Method for enriching milk extracellular vesicles

Abstract

Disclosed is a method for obtaining a product enriched in extracellular vesicles (EV), the method comprising the steps of: i. Obtaining a liquid comprising EV; ii. Subjecting the EV-containing liquid to ultrafiltration (UF) to provide an EV-enriched UF retentate (UFR), and a UF permeate (UFP) wherein the enrichment is an increased number of EVs in the UFR as compared to total protein levels in the EV-containing liquid; wherein a step of chelating a divalent cation with EDTA is not used in the preparation of the EV-containing liquid, preferably wherein EDTA is not used to prepare the EV-containing liquid.

Description

Technical Field

[0001] This invention relates to a method for enriching extracellular vesicles (EVs), and more particularly to a method for obtaining milk streams enriched with milk-derived extracellular vesicles (mEVs), more preferably bovine mEVs. The invention further relates to a product comprising such an mEV-enriched milk stream, and to the use of such an mEV-enriched milk stream. Background Technology

[0002] US 10,729,159 (US 2019 / 0150474) relates to methods for purifying exosomes, which involve using a whey composition as an exosome source. Exosomes are separated by subjecting the whey composition to a first ultrafiltration to obtain a first permeate and a first residue. The first residue can then be subjected to a second ultrafiltration to obtain a second permeate and a second residue. During the second ultrafiltration, the first residue can be treated with carbon dioxide. The second residue can then be subjected to a third ultrafiltration to obtain a third permeate and a third residue. Optionally, the third permeate can then be dried to obtain an exosome powder.

[0003] Moleirinho et al. disclosed the challenges in biotherapeutic particle manufacturing, including upstream and downstream steps and manufacturing platforms (Moleirinho, MG, (2019) Current challenges in biotherapeutic particle manufacturing. Expert Opinion on Biological Therapy, 20(5), 451–465. https: / / doi.org / 10.1080 / 14712598.2020.1693541).

[0004] BJ Benedikter et al. disclosed a method for isolating extracellular vesicles from cell culture media for compositional and functional studies (BJ Benedikter et al., Sci Rep [Scientific Reports] 7, 15297 (2017). https: / / doi.org / 10.1038 / s41598-017-15717-7).

[0005] The term "extracellular vesicle" or "EV" is used herein as a general term for lipid bilayer-bound particles released from cells, and unlike cells, EVs cannot replicate. EVs range in diameter from near the physically smallest possible monolayer liposome (approximately 20–30 nm) to 10 micrometers or larger, although the vast majority of EVs are smaller than 200 nm. They carry loads of proteins, nucleic acids, lipids, metabolites, and even organelles from their parent cells. Most cells studied to date (including some bacterial, fungal, and plant cells surrounded by cell walls) are thought to release EVs. A wide variety of EV subtypes have been proposed, defined in various ways based on size, biological origin, load, cellular origin, and function.

[0006] As used herein, the term "intact extracellular vesicle" refers to an extracellular vesicle (EV) in which the vesicle membrane has not ruptured and / or otherwise degraded, and therefore the vesicle size can be determined using methods described elsewhere herein. Endogenous loadings, i.e., bioactive agents, therapeutic agents (e.g., miRNAs), and / or other biomolecules inherently present in milk-derived extracellular vesicles (mEVs), are retained in the intact EV in their active form.

[0007] Extracellular vesicles (one or more EVs) comprise exosomes (< 100 nm) and microvesicles (100 nm–10 micrometers). EVs are present in biological fluids and participate in a variety of physiological and pathological processes. EVs are considered an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and genetic material.

[0008] Numerous studies have provided compelling evidence that vesicles (EVs) participate in the regulation of immune responses, acting as both enhancers and weakeners of the immune system, depending on the vesicle's origin and type. Studies have demonstrated the anti-inflammatory effects of milk-derived EVs (using human breast milk, bovine colostrum, and commercially available pasteurized cow milk) in in vitro systems and in animal models (therapeuticly). Strategies targeting the gut, particularly its microbiome, are under investigation and hold promise as therapeutic interventions for these diseases. In recent years, the use of milk-derived EVs as standalone drugs or as drug carriers has been frequently suggested. Due to their composition, milk-derived EVs exhibit high biocompatibility and limited immunogenicity, even across species. Milk-derived EVs have been shown to remain intact after absorption when taken up in the gastrointestinal tract, demonstrating excellent stability. These characteristics make milk-derived EVs well-suited as drug carriers, and these EVs themselves possess substantial immunomodulatory functions, and these vesicles can act as therapeutic agents even without a loading. Therefore, efficient and scalable methods for isolating or at least enriching milk streams containing EVs are desired. Such approaches facilitate sustainable ways of addressing and / or treating problems or deficiencies in a subject's immune response, and contribute to sustainable and environmentally friendly drug delivery systems.

[0009] Research in the field of milk-derived extracellular matrix (mEV) has progressed rapidly in the past few years; however, a standard protocol for reproducible and cost-effective mEV (or exosome) isolation has not yet been established.

[0010] Initially, exosome isolation relied primarily on ultracentrifugation, considered the gold standard. Other isolation methods have been developed; however, each has its own limitations and cannot isolate only exosomes. Exosomes can be contaminated with proteins, for example. Improved EV isolation methods may not only affect the quantity and purity of recovered EVs but may also lead to the isolation of specific EV populations with different sizes and functional characteristics and carrying certain RNA, protein, and lipid profiles. Therefore, developing scalable methods to isolate EVs in a rapid, efficient, reproducible, cost-effective, and clinically friendly manner remains a challenge (L. del Pozo-Acebo et al., Int. J. Mol. Sci. [International Journal of Molecular Sciences] 2021, 22, 1105. https: / / doi.org / 10.3390 / ijms22031105).

[0011] Ultracentrifugation has been used for EV separation, but it has several drawbacks, such as co-separation of non-exosome impurities, low reproducibility, potential damage to exosomes, and low sample throughput.

[0012] Body fluids contain numerous nanoparticles (some of which are non-vesicular nanoparticles) with sizes similar to those of exosomes co-eluted with EVs when size exclusion chromatography (SEC) is applied. A major drawback of SEC is the limited amount of EV recovered per unit volume. Therefore, there is a desire to further improve mEV / exosome enrichment methods.

[0013] Therefore, an object of the present invention is to provide a method for enriching extracellular vesicles (EVs), particularly a method for obtaining milk streams enriched with milk-derived extracellular vesicles (mEVs), more preferably bovine mEVs. Another object of the present invention is to provide an EV enrichment method capable of producing large quantities of mEVs, preferably in a method scalable to greater than 100 liters (e.g., >1000 liters) and / or capable of operating in continuous mode.

[0014] Another object of the present invention is to provide a mEV-enriched milk stream that can be used in nutritional products such as infant formula or food supplements. Alternatively, the mEV-enriched milk stream can be used in nutritional products such as sports nutrition products or adult nutrition products to enhance muscle performance in subjects who need to improve their physical performance.

[0015] The present invention also relates to a method for alleviating chronic fatigue in a subject who is recovering from or has recovered from a viral infection, the method comprising administering to the subject a product containing enriched extracellular vesicles of intact bovine mEV.

[0016] Another object of the present invention is to use the mEV-enriched products obtained as in the methods of the invention in a convenient manner to improve mitochondrial function and thereby improve muscle performance in subjects requiring improved physical performance. These methods can be used to prevent or treat conditions characterized by reduced reserve respiratory capacity, including sarcopenia and chronic or acute cardiac injury, for example, as described in European application EP 23165184.5.

[0017] These and other advantages of the method of the present invention will become more apparent from the detailed description. Summary of the Invention

[0018] In a first aspect, the present invention relates to a method for obtaining a product enriched with extracellular vesicles (EVs), the method comprising the steps of: i. Obtain a liquid containing EV; ii. The liquid containing EVs is subjected to ultrafiltration (UF) to provide EV-enriched UF reflux (UFR) and UF permeate (UFP), preferably wherein the UF is performed in percolation (DF) mode; wherein enrichment is an increase in the number of EVs in the UFR relative to the total protein level compared to the liquid containing EVs, and wherein the step of chelating divalent cations with EDTA is not used in the preparation of the liquid containing EVs, preferably wherein EDTA is not used to prepare the liquid containing EVs as defined in claim 1. As used herein, an increase in the number of EVs in the product relative to the total protein level means a reduction in the amount of protein per EV. For the avoidance of doubt, the UFP obtained in step ii. is EV-depleted compared to the liquid containing EVs.

[0019] In another aspect, the present invention relates to the use of products enriched with EVs as obtained by the methods of the present invention in food products or pharmaceuticals. The present invention also relates to such food products or pharmaceuticals comprising the products enriched with said EVs.

[0020] In another aspect, the present invention relates to synthetic food products of the present invention for use in increasing muscle growth in subjects.

[0021] In another aspect, the present invention relates to a composition comprising extracellular vesicles (EVs) and proteins, wherein the amount of protein is less than 4.0 fg / EV, preferably less than 3.0 fg / EV, and more preferably less than 2.0 fg / EV. Attached Figure Description

[0022] exist Figure 1 The image shows the emulsion EV separation selectivity of the ceramic membrane at different transmembrane pressures (TMP) of 0.50, 1.0, 1.5, and 2.0 bar. The upper line corresponds to a 0.2-micron filter, and the lower line corresponds to a 0.1-micron filter.

[0023] exist Figure 2 The emulsion EV separation selectivity of polymer membranes at different transmembrane pressures (TMP) of 0.50, 1.0, 1.5, and 2.0 bar is shown. The selectivity of the 500 kD filter is 1.0 at all TMPs, while the selectivity of the 800 kD filter increases from -0.5 to 1.5 as the TMP increases from low to high.

[0024] exist Figure 3 In the study, the selectivity of the 0.2-micron ceramic membrane in mEV separation was plotted as a function of pH and TMP. Optimal selectivity (5.8) was obtained at a pH of 5.2 and a TMP of 1.66 bar.

[0025] The selectivity of the polymer 800 kD micron membrane in mEV separation was plotted as a function of pH and TMP. Figure 4 The optimal selectivity (5.8) was obtained at pH 6.3 and TMP 1.00.

[0026] The results of percolation using a polymer 800 kD membrane are as follows: Figure 5 As shown in the figure. Percolation models were constructed at pH 6.3 and 4.1 with a crossflow of 1 m / s. The retention rates found in the experimental design were extrapolated to the linear model. This was done to investigate the loss of EV during percolation compared to protein loss. The model in Figure 5 The diagram illustrates how long each perfiltration run must take to remove all proteins and the EV loss per run. At pH 6.3, the EV loss is 13.9%, compared to 7.2% at pH 4.1. It should be noted that the perfiltration time required to remove all proteins at pH 6.3 is significantly shorter (2 hours 25 minutes vs. 55 minutes, respectively) compared to pH 4.1. pH 6.3 is considered superior to pH 4.1 due to this shorter time. Detailed Implementation

[0027] In a first aspect, the present invention relates to a method for obtaining a product enriched with extracellular vesicles (EVs), the method comprising the steps of: i. Obtain a liquid containing EV; ii. subject the liquid containing EV to ultrafiltration (UF) to provide EV-enriched UF effluent (UFR) and UF permeate (UFP). The number of EVs in the product is increased compared to the total protein level, and the step of chelating divalent cations with EDTA is not used in the preparation of the EV-containing liquid, preferably wherein EDTA is not used in the preparation of the EV-containing liquid. For the avoidance of doubt, the UFP obtained in step ii. depletes the EV compared to the EV-containing liquid. Preferably, the UF is performed in percolation (DF) mode. The exclusion of EDTA in the method according to the invention makes the EV product obtained in this method suitable for obtaining EDTA-free EVs, which is desirable when it is desired that the product be EDTA-free (e.g., to maintain a dose below the recommended daily dose defined by the FAO or the European Food Safety Authority (EFSA) or other regulatory bodies).

[0028] In the ultrafiltration (UF) step, the liquid containing EV is concentrated. Among other things, ultrafiltration results in the removal of water, lactose, and minerals. The UF residue (UFR) enriches the EV. In one embodiment, UF filtration is stopped when the volume of the UFR is less than 30% of the starting volume (i.e., the volume of the EV-containing liquid that has undergone UF), preferably when it is less than 22% of the starting volume, more preferably less than 20%. Most preferably, UF filtration is stopped when the volume of the UFR is less than 17.5% of the starting volume.

[0029] In one embodiment, the UF step is tangential flow filtration because it increases the yield of EVs.

[0030] In a preferred embodiment, the method of the present invention does not involve carbon dioxide treatment. In another embodiment, the method of the present invention includes only one ultrafiltration step. Preferably, the method of the present invention includes only one ultrafiltration step and does not involve carbon dioxide treatment, as this avoids unnecessary method complexity and cost.

[0031] To obtain a higher purity of the enriched EV product, an additional filtration step can be added to the method of the present invention. Preferably, the filtration step is a size exclusion chromatography (SEC) step. Such higher purity may be required when the enriched EV product is used in pharmaceutical applications or in food products for subjects with weakened or underdeveloped immune systems. In one embodiment, the additional filtration step is to use an agarose gel filtration matrix. Preferably, a dextran fraction range of 50,000-30,000,000 Da is used, more preferably 100,000-20,000,000 Da. In one embodiment, the method of the present invention includes an additional step iii. performed after step ii., wherein step iii. is the filtration step, preferably a size exclusion chromatography (SEC) step, more preferably an SEC step in a simulated moving bed (SMB) chromatography setting. The combination of ultrafiltration and SEC has not previously been used to prepare emulsion fractions enriched with mEV. As illustrated in the example, this combination of steps produces a composition comprising EV and protein, wherein the amount of protein is less than 4.0 fg / EV, preferably less than 3.0 fg / EV, and more preferably less than 2.0 fg / EV.

[0032] Simulated moving bed (SMB) chromatography originated in the petrochemical and mineral industries. Today, the pharmaceutical industry uses SMB chromatography to separate enantiomers from racemic mixtures. SMB chromatography has been used to separate monosaccharides from fructose-glucose solutions and for large-scale separation of disaccharides like sucrose from beet or cane syrup. SMB chromatography has also been used to purify trisaccharides (like 2'-fucosylated lactose) from fermentation, biocatalysis, or chemical synthesis (e.g., in EP 2857410 A1).

[0033] Simulated moving bed (SMB) chromatography was developed as a continuous separation process similar to continuous chemical separation processes such as distillation. In distillation, a countercurrent is established between the liquid and gas phases, which allows for continuous application of the feed and the extraction of one or more products. Furthermore, in theory, countercurrent chromatography should achieve separation superior to conventional cross-current operations. However, countercurrent chromatographic operations require the mobile and stationary phases to move in opposite directions. Therefore, SMB chromatography was developed as a practical solution to the difficulties associated with the concept of moving solid chromatographic materials in continuous chromatographic separation processes.

[0034] The classic SMB concept involves four distinct zones and four external application streams: a feed stream containing the components to be separated, a desorbent or mobile phase stream, an extractant stream, and a raffinate stream (where the raffinate stream represents one or more components with lower retention). These streams divide the SMB system into four distinct zones (each zone or section may contain one or more columns) with the following objectives: Zone I is required for solid-phase regeneration; Zone II is for the desorption of materials with lower desorption strength; Zone III is for the adsorption of strongly adsorbed materials; and finally, Zone IV is for the adsorption of materials with lower adsorption capacity. Therefore, the more strongly adsorbed components establish a concentration wave in Zone II and are transported to the extractant port, while the weaker adsorbed components migrate towards the raffinate port.

[0035] In principle, zones I and IV (regeneration zones) are used for solid phase regeneration, while zones II and III (separation zones) can be considered as the actual separation zones of the system. In addition to the four liquid flows and the resulting zones, the system also contains a recirculation pump (for closed-loop operation) for the mobile phase (desorbent), which forces the mobile phase through a fixed zone in one direction. Countercurrent flow is then achieved through the periodic shifting and continuous supply or removal of feed, desorbent, and product from one column to the next in the system. Besides the classic closed-loop 4-zone SMB system, an open-loop 3-zone system can also be used. The 3-zone open-loop system is economical when fresh solvents are relatively inexpensive (e.g., when using water or water / ethanol as the mobile phase). By using a 3-zone open-loop configuration, liquid phase regeneration is no longer required, thus making zone IV unnecessary.

[0036] In addition to the classic SMB system for separating two-component mixtures, eight-zone closed-loop or five-zone open-loop SMB systems have been developed for separating more than two components. Due to the continuous operation mode and the possibility of using relatively large column sizes and recirculating mobile phases, SMB systems can, in principle, be scaled up to production volumes of hundreds of tons. Technicians will be able to easily calculate SMB conditions based on the results of gel filtration experiments.

[0037] In another embodiment, the liquid comprising EV in the method of the present invention is a milk fraction, preferably a skimmed milk fraction.

[0038] In another embodiment of the method of the invention, the liquid containing EV is whey. Such whey can be obtained from any kind of milk (such as, for example, cow's milk). "Cow" refers to an animal in a herd and includes antelope, sheep, goats, domestic cattle, buffalo, and bison, and preferably refers to a domesticated herd (including sheep, goats, domestic cattle, and buffalo). Alternatively, the liquid containing EV may be obtained from the milk of cows, goats, sheep, camels, buffalo, or horses; cow's milk whey (such as yogurt whey or cheese whey) is preferred. In yet another embodiment, the whey is cheese whey, preferably cow's milk cheese whey, with cow's milk cheese whey being the most preferred.

[0039] In another embodiment of the method of the present invention, the UF is performed at a transmembrane pressure (TMP) between 10 and 600 kPa, preferably between 50 and 200 kPa, more preferably between 70 and 150 kPa, and most preferably between 80 and 140 kPa. TMP can also be expressed in bar or atmosphere (atm), using a conversion of one bar equal to 100 kPa and 0.99 atm.

[0040] In one embodiment, the dry matter content of the liquid containing EV in the method of the present invention is between 0.1% and 15%, preferably between 1.0% and 15%, more preferably between 2.0% and 10%, particularly preferably between 3.0% and 9.0%, and most preferably between 5.0% and 8.0%. Such a desired dry matter content can be obtained by diluting the EV-containing product with water or preferably an aqueous solution of milk minerals, more preferably by diluting the EV-containing product with a simulated milk ultrafiltrate (SMUF) (Jenness, R. and Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk and Dairy Journal, 16(3), 153–164). The use of a milk mineral solution or SMUF is particularly preferred when the EV-containing liquid is a milk fraction (such as whey protein fraction). Not wanting to be bound by any theory, it is believed that such a milk mineral solution or SMUF helps to keep EVs in their natural state.

[0041] In another embodiment of the method of the present invention, the protein content of the liquid containing EV is between 5 and 50 g / L, preferably between 10 and 40 g / L, more preferably between 15 and 35 g / L, particularly preferably between 19 and 29 g / L, and most preferably between 21 and 27 g / L.

[0042] In another embodiment of the method of the invention, the pH of the liquid containing EVs is higher than 5.0, preferably higher than 5.2. Such a pH of 5.0 or 5.2 or higher is preferred because the isoelectric points of α-lactalbumin and β-lactoglobulin are near these pH values. At lower pH values, these proteins will become neutral or positively charged, which causes them to aggregate, resulting in poor separation. The preferred maximum pH of the liquid containing EVs is pH = 8.0. Preferably, the pH of the liquid containing EVs is between 5.2 and 8.0, more preferably between 6.0 and 7.5, and most preferably between 6.0 and 6.6.

[0043] In a preferred embodiment, the UF step in the method of the present invention is performed using a polymer membrane, preferably a polymer spiral wound membrane. Particularly preferred are polyvinylidene fluoride (PVDF) membranes, especially spiral wound PVDF membranes. Alternatively, hydrophilic polyethersulfone (PES) membranes may be used.

[0044] Ultrafiltration is preferably performed using a 1-20 kDa membrane, more preferably a 5-10 kDa membrane. In one embodiment, the UF step is performed using a polymer membrane with a cutoff size between 600 kD and 1000 kD, preferably between 700 kD and 900 kD, and more preferably between 750 kD and 850 kD.

[0045] When using a polymer membrane, the ultrafiltration temperature is preferably in the range of 5°C-30°C, more preferably 10°C-20°C, more preferably 13°C-17°C, and most preferably 16°C-17°C.

[0046] Alternatively, in another embodiment of the method of the present invention, the UF step is performed using a ceramic membrane. Preferably, the cutoff size of the ceramic membrane is between 0.15 and 0.25 micrometers, more preferably between 0.19 and 0.21 micrometers. When using a ceramic membrane, the ultrafiltration temperature is preferably between 30°C and 80°C, preferably between 40°C and 70°C, more preferably between 50°C and 60°C.

[0047] In yet another embodiment, the amount of protein in the EV-enriched product obtained by the method of the present invention is less than 4.0 fg / EV, preferably less than 3.0 fg / EV, and more preferably less than 2.0 fg / EV.

[0048] In one aspect, the present invention relates to a composition comprising extracellular vesicles (EVs) and proteins, wherein the amount of protein is less than 4.0 fg / EV, preferably less than 3.0 fg / EV, and more preferably less than 2.0 fg / EV.

[0049] In one aspect, the present invention relates to products enriched in extracellular fluids, as obtained by the method of the present invention.

[0050] On the other hand, the EV-enriched product obtained by the method of the present invention can be used in food products or pharmaceuticals. Preferably, it is used in synthetic food products.

[0051] In another aspect, the present invention relates to a synthetic food product or medicine comprising a product enriched with EVs obtained by the methods of the present invention. Such a product may be used to enhance muscle performance in a subject and / or to increase muscle growth in a subject, for example, as described in European application EP 23165184.5 (which is incorporated herein by reference).

[0052] As used herein, a “synthetic composition” is an artificially prepared composition containing at least one compound produced by in vitro chemical and / or biological and / or physical means (e.g., by chemical reaction, enzymatic reaction, or by fractionation process). An example of such a fractionation process is the separation of milk into different fractions (e.g., fat and protein fractions). For the avoidance of doubt, the synthetic composition is not manufactured in a human or animal body.

[0053] Products enriched with mEVs can be pasteurized to provide storage stability. For example, products enriched with mEVs can be heated (e.g., at about 70°C for about 15 seconds) to ensure microbial stability in order to obtain pasteurized fractions. Other pasteurization conditions will be apparent to those skilled in the art and can be employed.

[0054] With or without pasteurization, products enriched with mEVs can be used as is or undergo additional processing steps to provide the desired physical form.

[0055] In a particularly preferred embodiment, mEV is an exosome.

[0056] Extracellular vesicles may rupture during their separation and / or enrichment. Therefore, in one embodiment, the mEV in the synthetic composition of the present invention comprises whole bovine mEV, preferably wherein the bovine mEV is derived from a whey-containing bovine milk fraction.

[0057] Transmission electron microscopy (TEM) can be used to assess the presence of mEVs in products enriched with mEVs. TEM is a technique that can be used for the direct visualization of nanostructures such as mEVs. Uranyl acetate can be used as a negative dye to study the effect of heat treatments (such as pasteurization, evaporation, spray drying, and freeze drying) on ​​the mEV structure of mEVs in products. In short, uranyl acetate acts as a negative dye that stains the background and leaves intact vesicle structures (such as intact extracellular vesicles) unstained and highly visible (as shown in WO 2022146743).

[0058] In one embodiment, the mEV-enriched product of the present invention comprises at least 0.001 wt% mEV, as determined relative to the dry weight of the product. In another specific embodiment, the mEV-enriched product comprises at least about 0.001 wt%, 0.01 wt%, 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt% mEV, as determined relative to the dry weight of the product. In yet another embodiment, the mEV-enriched product comprises at least about 10 8 Products enriched with mEVs per gram, such as those measured via nanoparticle tracking, are described. In short, nanoparticle tracking analysis (NTA) can be used to determine the diameter and concentration of mEVs. The principle of NTA is based on the characteristic motion of nanoscale particles in solution according to Brownian motion. The trajectory of the particles within a defined volume is recorded by a camera that captures scattered light when the particles are illuminated with a laser. The size of each tracked particle is determined using the Stokes-Einstein equations. In addition to particle size, this technique also allows for the determination of particle concentration.

[0059] In one embodiment, the mEV-enriched product of the present invention contains at least 0.1 wt% mEV, as determined relative to the dry weight of the product.

[0060] In another specific embodiment, the mEV-enriched product of the present invention comprises about 10 8 To about 10 14 A product enriched with mEV per gram. In yet another more specific embodiment, the product enriched with mEV comprises approximately 10 mEV / gram. 9 To about 10 13 The product enriched with mEV per gram. In another specific embodiment, the mEV-enriched product contains at least about three times the number of mEVs compared to a milk fraction containing raw whey. In another specific embodiment, the mEV-enriched product contains 3 to 50 times the number of mEVs compared to a milk fraction containing raw whey (e.g., cheese whey or yogurt whey).

[0061] In yet another embodiment of the invention, more than 90% of the Newton mEV has a diameter of about 10 nanometers to about 250 nanometers.

[0062] In yet another embodiment, at least 50 wt% of the mEV is complete, preferably wherein at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the mEV is complete. Preferably, the level of complete mEV is determined relative to the level of mEV in the source material (e.g., emulsion fraction) used to prepare the composition of the present invention.

[0063] In another embodiment, the product enriched with mEV is used for oral administration, preferably wherein the composition is a powder, liquid, or stick.

[0064] The compositions of the present invention may further comprise one or more components selected from the group consisting of protein fractions, carbohydrate fractions, and fat fractions. Optionally, the compositions comprise one or more nutrients selected from the group consisting of vitamins and minerals.

[0065] The product enriched with mEV can be the sole protein source in the nutritional composition of the present invention. However, the nutritional composition may include other protein sources, namely protein fractions. In one embodiment, the protein fractions include whole egg powder, egg yolk powder, egg white powder, whey protein, whey protein concentrate, whey protein isolate, whey protein hydrolysate, acidic casein, casein isolate, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, nonfat milk powder, skimmed condensed milk, whole cow's milk, partially or completely skimmed milk, coconut milk, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein... Protein isolates, pea protein hydrolysates, rice protein concentrates, rice protein isolates, rice protein hydrolysates, broad bean protein concentrates, broad bean protein isolates, broad bean protein hydrolysates, collagen, collagen isolates, meat protein, potato protein, chickpea protein, low-erucic acid canola protein, mung bean protein, quinoa protein, amaranth protein, chia seed protein, flaxseed protein, earthworm protein, insect protein, one or more amino acids and / or their metabolites, or combinations of two or more thereof.

[0066] The amino acid can be described as one of the free amino acids, or a mixture of such amino acids, and can be any amino acid known for use in nutritional products. The amino acid can be naturally occurring or synthetic. In a particular embodiment, one or more amino acids and / or their metabolites comprise one or more branched-chain amino acids or their metabolites. Examples of branched-chain amino acids include arginine, glutamine, leucine, isoleucine, and valine. In another particular embodiment, one or more branched-chain amino acids or their metabolites comprise α-hydroxyisocaproic acid (HICA, also known as squalane), ketoisocaproic acid (KIC), β-hydroxy-β-methylbutyric acid (HMB), and combinations of two or more thereof.

[0067] The nutritional composition may contain a protein fraction in an amount of about 1 wt% to about 50 wt% (e.g., about 1 wt% to about 30 wt%) of the nutritional composition. More specifically, the protein may be present in an amount of about 1 wt% to about 25 wt% of the nutritional composition (including about 1 wt% to about 20 wt%, about 2 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 10 wt%, about 10 wt% to about 25 wt%, or about 10 wt% to about 20 wt%). Even more specifically, the protein may comprise about 1 wt% to about 5 wt% of the nutritional composition, or about 20 wt% to about 30 wt% of the nutritional composition. Alternatively, in yet another embodiment, the nutritional product is a high-protein product that contains protein fractions in an amount of about 20 wt% to about 90 wt%, preferably 30 wt% to 80 wt%, more preferably 35 wt% to 75 wt%.

[0068] As used herein, carbohydrate fractions may comprise one or more selected from the group consisting of maltodextrin, starch, dextrose, dextrin, lactose, galactooligosaccharides, fructooligosaccharides, human milk oligosaccharides (HMOs), and galactomannan. Examples of starches that may be used include hydrolyzed starch, modified starch, corn starch, and hydrolyzed corn starch.

[0069] The nutritional composition may contain carbohydrates in an amount of about 5 wt% to about 75 wt% of the nutritional composition. More specifically, carbohydrates may be present in an amount of about 5 wt% to about 70 wt% of the nutritional composition (including about 5 wt% to about 65 wt%, about 5 wt% to about 50 wt%, about 5 wt% to about 40 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 10 wt% to about 65 wt%, about 20 wt% to about 65 wt%, about 30 wt% to about 65 wt%, about 40 wt% to about 65 wt%, about 40 wt% to about 70 wt%, or about 15 wt% to about 25 wt%).

[0070] Fat fractions may include milk fat, butter, anhydrous milk fat, algae oil, low-erucic acid rapeseed oil, flaxseed oil, borage oil, safflower oil, high-oleic safflower oil, high-gamma-linolenic acid (GLA) safflower oil, corn oil, soybean oil, sunflower seed oil, high-oleic sunflower seed oil, cottonseed oil, coconut oil, fractionated coconut oil, medium-chain triglyceride (MCT) oil, palm oil, palm kernel oil, palm oil extract, long-chain polyunsaturated fatty acids, or combinations of two or more thereof.

[0071] The nutritional composition may contain fat in an amount of about 0.5 wt% to about 30 wt% of the nutritional composition. More specifically, fat may be present in an amount of about 0.5 wt% to about 10 wt%, or about 1 wt% to about 30 wt% (including about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 5 wt%, about 3 wt% to about 30 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 10 wt%, or about 10 wt% to about 20 wt%) of the nutritional composition.

[0072] In one embodiment, the nutritional composition is a liquid nutritional composition and, based on the weight of the nutritional composition, comprises about 1 wt% to about 15 wt% of protein, about 0.5 wt% to about 10 wt% of fat, and about 5 wt% to about 30 wt% of carbohydrates.

[0073] In another embodiment, the nutritional composition is a powdered nutritional composition and, based on the weight of the nutritional composition, contains about 10 wt% to about 30 wt% of protein, about 5 wt% to about 15 wt% of fat, and about 30 wt% to about 65 wt% of carbohydrates.

[0074] In a particular embodiment, the nutritional composition comprises at least one protein containing milk protein concentrate and / or soy protein isolate, at least one fat containing milk fat, low-erucic acid rapeseed oil, corn oil, coconut oil and / or marine oil, and at least one carbohydrate containing maltodextrin, sucrose, lactose, galactooligosaccharides and / or fructooligosaccharides.

[0075] The nutritional composition may also contain one or more components that modify the physical, chemical, aesthetic, or processing characteristics of the nutritional composition, or that act as additional nutritional components. Non-limiting examples of additional components include preservatives, emulsifiers (e.g., lecithin), buffers, sweeteners including artificial sweeteners (e.g., saccharin, aspartame, acesulfame potassium, sucralose), colorants, flavoring agents, thickeners, stabilizers, etc.

[0076] In certain embodiments, the nutrient composition has a neutral pH, i.e., about 6 to 8, or more specifically about 6 to 7.5. In more specific embodiments, the nutrient composition has a pH of about 6.5 to 7.2, or more specifically about 6.8 to 7.1.

[0077] Nutritional compositions can be formed using any technique known in the art. In one embodiment, a nutritional composition can be formed by: (a) preparing an aqueous solution comprising proteins and carbohydrates; (b) preparing an oil blend comprising fats and oil-soluble components; and (c) mixing the aqueous solution and the oil blend together to form an emulsified liquid nutritional composition. The complete mEV can be added at any time desired during the process, e.g., to the aqueous solution or the emulsified blend. The complete mEV can be dry-blended with one or more dry ingredients in powder form, for example, for combination addition to a liquid composition, or if a powdered nutritional product is desired.

[0078] In a particular embodiment, the nutritional composition is applied in powder form. In another particular embodiment, the nutritional composition is applied in liquid form. The nutritional composition may be administered to the subject in either form.

[0079] When the nutritional composition is in powder form, for example, a portion of about 40 g to about 60 g (such as 45 g or 48.6 g or 50 g) will be applied as a powder or reconstituted in about 1 ml to about 500 ml of liquid.

[0080] When the nutritional composition is in liquid form (e.g., reconstituted from powder or formulated into a ready-to-drink product), a portion ranges from about 1 ml to about 500 ml, including about 110 ml to about 500 ml, about 110 ml to about 417 ml, about 120 ml to about 500 ml, about 120 ml to about 417 ml, about 177 ml to about 417 ml, about 207 ml to about 296 ml, about 230 ml to about 245 ml, about 110 ml to about 237 ml, about 120 ml to about 245 ml, about 110 ml to about 150 ml, and about 120 ml to about 150 ml. In specific embodiments, the portion is about 1 ml, or about 100 ml, or about 225 ml, or about 237 ml, or about 500 ml.

[0081] In certain embodiments, the nutritional composition comprising bovine mEV is administered to the subject once or more daily or weekly. In certain embodiments, the nutritional composition is administered to the subject about 1 to 6 times daily or weekly, or about 1 to 5 times daily or weekly, or about 1 to 4 times daily or weekly, or about 1 to 3 times daily or weekly. In certain embodiments, the nutritional composition is administered once or twice daily for a period of at least one week, at least two weeks, at least three weeks, or at least four weeks.

[0082] The concentrations and relative amounts of protein, carbohydrate, and fat fractions in the nutritional composition can vary significantly depending on, for example, the specific dietary needs of the intended user. In a particular embodiment, based on the weight of the nutritional composition, the composition comprises about 2 wt% to about 20 wt% of a protein source, about 5 wt% to about 30 wt% of a carbohydrate source, and about 0.5 wt% to about 10 wt% of a fat source; more specifically, such a composition is in liquid form. In another particular embodiment, based on the weight of the nutritional composition, the composition comprises about 10 wt% to about 25 wt% of a protein source, about 40 wt% to about 70 wt% of a carbohydrate source, and about 5 wt% to about 20 wt% of a fat source; more specifically, such a composition is in powder form.

[0083] In one aspect, the present invention relates to the synthetic nutritional compositions of the present invention for enhancing muscle performance in subjects and / or for increasing muscle growth in subjects, preferably for subjects who require improvement in physical performance.

[0084] In another aspect, the present invention relates to the use of the compositions of the invention for enhancing the muscle performance of a subject and / or increasing muscle growth in a subject, preferably for a subject who requires improvement in physical performance.

[0085] In another aspect, the present invention relates to the use of the compositions of the present invention in the manufacture of medicaments for enhancing muscle performance and / or increasing muscle growth.

[0086] In one embodiment, the daily dose of the composition used for purposes of the present invention is between 0.01 and 30 g mEV per day, preferably between 0.1 and 20 g mEV per day. More preferably, the daily dose is between 0.01 and 30 g mEV per day and between 0.01 and 30 g galactose per day, and even more preferably, the daily dose is between 0.1 and 20 g mEV per day and between 1.0 and 25 g galactose per day.

[0087] It should be noted that, as used in the specification and appended claims, unless the context clearly indicates otherwise, the singular forms “a” and “the” include a plural of indicators. For example, a component referred to in the singular is intended to comprise multiple components.

[0088] It will be understood that, unless otherwise defined, any reference to weight, weight ratio, etc. in this disclosure refers to dry matter, and in particular, the dry matter of the composition.

[0089] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0090] With regard to the term "includes" or "including" as used in the specification or claims, it is intended to include additional elements or steps in a manner similar to the interpretation of the term "comprising" as it is used as a transitional word in the claims.

[0091] As used herein, the term “comprising” is synonymous with “including” or “containing”, is open-ended, and does not exclude any additional undescribed elements, components, or method steps; while the term “consisting of” is a closed term and does not include any additional elements, steps, or components not expressly described.

[0092] Furthermore, the use of the term "or" (e.g., A or B) is intended to mean "A or B or both". The term "A or B only, not both" is used when "only A or B, not both" is expected. Therefore, the use of the term "or" in this document is inclusive, not exclusive. When the terms "and" and "or" are used together, as in "A and / or B", this means A or B as well as A and B.

[0093] Throughout this application, where publications are cited, the disclosures of those publications are incorporated herein by reference in their entirety to more fully describe the level of technology to which this invention pertains.

[0094] Unless otherwise expressly indicated by example or otherwise, all numerical quantities indicating amounts of material or reaction and / or conditions of use in this specification shall be understood to be modified by the word “about” when describing the broadest scope of the invention. It is generally preferred to practice within the stated numerical limits. Furthermore, unless explicitly stated otherwise: percentages, “parts,” and ratios are by weight; descriptions of a group or class of materials suitable for or preferably used for a given purpose relating to the invention mean that mixtures of any two or more members of that group or class are equally suitable or preferred; components described in chemical terms refer to components added to any combination specified in the specification and do not necessarily exclude chemical interactions between components of the mixture once mixed; the first definition of acronyms or other abbreviations applies to all subsequent uses of the same abbreviation herein, with necessary minor adjustments to the normal grammatical variations applicable to the initially defined abbreviation; and, unless explicitly stated otherwise, the measurement of a property is determined by the same technique as the same property previously or later referenced.

[0095] It should also be understood that the present invention is not limited to the specific embodiments and methods described herein, as specific components and / or conditions can certainly vary. Furthermore, the terminology used herein is for the purpose of describing specific embodiments of the invention only and is not intended to be limiting in any way.

[0096] The invention is described below with reference to the following non-limiting examples.

[0097] Example

[0098] While the invention has been described by way of examples, and while the examples have been described in considerable detail, such description is not intended to limit the scope of the appended claims or in any way to such details. Further advantages and modifications will readily become apparent to those skilled in the art. Therefore, the invention is not limited in its broader aspects to the specific details shown and described, representative compositions and methods, or illustrative examples. Thus, deviations from such details may be made without departing from the spirit or scope of the overall inventive concept.

[0099] The goal of these filtration experiments is to obtain as much mEV as possible from the permeate.

[0100] Retention rate (R) and selectivity (S) will be used to determine the effectiveness of the settings used during filtering.

[0101]

[0102] Cp = Concentration in the permeate

[0103] Cr = Concentration in the residue

[0104] Selective display of the effectiveness of EV and protein separation.

[0105]

[0106] R EV = EV retention rate

[0107] R 蛋白质 = Protein retention rate

[0108] In addition to retention and selectivity, flux is another parameter used. It indicates when equilibrium is reached in the system and how the membrane responds to changes that occur during the experiment.

[0109] The permeate flux is calculated using Equation 3.

[0110]

[0111] Permeate flux (l / h / m) 2 )

[0112] L = Collection volume (liters)

[0113] t = Collection time (h)

[0114] A = Area of ​​the membrane (m²) 2 )

[0115] Example 1A: Comparison of mEV selectivity under different membranes and UF conditions

[0116] In this example, four different membranes were tested under different conditions.

[0117] The membrane being tested

[0118] Starting materials

[0119] All experiments were conducted using 80% acidic whey protein concentrate (HCW80) from FrieslandCampina. First, HCW80 was diluted with Milli-Q water to a dry matter content of 6.5% ± 0.2% and a protein concentration of 24.0 g / L ± 1.0 g / L (p = 0.05).

[0120] Adjust the pH to the desired level using a 10% sulfuric acid aqueous solution.

[0121] Test conditions ceramic membrane

[0122] pH 5.2

[0123] Crossflow 6 m / s

[0124] 55°C

[0125] Transmembrane pressures: 0.5, 1.0, 1.5 and 2.0 bar.

[0126] Test conditions polymer membrane

[0127] pH 5.2

[0128] Crossflow 0.25 m / s

[0129] 15°C

[0130] Transmembrane pressures of 0.5, 1.0, 1.5 and 2.0 bar.

[0131] In each experiment, transmembrane pressure (TMP) was applied in cyclic mode for 45 minutes to reach equilibrium. After 45 minutes, samples of residual material and permeate were removed.

[0132] result

[0133] Ceramic membrane

[0134] For all tested TMP levels, except at 2.0 bar, the 0.2-micron ceramic membrane exhibited better selectivity compared to the 0.1-micron membrane. At TMP levels of 0.5 and 1.0 bar, the 0.2-micron membrane showed approximately twice the selectivity of the 0.1-micron membrane. The 0.2-micron membrane had a selectivity of 3.76 at 1.5 bar, while the 0.1-micron membrane had a selectivity of 1.29 at the same pressure. At 2.0 bar TMP, both membranes showed equal selectivity of 1.35. Figure 1 As shown.

[0135] polymer film

[0136] For all tested TMP values, the 500 kD membrane exhibited approximately the same selectivity (around 1.0), therefore this membrane could not separate mEVs. Conversely, the 800 kD membrane showed increasing selectivity with increasing TMP, with a maximum value of 1.54. Figure 2 As shown.

[0137] Ceramic 0.2-micron membranes and polymer 800 kD membranes were chosen to continue because they offer higher selectivity and protein throughput.

[0138] Example 1B: Comparing mEV selectivity under UF conditions

[0139] The effects of pH, TMP, and crossflow on selectivity were tested.

[0140] Ceramic 0.2 microns, pH and TMP

[0141] like Figure 3 As shown, the highest selectivity is obtained at a pH of 5.2. Without being bound by theory, it is assumed that whey protein aggregates form at lower pH levels. Specifically, at a pH of 5.2, TMP between 1.5 and 1.75 bar is preferred because the highest selectivity (> 4.0) is obtained at that point.

[0142] Polymer 800 kD, pH and TMP

[0143] When using a polymer 800 kD membrane, such as Figure 4 As shown, pH has a significant impact on selectivity. When combined with 1.0 bar TMP, selectivity exceeds 16 at pH 6.3, while at higher TMP levels, selectivity is much lower. Without being bound by theory, it is assumed that whey protein aggregates form at lower pH levels, leading to reduced flux and increased protein retention.

[0144] Polymer 800 kD – Crossflow velocity

[0145] When using a polymer 800 kD membrane, approximately 80% reduction in selectivity was achieved at all pH levels when the crossflow velocity was reduced from 1 m / s to 0.25 m / s.

[0146] Example 1C: Comparison of mEV selectivity – Percolation

[0147] This setting shows the effect of UF in percolation mode on selectivity.

[0148] During percolation, simulated milk ultrafiltrate (SMUF) (Jenness, R. and Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk and Dairy Journal, 16(3), 153–164) was used as the percolation medium.

[0149] Percolation settings: pH 6.3, crossflow 1 m / s, temperature 15°C, TMP 1.0 bar.

[0150] As previous experiments have shown, the highest selectivity was obtained with an 800 kD polymer membrane. The membrane was also tested at different pH levels in percolation mode (DF) at a crossflow rate of 1 m / s. Percolation models were constructed at pH 6.3 and pH 4.1. This model was used to determine how long each percolation run must be to separate all protein material from the EV and how much EV will be lost. This was in... Figure 5 As shown in the image.

[0151] At pH 6.3, EV loss was 13.9%, while at pH 4.1, EV loss would be 7.2%. However, at pH 6.3, all proteins would separate from the EV fraction after 55 minutes, while at pH 4.1, this would take 2 hours and 25 minutes.

[0152] Based on these results, a pH of 6.3 is preferred for the UF step when used in DF mode.

[0153] Example 2: Pilot Plant – Large Scale

[0154] The experiment was conducted in a pilot plant using a polymer 800 kD membrane.

[0155] Starting material: Whey acid (HCW80) (FrieslandCampina).

[0156] Target

[0157] The goal of this experiment is to enrich whey fractions containing extracellular vesicles (EVs) by using cross-flow filtration. Using this technique, proteins permeate through the filter, while the EVs remain in the residue.

[0158] Materials and Methods

[0159] Dilute HCW80 (FrieslandCampina, 31.45 L) with water to a dry matter content of 6.5% (150 L). Set the pH to 6.3 using HCl.

[0160] membrane

[0161] Polymer 800 kD spiral wound type (800 kD spiral wound type)

[0162] method

[0163] Considering the pressure limit, use the highest possible crossflow.

[0164] Transmembrane pressure: 1 bar

[0165] Temperature: 15°C

[0166] Start filtration in circulation mode with 150 liters of diluted HCW80 to establish equilibrium. After 45 minutes, filtration begins.

[0167] Collect all permeate fractions in 10-liter increments. During the collection of each fraction, remove a sample from each fraction along with a sample of the residual permeate.

[0168] The experiment was terminated when the osmotic residue was concentrated to 25 liters. This 25 liters was stored as a fraction containing EV. The EV purity of this fraction is given in Example 3. The protein concentration in the osmotic residue was 120 mg / ml, thus providing a total protein content of 3.0 kg in 25 liters. The total EV content was 3.3 × 10^14. This is 9.1 fg protein / EV. Other executions of this experiment yielded amounts of 6.37–9.2 fg protein / EV.

[0169] Example 3 Size Exclusion Chromatography

[0170] Gel filtration was used for size-based separation of materials. Sepharose CL-2B (bead diameter 60–200 μm; fractionation range 100,000–20,000,000 (dextran) and 70,000–40,000,000 (globulin), Sigma-Aldrich) was used as the column material, and PBS (50 mM NaH₂PO₄, 0.15 M NaCl) at pH 7.4 was used as the mobile phase. The column volume was 10 ml. EV-containing liquid (500 μL) was subjected to size exclusion chromatography on such a gel filtration column to further separate EVs from residual protein material.

[0171] In methods similar to those described by Webber et al. (Journal of Extracellular Vesicles 2013, 2: 19861 http: / / journalofextracellularvesicles.net / underindex.php / jev / article / view / 19861), nanoparticle tracking analysis is used to determine the purity of EVs by femtogram (fg) protein / EV to determine the number of EVs.

[0172] Using this method, the EV purity (fg protein / EV) of acidified whey (HCW80 before ultrafiltration, the starting material of Example 2), the UF-filtered whey residue (UFR) obtained as in Example 2, and the UFR subsequently subjected to SEC (UFR-SEC) was determined. The results are shown below:

[0173] Higher EV purity, such as a low protein / EV ratio, is desirable because it allows EV administration without affecting overall protein levels. Furthermore, it allows the use of EVs in applications where low protein levels are desired, such as in medical applications.

Claims

1. A method for obtaining a product enriched with extracellular vesicles (EVs), the method comprising the following steps: i. Obtain a liquid containing EV; ii. subject the liquid containing EV to ultrafiltration (UF) to provide EV-enriched UF effluent (UFR) and UF permeate (UFP), preferably wherein the UF is performed in percolation (DF) mode; The enrichment is defined as an increase in the number of EVs in the UFR compared to the total protein level, compared to the liquid containing EVs. The preparation of the EV-containing liquid does not involve the step of chelating divalent cations with EDTA, and preferably, EDTA is not used in the preparation of the EV-containing liquid; and The protein content in the enriched EV product is less than 4.0 fg / EV, preferably less than 3.0 fg / EV, and more preferably less than 2.0 fg / EV.

2. The method of claim 1, further comprising a step iii. performed after step ii., wherein step iii. is a filtration step, preferably a size exclusion chromatography (SEC) step, more preferably a simulated moving bed (SMB) chromatography setting, of the SEC step.

3. The method of claim 1 or 2, wherein the liquid containing EV is a milk fraction, preferably a skimmed milk fraction.

4. The method as described in any of the preceding claims, wherein the liquid containing EV is whey, preferably bovine whey.

5. The method as described in any of the preceding claims, wherein the UF is performed at a transmembrane pressure between 50 and 200 kPa, more preferably between 70 and 150 kPa.

6. The method as described in any of the preceding claims, wherein the dry matter content of the liquid containing EV is between 0.1% and 15%, preferably between 1.0% and 15%, more preferably between 2.0% and 10%, particularly preferably between 3.0% and 9.0%, and most preferably between 5.0% and 8.0%.

7. The method as claimed in any of the preceding claims, wherein the pH of the liquid containing EV is higher than 5.2, preferably between 5.2 and 8.0, more preferably between 6.0 and 7.5, and most preferably between 6.0 and 6.

6.

8. The method as described in any of the preceding claims, wherein the UF step is performed using a polymer film, preferably a polymer spiral wound film.

9. The method of claim 8, wherein the cutoff size of the polymer membrane is between 600 kD and 1000 kD.

10. The method of any one of claims 1 to 7, wherein the UF step is performed using a ceramic membrane; preferably wherein the cut-off size of the ceramic membrane is between 0.15 and 0.25 micrometers, more preferably between 0.19 and 0.21 micrometers.

11. The method of any one of claims 1-10, wherein the liquid containing EV is acidic whey.

12. A composition comprising extracellular vesicles (EVs) and proteins, wherein the amount of protein is less than 4.0 fg / EV, preferably less than 3.0 fg / EV, more preferably less than 2.0 fg / EV.

13. Use of the EV-enriched product obtained by the method of any one of claims 1-11 or the composition of claim 12 in a food product or a pharmaceutical product, preferably in a synthetic food product.

14. A product enriched with extracellular vesicles obtained by the method of any one of claims 1-11.

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