous access to heparin and a myriad of native and functional proteins and peptides from the mammalian intestinal mucosa of the slaughtered animal.

[0040] In the following paragraphs, specific embodiments of the type and number and sequence of these physical and / or chemical means applied are illustrated. The accompanying examples also serve to illustrate the yield of the method for obtaining a fraction containing a notable amount of heparin, while obtaining a particular enzyme of interest in a native and functional state in notable yield.

[0041] In certain embodiments of the method of the first aspect, the mammalian intestinal mucosa is porcine. Other mammalian species, including non-human, are also useful.

[0042] In another particular embodiment of the method, the preservative and antioxidant in step (i) is sodium metabisulfite added to a concentration of 4% w / w, preferably 2% w / w. Equivalent substances can be selected as preservatives and antioxidants.

[0043] In another particular embodiment, optionally in combination with any of the embodiments of the method above or below, (ii) dilute the preserved mucosa with up to 50 volumes of deionized water, more particularly with 1 to 25 volumes of deionized water, even more particularly with 1 to 15 volumes of deionized water, and even more particularly with 2 to 4 volumes of deionized water. In a more particular embodiment, the deionized water is replaced with an equal volume of a buffer solution having a pH comprised between 5 and 9, preferably a pH of 7.

[0044] In a more particular embodiment of the method, in step (ii), the mucosa is diluted with either deionized water or a buffer solution, more particularly with both, containing one or more surfactants. In even more particular embodiments, the surfactant is a non-ionic surfactant or a zwitterionic surfactant. Examples of commercially available surfactants are selected from the group consisting of Tween 20®, Tween 80, Triton X-100 or X-114, deoxycholic acid, Brij 35 or 58, BigCHAP or deoxy BigCHAP, MEGA-8, MEGA-9 or MEGA-10, octyl β-glucoside, and combinations thereof.

[0045] Steps (iii), (iv), (va), (vb), (vb1), (vb4) and (vb5) are equivalent to steps (iiia), (iva), (v.a'), (v.b'), (vb1'), (vb4a) and (vb5a), respectively. All embodiments defined herein for steps (iii), (iv), (va), (vb), (vb1), (vb4) and (vb5) also apply to steps (iiia), (iva), (v.a'), (v.b'), (vb1'), (vb4a) and (vb5a), respectively.

[0046] In certain embodiments, step (iii) or step (iiia) is carried out at a temperature of about 35° C. or less, or alternatively about 30° C. or less, or alternatively about 25° C. or less, or alternatively about 20° C. or less, or alternatively about 15° C. or less, or alternatively about 10° C. or less.

[0047] In another particular embodiment, step (iii) or step (iiia) is carried out at a temperature between 0° C. and 35° C., or alternatively between 0° C. and 30° C., or alternatively between 0° C. and 25° C., or alternatively between 0° C. and 20° C., or alternatively between 0° C. and 15° C., or alternatively between 0° C. and 10° C. In yet another particular embodiment, step (iiia) is carried out at a temperature between 0° C. and 8° C. In yet another particular embodiment, step (iii) is carried out at a temperature between 0° C. and 8° C.

[0048] In another particular embodiment of the method, the homogenization step (iii) is carried out by a technique selected from the group consisting of mechanical or physical cell lysis of the mammalian intestinal mucosa by mechanical disruption, fluid shear forces, high pressure or cavitation.

[0049] In another particular embodiment of the method, homogenization step (iiia) is carried out by a technique selected from the group consisting of mechanical or physical cell lysis of the mammalian intestinal mucosa by mechanical disruption, fluid shear forces, high pressure or cavitation. In another particular embodiment, homogenization step (iiia) is carried out in the presence of a detergent or a hydrolytic enzyme such as a phospholipase to detach proteins bound to membranes or cellular structures.

[0050] In a more particular embodiment, said homogenization step (iii) is carried out by mechanical disruption of the mucosal cells with a rotor-stator homogenizer at a g-force of at least 180 g, preferably 1700 g, for a minimum of 30 seconds, preferably 2 to 4 minutes.

[0051] In a particular embodiment, homogenization step (iiia) is performed by mechanical disruption of the mucosal cells with a rotor-stator homogenizer, more particularly at a g-force of at least about 150 g, about 180 g, about 500 g, about 750 g, about 1000 g, about 1250 g, about 1500 g, about 1700 g, about 1750 g or about 2000 g, more particularly between 1500 and 2000 g. More particularly, homogenization is performed at about 1700 g.

[0052] In certain embodiments, the homogenization step (iiia) is carried out for at least about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230 or about 240 seconds. More particularly, the homogenization is carried out for 120-240, 130-230, 140-220, 150-210, 160-200 or 170-190 seconds, even more particularly for 120-240 seconds.

[0053] In a more particular embodiment, the homogenization step (iiia) is carried out by mechanical disruption of the mucosal cells with a rotor-stator homogenizer at a g-force of at least 180 g, preferably 1700 g, for a minimum of 30 seconds, preferably 2 to 4 minutes.

[0054] Step (iva) comprises separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iiia) by one or more physical and / or chemical means selected from the group consisting of centrifugation, filtration and / or ultrafiltration, optionally using detergents, and combinations thereof, to obtain a pellet (P) containing heparin and a supernatant fraction (SN) containing the proteins and mucosal proteins and peptides.

[0055] The separation step (iva) may be carried out by methods well known in the art. The skilled artisan will be able to determine the exact separation conditions depending on the protein of interest.

[0056] Yet another particular embodiment of the method of the invention comprises performing a step (iv) of separation by centrifugation to obtain a pellet fraction containing heparin (P) and a supernatant fraction containing mucosal proteins and peptides, some of which are in solution and others in suspension (SN). These (P) and (SN) fractions are further processed in respective steps (vP) or (va) and (v.SN) or (vb) (both notations are interchangeable) as described above in the first aspect.

[0057] In a particular embodiment, the centrifugation in (iva) is carried out at a g-force of at least about 150 g, about 500 g, about 750 g, about 1000 g, about 1250 g, about 1500 g, about 1750 g, about 2000 g, about 2250 g, about 2500 g, about 2750 g, about 3000 g, about 3100 g, about 3200 g, about 3300 g, about 3400 g or about 3500 g, in particular 150-3500 g, 500-3400 g, 750-3300 g, 1000-3200 g, 1250-3100 g, 1500-3000 g, 1750-2750 g, 2000-2500 g, more particularly 3000-3500 g.

[0058] In certain embodiments, the centrifugation in (iva) lasts for at least about 5, about 10, about 15, about 20, about 25, about 30 or about 35 minutes, in particular for 5-35, 10-30 or 15-25 minutes, and more particularly for 20-30 minutes.

[0059] In a particular embodiment, the centrifugation in (iva) is carried out at a temperature of about 40°C, about 37°C, about 35°C, about 30°C, about 25°C, about 20°C, about 15°C, about 10°C, about 8°C, about 5°C, about 2°C or below about 0°C, in particular at a temperature of 0-40°C or 2-37°C or 5-35°C or 10-30°C or 15-25°C, more particularly the centrifugation in (iva) is carried out at a temperature of 0-25°C, even more particularly at a temperature of 0-8°C.

[0060] In a most particular embodiment, the centrifugation in (iv) is carried out at a g-force of at least 150 g, preferably 3000 g, and at a temperature below 40° C., in particular below 25° C., more preferably below 8° C., for at least 5 minutes, preferably 20 minutes, to obtain a solid phase (hence the pellet phase (P)) comprising the heparin, and a liquid phase (hence the supernatant phase (SN)) comprising the mixture of mucosal proteins and peptides, some of which are liquid and others are in suspension, as described above.

[0061] In a most particular embodiment, the centrifugation in (iva) is carried out at a g-force of at least 150 g, preferably 3000 g, and at a temperature below 40° C., in particular below 25° C., more preferably below 8° C., for at least 5 minutes, preferably 20 minutes, to obtain a solid phase (hence the pellet phase (P)) comprising the heparin, and a liquid phase (hence the supernatant phase (SN)) comprising the mixture of mucosal proteins and peptides, some of which are liquid and others are in suspension, as described above.

[0062] In a more particular embodiment, the centrifugation in (iva) is carried out at a g-force of 3000-3500 g and at a temperature of 0-40° C. or 0-37° C. or 0-25° C. or 0-8° C. for 15-35 minutes, in particular 20-30 minutes.

[0063] In a more particular embodiment, the centrifugation in (iva) is carried out at a g-force of about 3134 g and at a temperature of less than about 8° C. for about 20 minutes.

[0064] In a more particular embodiment, the centrifugation in (iva) is carried out at a g-force of about 3300 g and at a temperature of less than about 37° C., particularly 0-25° C., for about 30 minutes.

[0065] Another particular embodiment of the method of the invention also comprises a separation step (iva) by filtration or ultrafiltration to obtain a pellet (retentate) fraction containing the heparin and a supernatant (permeate) fraction containing the mucosal proteins and peptides, some of which are in solution and others in suspension. These (P) and (SN) fractions are further processed in respective steps (vP) or (v.a') and (v.SN) or (v.b') (both notations are interchangeable) as described above in the first aspect.

[0066] In a particular embodiment, the supernatant (SN) obtained in step (iva) comprises one or more of alkaline phosphatase (also referred to herein as ALP), mucin-2 (also referred to herein as MUC2) and lysozyme (also referred to herein as LYZ), more particularly alkaline phosphatase, mucin-2 and lysozyme. More particularly, the SN comprises one or more of glucagon-like peptide-1 (also referred to herein as GLP-1), thymosin β4, vasoactive intestinal peptide, lysozyme, lactotransferrin, regenerating islet-derived protein 3 (Reg III), regenerating islet-derived protein IV (Reg IV), matrix metalloproteinase-9, collagenase type IV of 72 kDa, interstitial collagenase, bactericidal permeability-enhancing protein, pulmonary surfactant-associated protein D, antimicrobial protein PR-39, angiogenin, alkaline phosphatase, mucin-2, trypsin, aminopeptidase, carboxypeptidase, catalase, triacylglycerol lipase, phospholipase, acid sphingomyelinase-like phosphodiesterase, α-amylase, α-galactosidase, sucrase-isomaltase, peroxiredoxin-6, superoxide dismutase, apolipoprotein A1, annexin-5, galectin-1, diazepam binding inhibitor, immunoglobulins, gastrotropins and hemoglobin, and more particularly all of them.

[0067] As mentioned above, in steps (vP) or (va), the pellet (P) of the embodiment before the centrifugation step is applied is subjected to an alkaline proteolysis process with proteases to obtain heparin and protein hydrolysates, with the aim of recovering the majority of the heparin of the homogenate in very high purity, followed by a precipitation step, an optional extraction step and an optional chromatography step to purify the heparin.

[0068] This further step of alkaline proteolysis process and heparin purification is equivalent to a standard known as a master file batch for the extraction of heparin and provides pharmaceutical grade heparin as well as a separate fraction containing protein hydrolysates that are typically used as a feed additive (as described in the Background section and in the previous paragraph).

[0069] The method of the present invention therefore provides heparin and protein hydrolysates extracted from mammalian intestinal mucosa, which are obtainable by a method defined in the first aspect and embodiments thereof comprising an alkaline proteolysis process step.

[0070] In parallel with the extraction of heparin in step (v.SN) or (vb), in another particular embodiment of the first aspect, also optionally in combination with any of the above or below embodiments, a supernatant fraction (SN) is obtained comprising a mixture of solubilized and / or suspended mucosal proteins and peptides when the centrifugation step in (iv) for obtaining it is carried out, and is subjected to one or more of successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and / or ion exchange chromatography steps (the order or sequence of these steps is interchangeable) in order to separate the proteins and peptides according to any of their solubility, isoelectric point and molecular weight.

[0071] This purification process of SN (i.e., step (v.SN)) involves, in some embodiments, the separation of all soluble proteins and peptides from the insoluble or suspended ones. The SN is then subjected to a diafiltration process through cassettes or cartridges with a cutoff of 300 kDa to 0.45 μm, preferably 0.1 μm.

[0072] The retentate (SNr) of this diafiltration step contains small particles, lipid residues and all water-insoluble or partially water-soluble mucosal proteins and peptides, the latter of which bind to other cellular structures such as cell membranes or organelles or other insoluble proteins to form aggregates that are not pelleted in the centrifugation in step (iv) of the method and are therefore not filterable (i.e., diafilterable).

[0073] On the contrary, the permeate (SNp) of this diafiltration step contains all water-soluble proteins and peptides having a molecular weight below the cut-off of the filter applied in the diafiltration step described above.

[0074] In a further purification step, fraction SNp is further diafiltered and concentrated through a filter with a cut-off of 1–5 kDa, with the aim to retain all soluble proteins and peptides contained in SNp and to obtain an enriched fraction of soluble proteins and peptides (SNpr).

[0075] In a more particular embodiment, when SNp is diafiltered to obtain SNpr, diafiltration is performed using a tangential flow filtration device, in an even more particular embodiment, diafiltration is performed by replacing the permeate with at least 4 volumes, preferably 5-10 volumes, of deionized water or a suitable buffer.

[0076] In a particular embodiment, optionally in combination with any of the above or below embodiments, the SNp (or SNpr) containing the solubilized mucosal proteins and peptides is then subjected to a further separation technique according to the isoelectric point of the proteins and peptides. In other words, the SNp (or SNpr) containing the solubilized mucosal proteins and peptides is subjected to one or more other steps of successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and / or ion exchange chromatography steps (the order or sequence of these steps is interchangeable) that allow the separation of said proteins and peptides according to their isoelectric point, which is also included in step (vb).

[0077] In a particular embodiment of step (vb), the SNp obtained as indicated in the previous paragraph and containing soluble mucosal proteins and peptides is preferably adjusted to a pH of 7.0 before being loaded in downflow onto a cation exchange chromatography column to obtain a bound fraction (SNpbf) and an unbound fraction (SNpubf), and the bound fraction is further eluted in upflow direction, then diafiltered and concentrated using a tangential flow system with a filter having a cut-off of 1-5 KDa, preferably 3 KDa, to obtain a basic fraction (also referred to herein as "basic fraction" or BF) containing basic proteins and peptides. Alternatively, the cation exchange chromatography can be performed in batch by adding 5-15% (w / w), preferably 10%, of a strong cation exchange resin to the fraction.

[0078] This step of cation exchange chromatography, particularly strong cation exchange chromatography, results in the enrichment of all basic proteins in the SNp. In a particular embodiment, elution and diafiltration of the bound fraction (SNpbf) is performed with phosphate buffer (50 mM) at a pH of 6.5-7.5.

[0079] In another particular embodiment of the method, when SNp is subjected to a cation exchange chromatography column, the unbound fraction (SNpubf) is preferably adjusted to a pH of 7.0 before being loaded onto an anion exchange chromatography column and further eluted in upflow direction, then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa, preferably 3 KDa, to obtain an acidic fraction (also referred to herein as "acidic fraction" or AF) containing acidic proteins and peptides.

[0080] This further step of anion exchange chromatography, particularly strong anion exchange chromatography, results in the enrichment of all acidic proteins in the SNp. In a particular embodiment, elution and diafiltration of the bound fraction is performed with phosphate buffer (50 mM) at a pH of 6.5 to 7.5.

[0081] Alternatively, acidic proteins and peptides can be captured before basic proteins and peptides.

[0082] In more particular embodiments, acidic proteins and peptides are captured before basic proteins and peptides.

[0083] When an ion exchange chromatography step is used, the rationale behind this separation is the isoelectric point of the protein or peptide, which provides a positively or negatively charged protein at a given pH.

[0084] Once either the acidic or basic fractions are obtained, these fractions are, in another particular embodiment, subjected to further fractionation steps by diafiltration, salting out methods, gel permeation chromatography, cation and anion exchange chromatography, and combinations thereof.

[0085] In another particular embodiment, either the basic or acidic fraction is further subjected to fractionation by diafiltration, salting out methods, protein precipitation, solvent extraction, cation and anion exchange chromatography, size exclusion chromatography, affinity chromatography and combinations thereof.

[0086] Alternatively, all these fractionation steps can be performed in a different order.

[0087] This subsequent fractionation yields purified fractions, even fractions that contain only one of the native proteins or peptides from the mammalian intestinal mucosa in a buffered aqueous solution.

[0088] In particular embodiments of the method, a series of tangential flow diafiltration and associated concentration of the permeate results in fractionation by molecular weight of both the basic and acidic fractions, which in even more particular embodiments are fractionated into the following molecular weight ranges: ·Over 300Kda 100~300Kda 50~100Kda 30~50Kda 10~30Kda 5~10Kda 1~5Kda It is characterized by:

[0089] In a more particular embodiment, where the process of the invention comprises further fractionation by molecular weight of the obtained basic and acidic fractions as set out in the previous embodiment, both the basic and acidic fractions from said step are further fractionated by molecular weight by gel permeation chromatography.

[0090] In another particular embodiment of the method, both the basic and the acidic fractions are fractionated by salting out methods, more particularly by addition of ammonium sulfate or by addition of organic solvents such as acetone or butanol.

[0091] "Salting out method" is to be understood as a process of precipitation of proteins due to the presence of salts in the medium, which reduces the solubility of the particular protein.

[0092] In yet another specific embodiment of the method, both the basic and acidic fractions are loaded onto weak cation and anion exchange chromatography columns, respectively, and single (isolated) proteins and peptides are eluted by ionic strength or pH gradient.

[0093] In another embodiment of the invention, the SNr fraction is processed to obtain products containing enzymatic activities of chemical, nutraceutical or pharmaceutical interest.

[0094] In certain embodiments, the SNr is treated to release water-soluble proteins, with or without enzymatic activity, from their attachment to membranes or other cellular structures.

[0095] In a particular embodiment, the SNr is homogenized (with or without detergents) and / or treated with hydrolases to release the bound proteins from the membrane or cellular structures. In a particular embodiment, the hydrolases include one or more of phospholipases and proteases. All released proteins and peptides are further purified by the same methods used for SNp in (vb) a series of one or more of successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and / or ion exchange chromatography steps (i.e. (v.SN)).

[0096] As shown in the following examples, this method in its first aspect in all its variations allows the production of specific fractions containing the enzymes of interest in active form, thus allowing the mixture of enzymes from mammalian intestinal mucosa to function according to their activity.

[0097] In a particular embodiment of the method of the first aspect, the method for the simultaneous obtaining of heparin and native proteins and peptides by fractionation of mammalian intestinal mucosa comprises: (i) adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa to obtain preserved mucosa, or storing the extract at a temperature that allows the proteins, peptides and heparin to be preserved in a native state; (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa; (iii) subjecting the diluted mucosa of step (ii) or the preserved mucosa of (i) to homogenization at a temperature below 40° C. to lyse the cell membranes of the mucosal cells and obtain a stable homogenate of the mucosa containing heparin and mucosal proteins and peptides; (iv) separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iii) by one or more physical and / or chemical means selected from the group consisting of centrifugation, filtration and / or ultrafiltration, optionally using a detergent, and combinations thereof, to obtain a pellet phase (P) containing heparin and a supernatant (SN) fraction containing mucosal proteins and peptides; (va) subjecting the pellet fraction (P) of (iv) containing heparin to an alkaline proteolysis process with a proteolytic enzyme to obtain a mixture of heparin and protein hydrolysates, and subjecting the heparin-containing mixture to one or more precipitation steps, and optionally one or more extraction steps, and one or more chromatography steps to purify said heparin from the protein hydrolysates; and (vb1) subjecting the supernatant (SN) containing the mixture of solubilized and / or suspended mucosal proteins and peptides to filtration through a filter with a maximum cutoff of 100 μm in order to retain small particles and lipid residues not pelleted in step (iv) to obtain a filtered supernatant containing the majority of the mucosal proteins and peptides; (vb2) diafiltration of the filtered supernatant through a filter having a cut-off of less than 0.8 μm to obtain a permeate (SNp) containing water-soluble mucosal proteins and peptides and a retentate (SNr) containing water-insoluble proteins and peptides (i.e. suspended in the crude supernatant SN); (vb3) optionally further diafiltration of the permeate (SNp) to concentrate soluble proteins and peptides (SNpr); (vb4) subjecting SNp (or SNpr) to a cation exchange chromatography column to obtain a bound fraction (SNpbf) and an unbound fraction (SNpubf) and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa to obtain a filtered supernatant basic fraction containing basic proteins and peptides; and (vb5) subjecting the unbound fraction (SNpubf) to an anion exchange chromatography column, further eluting it, and diafiltering and concentrating it using a tangential flow system with a filter having a cut-off of 1-5 KDa to obtain a filtered supernatant acidic fraction containing acidic proteins and peptides. (The order or sequence of cation and anion exchange chromatography is interchangeable).

[0098] In a specific embodiment, step (iv) of separating the heparin fraction (P) from the supernatant fraction (SN) is carried out by centrifugation of the stable homogenate obtained in step (iii).

[0099] In another more particular embodiment, optionally in combination with any of the above or below embodiments, step (v.SN) or (vb) further comprises subjecting either the basic or acidic fractions obtained according to steps (vb4) and (vb5) to a further fractionation step, such as fractionation by molecular weight using further diafiltration, salting out methods, further cation and anion exchange chromatography and combinations thereof. This latter embodiment is aimed at achieving high purification of the peptides and proteins, respectively, in the fractions, if necessary.

[0100] In an even more particular embodiment of the method comprising further fractionation by molecular weight of either the basic or acidic fractions of (vb4) and (vb5) in step (vb), in step (vb6) the peptides and proteins are fractionated according to molecular weight ranges selected from above 300 KDa, 100-300 KDa, 50-100 KDa, 30-50 KDa, 10-30 KDa, 5-10 KDa and 1-5 KDa.

[0101] In this manner, tangential flow can be used to first concentrate the sample and filter out low molecular weight compounds (i.e., water and other compounds), and then further selected fractions can be used to recover specific peptides and proteins within a known molecular weight range.

[0102] In a particular embodiment, optionally in combination with any of the above or below embodiments, the retentate (SNr) of (vb2) is further treated in substep (vb7) with a homogenization step (with or without detergents) and / or with hydrolases such as one or more of phospholipases and proteases to detach the bound proteins of interest from the membrane or other cellular structures. All released proteins and peptides are further purified in the same manner as used for SNp and disclosed in steps (vb3) to (vb6). The homogenization step of the retentate (SNr) is in a particular embodiment carried out in the same manner as the homogenization of step (iii) in the method. It is therefore carried out using a technique selected from the group consisting of mechanical disruption, fluid shear forces, high pressure or cavitation mechanical or physical cell lysis of mammalian intestinal mucosa.

[0103] In another embodiment, the method of the present invention further comprises: (vb4') subjecting the supernatant of step (vb1') to an anion exchange chromatography column to obtain a bound fraction (SNbf') and an unbound fraction (SNubf') and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cut-off of 1-5 KDa to obtain an acidic fraction of the supernatant containing acidic proteins and peptides. Further includes:

[0104] In another embodiment, the method of the present invention further comprises: (vb5') subjecting the unbound fraction (SNubf') of step (vb4') to a cation exchange chromatography column to obtain a bound fraction (SNubfbf') and an unbound fraction (SNubub'), and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa to obtain a supernatant basic fraction containing basic proteins and peptides. Further includes:

[0105] In another embodiment, the method of the present invention further comprises: (vb4a) subjecting the supernatant of step (vb1') to a cation exchange chromatography column to obtain a bound fraction (SNbf) and an unbound fraction (SNubf), and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa to obtain a basic fraction of the supernatant containing basic proteins and peptides. Further includes:

[0106] In another embodiment, the method of the present invention further comprises: (vb5a) subjecting the unbound fraction (SNubf) of step (vb4a) to an anion exchange chromatography column to obtain a bound fraction (SNubfbf) and an unbound fraction (SNubub), and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa to obtain a supernatant acidic fraction containing acidic proteins and peptides. Further includes:

[0107] In a more particular embodiment, anion exchange chromatography is performed before cation exchange chromatography.

[0108] In another embodiment, the method of the present invention further comprises: (vb2) diafiltration of the supernatant of step (vb1') through a filter having a cut-off of less than 0.8 μm to obtain a permeate (SNp) containing water-soluble mucosal proteins and peptides and a retentate (SNr) containing water-insoluble proteins and peptides; and (vb3) Optionally, diafiltration of the permeate (SNp) to concentrate soluble proteins and peptides (SNpr). Further includes:

[0109] In those embodiments comprising step (vb2), the subsequent step (vb4') or (vb4a) is carried out using the supernatant SNp or SNr, in particular the supernatant SNp, of step (vb2). In those embodiments comprising steps (vb2) and (vb3), the subsequent step (vb4') or (vb4a) is carried out using the supernatant SNp or SNpr, in particular the supernatant SNp, of step (vb3).

[0110] In a particular embodiment, the method for obtaining proteins and peptides in native state by fractionation of mammalian intestinal mucosa comprises steps (i), (ii), (iiia), (iva), (vb1') and (vb4') as defined herein. More particularly, it comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3) and (vb4') as defined herein.

[0111] In a particular embodiment, the method for obtaining proteins and peptides in native state by fractionation of mammalian intestinal mucosa comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb4a) and (vb5a) as defined herein. More particularly, it comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3), (vb4a) and (vb5a) as defined herein.

[0112] In a particular embodiment, the method for obtaining proteins and peptides in native state by fractionation of mammalian intestinal mucosa comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb4') and (vb5') as defined herein. More particularly, it comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3), (vb4') and (vb5') as defined herein.

[0113] In a particular embodiment, the method for obtaining proteins and peptides in native state by fractionation of mammalian intestinal mucosa comprises steps (i), (ii), (iiia), (iva), (vb1') and (vb4a) as defined herein. More particularly, it comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3) and (vb4a) as defined herein.

[0114] In a particular embodiment, the method for obtaining proteins and peptides in native state by fractionation of mammalian intestinal mucosa comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb4a), (vb5a) and (vba) as defined herein. More particularly, it comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3) (vb4a), (vb5a) and (vba) as defined herein.

[0115] In a particular embodiment, the method for obtaining proteins and peptides in native state by fractionation of mammalian intestinal mucosa comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb4'), (vb5') and (vba) as defined herein. More particularly, it comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3)(vb4'), (vb5') and (vba) as defined herein.

[0116] As mentioned above, another aspect of the present invention relates to compositions comprising mammalian intestinal mucosa proteins and peptides (i.e. mammalian intestinal mucosa fractions / extracts) obtainable by the method defined in the first aspect or any of its embodiments.

[0117] Therefore, this is (i) adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa to obtain preserved mucosa, or storing the extract at a temperature that allows the proteins, peptides and heparin to be preserved in a native state; (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa; (iii) subjecting the diluted mucosa of step (ii) or the preserved mucosa of (i) to homogenization at a temperature below 40° C. to lyse the cell membranes of the mucosal cells and obtain a stable homogenate of the mucosa containing heparin and mucosal proteins and peptides; (iv) separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iii) by one or more physical and / or chemical means selected from the group consisting of centrifugation, filtration and / or ultrafiltration, optionally using a detergent, and combinations thereof, to obtain a pellet phase (P) containing heparin and a supernatant (SN) fraction containing mucosal proteins and peptides; (va) subjecting the pellet fraction (P) of (iv) containing heparin to an alkaline proteolysis process with a proteolytic enzyme to obtain a mixture of heparin and protein hydrolysates, and subjecting the heparin-containing mixture to one or more precipitation steps, and optionally one or more extraction steps, and one or more chromatography steps to purify said heparin from the protein hydrolysates; and (vb1) subjecting the supernatant (SN) containing the mixture of solubilized and / or suspended mucosal proteins and peptides to filtration through a filter with a maximum cutoff of 100 μm in order to retain small particles and lipid residues not pelleted in step (iv) to obtain a filtered supernatant containing the majority of the mucosal proteins and peptides; (vb2) diafiltration of the filtered supernatant through a filter having a cut-off of less than 0.8 μm to obtain a permeate (SNp) containing water-soluble mucosal proteins and peptides and a retentate (SNr) containing water-insoluble proteins and peptides (i.e. suspended in the crude supernatant SN); (vb3) optionally further diafiltration of the permeate (SNp) to concentrate soluble proteins and peptides (SNpr); (vb4) subjecting SNp (or SNpr) to a cation exchange chromatography column to obtain a bound fraction (SNpbf) and an unbound fraction (SNpubf) and eluting the bound fraction, which is then diafiltered and concentrated using a tangential flow system with a filter having a cutoff of 1-5 KDa to obtain a filtered supernatant basic fraction containing basic proteins and peptides; and (vb5) subjecting the unbound fraction (SNpubf) to an anion exchange chromatography column, further eluting it, and diafiltering and concentrating it using a tangential flow system with a filter having a cut-off of 1-5 KDa to obtain a filtered supernatant acidic fraction containing acidic proteins and peptides; (The order or sequence of cation and anion exchange chromatography is interchangeable.) The present invention can be disclosed as a composition comprising mammalian intestinal mucosa proteins and peptides (i.e., a mammalian intestinal mucosa fraction / extract), obtainable by a method comprising the steps of:

[0118] In certain embodiments of the compositions comprising mammalian intestinal mucosa proteins and peptides, the mammalian intestinal mucosa proteins and peptides are selected from the group consisting of brush border enzymes, antimicrobial peptides, antioxidant enzymes, hormonal peptides, proteins involved in defense and / or healing processes in the intestinal mucosa, and combinations thereof, as revealed by literature, transcriptomic and proteomic analyses of porcine intestinal mucosa.

[0119] These compositions are thus isolated fractions or extracts of mammalian intestinal mucosa that contain the compound of interest.

[0120] In more particular embodiments of the compositions of the second aspect, they comprise brush border enzymes selected from glycosidases, peptidases, phosphatases, lipases and combinations thereof.

[0121] The brush border of the mammalian intestine, especially the small intestine, is the microvilli-covered surface of the epithelium where absorption takes place by microvilli with a diameter of about 100 nm and a length that varies from about 100 to 2000 nm. The brush border, especially of the inner wall of the intestine, is the site of terminal digestion of carbohydrates and proteins. The microvilli that make up the brush border have enzymes for this final part of digestion, fixed to their apical cell membrane as essential membrane proteins. These enzymes are present in the vicinity of transporters, which then allow the absorption of the digested nutrients.

[0122] In an even more particular embodiment, the composition comprises a phosphatase, more particularly selected from phytase, alkaline phosphatase and mixtures thereof.

[0123] In another particular embodiment, optionally in combination with the above or below embodiments of the compositions of the present invention, they comprise a glycosidase. Examples of particular glycosidases are selected from the group consisting of maltase, maltase-glucoamylase, sucrase-isomaltase, α-galactosidase, lactase, dextrinase, trehalase, lysozyme, and combinations thereof.

[0124] In another specific embodiment, optionally in combination with the above or below embodiments of the compositions of the invention, they comprise a peptidase selected from the group consisting of carboxypeptidases, aminopeptidases, endopeptidases, enteropeptidases and dipeptidases.

[0125] In another specific embodiment, optionally in combination with the above or below embodiments of the compositions of this invention, they comprise a lipase selected from the group consisting of triacylglycerol lipase, phospholipase, ceramidase and sphingomyelinase.

[0126] In another particular embodiment, the present invention provides one or more of the enzymes selected from alkaline phosphatase, phytase, α-galactosidase, lysozyme, aminopeptidase, carboxypeptidase, sucrase-isomaltase, maltase-glucoamylase, α-amylase, diamine oxidase, triacylglycerol lipase and phospholipase, and / or antioxidant enzymes selected from catalase, glutathione peroxidase, superoxide dismutase and combinations thereof, and / or mucosal proteins involved in mucosal defense and healing selected from mucin, actin, secretory immunoglobulin A, trefoil factor family proteins, thymosin beta 4, lactoferrin, apolipoprotein AI, annexin A5, annexin A1, galectin-1, and combinations thereof; and / or peptides having antibacterial activity selected from bactericidal permeability enhancing protein, derived from regenerating pancreatic islets 4, defensins, antimicrobial peptide PMAP-23, pulmonary surfactant associated protein D, derived from regenerating pancreatic islets 3, diazepam binding inhibitor, antimicrobial peptide 3910, antimicrobial peptide NK-lysine, liver-derived antimicrobial peptide 2, gastric inhibitory polypeptide (7-42), reactive oxygen species modulator 1 and combinations thereof; and / or A peptide having hormonal activity selected from peptide YY, GLP-1, GLP-2, gastrin, cholecystokinin, gastric inhibitory polypeptide, vasoactive intestinal peptide, neuropeptide Y, ghrelin, secretin, galanin, and combinations thereof The present invention relates to compositions comprising mucosal proteins and peptides, comprising:

[0127] As mentioned above, the present invention also relates to a composition comprising an acidic fraction of mammalian intestinal mucosa acidic proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1') and (vb4') as defined herein.

[0128] As mentioned above, the present invention also relates to a composition comprising an acidic fraction of mammalian intestinal mucosa acidic proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1'), (vb4a) and (vb5a) as defined herein.

[0129] In particular, the composition comprising the acidic fraction of mammalian intestinal mucosa acidic proteins and peptides comprises one or more of proglucagon, glucagon-like peptide-1 (GLP-1), thymosin β4 and vasoactive intestinal polypeptide. More particularly, the AF comprises proglucagon, glucagon-like peptide-1 (GLP-1), thymosin β4 and vasoactive intestinal polypeptide.

[0130] The present invention also relates to a composition comprising a basic fraction of mammalian intestinal mucosa basic proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1'), (vb4') and (vb5') as defined herein.

[0131] The present invention also relates to a composition comprising a basic fraction of mammalian intestinal mucosa basic proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1') and (vb4a) as defined herein.

[0132] In particular, the composition comprising the basic fraction of mammalian intestinal mucosa basic proteins and peptides comprises lysozyme.More particularly, it comprises one or more of lysozyme, lactotransferrin, collagenase type IV of 72 kDa, interstitial collagenase, matrix metalloproteinase-9, regeneration family member III / regeneration islet-derived protein 3 (Reg III), regeneration family member IV / regeneration islet-derived protein IV (Reg IV), antimicrobial protein PR-39, angiogenin, phosphoinositide phospholipase C, phospholipase D and pulmonary surfactant-associated protein D, and more particularly, it comprises all of them.

[0133] In a particular embodiment, the composition comprising the basic fraction of mammalian intestinal mucosa basic proteins and peptides is subdivided by washing with buffers of different concentrations. In a more particular embodiment, the washing is with NaCl buffers of different concentrations. More particularly, subfraction BF2 is obtained by washing the resin with a buffer of about 2% NaCl. In particular, BF2 comprises lysozyme, in particular with an activity of 150 U-FIP / mL. More particularly, BF2 comprises one or more of lysozyme, lactotransferrin, phosphoinositide phospholipase C, phospholipase D, antibacterial protein PR-39, regeneration family member 4 and angiogenin. In another particular embodiment, subfraction BF7 is obtained by further washing the resin with a buffer of about 7% NaCl. In certain embodiments, BF7 comprises one or more of 72 kDa type IV collagenase, interstitial collagenase, matrix metalloproteinase-9, regeneration family member III / regenerating islet-derived protein 3 (Reg III) and pulmonary surfactant associated protein D, and more particularly comprises all of them.

[0134] The present invention also relates to a composition S Nubub' comprising mammalian intestinal mucosa proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3), (vb4') and (vb5') as defined herein.

[0135] In particular, the SNubub' comprises alkaline phosphatase (ALP) and mucin-2 (MUC2). More particularly, the SNubub' comprises one or more of ALP, MUC2, aminopeptidase, carboxypeptidase, catalase, triacylglycerol lipase, phospholipase, acid sphingomyelinase-like phosphodiesterase, α-amylase, α-galactosidase, sucrase-isomaltase, peroxiredoxin-6, superoxide dismutase, apolipoprotein A1, annexin-5, galectin-1, diazepam binding inhibitor, immunoglobulin M, secretory immunoglobulin A, gastrotropin, thymosin β4, and hemoglobin, more particularly all of them.

[0136] In more specific embodiments, the SNubub' is further subdivided by successive filtration, ultrafiltration steps, solvent extraction, protein precipitation and / or gel permeation and affinity chromatography steps.

[0137] The present invention also relates to a composition S Nubub comprising mammalian intestinal mucosa proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1'), (vb4a) and (vb5a) as defined herein.

[0138] The present invention comprises steps (i), (ii), (iiia), (iva), (vb1'), (vb4') and (vb5') as defined herein for obtaining an SNubub' fraction, and (vba) subjecting the unbound fraction (SNubub') to an extraction treatment with an organic solvent to obtain an aqueous phase (SNububaq) The present invention also relates to a composition S Nububaq comprising mammalian intestinal mucosa proteins and peptides, which can be obtained by a method further comprising:

[0139] More particularly, the present invention also relates to a composition S Nububaq comprising mammalian intestinal mucosa proteins and peptides obtainable by a process comprising steps (i), (ii), (iiia), (iva), (vb1'), (vb2), (vb3), (vb4'), (vb5') and (vba) as defined herein.

[0140] In a particular embodiment, the SNubub' is subjected to an extraction treatment with an organic solvent (setp vba). More particularly, the organic solvent is selected from the group consisting of heptanol, pentanol, 4-tert-butylcatechol, limonene, cyclohexane and mixtures thereof, more particularly, it is 20-60% (w / w) butanol, even more particularly, it is about 40% (w / w) butanol. More particularly, the extraction treatment lasts for about 5 minutes and is performed at about 37°C.

[0141] In a particular embodiment, the aqueous phase of the resulting extract (SNububaq) contains alkaline phosphatase and mucin-2. In a more particular embodiment, Snububaq contains alkaline phosphatase with an activity of about 10.7 DEAU / mL. In a more particular embodiment, Snububaq contains one or more of alkaline phosphatase, mucin-2, immunoglobulin M, immunoglobulin A, sucrase-isomaltase, alpha-amylase, hemoglobin, aminopeptidase, maltase-glucoamylase, catalase, carboxypeptidase, alpha-galactosidase, peroxiredoxin-6, superoxide dismutase, acid sphingomyelinase-like phosphodiesterase, diazepam binding inhibitor, gastrotropin, apolipoprotein-1, annexin-5 and galectin-1, more particularly all of them.

[0142] In another particular embodiment, Snububaq is subjected to treatment with chemicals for precipitation, such as acetone, ammonium sulfate, PEG, aluminum and butanone, to obtain a precipitate and a supernatant (Snububaqsn). More particularly, the organic solvent is acetone, more particularly it is 20-40% (w / w) acetone, more particularly it is about 30% (w / w) acetone.

[0143] In another particular embodiment, the obtained supernatant (Snububaqsn) is further subjected to treatment with chemicals for precipitation, such as acetone, ammonium sulfate, PEG, aluminum and butanone, to obtain a precipitate (Snububaqsnp or ALPp) and a supernatant (Snububaqsnsn or MUC2sn). More particularly, the organic solvent is acetone, more particularly it is 50% to 99% (w / w) acetone, more particularly it is 60% to 90% (w / w) acetone, more particularly it is 80% acetone. In a particular embodiment, the acetone treatment is performed at a temperature of 25 to 45°C, more particularly in an embodiment 35 to 40°C, more particularly in an embodiment 37°C. In a more particular embodiment, the obtained precipitate (ALPp) contains ALP. In another particular embodiment, the obtained supernatant (MUCsn) contains MUC2.

[0144] In another embodiment, ALPp is further processed by affinity chromatography to obtain purified ALP.In particular, purified ALP is a pharmaceutical grade protein suitable for medical use.More specifically, purified ALP is obtained with a yield of about 38840 DEA units of alkaline phosphatase activity per kg mucosa (as known by those skilled in the art, 1 DEA unit hydrolyzes 1 μmol of p-nitrophenyl phosphate per minute at pH 9.8 and 37°C).

[0145] In another embodiment, MUC2sn is further purified by affinity chromatography to obtain a pharmaceutical grade protein suitable for medical use.In particular, the purified MUC2 is a pharmaceutical grade protein suitable for medical use.More particularly, the purified MUC2 is obtained with a yield of about 0.16 g N-acetylneuraminic acid per kg mucosa, which corresponds to a sialylated glycoprotein such as mucin-2. EXAMPLES

[0146] Example 1a: Transcriptome analysis of porcine intestinal mucosa

[0147] Transcriptome analysis was performed to survey the complete set of transcripts in the porcine intestinal mucosa and characterize the corresponding proteome.

[0148] material and method RNA extraction Nine samples of porcine intestinal mucosa were collected at the slaughterhouse and stored at -80°C until analysis. Total RNA was extracted using the RNeasy Plus Mini Kit (product no. 74134). RNA concentration was quantified by fluorescence and quality was confirmed by electrophoresis.

[0149] Library preparation and sequencing Transcripts were purified by poly(A) tail selection. Nine mRNA libraries were prepared using the Illumina TruSeq stranded mRNA kit. Libraries were amplified by PCR to confirm concentration and quality. Libraries were sequenced using an Illumina Novaseq 6000 in one lane (2 × 100 bp) of an SP flow cell (kit version: NV2864788-RGSBS).

[0150] Bioinformatics analysis Gene expression was examined using the reference genome of Sus scrofa 11.1 on iGenome (https: / / emea.support.illumina.com / sequencing / sequencing_software / igenome.html). Reads were mapped against the reference genome using Bowtie v2.0.5 (http: / / bowtie-bio.sourceforge.net / bowtie2 / index.shtml) and TopHat v2.0.6 (http: / / tophat.cbcb.umd.edu / ). Cufflink v2.1.1 (http: / / cufflinks.cbcb.umd.edu / ) was used to estimate the abundance of aligned reads and to test whole transcriptomes for differential expression and regulation. The Cummerbund v2.7.2 package (http: / / compbio.mit.edu / cummeRbund / ) was used to generate graphical displays of differential analyses.

[0151] result In total, we identified 120,814,076 transcript reads, of which 104,992,005 were mapped to the reference genome. From the mapped reads, we annotated over 16,000 genes and estimated their expression.

[0152] Example 2a: Proteomic analysis of the porcine intestinal mucosa samples of Example 2b

[0153] Proteomic analysis was performed to map the proteome of the porcine intestinal mucosa and complement the transcriptomic data.

[0154] material and method Protein extraction Samples of porcine intestinal mucosa were collected at the slaughterhouse and stored at -80°C until analysis. A representative sample was weighed (106.4 mg) into an Eppendorf tube at -20°C and TES buffer (10 mM Tris-HCl, 1 mM EDTA, 0.25 mM sucrose, pH 6.9) was added to obtain a concentration of 100 mg / mL. The sample was kept on ice and homogenized in a tissue homogenizer (TissueLyser LT, Qiagen) for 2 x 10 seconds using metal beads and the sample was kept on ice during homogenization. The homogenized sample was centrifuged at 4°C and 15.000 x g for 15 minutes (Eppendorf centrifuge) and the supernatant was collected. Protein concentration was determined using the BCA method with BSA as standard (Pierce BSA Protein Assay Kit). Protein concentration was determined to be 5 μg / μl.

[0155] Protein digestion and purification In all, four samples, each consisting of 10 μg protein, were reduced with DTT, alkylated with iodoacetamide, and digested with trypsin according to the "In-solution trypsin digestion and guanidination kit" (ThermoScientific). Two of these samples were guanidinated with O-methylisourea hemisulfate. Peptides from all four samples were then purified using "Peptide Desalting Spin Columns" (Pierce, see manufacturer's manual). After desalting, the samples were evaporated to dryness and dissolved in HPLC buffer A (2% acetonitrile, 0.1% formic acid in MilliQ water) before LC-MS analysis.

[0156] mass spectrometry Peptide samples were analyzed on a Bruker TimsTOFPro equipped with a Bruker NanoElute LC-system. Peptides were separated using a 90 min reversed-phase gradient with solvent A: 2% acetonitrile, 0.1% formic acid in MilliQ water and solvent B: acetonitrile, 0.1% formic acid. The reversed-phase column was a Bruker FIFTEEN (L=15 cm, ID=75 μm, C18, 1.9 μm, 120 Å). The mass spectrometer was run in DDA PASEF mode.

[0157] Database Search Data files were merged and searched in PEAKS XPro using the UniProt procine proteomics database (Proteomics ID UP000008227, download date: 2021.03.05) with the Mammalian SwissProt database as a contamination database. Error tolerance: precursor mass of 20 ppm, fragment ion of 0.05 Da. Fixed PTM: carbamidomethylation. Variable PTM: oxidation (M), acetylation (N-term), guanidination. False discovery rate (FDR) was set at 0.5%.

[0158] result Proteomic analysis of the porcine intestinal mucosa revealed 2810 identified protein groups at a false discovery rate (FDR) of 0.5%. Data from this experiment was cross-analyzed with data from transcriptomic analysis to confirm the global proteome of the porcine intestinal mucosa. From this, a list of candidate proteins was selected based on their abundance in the mucosa, their molecular properties and their potential commercial applications.

[0159] Example 2b: Industrial process for obtaining heparin and by-products (i.e. native proteins and peptides) from mammalian intestinal mucosa (I)

[0160] The method for the simultaneous isolation of heparin and selected native proteins and peptides from mammalian intestinal mucosa was carried out as follows.

[0161] The method generally involved the following steps: (i) Obtaining an aqueous extract of mammalian intestinal mucosa (i.e. isolating the intestinal mucosa using standard methods and dissolving / suspending it in water or an aqueous buffer), then adding sodium metabisulfite to said aqueous extract of mammalian intestinal mucosa (hereinafter referred to as "mucosa") to a concentration of 2%. Equivalent substances can be selected as preservatives and antioxidants. (ii) "Mucosa" was further diluted with 2 volumes of deionized water. This procedure yielded an intermediate (hereafter referred to as "diluted mucus"). Alternatively, diluted mucus could be obtained by diluting "mucosa" with an aqueous solution containing one of the following surfactants: Tween 20, Tween 80, Triton X-100 or X-114, deoxycholic acid, Brij 35 or 58, BigCHAP or deoxyBigCHAP, MEGA-8, MEGA-9 or MEGA-10, octyl β-glucoside. (iii) The diluted mucosa was subjected to homogenization with a rotor-stator homogenizer at a G force of 1700 g and a temperature of 2°C to 8°C for 2 to 4 minutes to lyse the cell membranes and obtain a stable homogenate of the mucosa (hereinafter referred to as "homogenate") containing heparin and mucosal proteins and peptides. Other homogenization modes can be performed by other techniques selected from the group consisting of mechanical or physical cell lysis of mammalian intestinal mucosa by mechanical disruption, fluid shear forces, high pressure or cavitation. Another alternative homogenization technique involves the addition of surfactants. (iv) The heparin and mucosal proteins and peptides contained in the "homogenate" obtained in step (iii) were separated by centrifugation to obtain a liquid and solid phase each containing different proportions of heparin and one or more of other mucosal proteins and peptides. Centrifugation was performed for 20 minutes at a force of 3134 g and a temperature of less than 8° C. to obtain a solid phase containing heparin (hereinafter referred to as "pellet" or P) and a liquid phase containing a mixture of mucosal proteins and peptides (hereinafter referred to as "supernatant" or SN).

[0162] (vP) The "pellet" was then subjected to standard extraction methods to obtain and purify heparin for pharmaceutical use (as mentioned above, this step can also be labeled (iv.a)). Briefly, the pellet was treated with alkaline proteolytic enzymes at 58 °C and pH 7.7-8 for 2 hours. After hydrolysis, the pH was lowered to 6.6 and the temperature was raised to 90 °C and maintained for 15 minutes to precipitate the fat. The product was centrifuged at 3134 g G force for 15 minutes, the supernatant was decanted and filtered through a 300 μm filter to separate the fat fraction. Anion exchange resin was then added to the supernatant to capture the heparin. The mixture was kept under stirring overnight, and the resin was separated from the liquid with a mesh filter and rinsed with osmotic water. Proteins were eluted from the resin with three successive additions of 3.5% (w / v) NaCl for 30 minutes each at 45 °C. Finally, heparin was eluted with three successive additions of 18%, 20% and 20% NaCl for 30 minutes each at 45° C. The three heparin eluates were combined and analyzed, giving an average heparin activity of 0.047 MIU per kg of mucosa (one unit of heparin is the amount of heparin required to maintain 1 mL of cat blood fluid at 0° C. for 24 hours, which corresponds to approximately 0.002 mg of pure heparin, as known by those skilled in the art).

[0163] (v.SN) (or as previously described (vb)) the "supernatant" was subjected to successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and ion exchange chromatography steps in order to separate proteins and peptides according to their solubility, isoelectric point and molecular weight (see specific steps (iv.SN.1) to (iv.SN.7) below).

[0164] (v.SN.1) The "supernatant" was coarsely filtered through a 100 μm cutoff filter in order to retain the lighter, and therefore non-pelletized, small particles and lipid residues. The intermediate obtained by this procedure is hereafter referred to as the "filtered supernatant".

[0165] (v.SN.2) The "filtered supernatant" was then diafiltered using a tangential flow filtration device with a filter having a cut-off of less than 0.1 μm. Diafiltration was performed by replacing the permeate with at least 4 volumes, preferably 5-7 volumes of buffer. The aim of this step was to recover both the retentate (SNr) and the permeate (SNp).

[0166] (v.SN.3) The permeate (SNp) was further diafiltered using a tangential flow filtration device with a filter having a cut-off of less than 3 kDa in order to concentrate it (SNpr).

[0167] (v.SN.4) Fraction SNpr was adjusted to a pH of 7.0 and then loaded in downflow onto a strong cation exchange chromatography column in order to bind and thereby concentrate all basic proteins and peptides present in the solution. The bound fraction (SNprb) was eluted in the upflow direction and then diafiltered with phosphate buffer (50 mM) at pH 7.0 and concentrated using a tangential flow system with a filter with a cut-off of 3 KDa. The intermediate mixture obtained by this procedure is hereafter referred to as the "basic fraction".

[0168] (v.SN.5) The unbound fraction (SNprub) of step (v.SN.4) was then adjusted to a pH of 7.0 before being loaded onto a strong anion exchange resin in order to bind and thus concentrate all the acidic proteins and peptides present in the solution. The bound fraction was eluted in the upflow direction and then diafiltered with a phosphate buffer (50 mM) at pH 7.0 and concentrated using a tangential flow system with a filter with a cut-off of 3 KDa. The intermediate obtained by this procedure is hereafter referred to as the "acidic fraction".

[0169] (v.SN.6) Both the "basic fraction" and the "acidic fraction" were independently subdivided by molecular weight by sequential diafiltration and concentration of the associated permeate. These fractions were divided into the following molecular weight ranges: ·Over 300Kda 100~300KDa 50~100KDa 30~50KDa 10~30KDa 5~10KDa 1~5KDa It was characterized by:

[0170] (v.SN.7) Both the "basic fraction" and the "acidic fraction" from step (v.SN.6) were further subdivided according to molecular weight by gel permeation chromatography.

[0171] (v.SN.8) Fraction SNr was further homogenized and comminuted according to steps (v.SN.3) to (v.SN.7). In this way, the following yields were obtained: alkaline phosphatase activity of 38.840 DEA units per kg mucosa (as known by those skilled in the art, 1 DEA unit hydrolyzes 1 μmol of p-nitrophenyl phosphate per minute at pH 9.8 and 37 °C) and lysozyme activity of 1.014.983 U-FIP per kg mucosa (as known by those skilled in the art, 1 FIP unit hydrolyzes 0.001 A per minute at pH 6.24 and 25 °C with a suspension of Micrococcus luteus as substrate in 2.6 ml of reaction mixture). 450 The results are shown in Table 1.

[0172] Alternatively, both the "basic fraction" and the "acidic fraction" could be subdivided by salting out methods such as by addition of ammonium sulfate or by addition of an organic solvent such as acetone.

[0173] Alternatively, the "basic fraction" and "acidic fraction" can be loaded onto weak cation and anion exchange columns, respectively, and single proteins and peptides can be eluted by ionic strength or pH gradient.

[0174] This example provides evidence of the ability of the process to simultaneously obtain one or more enzymes and heparin in suitable quantities and purity grades from mammalian intestinal mucosa. Using the process described as an example, enzymatic activities of 38.840 DEA units of alkaline phosphatase activity per kg mucosa and 1.014.983 U-FIP of lysozyme activity per kg mucosa were obtained, as well as heparin (a compound normally obtained from this tissue) with a heparin activity of 0.047 MIU per kg mucosa. These heparin values ​​are higher than those obtained by standard extraction processes of the prior art. Although the data are illustrated with alkaline phosphatase and lysozyme from SNr, the different steps, namely (v.SN.4) and (v.SN.5), also show how to separate and obtain all proteins according to their isoelectric points by choosing the appropriate pH and pH gradient. Other method steps were also applied, especially those based on the molecular weight of the proteins (see v.SN.6 and v.SN.7).

[0175] In fact, the methods proposed for the simultaneous access to heparin and to proteins and peptides in the native state by fractionation of mammalian intestinal mucosa are based on the properties of the proteins contained in the mucosa, mainly their isoelectric point at a given pH and / or their molecular weight and / or their solubility in water or in organic solvents.

[0176] Example 3a: Industrial process for obtaining heparin and by-products (i.e. native proteins and peptides) from mammalian intestinal mucosa (II)

[0177] The method included the following steps: (i) Obtaining an aqueous extract of mammalian intestinal mucosa (i.e. isolating the intestinal mucosa using standard methods and dissolving / suspending it in water or an aqueous buffer), then adding sodium metabisulfite to said aqueous extract of mammalian intestinal mucosa (hereinafter referred to as "mucosa") to a concentration of 2%. Equivalent substances can be selected as preservatives and antioxidants. (ii) "Mucosa" was further diluted with 2 volumes of deionized water. This procedure yielded an intermediate (hereafter referred to as "diluted mucus"). Alternatively, "diluted mucus" could be obtained by diluting "mucosa" with an aqueous solution containing one of the following surfactants: Tween 20, Tween 80, Triton X-100 or X-114, deoxycholic acid, Brij 35 or 58, BigCHAP or deoxyBigCHAP, MEGA-8, MEGA-9 or MEGA-10, octyl β-glucoside. (iii) The "diluted mucosa" was subjected to homogenization with a rotor-stator homogenizer at a G force of 1700 g and a temperature of 2°C to 8°C for 2 to 4 minutes to lyse the cell membranes and obtain a stable homogenate of the mucosa (hereinafter referred to as "homogenate") containing heparin and mucosal proteins and peptides. Other homogenization modes can be performed by other techniques selected from the group consisting of mechanical or physical cell lysis of mammalian intestinal mucosa by mechanical disruption, fluid shear forces, high pressure or cavitation. Homogenization can be performed in the presence of a surfactant. (iv) The heparin and mucosal proteins and peptides contained in the homogenate obtained in step (iiia) were separated by centrifugation to obtain a liquid and solid phase, each of which contains different proportions of heparin and one or more of the other mucosal proteins and peptides. Centrifugation was performed for 30 minutes at a g-force of 3300 g and a temperature below 37°C (preferably 0-25°C) to obtain a solid phase containing heparin (hereinafter referred to as "pellet" or P) and a liquid phase containing a mixture of mucosal proteins and peptides (hereinafter referred to as "supernatant" or SN). Alternatively, the separation of the "pellet" and "supernatant" can be achieved by tangential microfiltration through a 0.2 μm membrane. The SN contains, among other proteins and peptides, glucagon-like peptide-1 (GLP-1), thymosin β4, vasoactive intestinal peptide, lysozyme, lactotransferrin, regenerating islet-derived protein 3 (Reg III), regenerating islet-derived protein IV (Reg III), and the like, as confirmed by proteomic analysis. IV), matrix metalloproteinase-9, type IV collagenase of 72 kDa, interstitial collagenase, bactericidal permeability-enhancing protein, pulmonary surfactant-associated protein D, antimicrobial protein PR-39, angiogenin, alkaline phosphatase, mucin-2, trypsin, aminopeptidase, carboxypeptidase, catalase, triacylglycerol lipase, phospholipase, acid sphingomyelinase-like phosphodiesterase, α-amylase, α-galactosidase, sucrase-isomaltase, peroxiredoxin-6, superoxide dismutase, apolipoprotein A1, annexin-5, galectin-1, diazepam-binding inhibitor, immunoglobulins, gastrotropin, and hemoglobin. SN also contains exogenously derived phytases as confirmed by enzymatic assays.

[0178] (vP) The "pellet" was then subjected to standard extraction methods to obtain and purify heparin for pharmaceutical use (as mentioned above, this step can also be labeled (iv.a)). Briefly, the pellet was treated with enzymatic alkaline proteolysis at 58°C and pH 7.7-8 for 2 hours. After hydrolysis, the pH was reduced to 6.6 and the temperature was increased to 90°C and maintained for 15 minutes to precipitate the fat. The product was centrifuged at 3134 g g-force for 15 minutes, the supernatant was decanted and filtered through a 300 μm filter to separate the fat fraction. Anion exchange resin was then added to the supernatant at a rate of 0.1-1% w / w (preferably 0.2%) to capture the heparin. The mixture was kept under stirring overnight and the resin was separated from the liquid with a mesh filter and rinsed with osmotic water. Proteins were eluted from the resin with three successive additions of 3.5% (w / v) NaCl for 30 min each at 45° C. Finally, heparin was eluted with three successive additions of 18%, 20% and 20% NaCl for 30 min each at 45° C. The three heparin eluates were combined and analyzed, giving an average heparin activity of 0.047 MIU per kg mucosa (one unit of heparin is the amount of heparin required to maintain 1 mL of cat blood fluid at 0° C. for 24 hours, which corresponds to approximately 0.002 mg of pure heparin, as known by those skilled in the art).

[0179] (v.SN) (or (v.b') as described above) the "supernatant" was subjected to successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and ion exchange chromatography steps in order to separate proteins and peptides according to their solubility, isoelectric point and molecular weight (see specific steps (iv.SN.1) to (iv.SN.7) below).

[0180] The "supernatant" was coarsely filtered through a 100 μm cutoff filter in order to retain the smaller particles and lipid residues that were lighter and therefore not pelleted (v.SN.1). The intermediate obtained by this procedure is hereafter referred to as the "filtered supernatant" (SNf).

[0181] (v.SN.2) The "filtered supernatant" (SNf) was optionally diafiltered using a tangential flow filtration device with a filter having a cut-off of less than 0.1 μm. Diafiltration was performed by replacing the permeate with at least 4 volumes, preferably 5-7 volumes of buffer. The aim of this step was to recover both the retentate (SNr) and the permeate (SNp).

[0182] (v.SN.3) The permeate (SNp) was further diafiltered using a tangential flow filtration device with a filter having a cut-off of less than 3 kDa in order to concentrate it (SNpr).

[0183] (v.SN.4) Fraction SNpr (or fraction SNf), after adjustment to a pH of 7.0, was loaded in downflow onto a strong anion exchange chromatography column in order to bind and thereby concentrate all acidic proteins and peptides present in the solution. The bound fraction (SNprb) was eluted in the upflow direction and then diafiltered with phosphate buffer (50 mM) at pH 7.0 and concentrated using a tangential flow system with a filter with a cut-off of 3 KDa. Alternatively, anion exchange chromatography can be performed in batches by adding 0.5-1.5% (w / w) (preferably 1%) of strong anion exchange resin to the fraction. The intermediate mixture obtained by this procedure was hereafter called "acidic fraction" (AF). The "acidic fraction" (AF) contains in particular proteins and peptides identified by proteomic analysis (as shown in Example 3b).

[0184] (v.SN.5) The unbound fraction (SNprub) of step (v.SN.4) was then adjusted to a pH of 7.0 before being loaded onto a strong cation exchange column in order to bind and thus concentrate all basic proteins and peptides present in the solution. The bound fraction was eluted in the upflow direction and then diafiltered with phosphate buffer (50 mM) at pH 7.0 and concentrated using a tangential flow system with a filter with a cut-off of 3 KDa. Alternatively, the cation exchange chromatography can be carried out in batches by adding 5-15% (w / w) (preferably 10%) of strong cation exchange resin to the fraction. The intermediate obtained by this procedure is hereafter called the "basic fraction" (BF). The "basic fraction" (BF) contains in particular the proteins and peptides identified by proteomic analysis (shown in Example 2b). Two subfractions were eluted from the "basic fraction" using different NaCl concentrations. A subfraction (BF2) was eluted by washing the resin with 2% NaCl and characterized by enzymatic assays and proteomic analysis. It contained, among others, the proteins lysozyme with an activity of 150 U-FIP / mL, lactotransferrin, phosphoinositide phospholipase C, phospholipase D, antibacterial protein PR-39, refolding family member 4 and angiogenin. Another subfraction (BF7) containing the rest of the proteins was eluted from the "basic fraction" by washing the resin with 7% NaCl. Both subfractions were tested for antibacterial activity as detailed in Example 2c.

[0185] (v.SN.6) Both the "basic fraction" and the "acidic fraction" or subfractions thereof were independently subdivided by molecular weight by a series of diafiltrations and associated concentrations of the permeate. These fractions were divided into the following molecular weight ranges: ·Over 300Kda 100~300KDa 50~100KDa 30~50KDa 10~30KDa 5~10KDa 1~5KDa It was characterized by:

[0186] (v.SN.7) Both the "basic fraction" and the "acidic fraction" from step (v.SN.6) were further subdivided according to molecular weight by gel permeation chromatography.

[0187] (v.SN.8) The unbound fraction (SNprubub) of step (v.SN.5) contains in particular the proteins and peptides identified by proteomic analysis (shown in Example 2b). SNprubub can be used as an ingredient for feed and / or food applications or it can be further subdivided by successive filtration, ultrafiltration steps, solvent extraction, protein precipitation and / or gel permeation and affinity chromatography steps. In a specific case, SNprubub was treated with butanol 40% (w / w) to extract alkaline phosphatase and mucin-2 in the aqueous phase. This aqueous phase (SNprububaq) was characterized by enzymatic assays and proteomic analysis. It contains in particular the proteins and peptides identified by proteomic analysis (shown in Example 2b). In particular it contains alkaline phosphatase and mucin-2 with an activity of 10.7 DEA U / mL. SNprububaq was tested for antibacterial activity as described in Example 2c. To further purify its components, the SNprububaq phase was then treated with acetone 30% (w / w) and the resulting supernatant was treated with acetone 60% (w / w) to precipitate ALP and separate it from MUC2 remaining in the supernatant. ALP and MUC2 were then purified by affinity chromatography to obtain pharmaceutical grade proteins suitable for medical use. Alternatively, precipitation with ammonium sulfate, aluminum or a combination thereof was used in conjunction with the previous treatment. The supernatants of any of the derived fractions can be pretreated with phosphatidylinositol-specific phospholipase C to strip alkaline phosphatase and other membrane proteins to improve their separation and purification and to obtain higher yields.

[0188] (v.SN.8) Fraction SNr was further homogenized and comminuted according to steps (v.SN.3) to (v.SN.7).

[0189] In this way, the following yields were obtained: alkaline phosphatase activity of 38.840 DEA units per kg mucosa (as known by those skilled in the art, 1 DEA unit hydrolyzes 1 μmol of p-nitrophenyl phosphate per minute at pH 9.8 and 37° C.) and lysozyme activity of 1.014.983 U-FIP per kg mucosa (as known by those skilled in the art, 1 FIP unit hydrolyzes 0.001 A per minute at pH 6.24 and 25° C. using a suspension of Micrococcus luteus as substrate in 2.6 ml of reaction mixture). 450 The enzymes alkaline phosphatase and lysozyme were obtained by purification of the mucin from the sucrose solution (which produces a change in .mu.m). Mucin was isolated in significant yield as measured at 0.16 g N-acetylneuraminic acid per kg mucosa.

[0190] Alternatively, both the "basic fraction" and the "acidic fraction" could be subdivided by salting out methods such as by addition of ammonium sulfate or by addition of organic solvents such as butanol or acetone.

[0191] Alternatively, the "basic fraction" and "acidic fraction" can be loaded onto weak cation and anion exchange columns, respectively, and single proteins and peptides can be eluted by ionic strength or pH gradient.

[0192] This example provides evidence of the ability of the process to simultaneously obtain one or more enzymes and heparin in suitable quantities and purity grades from mammalian intestinal mucosa. In an example using the described process, enzyme activities of 38.840 DEA units of alkaline phosphatase activity per kg mucosa and 1.014.983 U-FIP of lysozyme activity per kg mucosa were obtained, and heparin (a compound normally obtained from this tissue) was also obtained with 0.047 MIU of heparin activity per kg mucosa, together with N-acetylneuraminic acid corresponding to 0.16 g of sialylated glycoprotein-like mucin-2 per kg mucosa. These heparin values ​​are higher than those obtained by standard extraction processes of the prior art. Although the data are exemplified by alkaline phosphatase, mucin-2 and lysozyme from SNr, the different steps, i.e. (v.SN.4) and (v.SN.5), also show how to separate and obtain all proteins according to their isoelectric points by choosing the pH and pH gradient. Other method steps were also applied, particularly those based on the molecular weight of the proteins (see v.SN.6 and v.SN.7).

[0193] Indeed, the methods proposed for the simultaneous access to heparin and to proteins and peptides in the native state by fractionation of mammalian intestinal mucosa are based on the properties of the proteins contained in the mucosa, mainly their isoelectric point at a given pH and / or their molecular weight and / or their solubility in water or in organic solvents.

[0194] Example 3b: Proteomic study of fractions obtained during the industrial process (II) of Example 2a

[0195] the purpose To identify and annotate proteins present in 13 lyophilized fractions of porcine intestinal mucosa and to estimate their relative abundance in each fraction.

[0196] method Sample processing Samples P01 to P13 Sample provided: Solution Digestive enzyme: Trypsin (0.1 μg / μL, Promega) Digestion protocol: Protocol_MS01_InSolTrypDig.doc Final peptide concentration / volume: 50 μL (3% ACN / 1% FA)

[0197] Sample analysis nanoLC conditions Injection volume: 5μL Chromatograph: Thermo Scientific Dionex Ultimate 3000 Trap columna: μ-precolumn 300 μm (inner diameter) × 5 mm PepMap100, 5 μm, 100 Å, C18 (Thermo Scientific) Analytical column: NanoEase MZ HSS T3 column (75 μm × 250 mm, 1.8 μm, 100 Å) (Waters) Eluents: A: 0.1% formic acid in H2O, B: 0.1% formic acid in CH3CN Gradient: 3%~35% B for 60 minutes + 35%~50% for 5 minutes + 50%~85% for 2 minutes ·Flow rate: 250nL / min

[0198] LC-MS coupling Ion source: Advion Triversa Nanomate on chip technology (Advion BioSciences, Ithaca, NY, USA) nESI Spray voltage: 1.7kV Discharge pressure: 0.5psi Mode: Positive

[0199] MS conditions ·Mass spectrometer: Orbitrap Fusion Lumos (trademark) Tribrid (Thermo Scientific) Spray voltage: 1.7kV Ion transfer tube temperature: 275℃ RF lens: 30% m / z range: 375~1500 a.m. Data-dependent mode: Fastest (strongest) DDA ·Fragmentation method: HCD Collision energy: 38% AGC: 1×104 Detection: Orbitrap (MS1 120k), Orbitrap (MS2 30k) Instrument acquisition software: Xcalibur software vs4.2.28.14 (Thermo Scientific)

[0200] Data analysis Database Search Search software #1 / version: MaxQuant[2] / v2.0.2.0 ·Search Node#1 Software#1:Andromeda Database: Uniprot Pig (release 2022_11) Enzyme: Trypsin with two cleavage defects (complete) Dynamic modifications: oxidation at M and P, acetylation at the N-terminus of proteins Static modification: Carbamidomethylation in C Precursor mass tolerance: 10ppm ·MS / MS mass tolerance: 0.02Da

[0201] iBAQ (intensity-based absolute quantification) values ​​are the raw intensity divided by the number of theoretical peptides. This normalization algorithm allows to estimate the amount of protein in each sample. Calculate IBAQ intensities[1] with MaxQuant[2] software.

[0202] result The fractions had different numbers of protein groups quantified (113 to 1905). Based on the results, we can conclude that the following proteins, especially with potential commercial applications, are isolated or enriched in the corresponding fractions:

[0203] Acidic fraction (AF): Pr-glucagon, glucagon-like peptide-1, thymosin β4, vasoactive intestinal polypeptide

[0204] Basic fraction (BF): lysozyme, lactotransferrin, collagenase type IV of 72 kDa, interstitial collagenase, matrix metalloproteinase-9, regeneration family member III / regeneration islet-derived protein 3 (Reg III), regeneration family member IV / regeneration islet-derived protein IV (Reg IV), antimicrobial protein PR-39, angiogenin, phosphoinositide phospholipase C, phospholipase D, and pulmonary surfactant-associated protein D.

[0205] SNprubub: alkaline phosphatase, mucin-2, immunoglobulin M, immunoglobulin A, sucrase-isomaltase, α-amylase, hemoglobin, aminopeptidase, maltase-glucoamylase, catalase, carboxypeptidase, α-galactosidase, peroxiredoxin-6, superoxide dismutase, acid sphingomyelinase-like phosphodiesterase, diazepam binding inhibitor, gastrotropin, apolipoprotein-1, annexin-5, galectin-1, triacylglycerol lipase, phospholipase, thymosin β4

[0206] SNprububaq: alkaline phosphatase, mucin-2, immunoglobulin M, immunoglobulin A, sucrase-isomaltase, α-amylase, hemoglobin, aminopeptidase, maltase-glucoamylase, catalase, carboxypeptidase, α-galactosidase, peroxiredoxin-6, superoxide dismutase, acid sphingomyelinase-like phosphodiesterase, diazepam binding inhibitor, gastrotropin, apolipoprotein-1, annexin-5, galectin-1

[0207] Example 3c: Screening of antibacterial and antifungal activity of porcine extracts obtained during the industrial process (II) of Example 2a against different pathogens

[0208] the purpose Evaluation of antibacterial and antifungal activity of nine fractions of porcine intestinal mucosa against three human pathogens, one plant pathogen and one plant pathogenic fungus

[0209] method pathogen Antibacterial and antifungal activity was tested against the pathogens in Table 1. [Table 1]

[0210] Inocula were obtained from pure cultures of growing strains and plated on solid Luria-Bertani (LB) medium for bacteria and potato dextrose agar (PDA) medium for fungi. Human pathogens (St and Sa) were incubated at 37°C for 24 h and fungi (Fo) at 23°C for 7 days. All bacterial suspensions were incubated at 2 × 10 7 The final concentration of CFU / ml was adjusted. For tests using fungi, the conidial suspension was diluted to 2 × 10 4 Conidia were prepared at a final concentration of / ml.

[0211] process The antibacterial activity (see slide 5 in the appendix) of the four fractions and two control samples was evaluated at a concentration of 0.5x. The antibacterial effect was compared to the reference antibiotic levofloxacin (50mg / L) for bacteria and to cycloheximide (50mg / L) for fungi. An untreated control in which the product was replaced with water (positive control for microbial growth) and a negative control containing medium without product or microbial suspension (to exclude possible contamination of the environment) were also included. Product controls in which each product was incubated without microbial suspension or medium (to eliminate product contamination) were included.

[0212] Assay The antibacterial activity of the different products against the five pathogens was determined by growth inhibition assay. Bacterial suspensions were prepared using sterile distilled water and 2 × 10 7Conidial suspensions were also prepared in sterile distilled water and inoculated on PDA medium at 2 × 10 4 The concentration of conidia / ml was adjusted. The growth inhibition assay consisted of mixing 100 μl of the product with 100 μl of bacterial / fungal suspension to obtain a final volume of 200 μl in each well of the microplate (1 × 10 7 Bacterial suspension with a final concentration of 1 x 10 CFU / ml 4 (Fungal suspension at a final concentration of conidia / ml). In the case of human pathogens (St and Sa), the microplates were incubated at 37° C. The fungi were incubated for 7 days at 20° C. with constant shaking.

[0213] The growth kinetics of the different pathogens in the presence of the products was analyzed in triplicates (three wells per product and pathogen) using an automated system (Bioscreen C MBR, Labsystems, Finland and Varioskan flash, Thermo Electron Corporation, USA) that allows the microbial growth to be monitored by absorbance readings at 600 nm, hourly (24 h) for bacteria and every 30 min (7 days) for fungi. The microbial growth was quantified by analyzing the area under the growth curve and calculating the percentage of growth inhibition for each pathogen treated with each product.

[0214] result The percent growth inhibition of each pathogen treated with each product is shown in Table 2. [Table 2]

[0215] Fractions AF, BF2 and SNprububaq show significant antibacterial potential.

[0216] Prior art documents Patent Literature ·China Patent No. 107236059 ·China Patent No. 106035980B ·China Patent No. 102839162B ·Russian Patent No. 2610669 · U.S. Patent Application Publication No. 20140369989 Non-patent literature ·Fontaine et al. (1985).Absence d'effet de la vitamina D sur la phytase et la phosphatase alcaline intestinales:relation avec l'absorption du phosphore phytique chez le porc.Reprod.Nutr.Develop., 25(4A), 717-727. ·Lackeyram et al. (2010). "Early weaning reduces expression of alkaline phosphatase in pigs." The Journal of Nutrition 140, pp.461-468

[0217] Terms For completeness, various aspects of the invention are set forth in the following numbered clauses.

[0218] Article 1. The following steps: (i) adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa to obtain preserved mucosa, or storing the extract at a temperature that allows the proteins, peptides and heparin to be preserved in a native state; (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa; (iii) subjecting the diluted mucosa of step (ii) or the preserved mucosa of (i) to homogenization at a temperature below 40° C. to lyse the cell membranes of the mucosal cells and obtain a stable homogenate of the mucosa containing heparin and mucosal proteins and peptides; (iv) separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iii) by one or more physical and / or chemical means selected from the group consisting of centrifugation, filtration and / or ultrafiltration, optionally using a detergent, and combinations thereof, to obtain a pellet (P) containing heparin and a supernatant fraction (SN) containing proteins and mucosal proteins and peptides; (va) subjecting the pellet fraction (P) of (iv) containing heparin to an alkaline proteolysis process with a proteolytic enzyme to obtain a mixture of heparin and protein hydrolysates, and subjecting the heparin-containing mixture to one or more precipitation steps, and optionally one or more extraction steps, and one or more chromatography steps to purify said heparin from the protein hydrolysates; and (vb) subjecting the supernatant (SN) of (iv) containing the mixture of solubilized and / or suspended mucosal proteins and peptides to one or more of the following successive filtration, ultrafiltration steps, protein precipitation and / or gel permeation and / or ion exchange chromatography steps in order to separate the proteins and peptides according to either their solubility, isoelectric point and / or molecular weight (the order or sequence of these steps may be interchangeable). 23. A method for simultaneously obtaining heparin and proteins and peptides in the native state by fractionation of mammalian intestinal mucosa, comprising:

[0219] Clause 2. The method of clause 1, wherein the separating step (iv) is carried out by centrifugation.

[0220] Clause 3. The method according to any of clauses 1 to 2, wherein the supernatant (SN) containing the mixture of mucosal proteins and peptides is subjected to filtration in step (vb) through a filter with a cutoff of up to 100 μm in order to retain small particles and lipid residues that were not pelleted in step (iv) to obtain a filtered supernatant containing the majority of the mucosal proteins and peptides.

[0221] Clause 4. The method according to clause 3, wherein the filtered supernatant is further diafiltered through a filter having a cut-off of less than 0.8 μm to obtain a permeate (SNp) containing mucosal proteins and peptides and a retentate (SNr) containing water insoluble proteins and peptides.

[0222] Clause 5. The method according to any of clauses 3-4, wherein the filtered supernatant containing mucosal proteins and peptides or either its permeate (SNp) or retentate (SNr) fraction is loaded onto a cation exchange chromatography column in downflow to obtain a bound fraction (bf) and an unbound fraction (ubf), and the bound fraction is further diluted in upflow direction, followed by diafiltration and concentration using a tangential flow system with a filter having a cut-off of 1-5 KDa to obtain a basic fraction of the filtered supernatant containing basic proteins and peptides.

[0223] Clause 6. The method according to clause 5, wherein the unbound fraction (ubf) is loaded onto an anion exchange chromatography column and further eluted in upflow direction, then diafiltered and concentrated using a tangential flow system with a filter having a cut-off of 1-5 KDa to obtain a filtered supernatant acidic fraction containing acidic proteins and peptides.

[0224] Clause 7. The method according to any of clauses 5-6, wherein either the basic or acidic fraction is further subjected to fractionation using diafiltration, salting out methods, cation and anion exchange chromatography and combinations thereof.

[0225] Clause 8. The method according to any one of clauses 4 to 7, wherein the retentate (SNr) containing the water insoluble proteins and peptides is treated, optionally in the presence of detergents and / or by homogenization with hydrolases, to detach bound proteins from membranes or cell structures before loading it downflow onto an ion exchange chromatography.

[0226] Clause 9. The method of clause 8, wherein the hydrolytic enzyme comprises one or more of a phospholipase and a protease.

[0227] Clause 10. The method according to any one of clauses 1 to 9, wherein in step (ii) the mucosa is diluted with deionised water containing one or more surfactants.

[0228] Clause 11. The method according to any of clauses 1 to 10, wherein the homogenization step (iii) is carried out by a technique selected from the group consisting of mechanical or physical cell lysis of the mammalian intestinal mucosa by mechanical disruption, fluid shear forces, high pressure or cavitation.

[0229] Clause 12. The method according to clause 11, wherein the homogenization step (iii) is carried out by mechanical disruption of the mucosal cells with a rotor-stator homogenizer at a g-force of at least 180 g, preferably 1700 g, for a minimum of 30 seconds, preferably 2 to 4 minutes.

[0230] Clause 13. The method according to any of clauses 1 to 10, wherein the homogenization step (iii) is carried out by chemical means comprising the addition of a surfactant to the diluted mucosa of step (ii).

[0231] Clause 14. A composition comprising mammalian intestinal mucosa proteins and peptides obtainable by the method defined in any of clauses 1 to 13.

[0232] Clause 15. The composition according to clause 14, wherein the mammalian intestinal mucosa proteins and peptides are selected from the group consisting of brush border enzymes, antimicrobial peptides, antioxidant enzymes, hormonal peptides, proteins involved in defense and / or healing processes in the intestinal mucosa and combinations thereof.

[0233] Clause 16. A method for obtaining heparin by fractionation of mammalian intestinal mucosa, comprising steps (i), (ii), (iii), (iv) and (va) as defined in clause 1.

[0234] Clause 17. A method for obtaining proteins and peptides in the native state by fractionation of mammalian intestinal mucosa, comprising steps (i), (ii), (iii), (iv) and (vb) as defined in clause 1.

Claims

1. The following steps: (i) A step of obtaining preserved mucosa by adding preservatives and antioxidants to an aqueous extract of mammalian intestinal mucosa, or a step of preserving the extract at a temperature that allows for the preservation of proteins, peptides and heparin in an undenatured state. (ii) Optionally, a step of diluting the preserved mucosa with up to 100 volumes of deionized water to obtain diluted mucosa. (iiia) A step of homogenizing the diluted mucosa from step (ii) or the preserved mucosa from step (i) at a temperature below 40°C and optionally in the presence of a surfactant and / or a hydrolytic enzyme such as phospholipase to dissolve the cell membranes of mucosal cells and obtain a stable homogenate of the mucosa containing heparin, mucosal proteins and peptides. (iva) Separating the heparin and mucosal proteins and peptides contained in the stable homogenate obtained in step (iiia) by one or more physical and / or chemical means selected from the group consisting of centrifugation and filtration (microfiltration and / or ultrafiltration) using a surfactant and combinations thereof, to obtain a pellet (P) or retaining liquid fraction containing heparin and a supernatant (SN) or permeate fraction containing mucosal proteins and peptides. (v.a') The pellet (P) or retained liquid fraction of (iva) containing heparin is subjected to an alkaline proteolytic process with a proteolytic enzyme to obtain a mixture of heparin and protein hydrolysate, and the mixture containing heparin is subjected to one or more precipitation steps, and optionally one or more extraction steps, and one or more chromatography steps to purify the heparin from the protein hydrolysate, and (v. b') A step of subjecting the supernatant (SN) or permeate of (iva), which contains a mixture of solubilized and / or suspended mucosal proteins and peptides, to one or more of the following steps in order to separate the proteins and peptides according to their solubility, isoelectric point and / or molecular weight: continuous filtration, ultrafiltration, solvent extraction, protein precipitation, enzymatic hydrolysis, gel permeation, ion exchange chromatography, size exclusion chromatography or affinity chromatography (the order or sequence of the steps is interchangeable). A method for simultaneously obtaining heparin, as well as undenatured proteins and peptides, from fractionation of mammalian intestinal mucosa.

2. The method according to claim 1, wherein the separation step (iva) is performed by centrifugal separation.

3. (v.b.1') The supernatant (SN) or permeate of step (v.b'), which includes a mixture of solubilized and / or suspended mucosal proteins and peptides, is subjected to filtration through a filter with a cutoff of up to 100 μm to retain small particles and lipid residues that were not pelletized in step (iva), in order to obtain a filtered supernatant containing the majority of the mucosal proteins and peptides. The method according to claim 1, further comprising:

4. (v.b.4') The supernatant from step (v.b.1') is subjected to an anion exchange chromatography column to obtain a bound fraction (SNbf') and an unbound fraction (SNubf'), the bound fraction is eluted, and then it is dialyzed and concentrated using a tangential flow system with a filter having a cutoff of 1 to 5 kDa to obtain an acidic fraction of the supernatant containing acidic proteins and peptides. The method according to claim 3, further comprising:

5. (v.b.5') The unbound fraction (SNubf') from step (v.b.4') is subjected to a cation exchange chromatography column to obtain a bound fraction (SNubfbf') and an unbound fraction (SNubub'), the bound fraction is eluted, and then the supernatant is dialyzed and concentrated using a tangential flow system with a filter having a cutoff of 1 to 5 kDa to obtain the basic fraction containing basic proteins and peptides. The method according to claim 4, further comprising:

6. (v.b.4a) The supernatant from step (v.b.1') is subjected to a cation exchange chromatography column to obtain a bound fraction (SNbf) and an unbound fraction (SNubf), the bound fraction is eluted, and then it is dialyzed and concentrated using a tangential flow system with a filter having a cutoff of 1 to 5 kDa to obtain a basic fraction of the supernatant containing basic proteins and peptides. The method according to claim 3, further comprising:

7. (v.b.5a) The unbound fraction (SNubf) from step (v.b.4a) is subjected to an anion exchange chromatography column to obtain a bound fraction (SNubfbf) and an unbound fraction (SNubub), the bound fraction is eluted, and then the supernatant acidic fraction containing acidic proteins and peptides is obtained by dialysfiltration and concentration using a tangential flow system with a filter having a cutoff of 1 to 5 kDa. The method according to claim 6, further comprising:

8. The method according to claim 4, wherein the basic or acidic fraction is further subjected to fractionation using dialysis filtration, salting out, protein precipitation, solvent extraction, cation and anion exchange chromatography, size exclusion chromatography, affinity chromatography, and combinations thereof.

9. The method according to claim 7, wherein the basic or acidic fraction is further subjected to fractionation using dialysis filtration, salting out, protein precipitation, solvent extraction, cation and anion exchange chromatography, size exclusion chromatography, affinity chromatography, and combinations thereof.

10. The method according to claim 1, wherein in step (ii), the mucous membrane is diluted with deionized water containing one or more surfactants.

11. The method according to claim 2, wherein in step (ii), the mucous membrane is diluted with deionized water containing one or more surfactants.

12. The method according to claim 1, wherein the homogenization step (iiiia) is carried out by a technique selected from the group consisting of mechanical fracture, fluid shear force, high pressure, or mechanical or physical cytolysis of mammalian intestinal mucosa by cavitation.

13. The method according to claim 12, wherein the homogenization step (iiia) is carried out by mechanically destroying the mucosal cells for at least 30 seconds, preferably 2 to 4 minutes, using a rotor-stator homogenizer with a g-force of at least 180 g, preferably 1700 g.

14. The method according to claim 2, wherein the homogenization step (iiia) is carried out by a technique selected from the group consisting of mechanical fracture, fluid shear force, high pressure, or mechanical or physical cytolysis of mammalian intestinal mucosa by cavitation.

15. The method according to claim 14, wherein the homogenization step (iiia) is carried out by mechanically destroying the mucosal cells for at least 30 seconds, preferably 2 to 4 minutes, using a rotor-stator homogenizer with a g-force of at least 180 g, preferably 1700 g.

16. The method according to claim 1, wherein the homogenization step (iiia) is carried out by chemical means including the addition of a surfactant to the diluted mucous membrane of step (ii).

17. The method of claim 2, wherein the homogenization step (iiia) is carried out by chemical means including the addition of a surfactant to the diluted mucous membrane of step (ii).

18. (v.b. 1') A step of subjecting the supernatant (SN) or permeate of step (v.b'), which includes a mixture of solubilized and / or suspended mucosal proteins and peptides, to filtration through a filter with a cutoff of up to 100 μm in order to retain small particles and lipid residues that were not pelletized in step (iva), in order to obtain a filtered supernatant containing the majority of the mucosal proteins and peptides. The method according to claim 17, further comprising:

19. A method for obtaining heparin by fractionation of mammalian intestinal mucosa, comprising steps (i), (ii), (iiia), (iva), and (v.a') as defined in claim 1.

20. A method for obtaining undenatured proteins and peptides by fractionation of mammalian intestinal mucosa, comprising steps (i), (ii), (iiia), (iva), and (v.b') as defined in claim 1.