Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L99/00—Compositions of natural macromolecular compounds or of derivatives thereof not provided for in groups C08L89/00 - C08L97/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
  • C08L5/10—Heparin; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2330/00—Production
  • C12N2330/10—Production naturally occurring

Definitions

• 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.
• 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".
• native proteins Proteins in their natural state with intact structure and function that is not altered by heat, chemicals, enzyme reaction, or other denaturants.
• 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.
• 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.
• Cibet Liyang technology Co Ltd discloses a method to obtain alkaline phosphatase from animal viscera (including pig intestine mucosa), in which mucosa is first cut and homogenized at 4 °C; 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 415 ll/rng 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 RU2610669. 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 (4°C-37°C), and they are carried out at physiological conditions (of pH and ionic force).
• the main aim is to recover the product of interest in the balance of a high yield, activity and high purity.
• 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.
• Heparin is obtained with a grade useful for pharmaceutical application (i.e. , at pharmaceutical grade according to regulations).
• 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.
• 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:
• step (iii) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than 40 °C, to lyse cell membrane of mucosa cells to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides;
• step (iv) separating heparin and the mucosa proteins and peptides contained in the stable homogenate obtained in step (iii) 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;
• a method for the simultaneous obtention of heparin, proteins and peptides in native state by fractionation of mammalian intestine mucosa comprising the following steps:
• step (iii) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than 25 °C, to lyse cell membrane of mucosa cells to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides;
• step (iv) generically separating heparin and the mucosa proteins and peptides contained in the stable homogenate obtained in step (iii) 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.
• 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.
• 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:
• step (iiia) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than 40 °C, 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;
• step (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 fraction comprising heparin (P) and proteins; and a supernatant fraction (SN) comprising mucosa proteins and peptides;
• 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 fraction comprising heparin (P) and proteins; and a supernatant fraction (SN) comprising mucosa proteins and peptides;
• 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.
• compositions including compounds of interest are obtained by the method.
• 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.
• 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: (v.b.T) submitting the supernatant (SN) of step (v.b’) comprising a mixture of solubilized and/or suspended mucosa proteins and peptides to filtration through an up to 100 pm cutoff filter in order to retain small particles and lipid residues which were not pelleted in step (iva), to obtain a filtered supernatant comprising most of the mucosa proteins and peptides; and
• step (v.b.4’) submitting the supernatant of step (v.b.T) 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 1 to 5 KDa to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides. or alternatively obtainable by a method comprising steps (i), (ii), (iiia), and (iva) as defined herein, further comprising:
• step (v.b.T) submitting the supernatant (SN) of step (v.b’) comprising a mixture of solubilized and/or suspended mucosa proteins and peptides to filtration through an up to 100 pm cutoff filter in order to retain small particles and lipid residues which were not pelleted in step (iva), to obtain a filtered supernatant comprising most of the mucosa proteins and peptides;
• step (v.b.4a) submitting the supernatant of step (v.b.T) 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 1 to 5 KDa, to obtain a basic fraction of the supernatant comprising basic proteins and peptides; and
• step (v.b.5a) submitting the unbound fraction (SNubf) of step (v.b.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 1 to 5 KDa, to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides.
• Another aspect of the invention relates to a basic fraction of intestine mammal mucosa basic proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v.b.T), and (v.b.4’) as defined herein to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides; and further comprising:
• composition SNubub’ comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.4’), and (v.b.5’) as defined herein.
• composition SNubub comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.4a), and (v.b.5a) as defined herein.
• the invention also provides, as will be illustrated in the examples, a fraction comprising heparin of pharmaceutical grade.
• a heparin complying with all the regulatory aspects according to health authorities.
• the specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in pmol min-1 mg-1). 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 pmol/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 pmol min-1 kg-1). This parameter gives a measurement of the quantity of enzyme extracted from the initial raw material.
• mammalian intestine mucosa 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 basement (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.
• 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:
• step (iii) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than 40 °C, to lyse cell membrane of mucosa cells to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides;
• step (iv) separating heparin and the mucosa proteins and peptides contained in the stable homogenate obtained in step (iii) 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;
• Steps (v.a) and (v.b) are also referenced in this description as (v.P) and (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.
• 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.a) or (v.b). 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.
• the mammalian intestine mucosa is the pig one.
• Other mammalian species, not including human, are also useful.
• the preservative and antioxidant agent in step (i) is sodium metabisulfite, added up to a concentration of 4% w/w, preferably 2% w/w.
• Equivalent substances can be chosen as a preservative and antioxidant agents.
• the preserved mucosa is diluted with up to 50 volumes of deionized water, more in particular is diluted with 1 to 25 volumes of deionized water, even more in particular with 1 to 15 volumes of deionized water, and also more in particular with 2 to 4 volumes of deionized water.
• a same volume of buffer at a pH comprised between 5 and 9 is used, preferably at 7.
• the mucosa in step (ii) is diluted either with deionized water or a buffer, and more in particular both comprising one or more detergents.
• the detergents are non-ionic detergents or zwitterionic detergents. Examples of commercially available detergents 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 beta-glucoside, and combinations thereof.
• Steps (iii), (iv), (v.a), (v.b), (v.b.1), (v.b.4) and (v.b.5) are equivalent to steps (iiia), (iva), (v.a’), (v.b’), (v.b.T), (v.b.4a) and (v.b.5a) respectively.
• All embodiments defined herein for steps (iii), (iv), (v.a), (v.b), (v.b.1), (v.b.4) and (v.b.5) also apply to steps (iiia), (iva), (v.a’), (v.b’), (v.b.T), (v.b.4a) and (v.b.5a) respectively.
• step (iii) or step (iiia) is performed at a temperature equal to or lower than about 35 °C, or alternatively equal to or lower than about 30 °C, or alternatively equal to or lower than about 25 °C, or alternatively equal to or lower than about 20 °C, or alternatively equal to or lower than about 15 °C, or alternatively equal to or lower than about 10 °C.
• step (iii) or step (iiia) is performed at a temperature from 0 to 35 °C, or alternatively from 0 to 30 °C, or alternatively from 0 to 25 °C, or alternatively from 0 to 20 °C, or alternatively from 0 to 15 °C, or alternatively from 0 to 10 °C.
• step (iiia) is performed at a temperature from 0 °C to 8 °C.
• step (iii) is performed at a temperature from 0 °C to 8 °C.
• 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.
• 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.
• 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.
• 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 180 g, preferably 1700 g, for a minimum of 30 seconds, preferably between 2 and 4 minutes.
• 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 150 g, about 180g, 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 in particular from 1500 to 2000 g. More in particular homogenization is carried out at about 1700 g.
• 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 in particular the homogenization is carried out for a period from 120 to 240, from 130 to 230, from 140 to220, from 150 to 210, from 160 to 200, or from 170 to 190 seconds, even more in particular from 120 to 240 seconds.
• 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 180 g, preferably 1700 g, for a minimum of 30 seconds, preferably between 2 and 4 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.
• 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.
• 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).
• P heparin
• SN supernatant fraction comprising mucosa proteins and peptides, some of them in solution and others in suspension
• these (P) and (SN) fractions are further processed in respective steps (v.P) or (v.a), and (v.SN) or (v.b), both notations being interchangeable.
• 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 3400g or about 3500 g; in particular from 150 to 3500 g, from 500 to 3400 g, from 750 to 3300 g, from 1000 to 3200 g, from 1250 to 3100 g, from 1500 to 3000 g, from 1750 to 2750 g, from 2000 to 2500 g; more in particular from 3000 to 3500 g.
• 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 from 5 to 35, from 10 to 30, or from 15 to 25 minutes; more in particular it lasts for from 20 to30 minutes.
• the centrifugation in (iva) is performed at a temperature equal to or lower than 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 about 0 °C, in particular at a temperature from 0 to 40 °C, or from 2 to 37 °C, or from 5 to 35 °C, or from 10 to 30 °C, or from 15 to 25 °C, more in particular the centrifugation in (iva) is performed at a temperature from 0 to 25 °C, even more in particular at a temperature from 0 to 8 °C.
• the centrifugation in (iv) is carried out at a g-force of at least 150 g, preferably at 3000 g during at least 5 minutes, preferably 20 minutes and at a temperature below 40°C, in particular below 25 °C, more preferably below 8 °C, 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)).
• a g-force of at least 150 g preferably at 3000 g during at least 5 minutes, preferably 20 minutes and at a temperature below 40°C, in particular below 25 °C, more preferably below 8 °C, 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
• the centrifugation in (iva) is carried out at a g-force of at least 150 g, preferably at 3000 g during at least 5 minutes, preferably 20 minutes and at a temperature below 40°C, in particular below 25 °C, more preferably below 8 °C, 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)).
• a g-force of at least 150 g, preferably at 3000 g during at least 5 minutes, preferably 20 minutes and at a temperature below 40°C, in particular below 25 °C, more preferably below 8 °C, 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
• the centrifugation in (iva) is carried out at a g-force from 3000 to 3500 g for a period of time from 15 to 35 minutes, particularly from 20 to 30 minutes, and at a temperature from 0 to 40 °C, or from 0 to 37 °C, or from 0 to 25 °C, or from 0 to 8 °C.
• the centrifugation in (iva) is carried out at a g-force of about 3134 g for a period of time about 20 minutes and at a temperature below about 8 °C.
• the centrifugation in (iva) is carried out at a g-force of about 3300 g for a period of time of about 30 minutes and at a temperature below about 37 °C, particularly from 0 to 25 °C.
• 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.
• a pellet (retentate) fraction comprising heparin
• a supernatant (permeate) fraction comprising mucosa proteins and peptides, some of them in solution and others in suspension.
• these (P) and (SN) fractions are further processed in respective steps (v.P) or (v.a’), and (v.SN) or (v.b’), both notations being interchangeable.
• the supernatant (SN) obtained in step (iva) comprises one or more of alkaline phosphatase (also referred herein as to ALP), mucin-2 (also referred herein as to MLIC2) and lysozyme (also referred herein as to LYZ), more particularly it comprises alkaline phosphatase, mucin-2 and lysozyme.
• alkaline phosphatase also referred herein as to ALP
• mucin-2 also referred herein as to MLIC2
• lysozyme also referred herein as to LYZ
• the SN further comprises one or more of glucagon like peptide-1 (also referred herein as to GLP-1), thymosin beta-4, vaso intestinal peptide, lysozyme, lactotransferrin, regenerating islet- derived protein 3 (Reg III), regenerating islet-derived protein IV (Reg IV), matrix metalloproteinase-9, 72 kDa type IV collagenase, interstitial collagenase, bactericidal permeability-increasing protein, pulmonary surfactant-associated protein D, antibacterial protein PR-39, angiogenin, alkaline phosphatase, mucin-2, trypsin, aminopeptidases, carboxypeptidases, catalase, triacylglycerol lipase, phospholipases, acid sphingomyelinase-like phosphodiesterase, alpha-amylase, alpha-galactosidase, sucrase- isomaltas
• 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.
• precipitation steps, optional extractive steps, and optional chromatography are carried out to purify heparin.
• 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.
• 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 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 300 kDa and 0.45 pm, preferably 0.1 pm.
• 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).
• the permeate of this diafiltration step 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.
• the fraction SNp is further diafiltered and concentrated through a filter having a cut-off between 1 and 5 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).
• 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 4 volumes of deionized water or a suitable buffer, preferably between 5 and 10 volumes.
• 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.
• 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.
• an SNp obtained as indicated in previous paragraphs and comprising the soluble mucosa proteins and peptides is, preferably after having adjusted the pH to 7.0, 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 1 to 5 KDa, preferably of 3 Kda, to obtain a basic fraction (also referred herein as to BASIC FRACTION or BF) comprising basic proteins and peptides.
• the cationic exchange chromatography can be done in batch, adding from 5 to 15% (w/w) of strong cationic exchange resin to the fraction, preferably 10%.
• a strong cationic exchange chromatography all basic proteins in the SNp are consequently concentrated.
• the elution of the bound fraction (SNpbf) and diafiltration is carried out through a phosphate buffer 50 mM at pH between 6.5 and 7.5.
• the unbound fraction (SNpubf) is, preferably after having adjusted the pH to 7.0, 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 1 to 5 Kda, preferably 3 Kda, to obtain an acidic fraction (also referred herein as to ACIDIC FRACTION or AF) comprising acidic proteins and peptides.
• an acidic fraction also referred herein as to ACIDIC FRACTION or AF
• anionic exchange chromatography in particular a strong anionic exchange chromatography, all acidic proteins in the SNp are consequently concentrated.
• the elution of the bound fraction and diafiltration is carried out through a phosphate buffer 50 mM at pH between 6.5 and 7.5.
• acidic proteins and peptides can be captured before the basic ones.
• the acidic proteins and peptides are captured before the basic ones.
• 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.
• 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.
• 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.
• 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:
• both the basic and acidic fractions from the step are further fractionated by molecular weight through a gel permeation chromatography.
• 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.
• 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.
• the SNr fraction is treated in order to obtain products containing enzymatic activity having chemical, nutraceutical or pharmaceutical interest.
• SNr is treated in order to release water-soluble proteins, having or not enzymatic activity, from their bonds to membranes or other cell structures.
• SNr is treated with homogenization (with or without detergent), an/or with hydrolytic enzymes to detach bound proteins from membranes or cell structures.
• 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.SN)).
• a method comprising the following steps: (i) adding to an aqueous extract of mammalian intestinal mucosa a preservative and an antioxidant agent to obtain a preserved mucosa, or preserving the extract at a temperature allowing preserving proteins, peptides and heparin in native state;
• step (iii) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than 40 °C, to lyse cell membrane of mucosa cells to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides;
• step (iv) separating heparin and the mucosa 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 detergents, and combinations thereof, to obtain a pellet phase (P) comprising heparin; and a supernatant (SN) fraction comprising mucosa proteins and peptides;
• step (v.b.1) submitting the supernatant (SN) comprising a mixture of solubilized and/or suspended mucosa proteins and peptides to filtration through an up to 100 pm cut-off filter in order to retain small particles and lipid residues which were not pelleted in step (iv), to obtain a filtered supernatant comprising most of the mucosa proteins and peptides;
• v.b.2 diafiltering the filtered supernatant through a filter having a cut-off of less than 0.8 pm to obtain a permeate (SNp) comprising the mucosa soluble in water proteins and peptides, and a retentate (SNr) with non-soluble in water proteins and peptides (i.e. , those that were suspended in the crude supernatant SN);
• SNp permeate
• SNr retentate
• 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).
• step (v.SN) or (v.b) further comprises submitting any of the basic or acidic fractions obtained according to steps (v.b.4) and (v.b.5) 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.
• step (v.SN) or (v.b) further comprises submitting any of the basic or acidic fractions obtained according to steps (v.b.4) and (v.b.5) 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.
• step (v.b) the further fractionation by molecular weight of any of the basic or acidic fractions of (v.b.4) and (v.b.5)
• the peptides and proteins are fractionated in a step (v.b.6) according to molecular weights ranges selected from more than 300 KDa, between 100 and 300 KDa, between 50 and 100 KDa, between 30 and 50 KDa, between 10 and 30 KDa, between 5 and 10 KDa, and between 1 and 5 KDa.
• 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.
• the low molecular weight compounds i.e., water and other compounds
• the retentate (SNr) of (v.b.2) is further treated in a sub-step (v.b.7) 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.b.3) to (v.b.6).
• 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.
• the method of the invention further comprises: (v.b.4’) submitting the supernatant of step (v.b.T) 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 1 to 5 KDa to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides.
• the method of the invention further comprises:
• step (v.b.5’) submitting the unbound fraction (SNubf’) of step (v.b.4’) 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 1 to 5 KDa to obtain a basic fraction of the supernatant comprising basic proteins and peptides.
• the method of the invention further comprises:
• step (v.b.4a) submitting the supernatant of step (v.b.T) 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 1 to 5 KDa, to obtain a basic fraction of the supernatant comprising basic proteins and peptides.
• the method of the invention further comprises:
• step (v.b.5a) submitting the unbound fraction (SNubf) of step (v.b.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 1 to 5 KDa, to obtain an acidic fraction of the supernatant comprising acidic proteins and peptides.
• the anionic exchange chromatography is performed before the cationic exchange chromatography.
• the method of the invention further comprises:
• step (v.b.2) diafiltering the supernatant of step (v.b.T) through a filter having a cut-off of less than 0.8 pm to obtain a permeate (SNp) comprising the mucosa soluble in water proteins and peptides, and a retentate (SNr) with non-soluble in water proteins and peptides; and (v.b.3) optionally diafiltering the permeate (SNp) to concentrate the soluble proteins and peptides (SNpr).
• SNp permeate
• SNr retentate
• next step (v.b.4’) or (v.b.4a) is performed with the supernatant SNp or SNr of step (v.b.2)., particularly with the supernatant SNp.
• next step (v.b.4’) or (v.b.4a) is performed with the supernatant SNp or SNpr of step (v.b.3), particularly with the supernatant SNp.
• 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.b.T), and (v.b.4’) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.2), (v.b.3), and (v.b.4’) as defined herein.
• 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.b.T), (v.b.4a) and (v.b.5a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.2), (v.b.3), (v.b.4a) and (v.b.5a) as defined herein.
• 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.b.T), (v.b.4’) and (v.b.5’) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.2), (v.b.3), (v.b.4’) and (v.b.5’) as defined herein.
• 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.b.T), and (v.b.4a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.2), (v.b.3) and (v.b.4a) as defined herein.
• 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.b.T), (v.b.4a), (v.b.5a) and (v.b.a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.2), (v.b.3) (v.b.4a), (v.b.5a) and (v.b.a) as defined herein.
• 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.b.T), (v.b.4’), (v.b.5’) and (v.b.a) as defined herein. More particularly, it comprises the steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.2), (v.b.3) (v.b.4’), (v.b.5’) and (v.b.a) as defined herein.
• compositions i.e., fractions/extracts of intestine mammal mucosa
• intestine mammal mucosa proteins and peptides obtainable by a method as defined in the first aspect or in any of its embodiments.
• composition i.e., a fraction/extract of intestine mammal mucosa
• intestine mammal mucosa proteins and peptides obtainable by a method comprising:
• step (ii) optionally diluting the preserved mucosa with up to 100 volumes of deionized water, to obtain a diluted mucosa; (iii) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than 40 °C, to lyse cell membrane of mucosa cells to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides;
• step (iv) separating heparin and the mucosa 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 detergents, and combinations thereof, to obtain a pellet phase (P) comprising heparin; and a supernatant (SN) fraction comprising mucosa proteins and peptides;
• step (v.b.1) submitting the supernatant (SN) comprising a mixture of solubilized and/or suspended mucosa proteins and peptides to filtration through an up to 100 pm cutoff filter in order to retain small particles and lipid residues which were not pelleted in step (iv), to obtain a filtered supernatant comprising most of the mucosa proteins and peptides;
• v.b.2 diafiltering the filtered supernatant through a filter having a cut-off of less than 0.8 pm to obtain a permeate (SNp) comprising the mucosa soluble in water proteins and peptides, and a retentate (SNr) with non-soluble in water proteins and peptides (i.e. , those that were suspended in the crude supernatant SN);
• SNp permeate
• SNr retentate
• 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.
• compositions are thus isolated fractions or extracts of the mammal intestine mucosa comprising the compounds of interest.
• 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 is the microvilli-covered surface of the epithelium where absorption takes place with microvilli of approximately 100 nm in diameter and varying from approximately 100 to 2000 nm in length.
• 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.
• the composition comprises phosphatases, more in particular selected from phytases, alkaline phosphatase and mixtures thereof.
• glycosidases optionally in combination with the embodiments above or below of the compositions of the invention, they comprise glycosidases.
• glycosidase are selected from the group consisting of maltase, maltase- glucoamylase, sucrase-isomaltase, alpha-galactosidase, lactase, dextrinase, trehalase, lysozyme and combinations thereof.
• compositions of the invention comprise peptidases selected from the groups consisting of carboxypeptidases, aminopeptidases, endopeptidases, enteropeptidase and dipeptidase.
• compositions of the invention comprise lipases selected from the groups consisting of triacylglycerol lipase, phospholipases, ceramidases and sphingomyelinases.
• the invention relates to a composition with mucosa proteins and peptides, which comprises: one or more of the enzymes selected from Alkaline Phosphatase, Phytase, Alphagalactosidase, Lysozyme, Aminopeptidase, Carboxipeptidase, Sucrase- isomaltase, Maltase-Glucoamylase, Alpha-Amylase, Diaminoxidase, Triacylglycerol lipase and Phospholipase; and/or antioxidant enzymes selected from Catalase, Glutathione peroxidase, Superoxide dismutase, and combinations thereof; and/or mucosa proteins participating in mucosa defence and healing selected from Mucins, Actin, Secretory Immunoglobulin A, Trefoil Factor Family proteins, Thymosin beta 4, Lactoferrin; Apolipoprotein A-l, Annexin A5, Annexin A1, Ga
• the invention 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.b.T), and (v.b.4’) as defined herein.
• the invention 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.b.T), (v.b.4a) and (v.b.5a) as defined herein.
• the AF comprises Pro-glucagon, Glucagon like peptide-1 (GLP-1), Thymosin beta-4 and Vasointestinal peptide.
• the invention 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.b.T), (v.b.4’), and (v.b.5’) as defined herein.
• the invention 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.b.T), and (v.b.4a), as defined herein.
• 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, 72 kDa type IV collagenase, Interstitial collagenase, Matrix metalloproteinase-9, Regenerating family member III I regenerating islet-derived protein 3 (Reg III), Regenerating family member IV I regenerating islet-derived protein IV (Reg IV), Antibacterial protein PR-39, Angiogenin, Phosphoinositide phospholipase C, Phospholipase D, and Pulmonary surfactant-associated protein D, even more particularly it comprises all of them.
• the composition comprising a basic fraction of intestine mammal mucosa basic proteins and peptides is subfractioned by washing with buffers of different concentrations.
• the washing is with buffers of different concentrations of NaCI.
• a subtraction BF2 is obtained by washing the resin with a buffer of about 2% NaCI.
• BF2 comprises lysozyme, in particular with an activity of 150 U-FIP/mL.
• BF2 comprises one or more of lysozyme, lactotransferrin, phosphoinositide phospholipase C, phospholipase D, antibacterial protein PR-39, regenerating family member 4 and angiogenin.
• a subtraction BF7 is obtained by further washing the resin with a buffer of about 7% NaCI.
• BF7 comprises one or more of 72 kDa type IV collagenase, Interstitial collagenase, Matrix metalloproteinase-9, Regenerating family member III / regenerating islet-derived protein 3 (Reg III) and Pulmonary surfactant-associated protein D, and even more particularly comprises all of them.
• the invention 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.b.T), (v.b.2), (v.b.3), (v.b.4’), and (v.b.5’) as defined herein.
• the SNubub’ comprises alkaline phosphatase (ALP) and mucin-2 (MLIC2). 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-6, superoxide dismutase, Apolipoprotein A1, Annexin-5, Galectin-1, Diazepam binding inhibitor, Immunoglobulin M, secretory Immunoglobulin A, Gastrotropin, Thymosin beta-4 and Hemoglobin, and more particularly comprises all of them.
• ALP alkaline phosphatase
• MLIC2 mucin-2
• the SNubub’ comprises one or more of ALP, MUC2, aminopeptid
• 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 invention also relates to a composition SNubub comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.4a), and (v.b.5a) as defined herein.
• 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.b.T), (v.b.4’), and (v.b.5’) as defined herein to obtain a SNubub’ fraction; and further comprising:
• composition SNububaq comprising intestine mammal mucosa proteins and peptides obtainable by a method comprising steps (i), (ii), (iiia), (iva), (v.b.T), (v.b.2), (v.b.3), (v.b.4’), (v.b.5’) and (v.b.a) as defined herein.
• SNubub’ is submitted to an extraction treatment with an organic solvent (setp v.b.a).
• the organic solvent is selected from the group consisting of heptanol, pentanol, 4-tert-Butylcatechol, limonene, cyclohexane, and mixtures thereof, more in particular it is butanol from 20 to 60% (w/w), even more in particular it is butanol at about 40% (w/w). More in particular, the extraction treatment lasts for about 5 minutes and is performed at about 37°C.
• the aqueous phase of the obtained extract comprises alkaline phosphatase and mucin-2.
• Snububaq comprises alkaline phosphatase with an activity of about 10.7 DEA U/rnL.
• Snububaq comprises one or more of Alkaline phosphatase, Mucin-2, Immunoglobulin M, Immunoglobulin A, Sucrase-lsomaltase, Alpha-amylase, Hemoglobin, Aminopeptidases, Maltase-glucoamylase, Catalase, Carboxypeptidases, Alpha-galactosidase, Peroxiredoxin-6, Superoxide dismutase, Acid sphingomyelinase-like phosphodiesterase, Diazepam binding inhibitor, Gastrotropin, Apolipoprotein-1, Annexin-5 and Galectin-1 , and more particularly comprises all of them.
• 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).
• a chemical agent for precipitation such as acetone, ammonium sulphate, PEG, Aluminium, and butanone to obtain a precipitate and a supernatant (Snububaqsn).
• the organic solvent is acetone, more in particular it is acetone from 20 to 40% (w/w), more in particular it is acetone around 30% (w/w).
• 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).
• 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).
• the organic solvent is acetone, more in particular it is acetone from 50 to 99% (w/w), more in particular it is acetone from 60% to 90% (w/w), more in particular it is acetone 80%.
• the acetone treatment is done at a temperature between 25 and 45 °C, in a more particular embodiment between 35 and 40 °C, in a more particular embodiment at 37 °C.
• the resulting precipitate (ALPp) comprises ALP.
• the resulting supernatant (MUCsn) comprises MUC2.
• the ALPp is further processed by means of affinity chromatography to obtain purified ALP.
• the purified ALP is a pharmaceutical grade protein suitable for medical applications.
• the purified ALP is obtained with a yield of around 38840 DEA Units of alkaline phosphatase activity per kg of mucosa (one DEA unit will hydrolyse 1 pmol of p-nitrophenyl phosphate per minute at pH 9.8 at 37°C, as known by the skilled person).
• the MUC2sn is further purified by means of affinity chromatography to obtain pharmaceutical grade proteins suitable for medical applications.
• 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 0.16 g of N-acetylneuraminic acid per kg of mucosa corresponding to sialylated glycoproteins like mucin-2.
• Example 1a Transcriptomic analysis of porcine intestinal mucosa
• a transcriptomic analysis was performed to explore the complete set of transcripts of pig intestinal mucosa and characterize the corresponding proteome.
• Transcripts were purified by poly(A)-tail selection. 9 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 6000 on one lane of SP flow-cell, 2x100bp (Kit version NV2864788-RGSBS).
• a total of 120,814,076 transcripts were read, and 104,992,005 of them were mapped against the reference genome. From the mapped reads, more than 16,000 genes were annotated and their expression was estimated.
• Example 2a Proteomic analysis of porcine intestinal mucosa samples of example 2b.
• 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 - 80°C until analysis.
• a representative sample was weight of at -20 °C (106.4mg) in an Eppendorf tube and TES buffer (Tris-HCI 10 mM, EDTA 1 mM, sucrose 0.25mM, pH 6.9) was added to give a concentration of 100mg/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 4 °C, 15.000 x g for 15 min (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 5 pg/pl.
• Peptide samples were analyzed on a Bruker TimsTOFPro equipped with a Bruker NanoElute LC-system. Peptides were separated using a 90 min reverse phase gradient, with solvent A: 2% acetonitrile, 0.1 % formic acid in MilliQ water, and solvent B: acetonitrile, 0.1 % formic acid.
• Proteomics analysis of porcine intestinal mucosa revealed 2810 identified protein groups, with false discovery rate (FDR) of 0.5%.
• FDR false discovery rate
• the data from this experiment was crossed with the data obtained from the transcriptom ic 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.
• Example 2b Industrial process (I) for obtaining heparin and by-side products (i.e. , native proteins and peptides) from mammalian intestine mucosa.
• a method for the simultaneous isolation of heparin and selected native proteins and peptides, from mammalian intestinal mucosa was performed as follows.
• aqueous extract of a mammalian intestinal mucosa was obtained (i.e., separating the mucosa of the intestine with standard methods and dissolving/suspending it in water or in an aqueous buffer); and then to said aqueous extract of mammalian intestinal mucosa (hereinafter called MUCOSA) sodium metabisulfite was added up to a concentration of 2%.
• MUCOSA mammalian intestinal mucosa
• Equivalent substances can be chosen as a preservative and antioxidant agent;
• MUCOSA was further diluted with 2 volumes of deionized water. The output of this procedure yielded an intermediate hereinafter called DILUTED MUCOSA.
• the diluted mucosa could have been obtained by diluting the MUCOSA with an aqueous solution, comprising one of the following detergents Tween 20, Tween 80, Triton X-100 orX-114, deoxycholic acid, Brij 35 or 58, BigCHAP or deoxy BigCHAP, MEGA 8, MEGA 9 or MEGA 10, Octyl betaglucoside;
• an aqueous solution comprising one of the following detergents Tween 20, Tween 80, Triton X-100 orX-114, deoxycholic acid, Brij 35 or 58, BigCHAP or deoxy BigCHAP, MEGA 8, MEGA 9 or MEGA 10, Octyl betaglucoside;
• DILUTED MUCOSA was submitted to homogenization by a rotor-stator homogenizer at the G-force of 1700 g, during 2 to 4 minutes at a temperature between 2°C and 8°C to lyse the cell membranes and to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides (herein after called HOMOGENATE).
• Alternative 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, shear fluid forces, high pressure or cavitation.
• Another alternative homogenization technique relates to the addition of detergents;
• Heparin and the mucosa proteins and peptides contained in the HOMOGENATE obtained in step (iii) were separated by centrifugation in order to obtain liquid and solid phases, each comprising different proportion of one or more of heparin, and other mucosa proteins and peptides.
• the centrifugation was carried out at a G-force of 3134 g for 20 minutes and at a temperature below 8°C to obtain: a solid phase containing heparin, (hereinafter called PELLET or P), and a liquid phase containing a mixture of mucosa proteins and peptides (hereinafter called SUPERNATANT or SN).
• the PELLET was then submitted to the standard extraction method to obtain and purify heparin for pharmaceutical applications (This step can also be labelled as (iv.a), as previously indicated).
• the pellet was treated with an enzymatic alkaline proteolysis at 58°C and pH 7.7-8 during 2 hours. After the hydrolysis, pH was decreased to 6.6 and temperature was increased to 90°C and maintained during 15 minutes to precipitate the fat.
• the product was centrifuged at a G-force of 3134 g for 15 minutes, and the supernatant was decantated and filtrated through a 300 pm filter to separate the fat fraction. Then, an anion exchange resin was added to the supernatant to capture the heparin.
• the FILTERED SUPERNATANT was then diafiltered by means of a tangential flow filtration equipment through a filter having a cut-off of less than 0.1 pm.
• the diafiltration was carried out by substituting the permeate with at least 4 volumes of a buffer, preferably between 5 and 7 volumes. The aim of this step was to recover both the retentate (SNr) and the permeate (SNp).
• the fraction SNpr after having adjusted the pH to 7.0, was loaded downflow on a strong cationic exchange chromatographic column in order to bind, and consequently concentrate, all the basic proteins and peptides present in the solution.
• the bound fraction (SNprb) was eluted with the upflow direction and then diafiltered through a phosphate buffer 50 mM pH 7.0 and concentrated by means of a tangential flow system through a filter having a cut-off of 3 KDa.
• the intermediate mixture obtained by this procedure was hereinafter called BASIC FRACTION.
• the unbound fraction (SNprub) of the step (v.SN.4) after having adjusted the pH to 7.0, was then loaded on a strong anionic exchange resin in order to bind, and consequently concentrate, all the acidic proteins and peptides present in the solution.
• the bound fraction was eluted with the upflow direction and then diafiltered through a phosphate buffer 50 mM pH 7.0 and concentrated by means of a tangential flow system through a filter having a cut-off of 3 KDa.
• the intermediate obtained by this procedure is hereinafter called ACIDIC FRACTION.
• 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.
• 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.
• an enzymatic activity of 38.840 DEA Units of alkaline phosphatase activity per kg of mucosa, and 1.014.983 U-FIP of lysozyme activity per kg of mucosa are obtained, while heparin, a compound usually obtained from this tissue is also obtained with 0,047 MIU of heparin activity per Kg of mucosa.
• 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.
• Example 3a Industrial process (II) for obtaining heparin and by-side products (i.e. , native proteins and peptides) from mammalian intestine mucosa
• the method included the following steps:
• aqueous extract of a mammalian intestinal mucosa was obtained (i.e., separating the mucosa of the intestine with standard methods and dissolving/suspending it in water or in an aqueous buffer); and then to said aqueous extract of mammalian intestinal mucosa (hereinafter called MUCOSA) sodium metabisulfite was added up to a concentration of 2%.
• MUCOSA mammalian intestinal mucosa
• MUCOSA was further diluted with 2 volumes of deionized water.
• DILUTED MUCOSA an intermediate hereinafter called DILUTED MUCOSA.
• the diluted mucosa could have been obtained by diluting the MUCOSA with an aqueous solution, comprising one of the following detergents 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 betaglucoside;
• DILUTED MUCOSA was submitted to homogenization by a rotor-stator homogenizer at the G-force of 1700 g, during 2 to 4 minutes at a temperature between 2°C and 8°C to lyse the cell membranes and to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides (herein after called HOMOGENATE).
• Alternative 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, shear fluid forces, high pressure or cavitation. Homogenization can be performed in the presence of detergents;
• Heparin and the mucosa proteins and peptides contained in the HOMOGENATE obtained in step (iiia) were separated by centrifugation in order to obtain liquid and solid phases, each comprising different proportion of one or more of heparin, and other mucosa proteins and peptides.
• the centrifugation was carried out at a G-force of 3300 g for 30 minutes and at a temperature below 37°C (preferably between 0 and 25°C) to obtain: a solid phase containing heparin, (hereinafter called PELLET or P), and a liquid phase containing a mixture of mucosa proteins and peptides (hereinafter called SUPERNATANT or SN).
• the separation of the PELLET and the SUPERNATANT can be achieved by tangential microfiltration through 0.2 .m membrane.
• the SN contains, among other proteins and peptides, glucagon like peptide-1 (GLP-1), thymosin beta-4, vaso intestinal peptide, lysozyme, lactotransferrin, regenerating islet-derived protein 3 (Reg III), regenerating islet-derived protein IV (Reg IV), matrix metalloproteinase-9, 72 kDa type IV collagenase, interstitial collagenase, bactericidal permeability-increasing protein, pulmonary surfactant- associated protein D, antibacterial protein PR-39, angiogenin, alkaline phosphatase, mucin-2, trypsin, aminopeptidases, carboxypeptidases, catalase, triacylglycerol lipase, phospholipases, acid sphingomyelinase-like phosphodiesterase, al
• the SN also contains phytase from exogenous origin, as confirmed by enzymatic assay.
• the PELLET was then submitted to the standard extraction method to obtain and purify heparin for pharmaceutical applications (This step can also be labelled as (iv.a), as previously indicated).
• the pellet was treated with an enzymatic alkaline proteolysis at 58°C and pH 7.7-8 during 2 hours. After the hydrolysis, pH was decreased to 6.6 and temperature was increased to 90°C and maintained during 15 minutes to precipitate the fat. The product was centrifuged at a G-force of 3134 g for 15 minutes, and the supernatant was decantated and filtrated through a 300 pm filter to separate the fat fraction.
• an anion exchange resin was added to the supernatant in a proportion between 0,1 and 1% w/w (preferably 0,2%) to capture the heparin.
• the mixture was kept under stirring overnight, and resin was separated from the liquid with a mesh filter, and rinsed with osmotized water. Proteins were eluted from the resin with three successive additions of NaCI 3.5% (w/v) at 45°C for 30 minutes each. Finally, heparin was eluted with three successive additions of NaCI 18%, 20%, and 20% at 45°C for 30 minutes each.
• heparin eluates were combined and analyzed yielding an average of 0,047 MIU of heparin activity per Kg of mucosa (one unit of heparin is the quantity of heparin required to keep 1 ml of cat's blood fluid for 24 hours at 0°C; it is equivalent approximately to 0.002 mg of pure heparin, as known by the skilled person).
• the FILTERED SUPERNATANT (SNf) was optionally diafiltered by means of a tangential flow filtration equipment through a filter having a cut-off of less than 0.1 pm.
• the diafiltration was carried out by substituting the permeate with at least 4 volumes of a buffer, preferably between 5 and 7 volumes. The aim of this step was to recover both the retentate (SNr) and the permeate (SNp).
• the bound fraction (SNprb) was eluted with the upflow direction and then diafiltered through a phosphate buffer 50 mM pH 7.0 and concentrated by means of a tangential flow system through a filter having a cut-off of 3 KDa.
• the anionic exchange chromatography can be done in batch, adding between 0,5 and 1 ,5% (w/w) (preferably 1%) of strong anionic exchange resin to the fraction.
• the intermediate mixture obtained by this procedure was hereinafter called ACIDIC FRACTION (AF).
• the ACIDIC FRACTION (AF) contains, among other, the proteins and peptides identified by the proteomic analysis (shown in example 3b).
• the unbound fraction (SNprub) of the step (v.SN.4) after having adjusted the pH to 7.0, was then loaded on a strong cationic exchange column in order to bind, and consequently concentrate, all the basic proteins and peptides present in the solution.
• the bound fraction was eluted with the upflow direction and then diafiltered through a phosphate buffer 50 mM pH 7.0 and concentrated by means of a tangential flow system through a filter having a cut-off of 3 KDa.
• the cationic exchange chromatography can be done in batch, adding between 5 and 15% (w/w) (preferably 10%) of strong cationic exchange resin to the fraction.
• BASIC FRACTION BF
• the BASIC FRACTION contains, among other, the proteins and peptides identified by the proteomic analysis (shown in example 2b).
• Two subtractions were eluted from the BASIC FRACTION using different NaCI concentrations.
• a subtraction (BF2) was eluted and characterized by enzymatic assay and proteomic analysis. It contained, among other proteins, lysozyme with an activity of 150 U-FIP/mL, lactotransferrin, phosphoinositide phospholipase C, phospholipase D, antibacterial protein PR-39, regenerating family member 4 and angiogenin.
• BF7 was eluted containing the rest of the proteins from the BASIC FRACTION. Both subtractions were tested for antimicrobial activity as detailed in Example 2c.
• the unbound fraction (SNprubub) of the step (v.SN.5) contains, among other, the proteins and peptides identified by the proteomic analysis (shown in example 2b).
• SNprubub can be used as an ingredient for feed and/or food applications, or it can be further fractionated by means of successive filtration, and ultrafiltration steps, solvent extraction, protein precipitation, and/or gel permeation and affinity chromatography steps.
• 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 assay and proteomic analysis. It contains, among other, the proteins and peptides identified by the proteomic analysis (shown in example 2b).
• SNprububaq was tested for antimicrobial activity as described in the example 2c.
• ALP and MUC2 were then purified by means of affinity chromatography to obtain pharmaceutical grade proteins suitable for medical applications. Alternatively, precipitation with ammonium sulphate, aluminium, or their combinations with the previous treatments can be used.
• the supernatant of any of its derived fractions can also be treated previously with phosphatidylinositol-specific phospholipase C to detach alkaline phosphatase and other membrane proteins in order to improve their separation and purification, and obtain higher yields.
• 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.
• 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.
• an enzymatic activity of 38.840 DEA Units of alkaline phosphatase activity per kg of mucosa, and 1.014.983 U-FIP of lysozyme activity per kg of mucosa are obtained, as well as 0,16 g of N-acetylneuraminic acid per kg of mucosa corresponding to sialylated glycoproteins like mucin-2, while heparin, a compound usually obtained from this tissue is also obtained with 0,047 MIU of heparin activity per Kg of mucosa.
• 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.
• Example 3b Proteomic study of fractions obtained during the industrial process (II) of example 2a.
• iBAQ Intensity Based Absolute Quantification
• Example 3c Screening of the antimicrobial and antifungal activity of porcine extracts obtained during the industrial process (II) of example 2a against different pathogens
• 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 24 h at 37 °C, and the fungus (Fo) for 7 days at 23 °C. 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 4 fractions and two control samples was evaluated at a concentration of 0.5X.
• the antimicrobial effect was compared with the reference antibiotic levofloxacin (50 mg/L) in the case of bacteria, and cycloheximide (50 mg/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 5 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.
• 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 100 pl of the product with 100 pl of the bacterial/fungal suspension, obtaining a final volume of 200 pl 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 37 °C in the case of the human pathogens (St and Sa).
• the fungus was incubated for 7 days at 20°C with constant shaking.
• the growth kinetics of the different pathogens in the presence of the products was analyzed in triplicate (3 wells per product and pathogen) using automated systems that allow microbial growth to be monitored through absorbance readings at 600 nm (Bioscreen C MBR, Labsystems, Finland and Varioskan flash, Thermo Electron Corporation, USA) every hour in the case of bacteria (for 24 h) and every 30 min in the case of fungus (for 7 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 fractions AF, BF2 and SNprububaq show significant antimicrobial potential.
• Clause 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:
• step (iii) submitting diluted mucosa of step (ii) or preserved mucosa of (i) to homogenization at a temperature of less than 40 °C, to lyse cell membrane of mucosa cells to obtain a stable homogenate of the mucosa comprising heparin and mucosa proteins and peptides;
• step (iv) separating heparin and the mucosa proteins and peptides contained in the stable homogenate obtained in step (iii) 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;
• step (v.b) submitting the supernatant (SN) of (iv) comprising a mixture of solubilized and/or suspended mucosa proteins and peptides 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/or molecular weight.
• Clause 2. The method according to clause 1, wherein step (iv) of separation is carried out by centrifugation.
• Clause 3 The method according to any of the clauses 1-2, wherein the supernatant (SN) comprising a mixture of mucosa proteins and peptides is submitted in step (v.b) to filtration through an up to 100 pm cut-off filter in order to retain small particles and lipid residues which were not pelleted in step (iv), to obtain a filtered supernatant comprising most of the mucosa proteins and peptides.
• Clause 4 The method according to clause 3, wherein the filtered supernatant is further diafiltered through a filter having a cut-off less than 0.8 pm to obtain a permeate (SNp) comprising the mucosa proteins and peptides, and a retentate (SNr) comprising nonsoluble in water proteins and peptides.
• SNp permeate
• SNr retentate
• Clause 5 The method according to any of the clauses 3-4, wherein the filtered supernatant comprising the mucosa proteins and peptides or any of its permeate (SNp) or retentate (SNr) fractions, is loaded downflow on a cationic exchange chromatographic column to obtain a bound fraction (bf) and an unbound fraction (ubf); 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 1 to 5 KDa, to obtain a basic fraction of the filtered supernatant comprising basic proteins and peptides.
• SNp permeate
• SNr retentate
• Clause 6 The method according to clause 5, wherein the unbound fraction (ubf) is loaded on an anionic exchange chromatographic 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 1 to 5 KDa, to obtain an acidic fraction of the filtered supernatant comprising acidic proteins and peptides.
• Clause 7 The method according to any of the clauses 5-6, wherein any of the basic or acidic fractions are further submitted to fractionation by means of diafiltration, salting out methods, cationic and anionic exchange chromatography, and combinations thereof.
• Clause 8 The method according to any of the clauses 4-7, wherein the retentate (SNr) comprising non-soluble in water proteins and peptides is, prior to its loaded downflow on an ionic exchange chromatography, treated with homogenization, optionally in the presence of detergents, and/or with hydrolytic enzymes, to detach bound proteins from membranes or cell structures.
• SNr retentate
• hydrolytic enzymes comprise one or more of phospholipases and proteases.
• Clause 10 The method according to any of the clauses 1-9, wherein in step (ii) the mucosa is diluted with deionized water comprising one or more detergents.
• 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.
• 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 180 g, preferably 1700 g, for a minimum of 30 seconds, preferably between 2 to 4 minutes.
• Clause 14 A composition comprising intestine mammal mucosa proteins and peptides obtainable by a method as defined in any of the clauses 1-13.
• Clause 15 The composition according to clause 14, wherein 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.
• Clause 16 A method for the obtention of heparin by fractionation of mammalian intestine mucosa, comprising the steps (i), (ii), (iii), (iv), and (v.a) as defined in clause 1.
• Clause 17 A method for the obtention of proteins and peptides in native state by fractionation of mammalian intestine mucosa, comprising the steps (i), (ii), (iii), (iv), and (v.b) as defined in clause 1.

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