Milk extracellular vesicle enrichment process using peg

Abstract

The invention relates to a process for obtaining a milk extracellular vesicle (EV)- enriched product from whey by mixing whey with a PEG solution providing a whey- PEG mixture; and centrifuging the whey-PEG mixture providing a pellet enriched in milk EV.

Description

[0001] Milk extracellular vesicle enrichment process using PEG

[0002] Field of the invention

[0003] The invention relates to a process for obtaining a milk extracellular vesicle (EV)- enriched product, in particular to a process for obtaining a milk stream enriched in milk-derived extracellular vesicles (mEV). The invention further relates to a milk EV enriched product obtainable in the process of the invention and to the use of such a product in a food product or in medicine.

[0004] Background of the invention

[0005] 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.

[0006] 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.

[0007] 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.

[0008] Many studies provided compelling evidence that extracellular vesicles (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 gastro-intestinal tract, stay intact after absorption, indicating excellent stability. These characteristics make milk-derived EVs very suitable as drug carriers, but also by themselves, these EVs already have a substantial immunoregulatory function, and even without loading, these vesicles can act as therapeutics. It is therefore desirable to have an efficient scalable method to isolate or at least increase the level of EV in a milk stream. Such a process contributes to a sustainable way to address and / or treat problems or defects in a subject’s immune response and contributes to a sustainable and environment-friendly drug carrier. Such a process likewise contributes to the applicability of EV for use in improving muscle growth, muscle recovery, and / or mobility.

[0009] Research in the field of milk-derived EV (also referred to a milk EV or 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.

[0010] 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. 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).

[0011] Two different methods are commonly applied to isolate EVs from milk: ultracentrifugation and size-exclusion chromatography (SEC). However, ultracentrifugation has several drawbacks, such as the co-isolation of non-exosomal impurities, low reproducibility, potential damage of exosomes and low-throughput of samples.

[0012] Body fluids contain many nanoparticles (some non-vesicular) in the same size range as EV that can co-elute with them. 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.

[0013] Milk is a potential source for EV as it is biologically safe and it is available in large quantities. A scalable process is thus desired to extract milk EV or enrich a milk fraction with EV.

[0014] It is therefore an object of the invention to provide a process for the enrichment of extracellular vesicles, 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 a 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. SUMMARY OF THE INVENTION

[0018] In a first aspect, the invention relates to a process for obtaining a milk extracellular vesicle (EV)-enriched product comprising the steps of i. obtaining whey from milk; ii. obtaining a polyethylene glycol (PEG) solution; iii. mixing the whey of i. with the PEG solution of ii providing a whey-PEG mixture; iv. centrifuging the whey-PEG mixture providing a pellet enriched in milk EV; and v. dissolving the pellet enriched in milk EV of iv. in a liquid and subjecting the dissolved pellet to a filtration step, providing a further purified EV-enriched product.

[0019] Preferably, wherein no step of chelating divalent cations with EDTA is used in the preparation of the whey, preferably wherein no EDTA is used to prepare the EV- enriched product.

[0020] In another aspect the invention relates to a milk EV-enriched product obtainable with the process of the invention and to the use of such a product in a food product or medicine. The invention also relates to 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] DETAILED DESCRIPTION

[0023] In a first aspect the invention relates to a process for obtaining a milk extracellular vesicle (EV)-enriched product comprising the steps of i. obtaining whey from milk; ii. obtaining a polyethylene glycol (PEG) solution iii. mixing the whey of i. with the PEG solution of ii. providing a whey-PEG mixture; iv. centrifuging the whey-PEG mixture providing a pellet enriched in milk EV and v. dissolving the pellet enriched in milk EV of iv. in a liquid and subjecting the dissolved pellet to a filtration step, providing a further purified EV-enriched product. PEG increases the number of hydrophobic interactions with EVs and between EVs, which leads to water exclusion and EV pellet formation after incubation and a single low-speed centrifugation step. The inventors surprisingly found that EV pellet formation using PEG starting from whey gives much better results than starting from milk or skimmed milk due to the low levels of casein and milk fat. As used herein, the pellet obtained in step iv may also be referred to a precipitate.

[0024] Preferably, wherein the process does not comprise a step of chelating divalent cations with EDTA, more preferably wherein no EDTA is used to prepare the milk EV- containing product. The preferred 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 needed e.g. for meeting regulatory requirements.

[0025] The inventors surprisingly found that when starting the EV enrichment from whey instead of starting from milk or from skimmed milk, the yield (i.e. amount, purity, and / or speed of the process) improves. The inventors surprisingly found that this improvement is caused by the absence (or low levels) of casein and fat in whey. As such there is little or no interference of fat or casein in the enrichment process. This contributes to the applicability of the claimed method to the larger scale with which the process may be executed.

[0026] So, in one embodiment the amount of whey used in the process of the invention is more than 1 litre, preferably more than 10 litres, more preferably more than 100 litre, even more preferably more than 1000 litre. As used herein, litre and liter are used interchangeably.

[0027] In order to obtain higher purities of the EV-enriched product, an additional filtration step may be added to the process of the invention wherein the pellet enriched in milk EV is dissolved in a liquid and subjected to the filtration. 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 v. to be executed after step iv. wherein the pellet enriched in milk EV is dissolved in a liquid and the dissolved pellet is subjected to a filtration step, providing a further purified EV-enriched product; preferably wherein the filtration step is a size exclusion chromatography (SEC) step, more preferably a SEC step in a Simulated Moving Bed (SMB) chromatography set-up. A suitable liquid to take up the pellet enriched in milk EV is PBS buffer. Phosphate-buffered saline (PBS) is a buffer solution (pH ~ 7.4) commonly used in biological research. It is a waterbased salt solution comprising disodium hydrogen phosphate, and sodium chloride. The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solutions match those of the human body. PBS buffer is well-known in the art and commercially available e.g. from SigmaAldrich or Merck.

[0028] 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' -fucosy I lactose - from fermentation, bio catalysis or chemical synthesis (e.g. in EP2857410 A1 ). 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 cross-current 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.

[0029] 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.

[0030] In principle, zones 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.

[0031] 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.

[0032] In another embodiment, the whey in the process of the invention is cheese whey, preferably bovine cheese whey. In yet another embodiment, the whey in the process of the invention is acid whey, preferably bovine acid whey. Whey is the liquid remaining after milk has been curdled and strained. It is a by-product of the manufacturing of cheese or casein and has several commercial uses. Sweet whey is a by-product resulting from the manufacture of rennet types of hard cheese, like Cheddar or Swiss cheese. Acid whey (also known as sour whey) is a by-product brought out during the making of acid types of dairy products, such as strained yogurt. The main constituents of whey are lactose and whey proteins and low levels of EV. Whey proteins consist of a-lactalbumin, p-lactoglobulin, serum albumin, immunoglobulins, and proteose peptones.

[0033] The whey as used in the process of the invention may be obtained from any kind of milk like for example from bovine milk, “bovine” is referring to an animal of the cattle group and includes the antelopes, sheep, goats, cattle, buffalo, and bison, bovine is preferably referring to the domestic cattle group including sheep, goats, cattle, and buffalo. Alternatively the whey is obtained from cow, goat, sheep, camel, buffalo or horse milk; bovine milk whey being preferred.

[0034] In still another embodiment of the process of the invention, the whey has one or more of i. a fat content of from 0.0% to 10.0% by weight as determined to the dry mass of the whey; preferably of from 0.0% to 5.0%, more preferably from 0.0% to 3.0%, most preferably from 0.0% to 2.0%; ii. a casein content of from 0.0% to 5.0% by weight as determined to the dry mass of the whey; preferably of from 0.0% to 3.0%, more preferably from 0.0% to 2.0%, most preferably from 0.0% to 1.0%.

[0035] Preferably the fat content is from 0.0% to 10.0% by weight and the casein content of from 0.0% to 5.0% by weight, as determined to the dry mass of the whey. More preferably the fat content is from 0.0% to 5.0% by weight and the casein content of from 0.0% to 3.0% by weight, as determined to the dry mass of the whey. The "fat content" of a composition corresponds to the weight of the fat components present in the composition relatively to the total weight of the composition. The fat content is expressed as a weight percentage. The fat content can be measured using the Rbse- Gottlieb principle as described in ISO 23318:2022(E); which is a gravimetric method including hydrolysis and extraction by petroleum ether. The casein content is determined by the difference between the total nitrogen (Ntot) content and the noncasein nitrogen (NCN) using the Kjeldahl method.

[0036] In one embodiment the whey as used 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 whey with water or a milk mineral solution, 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.

[0037] 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.

[0038] In yet another embodiment of the process of the invention the whey has a pH below 6.0, preferably below 5.0. Preferably, the pH of the whey is between 3.0 and 5.0. In still another embodiment, the amount of protein per EV particle in the milk EV enriched product is below 50 fg / particle, preferably below 25 fg / particle, more preferably below 15 fg / particle, most preferably below 10 fg / particle.

[0039] Starting from whey, the mild process of the invention includes some other whey protein material. In one embodiment, the milk EV-enriched product is comprising active TGF beta.

[0040] Preferably, the PEG solution is an aqueous PEG solution. In one embodiment the aqueous PEG solution has a concentration of between 10 and 100 g PEG 1 100ml, preferably between 30 and 70 g PEG / 100ml, more preferable between 40 and 60 g / 100ml, most preferably the aqueous PEG solution has a concentration of 50 g PEG / 100ml.

[0041] The molecular mass of the PEG is preferably between 1 ,000 and 12,000, more preferably between 2,000 and 10,000, particularly preferably between 4,000 and 8,000, most preferably the PEG is PEG6000.

[0042] In still another embodiment of the process of the invention, the volume ratio of the PEG solution to whey is between 1 to 0.5 and 1 to 40; preferably between 1 to 0.8 and 1 to 20; more preferably between 1 to 2 and 1 to 8; most preferably 1 to 4.

[0043] The centrifugation (step iv) is preferably performed between 1000 and 2000 g; more preferably it is performed between 1000 and 2000 g and at a temperature of between 2 and 6 °C, even more preferably at a temperature of 4 °C. In another aspect, the invention relates to an EV-enriched product as obtained in the process of the invention.

[0044] In yet another aspect, the invention relates to the use of such a product in a food product or in medicine. Preferably it is used in a synthetic food product.

[0045] 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 (March 29, 2023) which is enclosed herein by reference. 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.

[0046] 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.

[0047] 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. In a particularly preferred embodiment, the mEV is an exosome.

[0048] 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.

[0049] 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 WO2022 146743).

[0050] 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 nanotracking procedure.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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. In another embodiment, the mEV-enriched product is for oral administration, preferably wherein the composition is a powder, a liquid or a bar.

[0057] 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.

[0058] 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. 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.

[0059] 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 alphahydroxy-isocaproic acid (HICA, also known as leucic acid), keto isocaproate (KIC), beta-hydroxy-beta-methylbutyrate (HMB), and combinations of two or more thereof. 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%. 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 hydrolysed starch, modified starch, corn-starch, and hydrolysed corn-starch. 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] In one embodiment the daily dose of the composition for use of the invention is between 0.01 to 30 g of dry weight mEV per day, preferably between 0.1 and 20 g of dry weight mEV per day. More preferably, the daily dose is between 0.01 and 30 g of dry weight 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 of dry weight mEV per day and between 1 .0 and 25 g galactose per day.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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. 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.

[0084] 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.

[0085] 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. The goal of these experiment was to get as much of the mEV in the pellet.

[0088] Starting material

[0089] Whey Protein Concentrate (80%) (HCW80) (acid whey) 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).

[0090] The desired pH was adjusted using an aqueous 10% sulphuric acid solution.

[0091] PEG solution

[0092] PEG-6000 was dissolved in Milli Q water (25 g / 50 ml) and stored at 4°C till further use.

[0093] Example 1A

[0094] 1 part of PEG 6000 solution was mixed with 4 parts of acid whey at a pH of 4.2 and allowed to incubate overnight at 4°C. The mixture was transferred into a centrifuge tube and centrifugated at 1500 g for 30 minutes at 4°C. The supernatant was discarded and the pellet was allowed to dry.

[0095] Next, the pellet was dissolved in PBS (Merck) and filtered over a 0.22 urn filter to remove any remaining non-dissolved particles. The filtrate was stored at 4°C. The results of this purification are shown in Table 1 , entry pH 4.2 -> PEG together with the EV contents of the starting material HCW80.

[0096] Example 1B

[0097] The same experiment as Example 1 A was repeated except that the pH was set at 6.8.

[0098] The results of this purification are shown in Table 1 (entry: pH 6.8 -> PEG).

[0099] Example 2 Size Exclusion Chromatography (SEC)

[0100] 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.

[0101] 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 using a micro-BCA kit (Journal of Extracellular Vesicles 2013, 2: 19861 http: / / journalofextracellularvesicles.net / under index. php / jev / article / view / 19861 ).

[0102] Using this protocol, the EV purity (fg protein / EV) was determined of acid whey (HCW80 prior to ultrafiltration), Centrifuged PEG- acid whey retentate pellet as obtained in Example 1 , and UFR followed by SEC (UFR-SEC). The results of this purification are shown in Table 1 (entry pH 4.2 ->PEG ->SEC).

[0103] These experiments show that PEG precipitation at pH 4.2 results in a better EV purity as compared to pH 6.8 which is reflected in the lower protein levels per EV (2.92 vs 4.11 fg / EV for pH 4.2 and 6.8, respectively). Executing the process at pH 4.2 has as a further benefit that the level of TGFb is higher. TGFb is known for its positive health effects (e.g. EP1218410). Alternatively, the variation in pH may be used in different embodiments of the invention in order to obtain an EV fraction with high or low levels of TGFb. Table 1 EV contents and purification results

[0104] The optional SEC step reduces the level of TGFb. However, the level of protein per EV is significantly reduced from 2.92 to 0.23 fg protein / EV.

Claims

CLAIMS1 . Process for obtaining a milk extracellular vesicle (EV)-enriched product comprising the steps of i. obtaining whey from milk; ii. obtaining a polyethylene glycol (PEG) solution iii. mixing the whey of i. with the PEG solution of ii. providing a whey-PEG mixture; iv. centrifuging the whey-PEG mixture providing a pellet enriched in milk EV; and v. dissolving the pellet enriched in milk EV of iv. in a liquid and subjecting the dissolved pellet to a filtration step, providing a further purified EV-enriched product.

2. The process of claim 1 ; wherein the filtration step in step v. is a size exclusion chromatography (SEC) step.

3. The process of claim 1 or 2 wherein the whey is cheese whey, preferably bovine cheese whey.

4. The process of claim 1 or 2 wherein the whey is acid whey, preferably acid whey from bovine milk.

5. The process of any of the preceding claims wherein the whey has one or more of i. a fat content of from 0.0% to 10.0% by weight as determined to the dry mass of the whey preferably of from 0.0% to 5.0%, more preferably from 0.0% to 3.0%, most preferably from 0.0% to 2.0%; ii. a casein content of from 0.0% to 5.0% by weight as determined to the dry mass of the whey preferably of from 0.0% to 3.0%, more preferably from 0.0% to 2.0%, most preferably from 0.0% to 1 .0%.

6. The process of any of the preceding claims wherein the amount of protein per EV particle in the milk EV enriched product is below 50 fg / particle, preferably below 25 fg / particle.

7. The process of any of the preceding claims wherein the amount of protein per EV particle in the milk EV enriched product is below 15 fg / particle, preferably below10 fg / particle.

8. The process of any of the preceding claims wherein the EV-enriched product is comprising active TGF beta.

9. The process of any of the preceding claims wherein the PEG solution is an aqueous PEG solution, preferably wherein the aqueous PEG solution of the polymer has a concentration of 50 gr PEG 1 100ml.

10. The process of any of the preceding claims wherein the PEG has an average molecular mass of between 1 ,000 and 12,000, preferably between 2,000 and 10,000, more preferably between 4,000 and 8,000, most preferably wherein the PEG is PEG6000.11 . The process of any of the preceding claims wherein the volume ratio of the PEG solution to whey is between 1 to 0.5 and 1 to 40; preferably between 1 to 0.8 and 1 to 20; more preferably between 1 to 2 and 1 to 8; most preferably 1 to 4.

12. The process of any of the preceding claims wherein the centrifugation is performed at between 1000 and 2000 g.

13. Use of a milk EV-enriched product obtainable with the process of any of the preceding claims in a food product or medicine.

14. The use of claim 13 wherein the food product is a synthetic food product.

15. A synthetic food product or medicine comprising the milk EV-enriched product of claim 13.

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