Mammary cell extracellular vesicle enrichment method using peg

Abstract

The present invention relates to a method for obtaining a product enriched in milk extracellular vesicles (EVs) from whey, by mixing whey with a PEG solution, thereby providing a whey-PEG mixture; and centrifuging the whey-PEG mixture, thereby providing a pellet enriched in milk EVs.

Description

Technical Field

[0001] This invention relates to a method for obtaining a product enriched with milk extracellular vesicles (EVs), and more particularly to a method for obtaining milk streams enriched with milk-derived extracellular vesicles (mEVs). The invention further relates to a product enriched with milk EVs obtainable in the method of the invention, and the use of such a product in food products or pharmaceuticals. Background Technology

[0002] The term "extracellular vesicle" or "EV" is used herein as a general term for lipid bilayer-bound particles released from cells, and unlike cells, EVs cannot replicate. EVs range in diameter from near the physically smallest possible monolayer liposome (approximately 20–30 nm) to 10 micrometers or larger, although the vast majority of EVs are smaller than 200 nm. They carry loads of proteins, nucleic acids, lipids, metabolites, and even organelles from their parent cells. Most cells studied to date (including some bacterial, fungal, and plant cells surrounded by cell walls) are thought to release EVs. A wide variety of EV subtypes have been proposed, defined in various ways based on size, biological origin, load, cellular origin, and function.

[0003] As used herein, the term "intact extracellular vesicle" refers to an extracellular vesicle (EV) in which the vesicle membrane has not ruptured and / or otherwise degraded, and therefore the vesicle size can be determined using methods described elsewhere herein. Endogenous loadings, i.e., bioactive agents, therapeutic agents (e.g., miRNAs), and / or other biomolecules inherently present in milk-derived extracellular vesicles (mEVs), are retained in the intact EV in their active form.

[0004] Extracellular vesicles (one or more EVs) comprise exosomes (< 100 nm) and microvesicles (100 nm–10 micrometers). EVs are present in biological fluids and participate in a variety of physiological and pathological processes. EVs are considered an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and genetic material.

[0005] Numerous studies have provided compelling evidence that extracellular vesicles (EVs) are involved in the regulation of immune responses, acting as both enhancers and weakeners of the immune system, depending on the vesicle's origin and type. Studies have demonstrated the anti-inflammatory effects of milk-derived EVs (using human breast milk, as well as bovine colostrum and commercially available pasteurized cow milk) in in vitro systems and in animal models (therapeuticly). Strategies targeting the gut, particularly its microbiome, are under investigation and hold promise as therapeutic interventions for these diseases. In recent years, the use of milk-derived EVs as standalone drugs or as drug carriers has been frequently suggested. Due to their composition, milk-derived EVs exhibit high biocompatibility and limited immunogenicity, even across species. Milk-derived EVs have been shown to remain intact after absorption when taken up in the gastrointestinal tract, demonstrating excellent stability. These characteristics make milk-derived EVs well-suited as drug carriers, and these EVs themselves possess substantial immunomodulatory functions, and even without a loading, these vesicles can act as therapeutic agents. Therefore, efficient and scalable methods for isolating or at least increasing EV levels in milk streams are desired. This approach facilitates sustainable ways of addressing and / or treating problems or deficiencies in a subject's immune response, and promotes sustainable and environmentally friendly drug delivery systems. It also contributes to the suitability of EVs for improving muscle growth, muscle recovery, and / or mobility.

[0006] Research in the field of milk-derived EVs (also known as milk EVs or mEVs) has progressed rapidly in the past few years; however, a standard protocol for reproducible and cost-effective mEV (or exosome) isolation has not yet been established.

[0007] Initially, exosome isolation relied primarily on ultracentrifugation, considered the gold standard. Other isolation methods have been developed; however, each has its own limitations and cannot isolate only exosomes. Improved EV isolation methods may not only affect the quantity and purity of recovered EVs but may also lead to the isolation of specific EV populations with different sizes and functional characteristics, carrying certain RNA, protein, and lipid profiles. Therefore, developing scalable methods to isolate EVs in a rapid, efficient, reproducible, cost-effective, and clinically friendly manner remains a challenge (L. del Pozo-Acebo et al., Int. J. Mol. Sci. [International Journal of Molecular Sciences] 2021, 22, 1105. https: / / doi.org / 10.3390 / ijms22031105).

[0008] Two different methods are typically used to separate EVs from milk: ultracentrifugation and size exclusion chromatography (SEC). However, ultracentrifugation has several drawbacks, such as co-separation of non-exosome impurities, low reproducibility, potential damage to exosomes, and low sample throughput.

[0009] Body fluids contain numerous nanoparticles (some of which are non-vesicle nanoparticles) with sizes similar to those of EVs that can be co-eluted with them. A major drawback of SEC is the limited amount of EVs recovered per unit volume. Therefore, there is a desire to further improve mEV / exosome enrichment methods.

[0010] Milk is a potential source of EVs because it is biosafe and readily available in large quantities. Therefore, scalable methods are desired for extracting EVs from milk or enriching EV-containing milk fractions.

[0011] Therefore, an object of the present invention is to provide a method for enriching extracellular vesicles, particularly a method for obtaining milk streams enriched with milk-derived extracellular vesicles (mEVs), more preferably bovine mEVs. Another object of the present invention is to provide an EV enrichment method capable of producing large quantities of mEVs, preferably in a method scalable to greater than 100 liters (e.g., >1000 liters) and / or capable of operating in continuous mode.

[0012] Another object of the present invention is to provide a mEV-enriched milk stream that can be used in nutritional products such as infant formula or food supplements. Alternatively, the mEV-enriched milk stream can be used in nutritional products such as sports nutrition products or adult nutrition products to enhance muscle performance in subjects who need to improve their physical performance.

[0013] The present invention also relates to a method for alleviating chronic fatigue in a subject who is recovering from or has recovered from a viral infection, the method comprising administering to the subject a product containing enriched extracellular vesicles of intact bovine mEV.

[0014] Another object of the present invention is to use the mEV-enriched products obtained as in the methods of the invention in a convenient manner to improve mitochondrial function and thereby improve muscle performance in subjects requiring improved physical performance. These methods can be used to prevent or treat conditions characterized by reduced reserve respiratory capacity, including sarcopenia and chronic or acute cardiac injury, for example, as described in European application EP 23165184.5.

[0015] These and other advantages of the method of the present invention will become more apparent from the detailed description. Summary of the Invention

[0016] In a first aspect, the present invention relates to a method for obtaining a product enriched with extracellular vesicles (EVs) of milk cells, the method comprising the following steps: i. Obtaining whey from milk; ii. Obtain a polyethylene glycol (PEG) solution; iii. Mixing the whey from i. with the PEG solution from ii. to provide a whey-PEG mixture; iv. Centrifuge the whey-PEG mixture to provide a precipitate enriched with milk EVs; and v. Dissolve the precipitate of the enriched emulsion EV from iv. in a liquid and subject the dissolved precipitate to a filtration step to provide a further purified product of enriched EV.

[0017] Preferably, the step of chelating divalent cations with EDTA is not used in the preparation of whey, and preferably, EDTA is not used to prepare the product enriched with EV.

[0018] In another aspect, the present invention relates to a product enriched with milk EVs that can be obtained using the method of the present invention, and the use of such a product in food products or pharmaceuticals. The present invention also relates to food products or pharmaceuticals comprising the product enriched with EVs.

[0019] In another aspect, the present invention relates to synthetic food products of the present invention for use in increasing muscle growth in subjects. Detailed Implementation

[0020] In a first aspect, the present invention relates to a method for obtaining a product enriched with extracellular vesicles (EVs) of milk cells, the method comprising the following steps: i. Obtaining whey from milk; ii. Obtain a polyethylene glycol (PEG) solution; iii. Mixing the whey from i. with the PEG solution from ii. to provide a whey-PEG mixture; iv. Centrifuge the whey-PEG mixture to provide a precipitate enriched with milk EVs, and v. Dissolve the precipitate of the enriched emulsion EV from iv. in a liquid and subject the dissolved precipitate to a filtration step to provide a further purified product of enriched EV.

[0021] PEG increases the amount of hydrophobic interactions with EVs and with each other, leading to the expulsion of water and the formation of EV precipitate after incubation and a single low-speed centrifugation step. The inventors unexpectedly discovered that using PEG to form EV precipitate starting from whey was far more effective than starting from milk or skim milk, due to the lower levels of casein and milk fat. As used herein, the precipitate obtained in step iv may also be referred to as precipitate.

[0022] Preferably, the method does not include the step of chelating divalent cations with EDTA, and more preferably, EDTA is not used to prepare the milk-containing EV product. The preferred exclusion of EDTA in the method according to the invention makes the EV product obtained as described in this method suitable for obtaining EDTA-free EVs, which is desirable when EDTA-free products are required, for example, to meet regulatory requirements.

[0023] The inventors unexpectedly discovered that when EV enrichment begins with whey instead of milk or skim milk, the yield (i.e., the quantity, purity, and / or speed of the method) is improved. The inventors also unexpectedly discovered that this improvement is caused by the absence (or low levels) of casein and fat in whey. Therefore, there is little or no interference from fat or casein during the enrichment process. This facilitates the applicability of the claimed method to a larger scale where the process can be performed.

[0024] Therefore, in one embodiment, the amount of whey used in the method of the present invention is greater than 1 liter, preferably greater than 10 liters, more preferably greater than 100 liters, and even more preferably greater than 1000 liters. As used herein, liter and litre are used interchangeably.

[0025] To obtain a product enriched with higher purity EVs, an additional filtration step can be added to the method of the present invention, wherein the precipitate of enriched milk EVs is dissolved in a liquid and subjected to filtration. Preferably, the filtration step is a size exclusion chromatography (SEC) step. Such higher purity may be required when the enriched EVs product is used in pharmaceutical applications or as a food product for subjects with weakened or underdeveloped immune systems. In one embodiment, the additional filtration step is using an agarose gel filtration matrix. Preferably, a dextran fraction range of 50,000-30,000,000 Da is used, more preferably 100,000-20,000,000 Da. In one embodiment, the method of the present invention includes an additional step v. performed after step iv., wherein the precipitate of enriched milk EVs is dissolved in a liquid and the dissolved precipitate is subjected to a filtration step, thereby providing a product enriched with further purified EVs; preferably, the filtration step is a size exclusion chromatography (SEC) step, more preferably an SEC step in a simulated moving bed (SMB) chromatography setting. A suitable liquid for dissolving the precipitate of enriched milk EVs is PBS buffer. Phosphate-buffered saline (PBS) is a commonly used buffer solution in biological research (pH approximately 7.4). It is an aqueous salt solution containing disodium hydrogen phosphate and sodium chloride. The buffer helps maintain a constant pH. The osmotic pressure and ion concentration of the solution are matched to those of the human body. PBS buffer is well known in the art and is commercially available, for example, from Sigma-Aldrich or Merck.

[0026] Simulated moving bed (SMB) chromatography originated in the petrochemical and mineral industries. Today, the pharmaceutical industry uses SMB chromatography to separate enantiomers from racemic mixtures. SMB chromatography has been used to separate monosaccharides from fructose-glucose solutions and for large-scale separation of disaccharides like sucrose from beet or cane syrup. SMB chromatography has also been used to purify trisaccharides (like 2'-fucosylated lactose) from fermentation, biocatalysis, or chemical synthesis (e.g., in EP 2857410 A1).

[0027] Simulated moving bed (SMB) chromatography was developed as a continuous separation process similar to continuous chemical separation processes such as distillation. In distillation, a countercurrent is established between the liquid and gas phases, which allows for continuous application of the feed and the extraction of one or more products. Furthermore, in theory, countercurrent chromatography should achieve separation superior to conventional cross-current operations. However, countercurrent chromatographic operations require the mobile and stationary phases to move in opposite directions. Therefore, SMB chromatography was developed as a practical solution to the difficulties associated with the concept of moving solid chromatographic materials in continuous chromatographic separation processes.

[0028] The classic SMB concept involves four distinct zones and four external application streams: a feed stream containing the components to be separated, a desorbent or mobile phase stream, an extractant stream, and a raffinate stream (where the raffinate stream represents one or more components with lower retention). These streams divide the SMB system into four distinct zones (each zone or section may contain one or more columns) with the following objectives: Zone I is required for solid-phase regeneration; Zone II is for the desorption of materials with lower desorption strength; Zone III is for the adsorption of strongly adsorbed materials; and finally, Zone IV is for the adsorption of materials with lower adsorption capacity. Therefore, the more strongly adsorbed components establish a concentration wave in Zone II and are transported to the extractant port, while the weaker adsorbed components migrate towards the raffinate port.

[0029] In principle, zones I and IV (regeneration zones) are used for solid phase regeneration, while zones II and III (separation zones) can be considered as the actual separation zones of the system. In addition to the four liquid flows and the resulting zones, the system also contains a recirculation pump (for closed-loop operation) for the mobile phase (desorbent), which forces the mobile phase through a fixed zone in one direction. Countercurrent flow is then achieved through the periodic shifting and continuous supply or removal of feed, desorbent, and product from one column to the next in the system. Besides the classic closed-loop 4-zone SMB system, an open-loop 3-zone system can also be used. The 3-zone open-loop system is economical when fresh solvents are relatively inexpensive (e.g., when using water or water / ethanol as the mobile phase). By using a 3-zone open-loop configuration, liquid phase regeneration is no longer required, thus making zone IV unnecessary.

[0030] In addition to the classic SMB system for separating two-component mixtures, eight-zone closed-loop or five-zone open-loop SMB systems have been developed for separating more than two components. Due to the continuous operation mode and the possibility of using relatively large column sizes and recirculating mobile phases, SMB systems can, in principle, be scaled up to production volumes of hundreds of tons. Technicians will be able to easily calculate SMB conditions based on the results of gel filtration experiments.

[0031] In another embodiment, the whey in the method of the present invention is cheese whey, preferably cow cheese whey. In yet another embodiment, the whey in the method of the present invention is acid whey, preferably cow acid whey. Whey is the liquid remaining after milk coagulation and filtration. It is a byproduct of the manufacture of cheese or casein and has several commercial uses. Sweet whey is a byproduct of the manufacture of rennet-type hard cheeses (like cheddar or Swiss cheese). Acid whey (also known as acidic whey) is a byproduct of the manufacture of acidic types of dairy products (such as filtered yogurt). The main components of whey are lactose and whey protein, as well as low levels of EV. Whey protein consists of α-lactalbumin, β-lactoglobulin, serum albumin, immunoglobulins, and peptone.

[0032] The whey used in the method of this invention can be obtained from any kind of milk, such as, for example, cow's milk. "Cattle" refers to animals in a herd and includes antelope, sheep, goats, domestic cattle, buffalo, and bison; cattle preferably refers to domesticated cattle (including sheep, goats, domestic cattle, and buffalo). Alternatively, whey can be obtained from the milk of cows, goats, sheep, camels, buffalo, or horses; bovine whey is preferred.

[0033] In another embodiment of the method of the present invention, the whey has one or more of the following: i. A fat content of 0.0% to 10.0% by weight, as determined relative to the dry weight of whey; preferably 0.0% to 5.0%, more preferably 0.0% to 3.0%, and most preferably 0.0% to 2.0% by weight; ii. A casein content of 0.0% to 5.0% by weight, as determined relative to the dry weight of whey; preferably 0.0% to 3.0%, more preferably 0.0% to 2.0%, and most preferably 0.0% to 1.0%.

[0034] Preferably, as determined relative to the dry weight of whey, the fat content is from 0.0% to 10.0% by weight, and the casein content is from 0.0% to 5.0% by weight. More preferably, as determined relative to the dry weight of whey, the fat content is from 0.0% to 5.0% by weight, and the casein content is from 0.0% to 3.0% by weight. The “fat content” of the composition corresponds to the ratio of the weight of the fat component present in the composition to the total weight of the composition. Fat content is expressed as a weight percentage. Fat content can be measured using the Röse-Gottlieb principle, as described in ISO 23318:2022(E); this method is a gravimetric analysis method including hydrolysis and petroleum ether extraction. Casein content is determined using the Kjeldahl method by the difference between the total nitrogen (Ntot) content and the non-casein nitrogen (NCN).

[0035] In one embodiment, the whey used in the method of the present invention has a dry matter content between 0.1% and 15%, preferably between 1.0% and 15%, more preferably between 2.0% and 10%, particularly preferably between 3.0% and 9.0%, and most preferably between 5.0% and 8.0%. This desired dry matter content can be obtained by diluting the whey with water or a milk mineral solution, preferably by diluting the whey with simulated milk ultrafiltrate (SMUF) (Jenness, R. and Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk and Dairy Journal, 16(3), 153–164). The use of a milk mineral solution or SMUF is particularly preferred when the liquid containing EV is a milk fraction (such as whey protein fraction). Not wanting to be bound by any theory, it is believed that such a milk mineral solution or SMUF helps to keep EVs in their natural state.

[0036] In another embodiment of the method of the present invention, the protein content of the liquid containing EV is between 5 and 50 g / L, preferably between 10 and 40 g / L, more preferably between 15 and 35 g / L, particularly preferably between 19 and 29 g / L, and most preferably between 21 and 27 g / L.

[0037] In another embodiment of the method of the present invention, the pH of the whey is below 6.0, preferably below 5.0. Preferably, the pH of the whey is between 3.0 and 5.0.

[0038] In yet another embodiment, the amount of protein / EV particles in the product enriched with milk EV is less than 50 fg / particle, preferably less than 25 fg / particle, more preferably less than 15 fg / particle, and most preferably less than 10 fg / particle.

[0039] Starting with whey, the mild method of the present invention includes several other whey protein materials. In one embodiment, the product enriched with whey EVs contains active TGF β.

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

[0041] The molecular weight of 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, and most preferably PEG6000.

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

[0043] Centrifugation (step iv) is preferably carried out between 1000 g and 2000 g; more preferably, centrifugation is carried out between 1000 g and 2000 g and at a temperature between 2°C and 6°C, or even more preferably at a temperature of 4°C.

[0044] In another aspect, the present invention relates to a product enriched with EVs as obtained in the method of the present invention.

[0045] In another aspect, the present invention relates to the use of such a product in food products or pharmaceuticals. Preferably, it is used in synthetic food products.

[0046] In another aspect, the present invention relates to a synthetic food product or medicine comprising a product enriched with EVs obtained by the methods of the present invention. Such a product may be used to enhance muscle performance in a subject and / or to increase muscle growth in a subject, for example, as described in European application EP 23165184.5 (29 March 2023, which is incorporated herein by reference).

[0047] As used herein, a “synthetic composition” is an artificially prepared composition containing at least one compound produced by in vitro chemical and / or biological and / or physical means (e.g., by chemical reaction, enzymatic reaction, or by fractionation process). An example of such a fractionation process is the separation of milk into different fractions (e.g., fat and protein fractions). For the avoidance of doubt, the synthetic composition is not manufactured in a human or animal body.

[0048] Products enriched with mEVs can be pasteurized to provide storage stability. For example, products enriched with mEVs can be heated (e.g., at about 70°C for about 15 seconds) to ensure microbial stability in order to obtain pasteurized fractions. Other pasteurization conditions will be apparent to those skilled in the art and can be employed.

[0049] With or without pasteurization, products enriched with mEVs can be used as is or undergo additional processing steps to provide the desired physical form.

[0050] In a particularly preferred embodiment, mEV is an exosome.

[0051] Extracellular vesicles may rupture during their separation and / or enrichment. Therefore, in one embodiment, the mEV in the synthetic composition of the present invention comprises whole bovine mEV, preferably wherein the bovine mEV is derived from a whey-containing bovine milk fraction.

[0052] Transmission electron microscopy (TEM) can be used to assess the presence of mEVs in products enriched with mEVs. TEM is a technique that can be used for the direct visualization of nanostructures such as mEVs. Uranyl acetate can be used as a negative dye to study the effect of heat treatments (such as pasteurization, evaporation, spray drying, and freeze drying) on ​​the mEV structure of mEVs in products. In short, uranyl acetate acts as a negative dye that stains the background and leaves intact vesicle structures (such as intact extracellular vesicles) unstained and highly visible (as shown in WO 2022146743).

[0053] In one embodiment, the mEV-enriched product of the present invention comprises at least 0.001 wt% mEV, as determined relative to the dry weight of the product. In another specific embodiment, the mEV-enriched product comprises at least about 0.001 wt%, 0.01 wt%, 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt% mEV, as determined relative to the dry weight of the product. In another embodiment, the mEV-enriched product comprises at least about 10 8 Products enriched with mEVs per gram, such as those measured via nanoparticle tracking, are described. In short, nanoparticle tracking analysis (NTA) can be used to determine the diameter and concentration of mEVs. The principle of NTA is based on the characteristic motion of nanoscale particles in solution according to Brownian motion. The trajectory of the particles within a defined volume is recorded by a camera that captures scattered light when the particles are illuminated with a laser. The size of each tracked particle is determined using the Stokes-Einstein equations. In addition to particle size, this technique also allows for the determination of particle concentration.

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

[0055] In another specific embodiment, the mEV-enriched product of the present invention comprises about 10 8To about 10 14 A product enriched with mEV per gram. In yet another more specific embodiment, the product enriched with mEV comprises approximately 10 mEV / gram. 9 To about 10 13 The product enriched with mEV per gram. In another specific embodiment, the mEV-enriched product contains at least about three times the number of mEVs compared to a milk fraction containing raw whey. In another specific embodiment, the mEV-enriched product contains 3 to 50 times the number of mEVs compared to a milk fraction containing raw whey (e.g., cheese whey or yogurt whey).

[0056] In yet another embodiment of the invention, more than 90% of the Newton mEV has a diameter of about 10 nanometers to about 250 nanometers.

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

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

[0059] The compositions of the present invention may further comprise one or more components selected from the group consisting of protein fractions, carbohydrate fractions, and fat fractions. Optionally, the compositions comprise one or more nutrients selected from the group consisting of vitamins and minerals.

[0060] The product enriched with mEV can be the sole protein source in the nutritional composition of the present invention. However, the nutritional composition may include other protein sources. In one embodiment, the protein fraction includes whole egg powder, egg yolk powder, egg white powder, whey protein, whey protein concentrate, whey protein isolate, whey protein hydrolysate, acidic casein, casein isolate, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, nonfat milk powder, skimmed condensed milk, whole cow's milk, partially or completely skimmed milk, coconut milk, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein... Protein isolates, pea protein hydrolysates, rice protein concentrates, rice protein isolates, rice protein hydrolysates, broad bean protein concentrates, broad bean protein isolates, broad bean protein hydrolysates, collagen, collagen isolates, meat protein, potato protein, chickpea protein, low-erucic acid canola protein, mung bean protein, quinoa protein, amaranth protein, chia seed protein, flaxseed protein, earthworm protein, insect protein, one or more amino acids and / or their metabolites, or combinations of two or more thereof.

[0061] The amino acid can be described as one of the free amino acids, or a mixture of such amino acids, and can be any amino acid known for use in nutritional products. The amino acid can be naturally occurring or synthetic. In a particular embodiment, one or more amino acids and / or their metabolites comprise one or more branched-chain amino acids or their metabolites. Examples of branched-chain amino acids include arginine, glutamine, leucine, isoleucine, and valine. In another particular embodiment, one or more branched-chain amino acids or their metabolites comprise α-hydroxyisocaproic acid (HICA, also known as squalane), ketoisocaproic acid (KIC), β-hydroxy-β-methylbutyric acid (HMB), and combinations of two or more thereof.

[0062] The nutritional composition may contain a protein fraction in an amount of about 1 wt% to about 50 wt% (e.g., about 1 wt% to about 30 wt%) of the nutritional composition. More specifically, the protein may be present in an amount of about 1 wt% to about 25 wt% of the nutritional composition (including about 1 wt% to about 20 wt%, about 2 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 10 wt%, about 10 wt% to about 25 wt%, or about 10 wt% to about 20 wt%). Even more specifically, the protein may comprise about 1 wt% to about 5 wt% of the nutritional composition, or about 20 wt% to about 30 wt% of the nutritional composition. Alternatively, in yet another embodiment, the nutritional product is a high-protein product that contains protein fractions in an amount of about 20 wt% to about 90 wt%, preferably 30 wt% to 80 wt%, more preferably 35 wt% to 75 wt%.

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

[0064] The nutritional composition may contain carbohydrates in an amount of about 5 wt% to about 75 wt% of the nutritional composition. More specifically, carbohydrates may be present in an amount of about 5 wt% to about 70 wt% of the nutritional composition (including about 5 wt% to about 65 wt%, about 5 wt% to about 50 wt%, about 5 wt% to about 40 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 10 wt% to about 65 wt%, about 20 wt% to about 65 wt%, about 30 wt% to about 65 wt%, about 40 wt% to about 65 wt%, about 40 wt% to about 70 wt%, or about 15 wt% to about 25 wt%).

[0065] Fat fractions may include milk fat, butter, anhydrous milk fat, algae oil, low-erucic acid rapeseed oil, flaxseed oil, borage oil, safflower oil, high-oleic safflower oil, high-gamma-linolenic acid (GLA) safflower oil, corn oil, soybean oil, sunflower seed oil, high-oleic sunflower seed oil, cottonseed oil, coconut oil, fractionated coconut oil, medium-chain triglyceride (MCT) oil, palm oil, palm kernel oil, palm oil extract, long-chain polyunsaturated fatty acids, or combinations of two or more thereof.

[0066] The nutritional composition may contain fat in an amount of about 0.5 wt% to about 30 wt% of the nutritional composition. More specifically, fat may be present in an amount of about 0.5 wt% to about 10 wt%, or about 1 wt% to about 30 wt% (including about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 5 wt%, about 3 wt% to about 30 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 10 wt%, or about 10 wt% to about 20 wt%) of the nutritional composition.

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

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

[0069] In a particular embodiment, the nutritional composition comprises at least one protein containing milk protein concentrate and / or soy protein isolate, at least one fat containing milk fat, low-erucic acid rapeseed oil, corn oil, coconut oil and / or marine oil, and at least one carbohydrate containing maltodextrin, sucrose, lactose, galactooligosaccharides and / or fructooligosaccharides.

[0070] The nutritional composition may also contain one or more components that modify the physical, chemical, aesthetic, or processing characteristics of the nutritional composition, or that act as additional nutritional components. Non-limiting examples of additional components include preservatives, emulsifiers (e.g., lecithin), buffers, sweeteners including artificial sweeteners (e.g., saccharin, aspartame, acesulfame potassium, sucralose), colorants, flavoring agents, thickeners, stabilizers, etc.

[0071] In certain embodiments, the nutrient composition has a neutral pH, i.e., about 6 to 8, or more specifically about 6 to 7.5. In more specific embodiments, the nutrient composition has a pH of about 6.5 to 7.2, or more specifically about 6.8 to 7.1.

[0072] Nutritional compositions can be formed using any technique known in the art. In one embodiment, a nutritional composition can be formed by: (a) preparing an aqueous solution comprising proteins and carbohydrates; (b) preparing an oil blend comprising fats and oil-soluble components; and (c) mixing the aqueous solution and the oil blend together to form an emulsified liquid nutritional composition. The complete mEV can be added at any time desired during the process, e.g., to the aqueous solution or the emulsified blend. The complete mEV can be dry-blended with one or more dry ingredients in powder form, for example, for combination addition to a liquid composition, or if a powdered nutritional product is desired.

[0073] In a particular embodiment, the nutritional composition is applied in powder form. In another particular embodiment, the nutritional composition is applied in liquid form. The nutritional composition may be administered to the subject in either form.

[0074] When the nutritional composition is in powder form, for example, a portion of about 40 g to about 60 g (such as 45 g or 48.6 g or 50 g) will be applied as a powder or reconstituted in about 1 ml to about 500 ml of liquid.

[0075] When the nutritional composition is in liquid form (e.g., reconstituted from powder or formulated into a ready-to-drink product), a portion ranges from about 1 ml to about 500 ml, including about 110 ml to about 500 ml, about 110 ml to about 417 ml, about 120 ml to about 500 ml, about 120 ml to about 417 ml, about 177 ml to about 417 ml, about 207 ml to about 296 ml, about 230 ml to about 245 ml, about 110 ml to about 237 ml, about 120 ml to about 245 ml, about 110 ml to about 150 ml, and about 120 ml to about 150 ml. In specific embodiments, the portion is about 1 ml, or about 100 ml, or about 225 ml, or about 237 ml, or about 500 ml.

[0076] In certain embodiments, the nutritional composition comprising bovine mEV is administered to the subject once or more daily or weekly. In certain embodiments, the nutritional composition is administered to the subject about 1 to 6 times daily or weekly, or about 1 to 5 times daily or weekly, or about 1 to 4 times daily or weekly, or about 1 to 3 times daily or weekly. In certain embodiments, the nutritional composition is administered once or twice daily for a period of at least one week, at least two weeks, at least three weeks, or at least four weeks.

[0077] The concentrations and relative amounts of protein, carbohydrate, and fat fractions in the nutritional composition can vary significantly depending on, for example, the specific dietary needs of the intended user. In a particular embodiment, based on the weight of the nutritional composition, the composition comprises about 2 wt% to about 20 wt% of a protein source, about 5 wt% to about 30 wt% of a carbohydrate source, and about 0.5 wt% to about 10 wt% of a fat source; more specifically, such a composition is in liquid form. In another particular embodiment, based on the weight of the nutritional composition, the composition comprises about 10 wt% to about 25 wt% of a protein source, about 40 wt% to about 70 wt% of a carbohydrate source, and about 5 wt% to about 20 wt% of a fat source; more specifically, such a composition is in powder form.

[0078] In one aspect, the present invention relates to the synthetic nutritional compositions of the present invention for enhancing muscle performance in subjects and / or for increasing muscle growth in subjects, preferably for subjects who require improvement in physical performance.

[0079] In another aspect, the present invention relates to the use of the compositions of the invention for enhancing the muscle performance of a subject and / or increasing muscle growth in a subject, preferably for a subject who requires improvement in physical performance.

[0080] In another aspect, the present invention relates to the use of the compositions of the present invention in the manufacture of medicaments for enhancing muscle performance and / or increasing muscle growth.

[0081] In one embodiment, the daily dose of the composition used for purposes of the present invention is between 0.01 and 30 g dry weight mEV per day, preferably between 0.1 and 20 g dry weight mEV per day. More preferably, the daily dose is between 0.01 and 30 g dry weight mEV per day and between 0.01 and 30 g galactose per day, and even more preferably, the daily dose is between 0.1 and 20 g dry weight mEV per day and between 1.0 and 25 g galactose per day.

[0082] It should be noted that, as used in the specification and appended claims, unless the context clearly indicates otherwise, the singular forms “a” and “the” include a plural of indicators. For example, a component referred to in the singular is intended to comprise multiple components.

[0083] It will be understood that, unless otherwise defined, any reference to weight, weight ratio, etc. in this disclosure refers to dry matter, and in particular, the dry matter of the composition.

[0084] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0085] With regard to the term "includes" or "including" as used in the specification or claims, it is intended to include additional elements or steps in a manner similar to the interpretation of the term "comprising" as it is used as a transitional word in the claims.

[0086] As used herein, the term “comprising” is synonymous with “including” or “containing”, is open-ended, and does not exclude any additional undescribed elements, components, or method steps; while the term “consisting of” is a closed term and does not include any additional elements, steps, or components not expressly described.

[0087] Furthermore, the use of the term "or" (e.g., A or B) is intended to mean "A or B or both". The term "A or B only, not both" is used when "only A or B, not both" is expected. Therefore, the use of the term "or" in this document is inclusive, not exclusive. When the terms "and" and "or" are used together, as in "A and / or B", this means A or B as well as A and B.

[0088] Throughout this application, where publications are cited, the disclosures of those publications are incorporated herein by reference in their entirety to more fully describe the level of technology to which this invention pertains.

[0089] Unless otherwise expressly indicated by example or otherwise, all numerical quantities indicating amounts of material or reaction and / or conditions of use in this specification shall be understood to be modified by the word “about” when describing the broadest scope of the invention. It is generally preferred to practice within the stated numerical limits. Furthermore, unless explicitly stated otherwise: percentages, “parts,” and ratios are by weight; descriptions of a group or class of materials suitable for or preferably used for a given purpose relating to the invention mean that mixtures of any two or more members of that group or class are equally suitable or preferred; components described in chemical terms refer to components added to any combination specified in the specification and do not necessarily exclude chemical interactions between components of the mixture once mixed; the first definition of acronyms or other abbreviations applies to all subsequent uses of the same abbreviation herein, with necessary minor adjustments to the normal grammatical variations applicable to the initially defined abbreviation; and, unless explicitly stated otherwise, the measurement of a property is determined by the same technique as the same property previously or later referenced.

[0090] It should also be understood that the present invention is not limited to the specific embodiments and methods described herein, as specific components and / or conditions can certainly vary. Furthermore, the terminology used herein is for the purpose of describing specific embodiments of the invention only and is not intended to be limiting in any way.

[0091] The invention is described below with reference to the following non-limiting examples.

[0092] Example

[0093] While the invention has been described by way of examples, and while the examples have been described in considerable detail, such description is not intended to limit the scope of the appended claims or in any way to such details. Further advantages and modifications will readily become apparent to those skilled in the art. Therefore, the invention is not limited in its broader aspects to the specific details shown and described, representative compositions and methods, or illustrative examples. Thus, deviations from such details may be made without departing from the spirit or scope of the overall inventive concept.

[0094] The goal of these experiments is to obtain as much mEV as possible from the precipitate.

[0095] Starting materials

[0096] All experiments were conducted using whey protein concentrate (80%) (HCW80) (acidified whey) from FrieslandCampina. First, HCW80 was diluted with Milli-Q water to a dry matter content of 6.5% ± 0.2% and a protein concentration of 24.0 g / L ± 1.0 g / L (p = 0.05).

[0097] Adjust the pH to the desired level using a 10% sulfuric acid aqueous solution.

[0098] PEG solution

[0099] Dissolve PEG-6000 in Milli Q water (25 g / 50 ml) and store at 4°C until further use.

[0100] Example 1A

[0101] Mix 1 part PEG 6000 solution with 4 parts acid whey at pH 4.2 and allow to incubate overnight at 4°C. Transfer the mixture to a centrifuge tube and centrifuge at 1500 g for 30 minutes at 4°C. Discard the supernatant and allow the precipitate to dry.

[0102] Next, the precipitate was dissolved in PBS (Merck) and filtered through a 0.22 μm filter to remove any remaining undissolved particles. The filtrate was stored at 4°C.

[0103] The results of the purification are shown in Table 1, with entries for pH 4.2 -> PEG and EV content of the starting material HCW80.

[0104] Example 1B

[0105] Repeat the same experiment as in Example 1A, except that the pH was set at 6.8.

[0106] The results of this purification are shown in Table 1 (entry: pH 6.8 -> PEG).

[0107] Example 2: Size Exclusion Chromatography (SEC)

[0108] Gel filtration was used for size-based separation of materials. Sepharose CL-2B (bead diameter 60–200 μm; fractionation range 100,000–20,000,000 (dextran) and 70,000–40,000,000 (globulin), Sigma-Aldrich) was used as the column material, and PBS (50 mM NaH₂PO₄, 0.15 M NaCl) at pH 7.4 was used as the mobile phase. The column volume was 10 ml. EV-containing liquid (500 μL) was subjected to size exclusion chromatography on such a gel filtration column to further separate EVs from residual protein material.

[0109] In methods similar to those described by Webber et al. (Journal of Extracellular Vesicles, 2013, 2: 19861 http: / / journalofextracellularvesicles.net / underindex.php / jev / article / view / 19861) using a micro-bacillary BCA kit, nanoparticle tracking analysis is used to determine the purity of EVs by femtogram (fg) protein / EV to determine the number of EVs.

[0110] Using this protocol, the EV purity (fg protein / EV) of acid whey (HCW80 prior to ultrafiltration), the centrifuged PEG-acid whey osmotic residue precipitate obtained as in Example 1, and the subsequent SEC-refined UFR (UFR-SEC) was determined. The results of this purification are shown in Table 1 (entries: pH 4.2 -> PEG -> SEC).

[0111] These experiments demonstrate that PEG precipitation at pH 4.2 yields better EV purity compared to pH 6.8, reflected in lower protein levels / EV (2.92 fg / EV for pH 4.2 and 4.11 fg / EV for pH 6.8). Performing the method at pH 4.2 has a further benefit: higher TGFb levels. TGFb is known for its positive health effects (e.g., EP 1218410). Alternatively, pH variations can be used in different embodiments of the invention to obtain EV fractions with high or low TGFb levels.

[0112] Table 1. EV content and purification results

[0113] The optional SEC step reduced TGFb levels. However, the protein level / EV decreased significantly from 2.92 to 0.23 fg protein / EV.

Claims

1. A method for obtaining a product enriched with extracellular vesicles (EVs) of milk cells, the method comprising the following steps: i. Obtaining whey from milk; ii. Obtain a polyethylene glycol (PEG) solution; iii. The whey from i. is mixed with the PEG solution from ii. to provide a whey-PEG mixture; iv. Centrifuge the whey-PEG mixture to provide a precipitate enriched with milk EVs; as well as v. Dissolve the precipitate of the enriched emulsion EV from iv. in a liquid and subject the dissolved precipitate to a filtration step to provide a further purified product of enriched EV.

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

3. The method of claim 1 or 2, wherein the whey is cheese whey, preferably cow cheese whey.

4. The method of claim 1 or 2, wherein the whey is acid whey, preferably acid whey derived from cow's milk.

5. The method as described in any of the preceding claims, wherein the whey has one or more of the following: i. A fat content of 0.0% to 10.0%, preferably 0.0% to 5.0%, more preferably 0.0% to 3.0%, and most preferably 0.0% to 2.0% by weight, as determined relative to the dry weight of the whey; ii. A casein content of 0.0% to 5.0%, preferably 0.0% to 3.0%, more preferably 0.0% to 2.0%, and most preferably 0.0% to 1.0% by weight, as determined relative to the dry weight of the whey.

6. The method as described in any of the preceding claims, wherein the amount of protein / EV particles in the enriched milk EV product is less than 50 fg / particle, preferably less than 25 fg / particle.

7. The method as described in any of the preceding claims, wherein the amount of protein / EV particles in the enriched milk EV product is less than 15 fg / particle, preferably less than 10 fg / particle.

8. The method as described in any of the preceding claims, wherein the product enriched with EVs comprises active TGF β.

9. The method as claimed in any of the preceding claims, wherein the PEG solution is an aqueous PEG solution, preferably wherein the concentration of the aqueous PEG solution of the polymer is 50 gr PEG / 100 ml.

10. The method as claimed in any of the preceding claims, wherein the PEG has an average molecular weight between 1,000 and 12,000, preferably between 2,000 and 10,000, more preferably between 4,000 and 8,000, and most preferably wherein the PEG is PEG6000.

11. The method as claimed in any of the preceding claims, wherein the volume ratio of the PEG solution to whey is between 1:0.5 and 1:40; preferably between 1:0.8 and 1:20; more preferably between 1:2 and 1:8; and most preferably 1:

4.

12. The method as described in any of the preceding claims, wherein the centrifugation is performed between 1000 g and 2000 g.

13. Use of a product containing enriched milk EVs, obtainable using the method as described in any one of the preceding claims, in a food product or a pharmaceutical.

14. The use as described in claim 13, wherein the food product is a synthetic food product.

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

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