Patent Description:
Present invention relates to the field of obtention of heparin, proteins and peptides in their native form, and other compounds of interest, from slaughtered animals for consumption of meat.

In the processing of the several parts of slaughtered animals, mainly for food consumption, besides the parts used as food, remaining extracted viscera not commonly offered to meat consumers are further processed in order to obtain several products of interest for other fields. For example, it is widely known the method of obtention of heparin of high pure grade and useful in medicine from the mucosal tissue, in particular from the intestine mucosa. After the recovery of the mucosa, intestine tubular structures are mainly recycled for many different purposes, including the casing in sausage elaboration or even in medical applications such as implants.

In these processes for extracting heparin, for example, aggressive mechanical homogenization of mucosal tissues (i.e., intestine) is carried out at high temperatures (<NUM>-<NUM> or even higher temperatures) in the presence of proteolytic enzymes, and/or by means of salting out the homogenates. Next, precipitation steps, optional extractive steps, and chromatography are carried out. These methodologies give from the one side the heparin, in several purity grades depending on the steps of the methods, and from the other side, a protein hydrolysate, considered a by-product and which is commonly used in animal food as peptide supplementation. Examples of documents disclosing method of extraction of heparin and other by products from small intestinal mucosa include Chinese patent application <CIT>) and the Chinese patent <CIT>).

Proteins in their natural state with intact structure and function that is not altered by heat, chemicals, enzyme reaction, or other denaturants are named "native proteins". To date, in these methods of extraction of heparin, no proteins or peptides in native form are obtained, because high temperatures, enzymatic treatment, and mechanical stress reduce the same to the peptide hydrolysate. This hydrolysate is mostly made up of very small peptides and free amino acids not of high value. On the other hand, the regulations for the obtaining of heparin with the adequate purity grade and requirements imposed by health authorities for the medical application, do not allow many variations to the methodology for the obtaining of said compound.

Active enzymes and mixtures of active enzymes have also been obtained from mucosal tissues of slaughtered animals though. As a way of example, Chinese patent <CIT>) discloses a method to obtain alkaline phosphatase from animal viscera (including pig intestine mucosa), in which mucosa is first cut and homogenized at <NUM>; then it is precipitated with acid, extracted with butanol and dialyzed; further it is salted out with ammonium sulphate; and finally, the salted-out fraction is submitted to several chromatography columns to be further purified. An alkaline phosphatase product with a specific activity of <NUM> U/mg is so obtained. This method, however, does not allow the option of obtaining heparin or other enzymes according to prescribed regulations.

Proteolytic enzymes are obtained from fish digestive organs, for example following the method disclosed in Russian patent <CIT>. In this case, tissues are homogenized, filtered, and proteins are salted out in ammonium sulphate with a previous step of nucleic acids and lipids removal. Further, the precipitate is dissolved in a buffer and chromatography is performed.

All these methods to recover one or more enzymes in active form from animal mucosal tissues include a sequence of steps conceived to preserve enzyme integrity. They are performed at low and middle temperatures (<NUM>-<NUM>), and they are carried out at physiological conditions (of pH and ionic force).

In all the methods disclosed above, either for extraction of heparin or for the obtention of active enzymes, the main aim is to recover the product of interest in the balance of a high yield, activity and high purity.

Besides these methods departing from tissue of slaughtered animals, many enzymes are currently obtained using biotechnological methods including the expression of the enzymes in bioreactors. In these biotechnological methods, host cells (yeast, bacteria, etc.) are modified by means of recombinant gene technology to express the enzymes of interest when cultured. However, these methods are usually expensive, and they require of many reagents and energy suppliers to control growing conditions of the cells. Thus, they are usually considered methods of certain complexity.

Enzymes are used in several field of industry, as well as in the medical field. For example, cellulases and ligninases are used in biofuel industry, nucleases in the field of molecular biology, amylases and proteases in food processing, or xylanases in paper industry. Many enzymes are also used in clinical diagnostic methods. In the field of food supplementation and additivation of animal food, phytases have been employed to take profit of phosphorus of phytic acid usually contained in the mixture constituting fodders.

Albeit the existing methods, there is still a need of alternative methodologies to give value to products of slaughtered animals, obtained in the course of reproducible, non-complex and non-expensive processes, and allowing obtention of as much as possible reliable and effective by-products in high yields and complying with the regulations, if any. In the particular case of enzymes, there still remains a need in the field of additivation of food of active enzymes that can be obtained by reliable and economic processes.

With the aim of giving value to as many products as possible from discarded tissues of slaughtered animals, inventors propose a new method allowing obtaining simultaneously heparin and proteins and peptides in their native form, of mammalian intestine mucosa by a non-expensive but sustainable approach. Heparin is obtained with a grade useful for pharmaceutical application (i.e., at pharmaceutical grade according to regulations). Moreover, the proteins and peptides are obtained in their native state, thus, they are in their properly folded and assembled form with operative structure and function (e.g., enzymatic, structural, hormonal, etc).

Present invention is, thus, embedded in the context of the processing of slaughterhouse tissues, from which value-added products are obtained after tissue homogenization, such as heparin.

The new methodology includes homogenization of mammal tissue at mild conditions (cold temperature and physiological pH), which helps preserving stability and activity of isolated proteins and peptides. Several additional steps including fractionation steps and chromatography of homogenate material are also performed.

Thus, a first aspect of the invention is a method for the simultaneous obtention of heparin, proteins and peptides in native state by fractionation of mammalian intestine mucosa, comprising the following steps:.

Disclosed is, therefore, a method for the simultaneous obtention of heparin, proteins and peptides in native state by fractionation of mammalian intestine mucosa, comprising the following steps:.

Another aspect of the invention is a method for the simultaneous obtention of heparin, proteins and peptides in native state by fractionation of mammalian intestine mucosa, comprising the following steps:.

A further aspect of the invention is a method for the obtention of heparin by fractionation of mammalian intestine mucosa, comprising the steps (i), (ii), (iiia), (iva), and (v. a') as defined herein.

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

Several compositions including compounds of interest are obtained by the method. In particular compositions of or fractions from the mammalian intestine mucosa comprising purified acidic or basic proteins (i.e., enzymes) that are then useful in the common applications they are known for or for any new ones.

Thus, disclosed herein are compositions comprising an acidic fraction of intestine mammal mucosa acidic proteins and peptides which is obtainable by a method comprising steps (i), (ii), (iiia), and (iva) as defined herein, further comprising:.

or alternatively obtainable by a method comprising steps (i), (ii), (iiia), and (iva) as defined herein, further comprising:.

Also disclosed herein is a basic fraction of intestine mammal mucosa basic proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), and (v. <NUM>') as defined herein to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides; and further comprising:.

Also disclosed herein is a composition SNubub' comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>'), and (v. <NUM>') as defined herein.

Also disclosed herein is a composition SNubub comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. 4a), and (v. 5a) as defined herein.

Also disclosed herein is as will be illustrated in the examples, a fraction comprising heparin of pharmaceutical grade. Thus, a heparin complying with all the regulatory aspects according to health authorities.

In the context of this methodology, a mixture of native proteins of the mammalian intestine mucosa is obtained with a high yield, including the ones having high enzymatic activity.

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more. " Unless indicated otherwise, definite articles used herein, such as "the" also include the plural of the noun.

The term "about" or "around" as used herein refers to a range of values ± <NUM>% of a specified value. For example, the expression "about <NUM>" or "around <NUM>" includes ± <NUM>% of <NUM>, i.e. from <NUM> to <NUM>.

Enzyme activity, when indicated, is defined as the moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, (mainly pH and temperature). The SI unit is the katal, <NUM> katal = <NUM> mol s-<NUM>, but this is an excessively large unit. A more practical and commonly used value is enzyme unit (U) = <NUM>µmol min-<NUM>. <NUM> U corresponds to <NUM> nanokatals.

The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in µmol min-<NUM>-<NUM>). Specific activity gives a measurement of enzyme purity in the mixture. It is the micro moles of product formed by an enzyme in a given amount of time (minutes) under given conditions per milligram of total proteins. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal/kg, but a more practical unit is µmol/mg·min. Specific activity is a measure of enzyme processivity (the capability of enzyme to be processed), at a specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme.

The enzymatic activities of the enzymes obtained by the described method can also be expressed in units of activity per kilogram of mucosa (expressed in µmol min-<NUM>-<NUM>). This parameter gives a measurement of the quantity of enzyme extracted from the initial raw material.

The term "Mammalian intestine mucosa" according to this description is the term used in the terminology of the processing of animal sub-products in slaughterhouses, such as visceral material from animals. It includes all the material obtained after the scraping of intestines, namely the small intestine. From this scraping a clean tube/membrane is obtained, and the scrapped material is the so-called mammalian intestine mucosa, a complex mixture that comprises the intestine epithelium, the lamina propria (mucosa) and the muscularis mucosae (mucosa), the microvilli with the brush border and the lumen. This complex mixture is the one that in present application is, in some examples and embodiments, submitted to homogenization and further processed to obtain the enzymes of interest and heparin.

As previously indicated, a first aspect of the invention is a method for the simultaneous obtention of heparin, proteins, and peptides in native state by fractionation of mammalian intestine mucosa, comprising the following steps:.

b) are also referenced in this description as (v. SN), respectively, for referring to the managing or further processing of the (P) and (SN) fractions of (iv), which allow the simultaneous obtention of the heparin, and of the proteins and peptides in native state, of mammalian intestine mucosa.

"Simultaneous" is to be understood not only as processed at the same time with the required and adapted equipment, but also including a differential processing in time or place but providing from the same intestine mucosa the two compositions of interest. In other words, meanwhile heparin is obtained most of the other proteins and peptides of interest are preserved and also purified from another fraction also obtained from the same mucosa. This is genuine from the method of the invention, since until now in the process for the obtaining of heparin with a high yield from mammalian intestine mucosa, the proteolytic step applied only allowed to obtain a mixture of peptides besides the heparin; thus, non-functional or native proteins and peptides.

Present inventors have surprisingly found a combination of steps and pre-treatments that in combination allow obtaining both, heparin with a high yield and purity grade, as well as the native proteins and peptides (including functional enzymes) of the mammalian intestine mucosa.

The fractions obtained thereof contain the heparin or the proteins and peptides in several purification grades depending on the type, number and sequence of the physical and/or chemical means applied in step (iv) and the following (v. It will be understood that in any case the contents of each of the fractions are profitable products or compositions resulting from the method of the invention, which method allows the simultaneous obtention of heparin and a myriad of native and functional proteins and peptides from mammalian intestine mucosa of slaughtered animals.

In the paragraphs that follow, particular embodiments of the type and number and sequence of these physical and/or chemical means applied are illustrated. The accompanying examples serve also for the purpose of showing the yield of the method for obtaining fractions with noteworthy amounts of heparin while certain enzymes of interest, in native and functional state, are also obtained with noteworthy yields.

In a particular embodiment of the method of the first aspect, the mammalian intestine mucosa is the pig one. Other mammalian species, not including human, are also useful.

In another particular embodiment of the method, the preservative and antioxidant agent in step (i) is sodium metabisulfite, added up to a concentration of <NUM>% w/w, preferably <NUM>% w/w. Equivalent substances can be chosen as a preservative and antioxidant agents.

In another particular embodiment, optionally in combination with any of the method embodiments above or below, (ii) the preserved mucosa is diluted with up to <NUM> volumes of deionized water, more in particular is diluted with <NUM> to <NUM> volumes of deionized water, even more in particular with <NUM> to <NUM> volumes of deionized water, and also more in particular with <NUM> to <NUM> volumes of deionized water. In a more particular embodiment, instead of deionized water, a same volume of buffer at a pH comprised between <NUM> and <NUM> is used, preferably at <NUM>.

In a more particular embodiment of the method, in step (ii) the mucosa is diluted either with deionized water or a buffer, and more in particular both comprising one or more detergents. In even a more particular embodiment the detergents are non-ionic detergents or zwitterionic detergents. Examples of commercially available detergents are selected from the group consisting of Tween <NUM>®, Tween <NUM>, Triton X-<NUM> or X-<NUM>, deoxycholic acid, Brij <NUM> or <NUM>, BigCHAP or deoxy BigCHAP, MEGA <NUM>, MEGA <NUM> or MEGA <NUM>, octyl beta-glucoside, and combinations thereof.

Steps (iii), (iv), (v. <NUM>), (v. <NUM>) and (v. <NUM>) are equivalent to steps (iiia), (iva), (v. <NUM>'), (v. 4a) and (v. 5a) respectively. All embodiments defined herein for steps (iii), (iv), (v. <NUM>), (v. <NUM>) and (v. <NUM>) also apply to steps (iiia), (iva), (v. <NUM>'), (v. 4a) and (v. 5a) respectively.

In a particular embodiment, step (iii) or step (iiia) is performed at a temperature equal to or lower than about <NUM>, or alternatively equal to or lower than about <NUM>, or alternatively equal to or lower than about <NUM>, or alternatively equal to or lower than about <NUM>, or alternatively equal to or lower than about <NUM>, or alternatively equal to or lower than about <NUM>.

In another particular embodiment, step (iii) or step (iiia) is performed at a temperature from <NUM> to <NUM>, or alternatively from <NUM> to <NUM>, or alternatively from <NUM> to <NUM>, or alternatively from <NUM> to <NUM>, or alternatively from <NUM> to <NUM>, or alternatively from <NUM> to <NUM>. In yet another particular embodiment, step (iiia) is performed at a temperature from <NUM> to <NUM>. In yet another particular embodiment, step (iii) is performed at a temperature from <NUM> to <NUM>.

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

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 mammalian intestinal mucosa by mechanical disruption, shear fluid forces, high pressure or cavitation. In another particular embodiment, the homogeneization step (iiia) is carried out in the presence of detergents or hydrolytic enzymes such as phospholipases to detach bound proteins to membranes or cell structures.

In a more particular embodiment, said homogenization step (iii) is carried out by mechanical disruption of the mucosa cells by means of a rotor-stator homogenizer at the g-force of at least <NUM>, preferably <NUM>, for a minimum of <NUM> seconds, preferably between <NUM> and <NUM> minutes.

In a particular embodiment, homogenization step (iiia) is carried out by mechanical disruption of the mucosa cells by means of a rotor-stator homogenizer. More in particular at a g-force of at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM> or about <NUM>; more in particular from <NUM> to <NUM>. More in particular homogenization is carried out at about <NUM>.

In a particular embodiment, homogenization step (iiia) is carried out for at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM> seconds. More in particular the homogenization is carried out for a period from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to220, from <NUM> to <NUM>, from <NUM> to <NUM>, or from <NUM> to <NUM> seconds, even more in particular from <NUM> to <NUM> seconds.

In a more particular embodiment, the homogenization step (iiia) is carried out by mechanical disruption of the mucosa cells by means of a rotor-stator homogenizer at a g-force of at least <NUM>, preferably <NUM>, for a minimum of <NUM> seconds, preferably between <NUM> and <NUM> minutes.

Step (iva) comprises separating heparin and the mucosa proteins and peptides contained in the stable homogenate obtained in step (iiia) by one or more of 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 fraction comprising heparin (P) and proteins; and a supernatant fraction (SN) comprising mucosa proteins and peptides.

The separation step (iva) may be performed by methods well-known in the art. The skilled in the art will be able to determine the exact conditions of the separation depending on the proteins of interest.

Also, another particular embodiment of the method of the invention comprises carrying out step (iv) of separation by centrifugation in order to obtain a pellet fraction comprising heparin (P); and a supernatant fraction comprising mucosa proteins and peptides, some of them in solution and others in suspension (SN). As previously indicated in the first aspect, these (P) and (SN) fractions are further processed in respective steps (v. a), and (v. b), both notations being interchangeable.

In a particular embodiment, the centrifugation in (iva) is carried out at a g-force of at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM> or about <NUM>; in particular from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>; more in particular from <NUM> to <NUM>.

In a particular embodiment the centrifugation in (iva) lasts for at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM> minutes, in particular from <NUM> to <NUM>, from <NUM> to <NUM>, or from <NUM> to <NUM> minutes; more in particular it lasts for from <NUM> to30 minutes.

In a particular embodiment the centrifugation in (iva) is performed at a temperature equal to or lower than about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>, in particular at a temperature from <NUM> to <NUM>, or from <NUM> to <NUM>, or from <NUM> to <NUM>, or from <NUM> to <NUM>, or from <NUM> to <NUM>, more in particular the centrifugation in (iva) is performed at a temperature from <NUM> to <NUM>, even more in particular at a temperature from <NUM> to <NUM>.

In a most particular embodiment, the centrifugation in (iv) is carried out at a g-force of at least <NUM>, preferably at <NUM> during at least <NUM> minutes, preferably <NUM> minutes and at a temperature below <NUM>, in particular below <NUM>, more preferably below <NUM>, to obtain, as indicated, a solid phase containing heparin (thus, a pellet phase (P)); and a liquid phase containing a mixture of mucosa proteins and peptides, some of them in solution, others in suspension (thus, a supernatant phase (SN)).

In a most particular embodiment, the centrifugation in (iva) is carried out at a g-force of at least <NUM>, preferably at <NUM> during at least <NUM> minutes, preferably <NUM> minutes and at a temperature below <NUM>, in particular below <NUM>, more preferably below <NUM>, to obtain, as indicated, a solid phase containing heparin (thus, a pellet phase (P)); and a liquid phase containing a mixture of mucosa proteins and peptides, some of them in solution, others in suspension (thus, a supernatant phase (SN)).

In a more particular embodiment, the centrifugation in (iva) is carried out at a g-force from <NUM> to <NUM> for a period of time from <NUM> to <NUM> minutes, particularly from <NUM> to <NUM> minutes, and at a temperature from <NUM> to <NUM>, or from <NUM> to <NUM>, or from <NUM> to <NUM>, or from <NUM> to <NUM>.

In a more particular embodiment, the centrifugation in (iva) is carried out at a g-force of about <NUM> for a period of time about <NUM> minutes and at a temperature below about <NUM>.

In a more particular embodiment, the centrifugation in (iva) is carried out at a g-force of about <NUM> for a period of time of about <NUM> minutes and at a temperature below about <NUM>, particularly from <NUM> to <NUM>.

Also, another particular embodiment of the method of the invention comprises carrying out step (iva) of separation by filtration or ultrafiltration in order to obtain a pellet (retentate) fraction comprising heparin; and a supernatant (permeate) fraction comprising mucosa proteins and peptides, some of them in solution and others in suspension. As previously indicated in the first aspect, these (P) and (SN) fractions are further processed in respective steps (v. a'), and (v. b'), both notations being interchangeable.

In a particular embodiment, the supernatant (SN) obtained in step (iva) comprises one or more of alkaline phosphatase (also referred herein as to ALP), mucin-<NUM> (also referred herein as to MUC2) and lysozyme (also referred herein as to LYZ), more particularly it comprises alkaline phosphatase, mucin-<NUM> and lysozyme. More in particular the SN further comprises one or more of glucagon like peptide-<NUM> (also referred herein as to GLP-<NUM>), thymosin beta-<NUM>, vaso intestinal peptide, lysozyme, lactotransferrin, regenerating islet-derived protein <NUM> (Reg III), regenerating islet-derived protein IV (Reg IV), matrix metalloproteinase-<NUM>, <NUM> kDa type IV collagenase, interstitial collagenase, bactericidal permeability-increasing protein, pulmonary surfactant-associated protein D, antibacterial protein PR-<NUM>, angiogenin, alkaline phosphatase, mucin-<NUM>, trypsin, aminopeptidases, carboxypeptidases, catalase, triacylglycerol lipase, phospholipases, acid sphingomyelinase-like phosphodiesterase, alpha-amylase, alpha-galactosidase, sucrase-isomaltase, peroxiredoxin-<NUM>, superoxide dismutase, apolipoprotein A1, annexin-<NUM>, galectin-<NUM>, diazepam binding inhibitor, immunoglobulins, gastrotropin, and hemoglobin; and more particularly comprises all of them.

As indicated, in a step (v. a), and with the aim of recovering most of the heparin of the homogenate and with a high degree of purity, the pellet (P) of previous embodiments in which a step of centrifugation is applied, is submitted to an alkaline proteolytic process by means of proteolytic enzymes to obtain heparin and a protein hydrolysate. Next, precipitation steps, optional extractive steps, and optional chromatography are carried out to purify heparin.

This further steps of alkaline proteolytic process and heparin purification are equivalent to the standard ones, known as master file batch for the extraction of heparin, and that gives heparin of pharmaceutical grade and a separate fraction comprising a protein hydrolysate that is usually used as animal food additive (as explained in the background section and in previous paragraphs).

Thus, the method of the invention provides a heparin extracted from mammalian intestinal mucosa and a protein hydrolysate, which are obtainable by a method as defined in the first aspect and its embodiment including the alkaline proteolytic process step.

Parallel to the extraction of heparin, in a step (v. b) in another particular embodiment of the first aspect, also optionally in combination with any of the embodiments above or below, the supernatant fraction (SN) comprising solubilized and/or suspended a mixture of mucosa proteins and peptides when a centrifugation step in (iv) is carried out for its obtention, it is submitted to one or more of successive filtration, ultrafiltration steps, protein precipitation and/or gel permeation and/or to ion exchange chromatography steps, being exchangeable the order or sequence of the steps, in order to separate the proteins and peptides according to any of their solubility, isoelectric point and molecular weight.

This purification process of the SN (i.e., step (v. SN)) comprises, in some embodiments, a separation of all soluble proteins and peptides from the insoluble or suspended ones. The SN is submitted then to a diafiltration process through cassettes or cartridges having a cut-off between <NUM> kDa and <NUM>, preferably <NUM>.

The retentate of this diafiltration step (SNr) contains small particles, lipid residues and all the mucosa proteins and peptides which are insoluble in water, or partly water soluble. The latter are bound to other cell structures such as cell membranes or organelles or other insoluble proteins to form aggregates, and they were not pelleted in a centrifugation within step (iv) of the method and, thus are not filterable (i.e., diafilterable).

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

In a further purification step, the fraction SNp is further diafiltered and concentrated through a filter having a cut-off between <NUM> and <NUM> kDa with the aim of retaining all the soluble proteins and peptides contained in SNp, obtaining a concentrated fraction of soluble proteins and peptides (SNpr).

In a more particular embodiment, when the SNp is diafiltered to obtain the SNpr, the diafiltration is carried out by means of a tangential flow filtration equipment. In even a more particular embodiment, the diafiltration is carried out by substituting the permeate with at least <NUM> volumes of deionized water or a suitable buffer, preferably between <NUM> and <NUM> volumes.

In a particular embodiment, optionally in combination with any of the embodiments above or below, the SNp (or SNpr) comprising solubilized mucosa proteins and peptides is then submitted to additional separation techniques, based according to the isoelectric point of the proteins and peptides. In other words, SNp (or SNpr) comprising solubilized mucosa proteins and peptides is submitted to other of the also included in step (v. b) one or more of successive filtration, ultrafiltration steps, protein precipitation and/or gel permeation and/or to ion exchange chromatography steps, being exchangeable the order or sequence of the steps allowing the separation of said proteins and peptides by means based according to the isoelectric point of the proteins and peptides.

In a particular embodiment of step (v. b), an SNp obtained as indicated in previous paragraphs and comprising the soluble mucosa proteins and peptides is, preferably after having adjusted the pH to <NUM>, loaded downflow on a cationic exchange chromatographic column to obtain a bound fraction (SNpbf) and an unbound fraction (SNpubf); and the bound fraction is further eluted with the upflow direction and then diafiltered and concentrated by means of a tangential flow system through a filter having a cut-off from <NUM> to <NUM> KDa, preferably of <NUM> Kda, to obtain a basic fraction (also referred herein as to BASIC FRACTION or BF) comprising basic proteins and peptides. Alternatively, the cationic exchange chromatography can be done in batch, adding from <NUM> to <NUM>% (w/w) of strong cationic exchange resin to the fraction, preferably <NUM>%.

With this step of cationic exchange chromatography, in particular a strong cationic exchange chromatography, all basic proteins in the SNp are consequently concentrated. In a particular embodiment the elution of the bound fraction (SNpbf) and diafiltration is carried out through a phosphate buffer <NUM> at pH between <NUM> and <NUM>.

In another particular embodiment of the method, when the SNp is submitted to the cationic exchange chromatographic column, the unbound fraction (SNpubf) is, preferably after having adjusted the pH to <NUM>, loaded on an anionic exchange chromatography column, and further eluted with the upflow direction and then diafiltered and concentrated by means of a tangential flow system through a filter having a cut-off from <NUM> to <NUM> Kda, preferably <NUM> Kda, to obtain an acidic fraction (also referred herein as to ACIDIC FRACTION or AF) comprising acidic proteins and peptides.

With this further step of anionic exchange chromatography, in particular a strong anionic exchange chromatography, all acidic proteins in the SNp are consequently concentrated. In a particular embodiment the elution of the bound fraction and diafiltration is carried out through a phosphate buffer <NUM> at pH between <NUM> and <NUM>.

Alternatively, acidic proteins and peptides can be captured before the basic ones.

In a more particular embodiment, the acidic proteins and peptides are captured before the basic ones.

When a step of ion exchange chromatography is employed the rationale behind the separation is the isoelectric point of the proteins or the peptides that, at a predetermined pH gives a protein positively or negatively charged.

Once any of the acidic and basic fractions are obtained, these fractions are in another particular embodiment submitted to additional fractionation steps by means of diafiltration, salting out methods, gel permeation chromatography, cationic and anionic exchange chromatography and combinations thereof.

In another particular embodiment any of the basic or acidic fractions are further submitted to fractionation by means of diafiltration, salting out methods, protein precipitation, solvent extraction, cationic and anionic exchange chromatography, size exclusion chromatography, affinity chromatography and combinations thereof.

Alternatively, all these fractionation steps can be carried out in different order.

With this subsequent fractionation, purified fractions are obtained, even fractions with only one of the native proteins or peptides from the mammalian intestine mucosa in a buffered water solution.

In a particular embodiment of the method, both the basic and acidic fractions are fractionated by molecular weight through a series of tangential flow diafiltrations and concentrations of the related permeates. The fractions will be characterized, in even a more particular embodiment, by the following molecular weight ranges:.

In a more particular embodiment, when the method of the invention comprises the further fractionation of the obtained basic and acidic fractions by molecular weight as indicated in the previous embodiment, both the basic and acidic fractions from the step are further fractionated by molecular weight through a gel permeation chromatography.

In an alternative particular embodiment of the method, both the basic and acidic fractions are fractionated by salting out methods, such as more in particular through the addition of ammonium sulphate or by the addition of an organic solvent such as acetone or butanol.

A "salting out method" is to be understood as a step of precipitation of proteins due to the presence in the media of salts that lower the solubility of particular proteins.

In also another alternative particular embodiment of the method, both the basic and acidic fractions are loaded on weak cationic and anionic exchange chromatography columns, respectively, and the single (isolated) proteins and peptides, are eluted by means of ionic strength or pH gradient.

In another embodiment of the present invention, the SNr fraction is treated in order to obtain products containing enzymatic activity having chemical, nutraceutical or pharmaceutical interest.

In a particular embodiment, SNr is treated in order to release water-soluble proteins, having or not enzymatic activity, from their bonds to membranes or other cell structures.

In one particular embodiment, SNr is treated with homogenization (with or without detergent), an/or with hydrolytic enzymes to detach bound proteins from membranes or cell structures. In a particular embodiment the hydrolytic enzymes comprise one or more of phospholipases and proteases. All released proteins and peptides are further purified by the same methods used for SNp in the series of one or more of successive filtration, ultrafiltration steps, protein precipitation and/or gel permeation and/or to ion exchange chromatography steps of (v. b) (i.e., (v.

As will be illustrated in the examples below, with this method of the first aspect in all its variations, particular fractions containing enzymes of interest in active form were obtained. Thus, mixtures of enzymes from the mammal intestine mucosa that perform and function according to their activities.

In a particular embodiment of the method of the first aspect, for the simultaneous obtention of heparin, proteins and peptides in native state by fractionation of mammalian intestine mucosa, is a method comprising the following steps:.

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

In another more particular embodiment, optionally in combination with any of the embodiments above or below, step (v. b) further comprises submitting any of the basic or acidic fractions obtained according to steps (v. <NUM>) and (v. <NUM>) to further fractionation steps, such as fractionation by molecular weight, by means of additional diafiltration, salting out methods, additional cationic and anionic exchange chromatography, and combinations thereof. This later embodiment aims to achieve the high purification of each of the peptides and proteins in the fractions, if required.

In even a more particular embodiment of the method including in step (v. b) the further fractionation by molecular weight of any of the basic or acidic fractions of (v. <NUM>) and (v. <NUM>), the peptides and proteins are fractionated in a step (v. <NUM>) according to molecular weights ranges selected from more than <NUM> KDa, between <NUM> and <NUM> KDa, between <NUM> and <NUM> KDa, between <NUM> and <NUM> KDa, between <NUM> and <NUM> KDa, between <NUM> and <NUM> KDa, and between <NUM> and <NUM> KDa.

With the tangential flow the samples were, thus first concentrated filtering out the low molecular weight compounds (i.e., water and other compounds), and then by means of the further selected fractionations, particular peptides and proteins within a known molecular weight can be recovered.

In one particular embodiment, optionally in combination with any of the embodiments above or below, the retentate (SNr) of (v. <NUM>) is further treated in a sub-step (v. <NUM>) with an homogenization step (with or without detergent), and/or with hydrolytic enzymes, such as one or more of phospholipases and proteases to detach the bound proteins of interest from the membranes or other cell structures. All released proteins and peptides are further purified by the same methods used for SNp and disclosed in steps (v. <NUM>) to (v. The step of homogenization of the retentate (SNr) is, in a particular embodiment, carried out in the same way that homogenization of step (iii) in the method is carried out. Thus, it is carried out with a technique selected from the group consisting of mechanical or physical cell lysis of mammalian intestinal mucosa by mechanical disruption, shear fluid forces, high pressure or cavitation.

In another embodiment, the method of the invention further comprises:
(v. <NUM>') submitting the supernatant of step (v. <NUM>') to an anionic exchange chromatography column to obtain a bound fraction (SNbf') and an unbound fraction (SNubf'), and eluting the bound fraction and then diafilter and concentrate it by means of a tangential flow system through a filter having a cut-off from <NUM> to <NUM> KDa to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides.

In another embodiment, the method of the invention further comprises:
(v. <NUM>') submitting the unbound fraction (SNubf') of step (v. <NUM>') to a cationic exchange chromatography column to obtain a bound fraction (SNubfbf') and an unbound fraction (SNubub'), and eluting the bound fraction then diafilter and concentrate it by means of a tangential flow system through a filter having a cut-off from <NUM> to <NUM> KDa to obtain a basic fraction of the supernatant comprising basic proteins and peptides.

In another embodiment, the method of the invention further comprises:
(v. 4a) submitting the supernatant of step (v. <NUM>') to a cationic exchange chromatography column to obtain a bound fraction (SNbf) and an unbound fraction (SNubf), and eluting the bound fraction and then diafilter and concentrate it by means of a tangential flow system through a filter having a cut-off from <NUM> to <NUM> KDa, to obtain a basic fraction of the supernatant comprising basic proteins and peptides.

In another embodiment, the method of the invention further comprises:
(v. 5a) submitting the unbound fraction (SNubf) of step (v. 4a) to an anionic exchange chromatography column to obtain a bound fraction (SNubfbf) and an unbound fraction (SNubub), and eluting the bound fraction and then diafilter and concentrate it by means of a tangential flow system through a filter having a cut-off from <NUM> to <NUM> KDa, to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides.

In a more particular embodiment, the anionic exchange chromatography is performed before the cationic exchange chromatography.

In another embodiment, the method of the invention further comprises:.

In those embodiments comprising step (v. <NUM>), the next step (v. <NUM>') or (v. 4a) is performed with the supernatant SNp or SNr of step (v. , particularly with the supernatant SNp. In those embodiments comprising steps (v. <NUM>) and (v. <NUM>), the next step (v. <NUM>') or (v. 4a) is performed with the supernatant SNp or SNpr of step (v. <NUM>), particularly with the supernatant SNp.

In a particular embodiment, the method for the obtention proteins and peptides in native state by fractionation of mammalian intestine mucosa comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), and (v. <NUM>') as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. <NUM>), and (v. <NUM>') as defined herein.

In a particular embodiment the method for the obtention proteins and peptides in native state by fractionation of mammalian intestine mucosa comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. 4a) and (v. 5a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. <NUM>), (v. 4a) and (v. 5a) as defined herein.

In a particular embodiment the method for the obtention proteins and peptides in native state by fractionation of mammalian intestine mucosa comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>') and (v. <NUM>') as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. <NUM>), (v. <NUM>') and (v. <NUM>') as defined herein.

In a particular embodiment the method for the obtention proteins and peptides in native state by fractionation of mammalian intestine mucosa comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), and (v. 4a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. <NUM>) and (v. 4a) as defined herein.

In a particular embodiment the method for the obtention proteins and peptides in native state by fractionation of mammalian intestine mucosa comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. 5a) and (v. a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. 5a) and (v. a) as defined herein.

In a particular embodiment the method for the obtention proteins and peptides in native state by fractionation of mammalian intestine mucosa comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>'), (v. <NUM>') and (v. a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. <NUM>'), (v. <NUM>') and (v. a) as defined herein.

As indicated, also disclosed herein are compositions (i.e., fractions/extracts of intestine mammal mucosa) comprising intestine mammal mucosa proteins and peptides obtainable by a method as defined in the first aspect or in any of its embodiments.

Thus, this can be disclosed as a composition (i.e., a fraction/extract of intestine mammal mucosa) comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising:.

In a particular embodiment of said composition comprising intestine mammal mucosa proteins and peptides, the intestine mammal mucosa proteins and peptides are selected from the group consisting of brush border enzymes, antimicrobial peptides, antioxidant enzymes, hormone peptides, proteins participating in defence and/or healing processes in the intestine mucosa, and combinations thereof, as revealed by literature, transcriptomic, and proteomic analysis of the pig intestine mucosa.

These compositions are thus isolated fractions or extracts of the mammal intestine mucosa comprising the compounds of interest.

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

Brush border of the mammalian intestine, in particular of the small intestine, is the microvilli-covered surface of the epithelium where absorption takes place with microvilli of approximately <NUM> in diameter and varying from approximately <NUM> to <NUM> in length. In particular, the brush borders of the intestinal lining are the site of terminal carbohydrate and protein digestion. The microvilli that constitute the brush border have enzymes for this final part of digestion anchored into their apical plasma membrane as integral membrane proteins. These enzymes are found near to the transporters that will then allow absorption of the digested nutrients.

In even a more particular embodiment, the composition comprises phosphatases, more in particular selected from phytases, alkaline phosphatase and mixtures thereof.

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

In another particular embodiment, optionally in combination with the embodiments above or below of the compositions of the invention, they comprise peptidases selected from the groups consisting of carboxypeptidases, aminopeptidases, endopeptidases, enteropeptidase and dipeptidase.

In another particular embodiment, optionally in combination with the embodiments above or below of the compositions of the invention, they comprise lipases selected from the groups consisting of triacylglycerol lipase, phospholipases, ceramidases and sphingomyelinases.

In another particular embodiment, the disclosure relates to a composition with mucosa proteins and peptides, which comprises:.

As mentioned above, the disclosure also relates to a composition comprising an acidic fraction of intestine mammal mucosa acidic proteins and peptides which is obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), and (v. <NUM>') as defined herein.

As mentioned above, the disclosure also relates to a composition comprising an acidic fraction of intestine mammal mucosa acidic proteins and peptides which is obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. 4a) and (v. 5a) as defined herein.

In particular, the composition comprising an acidic fraction of intestine mammal mucosa acidic proteins and peptides comprises one or more of Pro-glucagon, Glucagon like peptide-<NUM> (GLP-<NUM>), Thymosin beta-<NUM> and Vasointestinal peptide. More in particular, the AF comprises Pro-glucagon, Glucagon like peptide-<NUM> (GLP-<NUM>), Thymosin beta-<NUM> and Vasointestinal peptide.

The disclosure also relates to a composition comprising a basic fraction of intestine mammal mucosa basic proteins and peptides which is obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>'), and (v. <NUM>') as defined herein.

The disclosure also relates to a composition comprising a basic fraction of intestine mammal mucosa basic proteins and peptides which is obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), and (v. 4a), as defined herein.

In particular, the composition comprising a basic fraction of intestine mammal mucosa basic proteins and peptides comprises lysozyme. More in particular, it comprises one or more of lysozyme, Lactotrasferrin, <NUM> kDa type IV collagenase, Interstitial collagenase, Matrix metalloproteinase-<NUM>, Regenerating family member III / regenerating islet-derived protein <NUM> (Reg III), Regenerating family member IV / regenerating islet-derived protein IV (Reg IV), Antibacterial protein PR-<NUM>, Angiogenin, Phosphoinositide phospholipase C, Phospholipase D, and Pulmonary surfactant-associated protein D, even more particularly it comprises all of them.

In a particular embodiment, the composition comprising a basic fraction of intestine mammal mucosa basic proteins and peptides is subfractioned by washing with buffers of different concentrations. In a more particular embodiment, the washing is with buffers of different concentrations of NaCl. More in particular, a subfraction BF2 is obtained by washing the resin with a buffer of about <NUM>% NaCl. In particular, BF2 comprises lysozyme, in particular with an activity of <NUM> U-FIP/mL. More in particular, BF2 comprises one or more of lysozyme, lactotransferrin, phosphoinositide phospholipase C, phospholipase D, antibacterial protein PR-<NUM>, regenerating family member <NUM> and angiogenin. In another particular embodiment, a subfraction BF7 is obtained by further washing the resin with a buffer of about <NUM>% NaCl. In a particular embodiment, BF7 comprises one or more of <NUM> kDa type IV collagenase, Interstitial collagenase, Matrix metalloproteinase-<NUM>, Regenerating family member III / regenerating islet-derived protein <NUM> (Reg III) and Pulmonary surfactant-associated protein D, and even more particularly comprises all of them.

The disclosure also relates to a composition SNubub' comprising intestine mammal mucosa proteins and peptides which is obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. <NUM>), (v. <NUM>'), and (v. <NUM>') as defined herein.

In particular, the SNubub' comprises alkaline phosphatase (ALP) and mucin-<NUM> (MUC2). More in particular, the SNubub' comprises one or more of ALP, MUC2, aminopeptidases, carboxypeptidases, catalase, triacylglycerol lipase, phospholipases, acid sphingomyelinase-like phosphodiesterase, alpha-amylase, alpha-galactosidase, sucrase-isomaltase, peroxiredoxin-<NUM>, superoxide dismutase, Apolipoprotein A1, Annexin-<NUM>, Galectin-<NUM>, Diazepam binding inhibitor, Immunoglobulin M, secretory Immunoglobulin A, Gastrotropin, Thymosin beta-<NUM> and Hemoglobin, and more particularly comprises all of them.

In more particular embodiments, SNubub' is further fractionated by means of successive filtration, and ultrafiltration steps, solvent extraction, protein precipitation, and/or gel permeation and affinity chromatography steps.

The disclosure also relates to a composition SNubub comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. 4a), and (v. 5a) as defined herein.

The disclosure also relates to a composition SNububaq comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>'), and (v. <NUM>') as defined herein to obtain a SNubub' fraction; and further comprising:
(v. a) submitting the unbound fraction (SNubub') to an extraction treatment with an organic solvent, obtaining an aqueous phase (SNububaq).

More particularly the invention also relates to a composition SNububaq comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v. <NUM>'), (v. <NUM>), (v. <NUM>), (v. <NUM>'), (v. <NUM>') and (v. a) as defined herein.

In a particular embodiment, SNubub' is submitted to an extraction treatment with an organic solvent (setp v. More in particular the organic solvent is selected from the group consisting of heptanol, pentanol, <NUM>-tert-Butylcatechol, limonene, cyclohexane, and mixtures thereof, more in particular it is butanol from <NUM> to <NUM>% (w/w), even more in particular it is butanol at about <NUM>% (w/w). More in particular, the extraction treatment lasts for about <NUM> minutes and is performed at about <NUM>.

In a particular embodiment, the aqueous phase of the obtained extract (SNububaq) comprises alkaline phosphatase and mucin-<NUM>. In a more particular embodiment, Snububaq comprises alkaline phosphatase with an activity of about <NUM> DEA U/mL. In a more particular embodiment, Snububaq comprises one or more of Alkaline phosphatase, Mucin-<NUM>, Immunoglobulin M, Immunoglobulin A, Sucrase-lsomaltase, Alpha-amylase, Hemoglobin, Aminopeptidases, Maltase-glucoamylase, Catalase, Carboxypeptidases, Alpha-galactosidase, Peroxiredoxin-<NUM>, Superoxide dismutase, Acid sphingomyelinase-like phosphodiesterase, Diazepam binding inhibitor, Gastrotropin, Apolipoprotein-<NUM>, Annexin-<NUM> and Galectin-<NUM>, and more particularly comprises all of them.

In another particular embodiment, the Snububaq is submitted to a treatment with a chemical agent for precipitation such as acetone, ammonium sulphate, PEG, Aluminium, and butanone to obtain a precipitate and a supernatant (Snububaqsn). More in particular the organic solvent is acetone, more in particular it is acetone from <NUM> to <NUM>% (w/w), more in particular it is acetone around <NUM>% (w/w).

In another particular embodiment, the resulting supernatant (Snububaqsn) is further submitted to a treatment with a chemical agent for precipitation such as acetone, ammonium sulphate, PEG, Aluminium, and butanone to obtain a precipitate (Snububaqsnp or ALPp) and a supernatant (Snububaqsnsn or MUC2sn). More in particular the organic solvent is acetone, more in particular it is acetone from <NUM> to <NUM>% (w/w), more in particular it is acetone from <NUM>% to <NUM>% (w/w), more in particular it is acetone <NUM>%. In a particular embodiment, the acetone treatment is done at a temperature between <NUM> and <NUM>, in a more particular embodiment between <NUM> and <NUM>, in a more particular embodiment at <NUM>. In a more particular embodiment, the resulting precipitate (ALPp) comprises ALP. In another particular embodiment, the resulting supernatant (MUCsn) comprises MUC2.

In another embodiment, the ALPp is further processed by means of affinity chromatography to obtain purified ALP. In particular the purified ALP is a pharmaceutical grade protein suitable for medical applications. More in particular, the purified ALP is obtained with a yield of around <NUM> DEA Units of alkaline phosphatase activity per kg of mucosa (one DEA unit will hydrolyse <NUM>µmol of p-nitrophenyl phosphate per minute at pH <NUM> at <NUM>, as known by the skilled person).

In another embodiment, the MUC2sn is further purified by means of affinity chromatography to obtain pharmaceutical grade proteins suitable for medical applications. In particular the purified MUC2 is a pharmaceutical grade protein suitable for medical applications. More in particular, the purified MUC2 is obtained with a yield of around <NUM> of N-acetylneuraminic acid per kg of mucosa corresponding to sialylated glycoproteins like mucin-<NUM>.

A transcriptomic analysis was performed to explore the complete set of transcripts of pig intestinal mucosa and characterize the corresponding proteome.

<NUM> samples of porcine intestinal mucosa were collected at the slaughterhouse and stored at -<NUM> until analysis. Total RNA was extracted using RNeasy Plus Mini Kit (ref: <NUM>). RNA concentration was quantified by fluorescence, and quality was verified by electrophoresis.

Transcripts were purified by poly(A)-tail selection. <NUM> mRNA libraries were prepared using Illumina TruSeq stranded mRNA kit. Libraries were amplified by PCR, and concentration and quality were verified. Libraries were sequenced using Illumina Novaseq <NUM> on one lane of SP flow-cell, 2x100bp (Kit version NV2864788-RGSBS).

Gene expression was studied using the reference genome of Sus scrofa11. <NUM> on iGenome (https://emea. com/sequencing/sequencing_software / igenome. Reads were mapped against the reference genome using Bowtie v2. <NUM> (http://bowtie-bio. sourceforge. net/bowtie2/index. shtml) and TopHat v2. <NUM> (http://tophat. Cufflink v2. <NUM> (http://cufflinks. edu/) was used to estimate abundance of aligned reads, and test for differential expression and regulation transcriptome-wide. The package Cummerbund v2. <NUM> (http://compbio. edu/cummeRbund/) was used to produce graphics representations of the differential analysis.

A total of <NUM>,<NUM>,<NUM> transcripts were read, and <NUM>,<NUM>,<NUM> of them were mapped against the reference genome. From the mapped reads, more than <NUM>,<NUM> genes were annotated and their expression was estimated.

A proteomic analysis was performed to map the proteome of the porcine intestinal mucosa and complement the transcriptomic data.

Samples of porcine intestinal mucosa were collected at the slaughterhouse and stored at - <NUM> until analysis. A representative sample was weight of at -<NUM> (<NUM>) in an Eppendorf tube and TES buffer (Tris-HCl <NUM>, EDTA <NUM>, sucrose <NUM>, pH <NUM>) was added to give a concentration of <NUM>/mL. The sample was kept on ice and homogenized 2x10sec in a tissue homogenisator (TissueLyser LT, Qiagen) using metal beads, keeping the sample on ice in-between homogenization. The homogenized sample was centrifuges at <NUM>, <NUM> x g for <NUM> (Eppendorf Centrifuge) and the supernatant was collected. The protein concentration was determined using the BCA method with BSA as standard (Pierce BSA Protein Assay Kit). The protein concentration was determined to <NUM>µg/µl.

In all, <NUM> samples of <NUM>µg protein each, where reduced with DTT, alkylated with iodoaceamide and digested with trypsin according to the "In-solution trypsin digestion and guanidination kit" (ThermoScientific). Two of the samples where guanidinated using O-methylisourea hemisulfate. Then, peptides were purified for all <NUM> samples using "Peptide Desalting Spin Columns" (Pierce, see manufacturers manual). After desalting, the samples were evaporated to dryness and dissolved in HPLC buffer A (<NUM>% acetonitrile, <NUM>% formic acid in MilliQ water) prior to LC-MS analysis.

Peptide samples were analyzed on a Bruker TimsTOFPro equipped with a Bruker NanoElute LC-system. Peptides were separated using a <NUM> reverse phase gradient, with solvent A: <NUM>% acetonitrile, <NUM>% formic acid in MilliQ water, and solvent B: acetonitrile, <NUM>% formic acid. The reverse phase column was a BrukerFIFTEEN (L=<NUM>, ID=<NUM>, C18, <NUM>, 120Å). The mass spectrometer was run in DDA PASEF mode.

Datafiles were merged and searched in PEAKS XPro using the UniProt procine proteomics database (Proteomics ID UP000008227, downloaded <NUM>. <NUM>) with the Mammalian SwissProt database as contamination database. Error tolerance: 20ppm precursor mass, <NUM>. 05Da fragment ion. Fixed PTM: carmidomethylation. Variable PTMs; oxidation (M), acetylation (N-tern), guanidination. False discovery rate (FDR) was set to <NUM>%.

Proteomics analysis of porcine intestinal mucosa revealed <NUM> identified protein groups, with false discovery rate (FDR) of <NUM>%. The data from this experiment was crossed with the data obtained from the transcriptomic analysis to conform a comprehensive proteome of the pig intestinal mucosa. From this, a list of candidate proteins was selected based on their abundance in the mucosa, their molecular characteristics, and their potential commercial application.

A method for the simultaneous isolation of heparin and selected native proteins and peptides, from mammalian intestinal mucosa, was performed as follows.

The method generically included the following steps:.

Alternatively, both BASIC and ACIDIC FRACTIONS could have been fractionated by salting out methods, such as through the addition of ammonium sulphate or by the addition of an organic solvent such as acetone.

Alternatively, the BASIC and ACIDIC FRACTIONS could have been loaded on weak cationic and anionic exchange column, respectively, and the single proteins and peptides, can be eluted by means of ionic strength or pH gradient.

This example provides evidence of the capability of the process to simultaneously obtain with proper amounts and purity grade the one or more enzymes and heparin from the mammalian intestine mucosa. As example with the described process an enzymatic activity of <NUM> DEA Units of alkaline phosphatase activity per kg of mucosa, and <NUM>. <NUM> U-FIP of lysozyme activity per kg of mucosa are obtained, while heparin, a compound usually obtained from this tissue is also obtained with <NUM>,<NUM> MIU of heparin activity per Kg of mucosa. These heparin values are higher than the ones obtained with the standard extraction process of the prior art. Data have been exemplified with alkaline phosphatase and lysozyme from SNr, but the different steps, namely (v. <NUM>) and (v. <NUM>) show also how to separate and to obtain all the proteins according to their isoelectric point, by choosing the appropriate pH and pH gradient. Other method steps, in particular those based on the molecular weight of the proteins were also applied (see v. <NUM> and v.

Indeed, the proposed method for the simultaneous obtention of heparin, proteins and peptides in native state by fractionation of mammalian intestine mucosa is based on the properties of the proteins contained in the mucosa, mainly on the isoelectric point at a predetermined pH, and/or their molecular weight, and/or solubility in water or organic solvents.

The method included the following steps:.

This way the enzymes alkaline phosphatase and lysozyme were obtained with the following yield: <NUM> DEA Units of alkaline phosphatase activity per kg of mucosa (one DEA unit will hydrolyse <NUM>µmol of p-nitrophenyl phosphate per minute at pH <NUM> at <NUM>, as known by the skilled person), and <NUM>. <NUM> U-FIP of lysozyme activity per kg of mucosa (one FIP unit will produce a change in A<NUM> nm of <NUM> per minute at pH <NUM> at <NUM>, using a suspension of Micrococcus lysodeikticus as substrate, in a <NUM> reaction mixture, as known by the skilled person). Mucin was isolated with a significant yield, as measured by <NUM>,<NUM> of N-acetylneuraminic acid per kg of mucosa.

Alternatively, both BASIC and ACIDIC FRACTIONS could also have been fractionated by salting out methods, such as through the addition of ammonium sulphate or by the addition of an organic solvent such as butanol or acetone.

This example provides evidence of the capability of the process to simultaneously obtain with proper amounts and purity grade the one or more enzymes and heparin from the mammalian intestine mucosa. As example with the described process an enzymatic activity of <NUM> DEA Units of alkaline phosphatase activity per kg of mucosa, and <NUM>. <NUM> U-FIP of lysozyme activity per kg of mucosa are obtained, as well as <NUM>,<NUM> of N-acetylneuraminic acid per kg of mucosa corresponding to sialylated glycoproteins like mucin-<NUM>, while heparin, a compound usually obtained from this tissue is also obtained with <NUM>,<NUM> MIU of heparin activity per Kg of mucosa. These heparin values are higher than the ones obtained with the standard extraction process of the prior art. Data have been exemplified with alkaline phosphatase, mucin-<NUM> and lysozyme from SNr, but the different steps, namely (v. <NUM>) and (v. <NUM>) show also how to separate and to obtain all the proteins according to their isoelectric point, by choosing the appropriate pH and pH gradient. Other method steps, in particular those based on the molecular weight of the proteins were also applied (see v. <NUM> and v.

To identify and annotate the proteins present in <NUM> freeze-dried fractions of porcine intestinal mucosa and to estimate their relative abundance within each fraction.

iBAQ (Intensity Based Absolute Quantification) values are the raw intensities divided by the number of theoretical peptides. This normalization algorithm is able to estimate protein amounts within each sample. IBAQ intensities [<NUM>] are calculated within MaxQuant [<NUM>] software.

Fractions had a varying number of protein groups quantified (from <NUM> to <NUM>). Based on the results we can conclude that, among others, the following proteins with potential commercial application are isolated or concentrated in the corresponding fractions:.

Interstitial collagenase, Matrix metalloproteinase-<NUM>, Regenerating family member III / regenerating islet-derived protein <NUM> (Reg III), Regenerating family member IV / regenerating islet-derived protein IV (Reg IV), Antibacterial protein PR-<NUM>, Angiogenin, Phosphoinositide phospholipase C, Phospholipase D, Pulmonary surfactant-associated protein D.

SNprubub: Alkaline phosphatase, Mucin-<NUM>, Immunoglobulin M, Immunoglobulin A, Sucrase-Isomaltase, Alpha-amylase, Hemoglobin, Aminopeptidases, Maltase-glucoamylase, Catalase, Carboxypeptidases, Alpha-galactosidase, Peroxiredoxin-<NUM>, Superoxide dismutase, Acid sphingomyelinase-like phosphodiesterase, Diazepam binding inhibitor, Gastrotropin, Apolipoprotein-<NUM>, Annexin-<NUM>, Galectin-<NUM>, Triacylglycerol lipase , Phospholipase, Thymosin beta-<NUM>.

Alkaline phosphatase, Mucin-<NUM>, Immunoglobulin M, Immunoglobulin A, Sucrase-Isomaltase, Alpha-amylase, Hemoglobin, Aminopeptidases , Maltase-glucoamylase , Catalase , Carboxypeptidases, Alpha-galactosidase, Peroxiredoxin-<NUM>, Superoxide dismutase, Acid sphingomyelinase-like phosphodiesterase, Diazepam binding inhibitor, Gastrotropin, Apolipoprotein-<NUM>, Annexin-<NUM>, Galectin-<NUM>.

Evaluation of the antimicrobial and antifungal activity of <NUM> fractions of porcine intestine mucosa against <NUM> human pathogenic bacteria, <NUM> phytopathogenic bacteria and <NUM> phytopathogenic fungus.

Antibacterial and antifungal activity were tested against the pathogens in Table <NUM>:.

The inoculums were obtained from pure cultures of growing strains, seeded in solid Luria-Bertani (LB) medium in the case of bacteria, and Potato dextrose agar (PDA) medium in the case of the fungus. The human pathogens (St, and Sa) were incubated for <NUM> at <NUM>, and the fungus (Fo) for <NUM> days at <NUM>. All bacterial suspensions were prepared to a final concentration of 2x107 CFU/ml. In the case of the test with the fungus, a suspension of conidia was prepared at a final concentration of 2x104 conidia/ml.

The antimicrobial activity of <NUM> fractions and two control samples (see annex slide <NUM>) was evaluated at a concentration of <NUM>. The antimicrobial effect was compared with the reference antibiotic levofloxacin (<NUM>/L) in the case of bacteria, and cycloheximide (<NUM>/L) in the case of the fungus. An untreated control was also included where the product was replaced with water (control positive for microbial growth), a negative control with culture medium without product nor microbial suspension (to rule out possible contamination of the environment). Product controls were included, where each product was incubated without microbial suspension or culture medium (to discard product contamination).

The antimicrobial activity of the different products against the <NUM> pathogens was determined by a growth inhibition assay. Bacterial suspensions were prepared with sterile distilled water and adjusted to a stock concentration of 2x107 CFU/ml in liquid LB medium. In the case of the fungus, the conidia suspension was also prepared with sterile distilled water and adjusted to a concentration of 2x104 conidia/ml in the PDA medium. The growth inhibition assay consisted of mixing <NUM>µl of the product with <NUM>µl of the bacterial/fungal suspension, obtaining a final volume of <NUM>µl in each well of the microplate (bacterial suspensions at a final concentration of 1x107 CFU/ml and fungal suspension at a final concentration of 1x104 conidia/ml). The microplates were incubated at <NUM> in the case of the human pathogens (St and Sa). The fungus was incubated for <NUM> days at <NUM> with constant shaking.

The growth kinetics of the different pathogens in the presence of the products was analyzed in triplicate (<NUM> wells per product and pathogen) using automated systems that allow microbial growth to be monitored through absorbance readings at <NUM> (Bioscreen C MBR, Labsystems, Finland and Varioskan flash, Thermo Electron Corporation, USA) every hour in the case of bacteria (for <NUM>) and every <NUM> in the case of fungus (for <NUM> days). Microbial growth was quantified by analyzing the area under the growth curve, calculating the percentage of growth inhibition for each pathogen treated with each product.

The percentage of growth inhibition for each pathogen treated with each product is shown in Table <NUM>.

Claim 1:
A method for the simultaneous obtention of heparin, proteins and peptides in native state by fractionation of mammalian intestine mucosa, comprising the following steps:
(i) adding to an aqueous extract of mammalian intestinal mucosa a preservative and antioxidant agent to obtain a preserved mucosa, or preserving the extract at a temperature allowing preserving proteins, peptides and heparin in native state;
(ii) optionally diluting the preserved mucosa with up to <NUM> volumes of deionized water, to obtain a diluted mucosa
(iiia) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than <NUM>, optionally in presence of detergents and/or hydrolytic enzymes such as phospholipases, to lyse cell membrane of mucosa cells to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides;
(iva) separating heparin and the mucosa proteins and peptides contained in the stable homogenate obtained in step (iiia) by one or more of physical and/or chemical means selected from the group consisting of centrifugation and filtration (microfiltration and/or ultrafiltration), optionally using detergents, and combinations thereof, to obtain a pellet (P) or retentate fraction comprising heparin and proteins; and a supernatant (SN) or permeate fraction comprising mucosa proteins and peptides;
(v.a') submitting the pellet (P) or retentate fraction of (iva) comprising heparin to an alkaline proteolytic process by means of proteolytic enzymes to obtain a mixture of heparin and a protein hydrolysate, submiting the mixture with heparin to one or more precipitation steps, and optionally to one or more of extractive steps, and to one or more of a chromatography to purify the said heparin from the protein hydrolysate; and
(v.b') submitting the supernatant (SN) or permeate of (iva) comprising a mixture of solubilized and/or suspended mucosa proteins and peptides to one or more of successive filtration, ultrafiltration steps, solvent extraction, protein precipitation, enzymatic hydrolysis, gel permeation, ion exchange chromatography, size exclusion chromatography, or affinity chromatography steps, being exchangeable the order or sequence of the steps, in order to separate the proteins and peptides according to any of their solubility, isoelectric point and/or molecular weight.