Milk extracellular vesicle enrichment process

Abstract

Process for obtaining an extracellular vesicle (EV)-enriched product comprising the steps of i. obtaining an EV-comprising liquid; ii. subjecting the EV-comprising liquid to ultrafiltration (UF) to provide an UF retentate (UFR) enriched in EV and an UF permeate (UFP), wherein the enrichment is an increase in the number of EV as compared to the total protein level in the UFR as compared to in the EV-comprising liquid; wherein no step of chelating divalent cations with EDTA is used in the preparation of the EV-comprising liquid, preferably wherein no EDTA is used to prepare the EV-comprising liquid.

Description

[0001] Milk extracellular vesicle enrichment process

[0002] Field of the invention

[0003] The invention relates to an extracellular vesicle (EV) enrichment process, in particular to a process for obtaining a milk stream enriched in milk- derived extracellular vesicles (mEV), more preferably bovine mEV. The invention further relates to a product comprising such a milk stream enriched in mEV and to the use of such a milk stream enriched in mEV.

[0004] Background of the invention

[0005] US 10,729,159 (US 2019 / 0150474) relates to exosome purification methods involving the use of a whey composition as an exosome source. Exosomes are isolated by subjecting the whey composition to a first ultrafiltration, yielding a first permeate and a first retentate. The first retentate may then be subjected to a second ultrafiltration, yielding a second permeate and a second retentate. During the second ultrafiltration, the first retentate may be treated with carbon dioxide. The second retentate may then be subjected to a third ultrafiltration, yielding a third permeate and a third retentate. The third permeate may then be optionally dried to yield an exosome powder.

[0006] Moleirinho et al disclose challenges in biotherapeutic particles manufacturing, including up and downstream steps and manufacturing platforms (Moleirinho, M. G., (2019) Current challenges in biotherapeutic particles manufacturing. Expert Opinion on Biological Therapy, 20(5), 451-465. https: / / doi.org / 10.1080 / 14712598.2020.1693541).

[0007] B. J. Benedikter et al disclose a method isolate extracellular vesicles from cell culture media for compositional and functional studies (B.J. Benedikter et al Sci Rep 7, 15297 (2017). https: / / doi.org / 10.1038 / S41598-017-15717-7).

[0008] The term "extracellular vesicle" or "EV" is herein defined as the generic term for lipid bilayer-delimited particles released from the cell and, unlike a cell, EVs cannot replicate. EVs range in diameter from near the size of the smallest physically possible unilamellar liposome (around 20-30 nanometres) to as large as 10 microns or more, although the vast majority of EVs are smaller than 200 nm. They carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell. Most cells that have been studied to date are thought to release EVs, including some bacterial, fungal, and plant cells that are surrounded by cell walls. A wide variety of EV subtypes have been proposed, defined variously by size, biogenesis pathway, cargo, cellular source, and function.

[0009] The term "intact extracellular vesicle" as used herein 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.

[0010] Extracellular vesicles (EV or EVs) comprise exosomes (<100 nm) and microvesicles (100 nm - 10 micron). EVs are present in biological fluids and are involved in multiple physiological and pathological processes. EVs are considered as an additional mechanism for intercellular communication allowing cells to exchange proteins, lipids and the genetic material.

[0011] Many studies provided compelling evidence that EVs are involved in the regulation of the immune response, acting as both enhancers and dampeners of the immune system, depending on the source and type of vesicle. Research has shown anti-inflammatory effects of milk-derived EVs, using human breast milk as well as bovine colostrum and store- bought pasteurized cow milk, in in vitro systems as well as therapeutically in animal models. Strategies to target the gut, and especially its microbiome, are under investigation and hold a promise as a therapeutic intervention for these diseases. The use of milk-derived EVs, either as stand-alone drug or as a drug carrier, is often suggested in recent years. 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 gastrointestinal 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 enrich a milk stream with EVs. 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.

[0012] Research in the field of milk-derived EV (mEV) has grown rapidly in the last few years, however a standard protocol for reproducible, cost- effective mEV (or exosome) isolation has not been established yet. Originally, exosome isolation relied basically on ultracentrifugation-based techniques which is considered the gold standard. Other isolation methods have been developed, however, each method has its own limitations and fails to isolate exclusively exosomes. The exosomes may, for example be contaminated by protein. Improved EV isolation methods may not only have an impact on the amount and purity of recovered EVs but may also result in the isolation of specific EV populations, with different sizes and functional characteristics and carrying certain RNA, protein and lipid profiles. Thus, the challenge remains to develop mass-scalable methods to isolate EVs in a rapid, efficient, reproducible, cost-effective and clinically friendly manner (L. del Pozo-Acebo et al, Int. J. Mol. Sci. 2021, 22, 1105. https: / / doi.org / 10.3390 / ijms22031105).

[0013] Ultracentrifugation has been used for EV isolation but it has several drawbacks, such as the co-isolation of non-exosomal impurities, low reproducibility, potential damage of exosomes and low-throughput of samples. Body fluids contain many nanoparticles (some non-vesicular) in the same size range as exosomes that co-elute with EVs when applying size exclusion chromatography (SEC). A main disadvantage of SEC is the limited quantity of EVs recovered by unit of volume. Hence further improvements to mEV / exosome enrichment processes are desired.

[0014] It is therefore an object of the invention to provide an extracellular vesicle (EV) enrichment process, in particular a process for obtaining a milk stream enriched in milk-derived extracellular vesicles (mEV), more preferably bovine mEV. It is another object of the invention to provide an EV enrichment process that is capable of producing large quantities of mEV, preferably in a process that is scalable to more than 100 litre such as >1000 litre scale and / or that can be run in an continuous mode.

[0015] It is another object of the invention to provide a milk stream enriched in mEV that may be used in a nutritional product such as an infant formula or food supplement. Alternatively, the mEV-enriched milk stream may be used in a nutrition product to enhance muscle performance in a subject in need of improved physical performance such as a sports nutrition product or adult nutrition product.

[0016] The present invention is also directed to providing a method of reducing chronic fatigue in a subject who is recovering or has recovered from a viral infection, comprising administering the extracellular vesicle enriched product comprising intact bovine mEV to the subject.

[0017] It is a further object of the invention to use the mEV-enriched product as obtained in the process of the invention in methods that are advantageous in providing a convenient manner to improve mitochondrial function, and thereby improve muscle performance, in a subject in need of improved physical performance. Such methods are useful in the prevention or treatment of conditions that are hallmarked by a reduction in spare respiratory capacity, including sarcopenia and chronic or acute cardiac damage for example as described in European Application EP23165184.5. These and additional advantages of the inventive methods will be more fully apparent in view of the detailed description.

[0018] SUMMARY OF THE INVENTION

[0019] In a first aspect, the invention relates to a process for obtaining an extracellular vesicle (EV)-enriched product comprising the steps of i. obtaining an EV-comprising liquid; ii. subjecting the EV-comprising liquid to ultrafiltration (UF) to provide an UF retentate (UFR) enriched in EV and an UF permeate (UFP), preferably wherein the UF is performed in diafiltration (DF) mode; wherein the enrichment is an increase in the number of EV as compared to the total protein level in the UFR. as compared to the EV-comprising liquid and wherein no step of chelating divalent cations with EDTA is used in the preparation of the EV-comprising liquid, preferably wherein no EDTA is used to prepare the EV-comprising liquid, as defined in claim 1. As used herein, an increase in the number of EV as compared to the total protein level in the product means a reduction in the amount of protein per EV. For the avoidance of doubt, the UFP as obtained in step ii. is depleted in EV as compared to the EV-comprising liquid.

[0020] In another aspect the invention relates to the use of the EV-enriched product as obtained in the process of the invention in a food product or medicine. The invention also relates to such a food product or medicine comprising said EV-enriched product.

[0021] In yet another aspect, the invention relates to the synthetic food product of the invention for use in increasing muscle growth in a subject.

[0022] In still another aspect, the invention relates to a composition comprising extracellular vesicles (EV) and protein wherein the amount of protein is less than 4.0 femtogram (fg) / EV, preferably less than 3.0 fg / EV, more preferably less than 2.0 fg / EV. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In Figure 1 the separation of milk EVs selectivity of the ceramic membranes is shown at different trans membrane pressures (TMP) of 0.50, 1.0, 1.5 and 2.0 bar. The upper line is for the 0.2 micron filter, the lower for the 0.1 micron filter.

[0024] In Figure 2 the separation of milk EVs selectivity of polymeric membranes is shown at different trans membrane pressures (TMP) of 0.50, 1.0, 1.5 and 2.0 bar. The 500 kD filter had a selectivity of 1.0 at each TMP while the selectivity of the 800 kD filter increased from -0.5 to 1.5 when going from low to high TMP.

[0025] In Figure 3 the selectivity of the ceramic 0.2 micron membrane in the separation of mEVs is plotted as a function of pH and TMP. The best selectivity (5.8) was obtained at a pH of 5.2 and at TMP of 1.66 Bar. The selectivity of the polymeric 800 kD micron membrane in the separation of mEVs is plotted as a function of pH and TMP in Figure 4. The best selectivity (5.8) was obtained at a pH of 6.3 and at TMP of 1.00 Bar. The results of a diafiltration performed with the polymeric 800 kD membrane are shown in Figure 5. A diafiltration model was made at pH 6,3 and 4,1 at a crossflow of 1 m / s. The retentions found in the design of experiment were extrapolated into a linear model. This was done to investigate the loss of EVs during the diafiltration as compared to the loss of proteins. This model is shown in Figure 5. It illustrates how long each diafiltration has to run to get rid of all proteins and the loss of EVs at each time. At pH 6.3 there was 13.9% loss of EVs as compared to 7.2% of EV loss at pH 4.1. It is noted that the diafiltration time needed to remove all the proteins is much shorter at pH 6.3 as compared to pH 4.1 (55 minutes versus 2 hours and 25 minutes, respectively). Due to this shorter time pH 6,3 was considered more preferable as compared to pH 4.1.

[0026] DETAILED DESCRIPTION

[0027] In a first aspect the invention relates to a process for obtaining an extracellular vesicle (EV)-enriched product comprising the steps of i. obtaining an EV-comprising liquid; ii. subjecting the EV-comprising liquid to ultrafiltration (UF) to provide an UF retentate (UFR) enriched in EV and an UF permeate (UFP); an increase in the number of EV as compared to the total protein level in the product and wherein no step of chelating divalent cations with EDTA is used in the preparation of the obtaining the EV-containing liquid, preferably wherein no EDTA is used to prepare the EV-containing liquid. For the avoidance of doubt, the UFP as obtained in step ii. is depleted in EV as compared to the EV-comprising liquid. Preferably, the UF is performed in diafiltration (DF) mode. The exclusion of the use of EDTA in the process according the invention renders the EV product as obtained in the process suitable for obtaining EV without EDTA which is desirable when a product without EDTA is desired e.g. for staying below the recommended daily dosage as defined by FAO or European Food Safety Authority (EFSA), or other regulatory authorities.

[0028] In the Ultrafiltration (UF) step the EV-comprising liquid is concentrated. Ultrafiltration results in among others removal of water, lactose and minerals. The UF retentate (UFR.) is enriched in EV. In one embodiment the 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-comprising liquid subjected to UF), preferably when it is less than 22%, more preferably less than 20% of the starting volume. Most preferably it is stopped when the UFR volume is less than 17.5% of the starting volume.

[0029] In one embodiment, the UF step is a tangential flow filtration as it increases the yield of EV.

[0030] In one preferred embodiment, the process of the invention does not involve carbon dioxide treatment. In another embodiment, the process of the invention comprises only one ultrafiltration step. Preferably, the process of the invention comprises only one ultrafiltration step and does not involve carbon dioxide treatment as this avoids unnecessary process complexity and process costs. In order to obtain higher purities of the EV-enriched product, an additional filtration step may be added to the process of the invention. Preferably, the filtration step is a size exclusion chromatography (SEC) step. Such higher purities may be required when the EV-enriched product is used in pharmaceutical applications or in food products for subjects with a weakened or not fully developed immune system. In one embodiment, the additional filtration step is using an agarose gel filtration base. Preferably using a dextran fractionation range of 50,000 - 30,000,000 Da, more preferably of 100,000 - 20,000,000. In one embodiment, the process of the invention comprises an additional step iii. to be executed after step ii. wherein step iii. is a filtration step, preferably a size exclusion chromatography (SEC) step, more preferably a SEC step in a Simulated Moving Bed (SMB) chromatography set-up. The combination of Ultrafiltration and SEC has not been used before in the preparation of a milk fraction enriched in mEVs. As shown in the example, this combination of steps results in a composition comprising EVs and protein wherein the amount of protein is less than 4.0 femtogram / EV, preferably less than 3.0 fg / EV, more preferably less than 2.0 fg / EV. Simulated moving bed (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' -fucosyllactose - from fermentation, biocatalysis or chemical synthesis (e.g. in EP2857410 Al).

[0031] Simulated moving bed (SMB) chromatography was developed as a continuous separation process analogous to continuous chemical separation processes such as rectification. In rectification, a counter current is established between the liquid and the gaseous phase, which allows then the continuous application of feed and withdrawal of product(s). In addition, counter-current chromatographic operations in theory should achieve separations superior than conventional crosscurrent operations. However, chromatographic counter-current operations would require the mobile and stationary phases to move in opposite directions. Thus, SMB chromatography was developed as a practical solution to the difficulties related to the concept of moving solid chromatography material in a continuous chromatographic separation process.

[0032] 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. Thus, stronger adsorbing components establish a concentration wave in zone II and are transported to the extract port whereas less strong adsorbing components migrate towards the raffinate port.

[0033] In principle, zone I and IV serve for regeneration of the solid phase (regeneration zones) whereas zones II and III can be regarded as the actual separation zones of the system (separation zones). In addition to the four liquid streams and resulting zones, the system contains (for the close loop operation) a recycling pump for the mobile phase (desorbent), passing the mobile phase through the fixed zones in one direction. Counter-current flow is then achieved by the periodical shifting and continuous supply or withdrawal of feed, desorbent, and products sequentially from one column to the next in the system. Besides the classical close loop 4 zones SMB system, open loop 3 zones systems can be used as well. The 3 zones open loop systems are economic in case fresh solvent is rather inexpensive e.g. in case water or water / ethanol is used as mobile phase. By using a 3 zones open loop configuration, the regeneration of the liquid phase is no more needed, thus making zone IV obsolete.

[0034] Besides the classical SMB systems for the separation of a two component mixture also eight-zone close loop or five zones open loop SMB systems have been developed for the separation of more than 2 components. Due to the continuous mode of operation and also the possibility of using rather large column sizes and recycling of the mobile phase SMB system can in principle scaled into production volumes of 100s of tons. The skilled person will readily be able to calculate SMB conditions based on the results of a gel filtration experiment.

[0035] In another embodiment, the EV-comprising liquid in the process of the invention is a milk fraction, preferably a skimmed milk fraction.

[0036] In still another embodiment of the process of the invention, the EV- comprising liquid is a whey. Such a whey 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. Alternatively, the EV-comprising liquid is obtained from cow, goat, sheep, camel, buffalo or horse milk; bovine milk whey being preferred, such as acid whey or cheese whey. In yet another embodiment the whey is a cheese whey, preferably bovine cheese whey, cow's milk cheese whey being most preferred.

[0037] In yet another embodiment of the process of the invention, the UF is performed with a transmembrane pressure (TMP) of between 10 and 600 kPa, preferably between 50 and 200 kilopascal (kPa), more preferably between 70 and 150 kPa, most preferably between 80 and 140 kPa. The TMP may also be expressed in bar or atmosphere (atm) using the conversion of one bar being equal to 100 kPa, being 0.99 atm.

[0038] In one embodiment the EV-comprising liquid in the process of the invention has a dry matter content of 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 %, most preferably between 5.0 and 8.0 %. Such a desired dry matter content may be obtained by diluting the EV-comprising product with water or preferably an aqueous milk mineral solution, more preferably by diluting it with Simulated Milk Ultra Filtrate (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). Use of 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.

[0039] In another embodiment of the process of the invention the EV-comprising liquid has a protein content of between 5 and 50 g / litre, preferably between 10 and 40 g / litre, more preferably between 15 and 35 g / litre, particularly preferably between 19 and 29 g / litre, most preferably between 21 and 27 g / litre.

[0040] In yet another embodiment of the process of the invention the EV- comprising liquid has a pH above 5.0, preferably above 5.2. Such a pH of 5.0 or 5.2 or higher is preferred as the iso-electric point of alfa lactalbumin and of beta lactoglobulin is around these pH values. At lower pH these proteins will become neutral or positively charged which causes them to aggregate resulting in a poorer separation. The preferred maximum pH of the EV-comprising liquid is pH=8.0. Preferably the EV- comprising liquid has a pH of between 5.2 and 8.0, more preferably between 6.0 and 7.5, most preferably a pH between 6.0 and 6.6.

[0041] In one preferred embodiment, the UF step in the process of the invention is performed using a polymeric membrane, preferably polymeric spiral wound membrane. Particularly preferred are polyvinylidene difluoride (PVDF) membranes, especially spiral wound PVDF membranes. Alternatively, hydrophilic polyethersulfone (PES) membranes may be used.

[0042] Ultrafiltration is preferably performed using 1-20 kDa membrane; preferably a 5-10 kDa membrane. In one embodiment the UF step is performed using a polymeric membrane with a cut-off size of between 600 kD and 1000 kD, preferably between 700 kD and 900 kD, more preferably between 750 kD and 850 kD.

[0043] When using a polymeric membrane, the ultrafiltration temperature is preferably in the range of 5-30°C, preferably 10-20°C, more preferably 13-17°C, most preferably 16-17°C.

[0044] Alternatively, in another embodiment of the process of the invention the UF step is performed using a ceramic membrane. Preferably the ceramic membrane has a cut-off size of between 0.15 and 0.25 micron, more preferably between 0.19 and 0.21 micron. When using a ceramic membrane, the ultrafiltration temperature is preferably between 30 and 80°C, preferably between 40 and 70°C, more preferably between 50 and 60°C.

[0045] In still another embodiment, the amount of protein in the EV-enriched product as obtained in the process of the invention is less than 4.0 femtogram / EV, preferably less than 3.0 fg / EV, more preferably less than 2.0 fg / EV.

[0046] In one aspect, the invention relates to a composition comprising extracellular vesicles (EV) and protein wherein the amount of protein is less than 4.0 femtogram / EV, preferably less than 3.0 fg / EV, more preferably less than 2.0 fg / EV.

[0047] In one aspect, the invention relates to an extracellular enriched product as obtained in the process of this invention. In another aspect, the EV-enriched product as obtained in the process of the invention may be used in a food product or in medicine. Preferably it is used in a synthetic food product.

[0048] In yet another aspect the invention relates to a synthetic food product or medicine comprising the EV-enriched product obtained in the process of the invention. Such a product may be used to enhance muscle performance in a subject and or for increasing muscle growth in a subject for example as described in European Application EP23165184.5 which is enclosed herein by reference.

[0049] As used herein, a "synthetic composition" is a composition which is artificially prepared and is containing at least one compound that is produced ex vivo chemically and / or biologically and / or physically, e.g. by means of chemical reaction, enzymatic reaction or by a fractionation process. An example of such a fractionation process is a process wherein bovine milk is separated into different fractions like a fat and protein fraction. For the avoidance of doubt, a synthetic composition is not made in vivo by man or animal.

[0050] The mEV-enriched product, may be pasteurized to provide storage stability. For example, the mEV-enriched product may be heated, for example, at about 70°C for about 15 seconds, to ensure microbiological stability in order to yield a pasteurized fraction. Other pasteurization conditions will be apparent to those skilled in the art and may be employed.

[0051] With or without pasteurization, the mEV-enriched product may be used as is or subjected to additional processing steps to provide a desired physical form.

[0052] In a particularly preferred embodiment, the mEV is an exosome. Extracellular vesicles may be ruptured during isolation and / or enrichment thereof. Accordingly, in one embodiment the mEV in the synthetic composition of the invention are comprising intact bovine mEV, preferably wherein the bovine mEV are sourced from a whey-containing bovine milk fraction. Transmission electron microscopy (TEM) may be used for purposes of assessing the presence of mEV in an mEV-enriched product. TEM is a technique which can be used for the direct visualization of nanosized structures, such as mEV. Uranyl acetate may be applied as a negative dye to study the impact of thermal treatments, such as pasteurization, evaporation, spray-drying, and freeze-drying, on the mEV structure of the mEV in the product. Briefly, the uranyl acetate acts as a negative dye, which stains the background and leaves the intact vesicular structures, such as intact extracellular vesicles, unstained and highly visible (as shown in WO2022146743).

[0053] In one embodiment, the mEV-enriched product of the 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 a further embodiment, the mEV-enriched product comprises at least about 108mEV per gram of the mEV-enriched product as measured by a nano tracking procedure. Briefly, nanoparticle tracking analysis (NTA) can be used to determine mEV diameter and concentration. The principle of NTA is based on the characteristic movement of nanosized particles in solution according to the Brownian motion. The trajectory of the particles in a defined volume is recorded by a camera that is used to capture the scatter light upon illumination of the particles with a laser. The Stokes- Einstein equation is used to determine the size of each tracked particle. In addition to particle size, this technique also allows determination of particle concentration.

[0054] In one embodiment, the mEV-enriched product of the invention comprises at least 0.1 wt% mEV as determined relative to the dry weight of the product.

[0055] In another specific embodiment, the mEV-enriched product of the invention comprises from about 108to about 1014mEV per gram of the mEV-enriched product. In yet a more specific embodiment, the mEV- enriched product comprises from about 109to about 1013mEV per gram of the mEV-enriched product. In another specific embodiment, the mEV enriched product contains at least about a three-fold increase in the number of mEV, as compared to a raw whey-containing bovine milk fraction. In another specific embodiment, the mEV-enriched product contains a 3-fold to 50-fold increase in the number of mEV, as compared to a raw whey-containing bovine milk fraction, for example cheese whey or acid whey.

[0056] In still another embodiment of the invention the diameter of greater than 90% of the bovine mEV is from about 10 nanometres to about 250 nanometres.

[0057] In yet another embodiment, at least 50 wt% of the mEV are intact, preferably wherein at least about 55, 60, 65, 70, 75, 80, 85, 90, or 95 % of the mEV are intact. Preferably, the level of intact mEV is determined relative to level of mEV in the source material e.g. milk fractions, used to prepare the composition of the invention.

[0058] In another embodiment, the mEV-enriched product is for oral administration, preferably wherein the composition is a powder, a liquid or a bar.

[0059] The composition of the invention may further comprise one or more selected from the group consisting of a protein fraction, a carbohydrate fraction, and a fat fraction. Optionally, the composition is comprising one or more nutrients selected from the group consisting of vitamins and minerals.

[0060] The mEV enriched product may be the sole source of protein in the nutritional composition of the invention. Nevertheless, additional protein sources can be included in the nutritional composition i.e. protein fraction. In one embodiment, the protein fraction comprises whole egg powder, egg yolk powder, egg white powder, whey protein, whey protein concentrates, whey protein isolates, whey protein hydrolysates, acid caseins, casein protein isolates, sodium caseinates, calcium caseinates, potassium caseinates, casein hydrolysates, milk protein concentrates, milk protein isolates, milk protein hydrolysates, non-fat dry milk, condensed skim milk, whole cow's milk, partially or completely defatted milk, coconut milk, soy protein concentrates, soy protein isolates, soy protein hydrolysates, pea protein concentrates, pea protein isolates, pea protein hydrolysates, rice protein concentrate, rice protein isolate, rice protein hydrolysate, fava bean protein concentrate, fava bean protein isolate, fava bean protein hydrolysate, collagen proteins, collagen protein isolates, meat proteins, potato proteins, chickpea proteins, canola proteins, mung proteins, quinoa proteins, amaranth proteins, chia proteins, hemp proteins, flax seed proteins, earthworm proteins, insect proteins, one or more amino acids and / or metabolites thereof, or combinations of two or more thereof.

[0061] The one or a mixture of amino acids, which may be described as free amino acids, can be any amino acid known for use in nutritional products. The amino acids may be naturally occurring or synthetic amino acids. In a specific embodiment, the one or more amino acids and / or metabolites thereof comprise one or more branched chain amino acids or metabolites thereof. Examples of branched chain amino acids include arginine, glutamine leucine, isoleucine, and valine. In another specific embodiment, the one or more branched chain amino acids or metabolites thereof comprise alpha-hydroxy-isocaproic acid (HICA, also known as leucic acid), keto isocaproate (KIC), beta-hydroxy-beta-methylbutyrate (HMB), and combinations of two or more thereof.

[0062] The nutritional composition may comprise a protein fraction in an amount from about 1 wt% to about 50 wt%, such as from about 1 wt% to about 30 wt% of the nutritional composition. More specifically, the protein may be present in an amount from 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% of the nutritional composition. Even more specifically, the protein comprises from about 1 wt% to about 5 wt% of the nutritional composition, or from about 20 wt% to about 30 wt% of the nutritional composition. Alternatively, in yet another embodiment, the nutritional product is a high protein product comprising a protein fraction in an amount from about 20 wt% to about 90 wt%, preferably from 30 wt% to 80 wt%, more preferably from 35 wt% to 75wt%.

[0063] As used herein, the carbohydrate fraction may comprise one or more selected from the group consisting of maltodextrin, starch, dextrose, dextrins, lactose, galactooligosaccharides, fructooligosaccharides, human milk oligosaccharides (HMOs), and galactomannan. Examples of starches that may be used include hydrolyzed starch, modified starch, cornstarch, and hydrolyzed cornstarch.

[0064] The nutritional composition may comprise carbohydrate in an amount from about 5 wt% to about 75 wt% of the nutritional composition. More specifically, the carbohydrate may be present in an amount from 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%, of the nutritional composition.

[0065] The fat fraction may comprise milk fat, cream, anhydrous milk fat, algal oil, canola oil, flaxseed oil, borage oil, safflower oil, high oleic safflower oil, high gamma-linolenic acid (GLA) safflower oil, corn oil, soy oil, sunflower oil, high oleic sunflower oil, cottonseed oil, coconut oil, fractionated coconut oil, medium chain triglycerides (MCT) oil, palm oil, palm kernel oil, palm olein, long chain polyunsaturated fatty acids, or combinations of two or more thereof.

[0066] The nutritional composition may comprise fat in an amount of from about 0.5 wt% to about 30 wt% of the nutritional composition. More specifically, the fat may be present in an amount from about 0.5 wt% to about 10 wt%, or from about 1 wt% to about 30 wt% of the nutritional composition, 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.

[0067] In one embodiment, the nutritional composition is a liquid nutritional composition and comprises from about 1 to about 15 wt% of protein, from about 0.5 to about 10 wt% fat, and from about 5 to about 30 wt% carbohydrate, based on the weight of the nutritional composition.

[0068] In another embodiment, the nutritional composition is a powder nutritional composition and comprises from about 10 to about 30 wt% of protein, from about 5 to about 15 wt% fat, and from about 30 wt% to about 65 wt% carbohydrate, based on the weight of the nutritional composition.

[0069] In a specific embodiment, the nutritional composition comprises at least one protein comprising milk protein concentrate and / or soy protein isolate, at least one fat comprising milk fat, canola oil, corn oil, coconut oil and / or marine oil, and at least one carbohydrate comprising maltodextrin, sucrose, lactose, galactooligosaccharides and / or fructooligosaccharides. The nutritional composition may also comprise one or more components to modify the physical, chemical, aesthetic, or processing characteristics of the nutritional composition or serve as additional nutritional components. Non-limiting examples of additional components include preservatives, emulsifying agents (e.g., lecithin), buffers, sweeteners including artificial sweeteners (e.g., saccharine, aspartame, acesulfame K, sucralose), colorants, flavorants, thickening agents, stabilizers, and so forth.

[0070] In specific embodiments, the nutritional composition has a neutral pH, i.e., a pH of from about 6 to 8 or, more specifically, from about 6 to 7.5. In more specific embodiments, the nutritional composition has a pH of from about 6.5 to 7.2 or, more specifically, from about 6.8 to 7.1.

[0071] The nutritional composition may be formed using any techniques known in the art. In one embodiment, the nutritional composition may be formed by (a) preparing an aqueous solution comprising protein and carbohydrate; (b) preparing an oil blend comprising fat and oil-soluble components; and (c) mixing together the aqueous solution and the oil blend to form an emulsified liquid nutritional composition. The intact mEV may be added at any time as desired in the process, for example, to the aqueous solution or to the emulsified blend. The intact mEV may be dry blended in powder form with one or more dry ingredients, for example, for combined addition to a liquid composition or if a powdered nutritional product is desirable.

[0072] In a specific embodiment, the nutritional composition is administered in the form of a powder. In another specific embodiment, the nutritional composition is administered in the form of a liquid. The nutritional composition can be administered to the subject in either form.

[0073] When the nutritional composition is a powder, for example, a serving size is from about 40 g to about 60 g, such as 45 g, or 48.6 g, or 50 g, to be administered as a powder or to be reconstituted in from about 1 ml to about 500 ml of liquid.

[0074] When the nutritional composition is in the form of a liquid, for example, reconstituted from a powder or manufactured as a ready-to-drink product, a serving ranges from about 1 ml to about 500 ml, including from about 110 ml to about 500 ml, from about 110 ml to about 417 ml, from about 120 ml to about 500 ml, from about 120 ml to about 417 ml, from about 177 ml to about 417 ml, from about 207 ml to about 296 ml, from about 230 m to about 245 ml, from about 110 ml to about 237 ml, from about 120 ml to about 245 ml, from about 110 ml to about 150 ml, and from about 120 ml to about 150 ml. In specific embodiments, the serving is about 1 ml, or about 100 ml, or about 225 ml, or about 237 ml, or about 500 ml. In specific embodiments, the nutritional compositions comprising bovine mEV are administered to a subject once or multiple times daily or weekly. In specific embodiments, the nutritional composition is administered to the subject from about 1 to about 6 times per day or per week, or from about 1 to about 5 times per day or per week, or from about 1 to about 4 times per day or per week, or from about 1 to about 3 times per day or per week. In specific 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.

[0075] The concentration and relative amounts of the protein fraction, carbohydrate fraction, and fat fraction in the nutritional compositions can vary considerably depending upon, for example, the specific dietary needs of the intended user. In a specific embodiment, the nutritional composition comprises a source of protein in an amount of about 2 wt% to about 20 wt%, a source of carbohydrate in an amount of about 5 wt% to about 30 wt%, and a source of fat in an amount of about 0.5 wt% to about 10 wt%, based on the weight of the nutritional composition, and, more specifically, such composition is in liquid form. In another specific embodiment, the nutritional composition comprises a source of protein in an amount of about 10 wt% to about 25 wt%, a source of carbohydrate in an amount of about 40 wt% to about 70 wt%, and a source of fat in an amount of about 5 wt% to about 20 wt%, based on the weight of the nutritional composition, and, more specifically, such composition is in powder form.

[0076] In one aspect the invention relates the synthetic nutritional composition of the invention for use in enhancing muscle performance in a subject and / or for use in increasing muscle growth in a subject, preferably in a subject in need of improved physical performance.

[0077] In yet another aspect, the invention relates to the use of the composition of the invention for enhancing muscle performance in a subject and / or the use of increasing muscle growth in a subject, preferably in a subject in need of improved physical performance. In still another aspect, the invention relates to the use of the composition of the invention in the manufacture of a medicament for enhancing muscle performance and / or increasing muscle growth.

[0078] In one embodiment the daily dose of the composition for use of the invention is between 0.01 to 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, 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.

[0079] It must be noted that, as used in the specification and the appended claims, the singular form "a", "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.

[0080] It will be understood that within this disclosure, any reference to a weight, weight ratio, and the like pertains to the dry matter, in particular the dry matter of the composition, unless defined otherwise.

[0081] Unless defined otherwise, 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 belongs.

[0082] To the extent that the term "includes" or "including" is used in the description or the claims, it is intended to be inclusive of additional elements or steps, in a manner similar to the term "comprising" as that term is interpreted when employed as a transitional word in a claim. As used herein, the term "comprising", which is synonymous with "including" or "containing", is open-ended, and does not exclude additional, unrecited element(s), ingredient(s) or method step(s), whereas the term "consisting of" is a closed term, which excludes any additional element, step, or ingredient which is not explicitly recited. Furthermore, to the extent that the term "or" is employed (e.g., A or B), it is intended to mean "A or B or both." When the "only A or B but not both" is intended, then the term "only A or B but not both" is employed. Thus, use of the term "or" herein is the inclusive, and not the exclusive use. When the term "and" as well as "or" are used together, as in "A and / or B" this indicates A or B as well as A and B.

[0083] Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

[0084] Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and / or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: percent, "parts of," and ratio values are by weight; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies, mutatis mutandis, to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.

[0085] It is also to be understood that this invention is not limited to the specific embodiments and methods described herein, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way. The invention is hereinafter illustrated with reference to the following, non-limiting, examples.

[0086] EXAMPLES

[0087] While the present invention has been illustrated by the description of embodiments thereof, and while the embodiments have been described in considerable detail, such descriptions are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the invention, in its broader aspects, is not limited to the specific details, the representative compositions and processes, or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.

[0088] The goal of these filtration experiment was to get as much of the mEV in the retentate.

[0089] Retention (R) and selectivity (S) will be used to determine the effect of the used settings during the filtration.

[0090] Retention ( / ?) = 1 — (Cp I Cr) (Formula 1)

[0091] Cp = Concentration in the permeate Cr = Concentration in the retentate

[0092] The selectivity shows how effective the separation of EVs and protein is.

[0093] Selectivity (s) = REV’S / R Protein (Formula 2)

[0094] REVS = Retention of the EVs

[0095] Rprotein = Retention of the proteins

[0096] In addition to the retention and selectivity, the flux is a parameter that is used. It indicates when an equilibrium has formed in the system and how the membrane is reacting to changes made during the experiment.

[0097] The permeate flux is calculated using Formula 3.

[0098] Permeate flux= LtA (Formula 3) Permeate flux (l / h / m2)

[0099] L = Amount collected (litre) t = Collection time (h)

[0100] A = Area of the membrane (m2)

[0101] Example 1 A Comparing the mEV selectivity of different membranes and UF conditions

[0102] In this example four different membranes were tested in different conditions.

[0103] Membranes tested

[0104] Ceramic 0,1 micron membrane Ceramic 0,2 micron membrane -Polymeric 500 kD membrane Polymeric 800 kD membrane

[0105] Starting material

[0106] Acid Whey Protein Concentrate (80%) (HCW80) from FrieslandCampina was used to conduct all experiments. First the HCW80 was diluted with Milli Q water to 6,5% ±0,2% dry matter content and a protein concentration of 24,0 g / l ± 1,0 g / l (p=0,05).

[0107] The desired pH was adjusted using an aqueous 10% sulphuric acid solution.

[0108] Test conditions Ceramic membranes pH 5,2

[0109] Crossflow 6 m / s

[0110] 55C°

[0111] Trans Membrane Pressure: 0.5, 1.0, 1.5 and 2.0 Bar.

[0112] Test conditions Polymeric membranes pH 5,2

[0113] Crossflow 0,25 m / s

[0114] 15 C°

[0115] Trans Membrane Pressure 0.5, 1.0, 1.5 and 2.0 Bar. In each experiment the Trans Membrane Pressure (TMP) was applied for 45 minutes in circulation mode to reach an equilibrium stage. After these 45 minutes a sample of the retentate and permeate was taken.

[0116] Results

[0117] Ceramic membrane

[0118] For all TMP tested, except for a TMP of 2.0 Bar, the 0.2 micron ceramic membrane had a better selectivity as compared to the 0.1 micron. At a TMP of 0.5 and 1.0 Bar the 0.2 micron had a selectivity which is about 2 times better than for the 0.1 micron membrane. The selectivity of the 0.2 micron membrane at 1.5 Bar was 3.76, while the 0.1 micron membrane had a selectivity of 1.29 at this pressure. At a TMP of 2.0 Bar, both membranes had an equal selectivity of 1.35, as shown in Figure 1. Polymeric membrane

[0119] The selectivity of the 500 kD membrane was about the same for all TMP tested (selectivity of about 1.0), so this membrane was not able to separate the mEVs. Instead, the 800 kD membrane showed a selectivity that increased with higher TMP with a maximum of 1.54, as shown in Figure 2.

[0120] The ceramic 0,2 micron and the polymeric 800 kD membrane were chosen to continue with due to their higher selectivity and higher protein fluxes.

[0121] Example IB Comparing the mEV selectivity of UF conditions

[0122] The effect on selectivity of pH, TMP and cross flow were tested.

[0123] Ceramic 0.2 micron, pH and TMP

[0124] As shown in Figure 3, the highest selectivity was obtained at a pH of 5.2. Without wishing to be bound to theory, it is assumed that at lower pH whey protein aggregates are formed. In particular at a pH of 5.2, a TMP of between 1.5 and 1.75 Bar is preferred as then the highest selectivity (>4.0) was obtained.

[0125] Polymeric 800 kD, pH and TMP Using the polymeric 800 kD membrane, , as shown in Figure 4, the pH had a major influence on the selectivity which was over 16 at a pH of 6.3 when combined with a TMP of 1.0 Bar while it was much lower at higher TMPs. Without wishing to be bound to theory, it is assumed that at lower pH whey protein aggregates are formed leading to a decrease in flux and increase in protein retention.

[0126] Polymeric 800 kD - Cross flow velocity

[0127] Using the polymeric 800 kD membrane, at all pH levels a decrease in selectivity of approximately 80% was obtained when decreasing the crossflow velocity from 1 m / s to 0,25 m / s.

[0128] Example 1C Comparing the mEV selectivity - diafiltration

[0129] In this set-up the effect on selectivity of performing the UF in diafiltration mode is shown.

[0130] During diafiltration Simulated Milk Ultra Filtrate (SMUF) was used as diafiltrate medium (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).

[0131] Diafiltration settings: pH 6.3, cross flow 1 m / s, Temperature 15 °C, TMP 1.0 Bar.

[0132] As shown in the previous experiments, the highest selectivity was obtained with the polymeric membrane of 800 kD. This membrane was also tested in Diafiltration mode (DF) at different pH at a cross flow of 1 m / s. A diafiltration model was made for a pH 6.3 and pH 4.1. The model was used to determine how long each diafiltration had to run to separate all protein material from the EV and how much EVs would be lost. This is shown in Figure 5.

[0133] At pH 6.3 there was 13.9% loss of EVs while the loss of EV would be 7.2% at pH 4.1. However, at pH 6.3 all proteins would be separated from the EV fraction after 55 minutes, while it would take 2 hours and 25 minutes at pH 4.1. Based on these results a pH of 6.3 was being preferred for the UF step also when used in DF mode.

[0134] Example 2 Pilot plant - large scale

[0135] This experiment was done in a pilot plant using the polymeric 800 kD membrane.

[0136] Starting material: Acid whey (HCW80) (FrieslandCampina).

[0137] Goal

[0138] The goal of this experiment was to enrich a milk fraction derived from acid whey with extracellular vesicles (EVs) by filtration using crossflow filtration. Using this technique, the proteins will permeate through the filter and the extracellular vesicles stay in the retentate.

[0139] Materials & Method

[0140] HCW80 (FrieslandCampina, 31.45 L) with a dry matter content of 31% was diluted with water to a dry matter content of 6.5% (150 L). The pH was set at 6.3 using HCI.

[0141] Membrane

[0142] Polymeric 800 kD spiral wound (Spiral wound 800kD)

[0143] Method

[0144] The highest possible crossflow was used considering pressure limits.

[0145] Transmembrane pressure: 1 bar

[0146] Temperature: 15°C

[0147] The filtration started with 150 litres of diluted HCW80 in circulation mode to establish an equilibrium. After these 45 minutes the filtration began.

[0148] All the permeate was collected in 10 litre fractions. A sample was taken of each fraction together with a sample of the retentate during collection of each fraction. When the retentate was concentrated to 25 litres, the experiment was finalized. These 25 litres were stored as the EV comprising fraction. EV purity of this fraction is given in Example 3. The protein concentration in the retentate was 120 mg / ml, providing a total amount of protein of 3.0 kg in the 25 litre. The total amount of EV was 3.3 x 10 14. This is 9,1 fg protein / EV. Other executions of this experiment resulted in an amount of 6.37-9.2 fg protein / EV.

[0149] Example 3 Size Exclusion Chromatography

[0150] Gel filtration was used to separate material based on size. Sepharose CL- 2B (bead diameter 60-200 pm; with a fractionation range of 100,000- 20,000,000 (Dextrans) and of 70,000-40,000,000 (Globular proteins) SigmaAldrich) was used as column material with PBS (50 mM NaH2PO4, 0.15 M NaCI) pH 7.4, as mobile phase. The column volume was 10 ml. EV-containing liquid (500 micro litre) was subjected to size-exclusion chromatography on such a gel-filtration column to further separate EVs from residual protein material.

[0151] Purity of the EVs was determined in femtogram (fg) protein / EV using Nanoparticle Tracking analysis to the determine the number of EV in a method analogous as described by Webber et al (Journal of Extracellular Vesicles 2013, 2: 19861 http: / / journalofextracellularvesicles.net / under index. php / jev / article / view / 19861).

[0152] Using this protocol, the EV purity (fg protein / EV) was determined of acid whey (HCW80 prior to ultrafiltration, starting material of Example 2), UF filtered acid whey retentate (UFR) as obtained in Example 2, and UFR followed by SEC (UFR-SEC). The results are shown below: A higher EV purity, e.g. a low level of protein per EV is desirable as it allows dosing of EVs without affecting overall protein levels. In addition it allows for usage of EVs in applications where low protein levels are desirable such as in medical applications.

Claims

CLAIMS1. Process for obtaining an extracellular vesicle (EV)-enriched product comprising the steps of i. obtaining an EV-comprising liquid; ii. subjecting the EV-comprising liquid to ultrafiltration (UF) to provide an UF retentate (UFR) enriched in EV and an UF permeate (UFP), preferably wherein the UF is performed in diafiltration (DF) mode; wherein the enrichment is an increase in the number of EV as compared to the total protein level in the UFR. as compared to in the EV-comprising liquid; wherein no step of chelating divalent cations with EDTA is used in the preparation of the EV-comprising liquid, preferably wherein no EDTA is used to prepare the EV-comprising liquid; and wherein the amount of protein in the EV-enriched product is less than 4.0 femtogram / EV, preferably less than 3.0 fg / EV, more preferably less than 2.0 fg / EV.

2. The process of claim 1 comprising an additional step iii. to be executed after step ii. wherein step iii. is a filtration step, preferably a size exclusion chromatography (SEC) step, more preferably a SEC step in a Simulated Moving Bed (SMB) chromatography set-up.

3. The process of claim 1 or 2 wherein the EV-comprising liquid is a milk fraction, preferably a skimmed milk fraction.

4. The process of any of the preceding claims wherein the EV-comprising liquid is a whey, preferably bovine milk whey.

5. The process of any of the preceding claims wherein the UF is performed with a transmembrane pressure of between 50 and 200 kilopascal (kPa), more preferably between 70 and 150 kPa.

6. The process of any of the preceding claims wherein the EV-comprising liquid has a dry matter content of 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 %, most preferably between 5.0 and 8.0 %.

7. The process of any of the preceding claims wherein the EV-comprising liquid has a pH above 5.2, preferably a pH of between 5.2 and 8.0, more preferably between 6.0 and 7.5, most preferably a pH between 6.0 and 6.6.

8. The process of any of the preceding claims wherein the UF step is performed using a polymeric membrane, preferably polymeric spiral wound membrane.

9. The process of claim 8 wherein the polymeric membrane has a cut-off size of between 600 kD and 1000 kD.

10. The process of anyone of claims 1 to 7 wherein the UF step is performed using a ceramic membrane; preferably wherein the ceramic membrane has a cut-off size of between 0.15 and 0.25 micron, preferably between 0.19 and 0.21 micron.

11. The process of anyone of claims 1-10 wherein the EV-comprising liquid is acid whey.

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

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

14. An extracellular vesicle-enriched product as obtained in the process of any one of claims 1-11.

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