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WO2025149372A1 - Procédé d'enrichissement en vésicules extracellulaires du lait à l'aide de peg - Google Patents

Procédé d'enrichissement en vésicules extracellulaires du lait à l'aide de peg

Info

Publication number
WO2025149372A1
WO2025149372A1 PCT/EP2024/088296 EP2024088296W WO2025149372A1 WO 2025149372 A1 WO2025149372 A1 WO 2025149372A1 EP 2024088296 W EP2024088296 W EP 2024088296W WO 2025149372 A1 WO2025149372 A1 WO 2025149372A1
Authority
WO
WIPO (PCT)
Prior art keywords
whey
milk
peg
product
enriched
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/088296
Other languages
English (en)
Inventor
Anouk Leonie FEITSMA
Alphons Adrianus Jacoba VAN DE LOO
Arnoldus Jacobus ARNTZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FrieslandCampina Nederland BV
Original Assignee
FrieslandCampina Nederland BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by FrieslandCampina Nederland BV filed Critical FrieslandCampina Nederland BV
Publication of WO2025149372A1 publication Critical patent/WO2025149372A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A23J3/08—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C21/00—Whey; Whey preparations
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
    • A23C9/146—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by ion-exchange
    • A23C9/1465—Chromatographic separation of protein or lactose fraction; Adsorption of protein or lactose fraction followed by elution
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/15—Reconstituted or recombined milk products containing neither non-milk fat nor non-milk proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
    • A23J1/205—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey from whey, e.g. lactalbumine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1276—Globules of milk; Constituents thereof

Definitions

  • intact extracellular vesicle refers to extracellular vesicles (EV) in which the vesicle membrane is not ruptured and/or otherwise degraded and as such a vesicle size may be determined using methods described elsewhere herein.
  • the endogenous cargo i.e. , the bioactive agents, therapeutics (e.g. miRNA), and/or other biomolecules which are inherently present in a milk-derived extracellular vesicle (mEV), are retained in intact EV in active form.
  • milk-derived EVs Due to its composition, milk-derived EVs are highly biocompatible and have limited immunogenicity even cross species. It has been demonstrated that milk-derived EVs, when taken up in the gastro-intestinal tract, stay intact after absorption, indicating excellent stability. These characteristics make milk-derived EVs very suitable as drug carriers, but also by themselves, these EVs already have a substantial immunoregulatory function, and even without loading, these vesicles can act as therapeutics. It is therefore desirable to have an efficient scalable method to isolate or at least increase the level of EV in a milk stream. Such a process contributes to a sustainable way to address and/or treat problems or defects in a subject’s immune response and contributes to a sustainable and environment-friendly drug carrier. Such a process likewise contributes to the applicability of EV for use in improving muscle growth, muscle recovery, and/or mobility.
  • milk-derived EV also referred to a milk EV or mEV
  • mEV milk-derived EV
  • SMB chromatography has its roots in the petrochemical and mineral industries.
  • Today, SMB chromatography is used by the pharmaceutical industry for the separation of enantiomers out of racemic mixtures.
  • SMB chromatography has already been used for the separation of the monosaccharide fructose from fructose-glucose solutions and for the separation of the disaccharide sucrose from sugar beet or sugar cane syrups on large-scale.
  • SMB chromatography has also been used for the purification of a trisaccharide - like 2' -fucosy I lactose - from fermentation, bio catalysis or chemical synthesis (e.g. in EP2857410 A1 ).
  • the classical SMB concept involves four different zones with four external applied streams: a feed stream containing the components to be separated, a desorbent or mobile phase stream, an extract and a raffinate stream (with the raffinate stream representing the less retained component(s).
  • These liquid streams divide the SMB system into four different zones (each zone or section can comprise one or more columns) with the following tasks: zone I is required for the regeneration of the solid phase, the purpose of zone II is the desorption of the less strongly desorbed material, the task of zone III is the adsorption of the strongly adsorbed material and finally the task of zone IV is the adsorption of the less adsorptive material.
  • zone I is required for the regeneration of the solid phase
  • zone II is the desorption of the less strongly desorbed material
  • the task of zone III is the adsorption of the strongly adsorbed material
  • the task of zone IV is the adsorption of the less adsorptive material.
  • the whey in the process of the invention is cheese whey, preferably bovine cheese whey.
  • the whey in the process of the invention is acid whey, preferably bovine acid whey.
  • Whey is the liquid remaining after milk has been curdled and strained. It is a by-product of the manufacturing of cheese or casein and has several commercial uses.
  • Sweet whey is a by-product resulting from the manufacture of rennet types of hard cheese, like Cheddar or Swiss cheese.
  • Acid whey also known as sour whey is a by-product brought out during the making of acid types of dairy products, such as strained yogurt.
  • Whey proteins consist of a-lactalbumin, p-lactoglobulin, serum albumin, immunoglobulins, and proteose peptones.
  • the whey as used in the process of the invention may be obtained from any kind of milk like for example from bovine milk, “bovine” is referring to an animal of the cattle group and includes the antelopes, sheep, goats, cattle, buffalo, and bison, bovine is preferably referring to the domestic cattle group including sheep, goats, cattle, and buffalo.
  • the whey is obtained from cow, goat, sheep, camel, buffalo or horse milk; bovine milk whey being preferred.
  • the fat content is from 0.0% to 10.0% by weight and the casein content of from 0.0% to 5.0% by weight, as determined to the dry mass of the whey. More preferably the fat content is from 0.0% to 5.0% by weight and the casein content of from 0.0% to 3.0% by weight, as determined to the dry mass of the whey.
  • the "fat content" of a composition corresponds to the weight of the fat components present in the composition relatively to the total weight of the composition.
  • the fat content is expressed as a weight percentage.
  • the fat content can be measured using the Rbse- Gottsch Kunststoff principle as described in ISO 23318:2022(E); which is a gravimetric method including hydrolysis and extraction by petroleum ether.
  • the casein content is determined by the difference between the total nitrogen (Ntot) content and the noncasein nitrogen (NCN) using the Kjeldahl method.
  • a milk mineral solution or of SMUF is particularly preferred when the EV-comprising liquid is a milk fraction such as a whey protein fraction. Without wishing to be bound to any theory, it is believed that such milk mineral solution or SMUF helps to keep the EVs in a native state.
  • PEG-6000 was dissolved in Milli Q water (25 g / 50 ml) and stored at 4°C till further use.
  • Example 1 A The same experiment as Example 1 A was repeated except that the pH was set at 6.8.
  • femtogram protein/EV using Nanoparticle Tracking analysis to the determine the number of EV in a method analogous as described by Webber et al using a micro-BCA kit (Journal of Extracellular Vesicles 2013, 2: 19861 http://journalofextracellularvesicles.net/ under index. php/jev/ article/view/19861 ).
  • the EV purity (fg protein /EV) was determined of acid whey (HCW80 prior to ultrafiltration), Centrifuged PEG- acid whey retentate pellet as obtained in Example 1 , and UFR followed by SEC (UFR-SEC). The results of this purification are shown in Table 1 (entry pH 4.2 ->PEG ->SEC).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Nutrition Science (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pediatric Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant l'obtention d'un produit enrichi en vésicules extracellulaires (VE) issues du lait à partir de lactosérum par mélange de lactosérum avec une solution de PEG de façon à obtenir un mélange lactosérum-PEG ; et par centrifugation du mélange lactosérum-PEG de façon à obtenir un granulé enrichi en VE du lait.
PCT/EP2024/088296 2024-01-09 2024-12-23 Procédé d'enrichissement en vésicules extracellulaires du lait à l'aide de peg Pending WO2025149372A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24150852.2 2024-01-09
EP24150852 2024-01-09

Publications (1)

Publication Number Publication Date
WO2025149372A1 true WO2025149372A1 (fr) 2025-07-17

Family

ID=89542154

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/088296 Pending WO2025149372A1 (fr) 2024-01-09 2024-12-23 Procédé d'enrichissement en vésicules extracellulaires du lait à l'aide de peg

Country Status (1)

Country Link
WO (1) WO2025149372A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1218410A1 (fr) 1999-10-06 2002-07-03 Campina B.V. Procede pour obtenir des preparations de facteurs de croissance (tgf-beta et igf-1) a partir de produits laitiers avec une faible contamination mutuelle croisee
EP2857410A1 (fr) 2013-10-04 2015-04-08 Jennewein Biotechnologie GmbH Procédé de purification de 2´-fucosyllactose utilisant la chromatographie à lit mobile simulé
WO2022146743A1 (fr) 2020-12-31 2022-07-07 Abbott Laboratories Procédés d'amélioration de rendement musculaire ou de réduction de fatigue chronique par administration d'exosomes dérivés du lait bovin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1218410A1 (fr) 1999-10-06 2002-07-03 Campina B.V. Procede pour obtenir des preparations de facteurs de croissance (tgf-beta et igf-1) a partir de produits laitiers avec une faible contamination mutuelle croisee
EP2857410A1 (fr) 2013-10-04 2015-04-08 Jennewein Biotechnologie GmbH Procédé de purification de 2´-fucosyllactose utilisant la chromatographie à lit mobile simulé
WO2022146743A1 (fr) 2020-12-31 2022-07-07 Abbott Laboratories Procédés d'amélioration de rendement musculaire ou de réduction de fatigue chronique par administration d'exosomes dérivés du lait bovin

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BLANS KRISTINE ET AL: "Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography", JOURNAL OF EXTRACELLULAR VESICLES, vol. 6, no. 1, 15 March 2017 (2017-03-15), pages 1 - 16, XP093004031, DOI: 10.1080/20013078.2017.1294340 *
JANINE STAM: "Isolation of extracellular vesicles with combined enrichment methods", JOURNAL OF CHROMATOGRAPHY B, vol. 1169, 27 February 2021 (2021-02-27), NL, pages 122604, XP093167484, ISSN: 1570-0232, DOI: 10.1016/j.jchromb.2021.122604 *
JENNESS, R., AND KOOPS, J.: "Preparation and properties of a salt solution which simulates milk ultrafiltrate", NETHERLANDS MILK AND DAIRY JOURNAL, vol. 16, no. 3, 1962, pages 153 - 164, XP055566467
L. DEL POZO-ACEBO ET AL., INT. J. MOL. SCI., vol. 22, 2021, pages 1105, Retrieved from the Internet <URL:https://doi.org/10.3390/ijms22031105>
LI XIAOPING ET AL: "Recent Advances on the Function and Purification of Milk Exosomes: A Review", FRONTIERS IN NUTRITION, vol. 9, 9 June 2022 (2022-06-09), XP093090308, DOI: 10.3389/fnut.2022.871346 *
SINGH ET AL: "Bovine Milk Derived Exosomal - Curcumin ExhibitingEnhanced Stability, Solubility, and Cellular Bioavailability", CLINICS IN ONCOLOGY, 8 February 2021 (2021-02-08), pages 1 - 8, XP093167437 *
WEBBER ET AL.: "using a micro-BCA kit", JOURNAL OF EXTRACELLULAR VESICLES, vol. 2, 2013, pages 19861, Retrieved from the Internet <URL:http://journalofextracellularvesicles.net/underindex.php/jev/article/view/19861>

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