Methods to reduce muscle atrophy and / or promote muscle regeneration

Abstract

The present invention provides a nutritional composition for use in a method to reduce muscle atrophy and / or promote muscle regeneration in subjects at risk of muscle atrophy. [Solution] The nutritional composition comprises (i) at least one of protein, fat, and carbohydrate, and (ii) exosomes isolated from milk having an intact lipid membrane and a natural miRNA agent retained within the exosome, and the method for promoting muscle regeneration comprises orally administering the nutritional composition.

Description

Technical Field

[0001] The present invention is directed to a method for reducing muscle atrophy and / or promoting muscle regeneration by orally administering a nutritional composition containing bovine milk-derived exosomes containing non-damaged exosomes.

Background Art

[0002] Skeletal muscle is the most abundant tissue in the body. The amount and functionality of skeletal muscle are major determinants of muscle strength, endurance, and physical ability throughout the lifespan. Skeletal muscle is a plastic tissue that shows changes in muscle mass and fiber size depending on physiological and pathological conditions.

[0003] Skeletal muscle mass is maintained by a delicate balance between protein synthesis and protein breakdown. Muscle is a highly adaptable tissue that responds quickly to anabolic stimuli such as physical activity or food intake. Conversely, prolonged starvation or inactivity is known to cause rapid muscle weakness. Muscle atrophy occurs when the rate of protein breakdown exceeds the rate of protein synthesis. This phenomenon occurs under a variety of conditions. For example, muscle fatigue is a common feature of poor prognosis and negative outcomes in diseases such as acquired immunodeficiency syndrome (AIDS), cancer, diabetes, chronic obstructive pulmonary disease (COPD), amyotrophic lateral sclerosis (ALS), non-alcoholic fatty liver disease (NAFLD), and burns. Catalytic states in which muscle weakness prevails over muscle growth also occur during human life. In adults, muscle mass typically begins to decline gradually from around 35 to 40 years of age, at a rate of 0.4 to 1.0% per year. This age-related decline in muscle mass and strength occurs, and when this muscle loss occurs in otherwise healthy aging individuals, it is called sarcopenia (muscle weakness). This decline in muscle mass and strength can increase the risk of developing metabolic diseases and / or physical disabilities, and can lead to the inability to maintain daily functioning. There is a consensus in the scientific community that muscle atrophy is associated with a variety of undesirable outcomes, including delayed recovery from illness, decreased quality of life, physical disability, decreased resting metabolic rate, decreased insulin sensitivity, delayed wound healing, and increased healthcare costs.

[0004] Skeletal muscle tissue responds to anabolic stimuli, namely dietary protein intake and physical activity, for protein synthesis. However, for individuals affected by injury, illness, and / or aging, it is not always possible to perform physical activity sufficient for protein synthesis to maintain or increase muscle mass. Therefore, it is desirable to develop nutritional intervention strategies that resist or mitigate the decline in muscle mass and strength, and / or promote muscle regeneration. [Overview of the project]

[0005] The present invention relates to a method for reducing muscle atrophy and / or promoting muscle regeneration in subjects at risk of muscle atrophy. An object of the present invention is to provide such a method, particularly suitable for subjects where it is not easy and / or possible to implement physical activity interventions for protein synthesis that are sufficient to maintain or increase muscle mass.

[0006] In one embodiment, the present invention relates to a method for reducing muscle atrophy and / or promoting muscle regeneration in subjects at risk of muscle atrophy. This method involves orally administering a nutritional composition containing at least one of proteins, fats, and carbohydrates, as well as bovine milk-isolated exosomes containing intact exosomes.

[0007] The method of the present invention has the advantage of providing a simple method for reducing muscle atrophy and / or promoting muscle regeneration in subjects at risk of muscle atrophy. This method may be performed continuously for a period of time necessary depending on the subject's risk of muscle atrophy. These and other advantages of the method of the present invention will be better understood from the detailed description provided herein.

[0008] Specific aspects of the present invention will be specifically illustrated with reference to the following drawings. [Brief explanation of the drawing]

[0009] [Figure 1] As described in Example 2, we have shown proteolysis with dexamethasone (C) and proteolysis in the presence of undamaged bovine milk isolated exosomes (Ex). [Figure 2] As described in Example 2, this shows Akt phosphorylated (C) myotubes incubated alone and incubated with isolated bovine milk exosomes. [Figure 3] As described in Example 2, the effects of various components on the transcriptional activity of the ubiquitin promoter are demonstrated. [Figure 4]As described in Example 2, the effects of various components on atrogin-1 protein levels are shown. [Figure 5] As described in Example 2, the effects of various components on FoxO transcription activity are demonstrated. [Figure 6] As described in Example 2, the effects of undamaged bovine milk-isolated exosomes and sonication-treated exosomes on myoblast cells Mef2 are shown, respectively. [Modes for carrying out the invention]

[0010] While the general concept of the present invention can take many different embodiments, specific embodiments of the invention are described in detail herein, and it should be understood that this disclosure should be considered as illustrating the principles of the general concept of the invention. Therefore, there is no intention to limit the general concept of the invention to the exemplary specific embodiments described herein.

[0011] In one embodiment, the present invention relates to a method comprising administering a nutritional composition. The term “nutritional composition” as herein includes all forms of nutritional compositions unless otherwise specified, such forms include nutritional liquids, emulsified liquids, and liquids obtained by reconstituting nutritional powders and nutritional solids, for example, by adding water, and such nutritional powders and nutritional solids include, but are not limited to, powder form. The nutritional composition is suitable for oral administration in humans.

[0012] All percentages, fractions, and ratios in this specification are based on the weight of the total composition unless otherwise specified. Such weights for the listed components are based on their activity levels unless otherwise specified, and therefore do not include solvents or by-products that may be present in commercially available materials.

[0013] The technical terms used herein are for the sole purpose of describing embodiments, but should not be construed as limiting the entire disclosure. Unless otherwise explicitly stated, "[a]", "[an]", "[the]", and "[at least one]" are used synonymously. Furthermore, as used herein and in the accompanying claims, the singular forms "[a]", "[an]", and "[the]" also include their plural forms unless the context explicitly states otherwise.

[0014] Throughout this specification, where a numerical range is defined for a particular property of the Invention, the Invention also relates to and expressly includes all specific subranges that fall within that range. Furthermore, throughout this specification, where a group of substances is defined for a particular property of the Invention, the Invention also relates to and expressly includes all specific subgroups that fall within that group. Any particular range or group should be understood as a concise way of referring to all individual members of the range or group, as well as all possible subranges or subgroups contained therein.

[0015] Various embodiments of the nutritional composition used in the methods of this disclosure may substantially contain the optional or selected components or features described herein, provided that the remaining nutritional composition always contains all the necessary components or features described herein. In this regard, and unless otherwise specified, the term “substantially contained” means that the selected nutritional product contains an optional component in a functionally effective amount, typically less than 1% by weight of the optional or selected essential components, including less than 0.5%, less than 0.1%, and zero%.

[0016] The methods and nutritional compositions described herein may include, consist of, or essentially consist of, any of the essential steps and elements described herein, as well as any additional or optional steps and elements described herein. Any combination of the methods or processing steps described herein may be carried out in any order unless otherwise specifically stated or unless the context referring to such combination clearly indicates otherwise.

[0017] Unless otherwise specified herein, exemplary embodiments, dependent embodiments, specific embodiments, and optional embodiments are all exemplary embodiments, dependent embodiments, specific embodiments, and optional embodiments, respectively, relative to all embodiments described herein.

[0018] In one embodiment, the present invention relates to a method for reducing muscle atrophy and / or promoting muscle regeneration in subjects at risk of muscle atrophy. In a particular embodiment, the subject is a human. In other embodiments, the subject is a non-human animal. For example, the subject may be an elderly person, for example, over 40, over 45, over 50, over 55, over 60, over 65, over 70, or older. As described above, older adults typically exhibit some degree of muscle loss, and it may be difficult to prevent such muscle loss through dietary protein intake and exercise alone. This age-related decline in muscle mass and strength that occurs in healthy aging individuals is called sarcopenia (muscle weakness).

[0019] In another embodiment, the subject may experience events related to muscle atrophy or events related to the risk of developing muscle atrophy. For example, the subject may have acquired immunodeficiency syndrome (AIDS), cancer including cancer cachexia, diabetes, chronic obstructive pulmonary disease (COPD), amyotrophic lateral sclerosis (ALS), non-alcoholic fatty liver disease (NAFLD), or burns (these conditions typically include muscle fatigue). In another embodiment, the subject may be in a state of prolonged malnutrition and / or receiving clinical treatment with corticosteroids.

[0020] The present invention involves orally administering a nutritional composition containing at least one of proteins, fats, and carbohydrates, as well as bovine milk isolate exosomes containing intact exosomes. The inventors unexpectedly discovered that intact bovine milk isolate exosomes affect specific cellular mechanisms that contribute to reducing muscle atrophy and / or promoting muscle regeneration.

[0021] As used herein, “bovine milk isolated exosomes” refers, unless otherwise specified, to exosomes substantially isolated from other bovine milk components such as lipids, cells, and cell debris, and concentrated to a higher concentration than that present in bovine milk. Milk exosomes are small solid particles “dissolved” in bovine milk, making up only a small percentage of the total solids in the milk. Isolation of exosomes as described herein prepares a liquid substance in which the exosomes originally present in milk are concentrated. Obviously, bovine milk isolated exosomes may also contain other milk solids of similar size to milk exosomes and may be isolated together with the exosomes (i.e., casein and other whey proteins). As used herein, “powdered exosomes” refers, unless otherwise specified, to a dry powder containing exosomes isolated from bovine milk. The isolated exosomes are dried to form a dry powder. As described above, the isolated liquid containing exosomes also contains co-isolated milk solids, so the powdered exosomes also contain such other milk solids in the resulting powder. In one embodiment, the isolated exosomes contain at least 10 wt% exosomes, at least 15 wt% exosomes, at least 20 wt% or at least 25 wt% exosomes, and a well-balanced amount of other bovine milk isolated components.

[0022] What is important is that the powdery exosomes of the present invention contain intact exosomes. Intact exosomes have no damage to their lipid membranes, and the contents of the exosomes remain retained within the exosomes. Intact bovine milk exosomes contain various bioactive substances, for example, multiple miRNAs that enhance the healthy functions of various organs, tissues, and systems. However, when the lipid membrane of an exosome ruptures, factors such as miRNAs tend to decompose rapidly, and their beneficial functions are lost. Milk exosomes provide a protective environment for miRNAs, but in many current exosome isolation techniques, in most cases, damage is caused to the exosome membrane, resulting in the decomposition of bioactive substances. In the present invention, intact isolated exosomes obtained from bovine milk using gentle treatment that does not disrupt the exosome membrane are utilized, so that the exosomes are kept in an intact state and the bioactive substances are maintained in the exosome structure.

[0023] There are various methods for confirming that exosomes are intact. One such method is the method using uranyl acetate staining. For example, staining is performed with 2% uranyl acetate for 5 minutes. Uranyl acetate stains exosomes "negatively"; that is, uranyl acetate stains only the inner compartment of the exosome when the exosome membrane is disrupted or damaged. Furthermore, exosomes with damaged membranes tend to aggregate and lose their typical spherical morphology (for example, when observed at 10,000 times magnification using transmission electron microscopy (TEM)).

[0024] In one embodiment, the isolated and concentrated exosomes are provided as a suspension liquid. In another embodiment, the suspension of concentrated exosomes is dried for the purpose of providing powdered exosomes. There are various methods available for the careful isolation of exosomes to avoid disruption of the lipid membrane. Fresh bovine milk, refrigerated bovine milk, thawed frozen bovine milk, or stored bovine milk may be used as the exosome source. In certain embodiments, isolating the exosomes includes isolating them immediately after obtaining the milk from the cow. In another embodiment, isolating the exosomes includes isolating them within about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days from the time the milk is obtained from the cow. In certain embodiments, the exosomes are isolated within about 10 days, or about 14 days from the time the milk is obtained from the cow. In another embodiment, the bovine milk may be frozen and then thawed and subjected to exosome isolation processing, and the milk is preferably milk frozen within about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days from the time the milk is obtained from the cow. The thawed milk is preferably processed immediately upon thawing. In a particular embodiment, fresh bovine milk is subjected to processing within about 5 days from the time the milk is obtained from the cow, or the thawed bovine milk subjected to processing is thawed from bovine milk frozen within about 5 days from the time the milk is obtained from the cow.

[0025] In one embodiment, a whey-containing bovine milk fraction, or specifically, cheese whey, is used as the exosome source. In a particular embodiment, the pH of the bovine milk product is lowered to, for example, about 3.0 - 4.6 to precipitate the milk solids, and the whey-containing bovine milk fraction is obtained by removing the milk solids. Such fractions are often produced as a by-product of cheese production and are called cheese whey.

[0026] In one embodiment, exosomes are isolated from bovine milk or a bovine milk product such as cheese whey by centrifuging to form a lipid fraction uppermost layer, a whey fraction intermediate layer, and a first precipitate of cells and debris. The whey fraction is separated from the lipid fraction and the first precipitate and subjected to one or more further centrifuges, e.g., at higher speeds and optionally for longer periods, to prepare a substantially clear whey fraction. Further fats, casein aggregates, and cell debris are removed, e.g., to prepare a substantially clear whey fraction. The substantially clear whey fraction is then microfiltered to remove any remaining cell debris. Next, the microfiltered whey fraction is subjected to additional centrifugation for the purpose of obtaining a second precipitate containing exosomes. Then, the second precipitate is carefully suspended in an aqueous medium for the purpose of obtaining an exosome suspension in which the second precipitate is dissolved without disrupting the exosome membranes. It is important to suspend the second precipitate in a gentle manner that does not destroy the exosome membrane. In a particular embodiment, the second precipitate is incubated in an aqueous medium such as sterile phosphate-buffered saline (PBS) or water for a long period of time, for example, at least 6 hours, at least 8 hours, at least 10 hours, or at least 12 hours, and up to 18, 24, 30, or 36 hours or more. For example, a swirling shaker may be used at a low speed, i.e., 500 rpm or less. Once the precipitate is completely suspended, the suspension can be added to the nutritional composition to form a suspension, or it may be dried to obtain powdered exosomes. In either drying step, care should be taken to avoid destroying the lipid membrane of the exosomes. In a particular embodiment, the drying step includes freeze-drying.

[0027] In certain embodiments of the present invention, the isolation of exosomes involves centrifugation at a specific rate, time, and / or temperature. While centrifugation can cause damage to the exosome membrane if performed with excessive force, the centrifugation times and rates described herein yield undamaged exosome isolates. In certain embodiments, bovine milk is centrifuged at less than about 15,000 G, e.g., about 12,000 G, at e.g., about 4°C, for about 15 minutes, to obtain the whey layer formed between the top layer of fat (lipids) and the cell debris precipitate. In certain embodiments, the whey fraction is also centrifuged two more times, e.g., at about 21,000 G, at e.g., about 4°C, for about 30 minutes, under conditions that preserve the exosomes in an undamaged form, with each subsequent centrifugation aimed at further removing fat and / or debris. A substantially clear whey fraction is obtained. For example, the substantially clear whey fraction is microfiltered using a 0.22 μm filter made of a hydrophilic material such as polyethersulfone with low protein adsorption, and the microfiltered whey is centrifuged at 100,000 G and 4°C for about 60 minutes to prepare a precipitate containing exosomes. In certain embodiments, the precipitate containing exosomes is dissolved by incubation in a swirling shaker for at least about 12 hours, or about 12 to about 36 hours, or about 15 to about 30 hours, or about 18 to about 24 hours. This dissolution is carried out under conditions that preserve the exosomes in an undamaged form, i.e., under conditions that the contents of the exosomes are retained within the exosomes without damaging the membrane.

[0028] If a powder form is desired for storage or handling, or if it is desired to be added to a composition containing other components, such as a nutritional composition, the exosomes may be dried. Any such drying is carried out under conditions that preserve the exosomes in an undamaged form. In certain embodiments of the present invention, isolated exosomes are dried, for example, by freeze-drying or spray-drying, for the purpose of preparing powdered exosomes under conditions that preserve the exosomes in an undamaged form. In certain embodiments of the present invention, the freeze-drying step includes exposing the exosome suspension to a temperature of -80°C and a vacuum of less than 0.3 mbar for a sufficient amount of time. In certain embodiments, the time may vary depending on the amount of liquid to be freeze-dried and the nature of the freeze-dryer, and may vary between about 5 and about 40 hours, more specifically between about 10 and about 30 hours, or between about 15 and about 25 hours. Importantly, it is necessary to completely dry the exosomes in the step. In one particular embodiment, as described in the examples, the freeze-drying time at a temperature of -80°C and a vacuum of less than 0.3 mbar was 24 hours or more.

[0029] Further embodiments of obtaining undamaged bovine milk isolation exosomes for use in the method of the present invention include variations in freeze-drying time, temperature, and pressure. In another embodiment of the method, the milk exosomes are maintained at a temperature of at least about -50°C, or at least about -60°C, or at least about -70°C, or at least about -80°C, placed under a vacuum of less than about 0.3 mbar, or less than about 0.2 mbar, or less than about 0.1 mbar, and maintained under these conditions for at least about 5, 10, 15, 20, 25, 30, 35, or 40 hours.

[0030] Both the exosome suspensions and powdered exosomes produced by such methods contain intact exosomes; that is, exosomes whose membranes are not ruptured and / or degraded, and whose contents are retained within the exosomes. In certain embodiments, at least about 50 wt% of the exosomes in the exosome suspension or powder form are intact. In further embodiments, at least about 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt% of the exosomes in the exosome suspension or powder form are intact.

[0031] In certain embodiments, more than 90% of the isolated exosomes have a diameter of approximately 10 nanometers to 250 nanometers, or approximately 20 to 200 nm, or approximately 50 to 150 nm.

[0032] Certain embodiments of the nutritional compositions described herein include proteins, carbohydrates, and / or fats, as well as bovine milk-isolated exosomes. For the purpose of providing the therapeutic benefits described herein, such as reducing muscle atrophy and / or increasing muscle regeneration, the nutritional compositions may contain any desired effective amount of bovine milk-isolated exosomes. In a particular embodiment, the nutritional composition contains about 0.001 to about 10 wt% of bovine milk-isolated exosomes in a liquid suspension or powder form, based on the weight of the composition; or more specifically, about 0.1 to about 5 wt% of bovine milk-isolated exosomes. When referring to the amount of bovine milk-isolated exosomes in a composition, it refers to the amount of the suspension or powder containing the exosomes added to the composition.

[0033] In the embodiments of the nutritional compositions described herein, proteins, carbohydrates, and / or fats of various sources and of various types can be used. In certain embodiments, the nutritional composition used in the method comprises proteins, carbohydrates, and fats.

[0034] In a particular embodiment of the nutritional composition, protein accounts for about 1 wt% to about 30 wt% of the nutritional composition. In a more specific embodiment, protein accounts for about 1 wt% to about 25 wt% of the nutritional composition, which includes accounting for about 1 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%, or about 10 wt% to about 20 wt% of the nutritional composition. In another particular embodiment, protein accounts for about 1 wt% to about 5 wt% of the nutritional composition. In a further particular embodiment, protein accounts for about 20 wt% to about 30 wt% of the nutritional composition.

[0035] The nutritional composition may contain one or more types of proteins. In the nutritional composition used in the method of the present invention, proteins from various sources and of various types can be utilized. For example, protein sources may include, but are not limited to, the following: intact proteins, hydrolyzed proteins, and partially hydrolyzed proteins derived from any suitable source, such as milk (e.g., casein, whey), animals (e.g., meat, fish), grains (e.g., rice, brown rice, corn, barley, etc.), vegetables (e.g., soybeans, peas, yellow peas, broad beans, chickpeas, canola, potatoes, mung beans, quinoa, amaranth, and ancient grains such as chia, hemp, flax, etc.), and combinations of two or more of these. The protein may also contain, together with the intact proteins, hydrolyzed proteins, and partially hydrolyzed proteins described herein, one or a mixture of amino acids (often referred to as free amino acids) and / or their metabolites, or combinations of one or more such amino acids and / or metabolites, which are known to be used in nutritional products. The amino acids may be natural or synthetic amino acids.

[0036] More specific examples of suitable proteins for use in the exemplary nutritional compositions described herein include, but are not limited to, the following: whole egg powder, egg yolk powder, egg white powder, whey protein, whey protein concentrate, whey protein isolate, whey protein hydrolysate, acid casein, casein protein isolate, sodium caseate, calcium caseate, potassium caseate, casein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, skim milk powder, concentrated skim milk powder, whole bovine milk, partially or completely skimmed milk, coconut milk, soy protein concentrate, soy protein isolate. , soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, rice protein concentrate, rice protein isolate, rice protein hydrolysate, broad bean protein concentrate, broad bean protein isolate, broad bean protein hydrolysate, collagen protein, collagen protein isolate, meat proteins such as beef protein isolate and / or chicken protein isolate, potato protein, chickpea protein, canola protein, mung bean protein, quinoa protein, amaranth protein, chia protein, hemp protein, flax protein, earthworm protein, insect protein, and combinations of two or more of these. Suitable amino acids may be natural or synthetic amino acids. In one embodiment, the nutritional composition includes one or more branched-chain amino acids (leucine, isoleucine, and / or valine) and / or one or more metabolites of branched-chain amino acids, such as leucic acid (also known as α-hydroxyisocaproic acid or HICA), ketoisocaproate (KIC), and / or β-hydroxy-β-methylbutyrate (HMB) as proteins. The nutritional composition may include any individual protein source or any combination of any of the various protein sources listed above.

[0037] In certain embodiments, carbohydrates are present in an amount of about 5 wt% to about 75 wt% of the nutritional composition. In more specific embodiments, carbohydrates are present in an amount of about 5 wt% to about 70 wt% of the nutritional composition, which includes being 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%, or about 15 wt% to about 25 wt% of the nutritional composition.

[0038] Suitable carbohydrates for use in the nutritional composition may be simple sugars, complex sugars, heteromorphic molecular species, or combinations thereof. Various carbohydrate sources are available, as long as they are suitable for use in the nutritional composition or compatible with any other selected components or features present in the nutritional composition. Non-limiting examples of suitable carbohydrates for use in the nutritional composition include: maltodextrin, hydrolyzed or processed starch, hydrolyzed or processed corn starch, glucose polymers such as polydextrose and dextrins, corn syrup, corn syrup solids, rice maltodextrin, rice-derived carbohydrates such as soft, powdery brown rice flour and brown rice syrup, sucrose, glucose, fructose, lactose, high-fructose corn syrup, honey, sugar alcohols (e.g., reduced maltose, erythritol, sorbitol), isomaltulose, sucromalt, pullulan, potato starch, corn starch, fructooligosaccharides, Galactooligosaccharides, oat fiber, soy fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, gellan gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, acacia gum, chitosan, arabinolactin, glucomannan, xanthan gum, alginic acid, pectin, low methoxypectin, high methoxypectin, cereal β-glucans, carrageenan, psyllium, Fibersol®, fruit puree, vegetable puree, isomaltoligosaccharides, monosaccharides, disaccharides, tapioca-derived carbohydrates, inulin, other digestible starches, and artificial sweeteners, and combinations of two or more of these. The nutritional composition may contain any individual carbohydrate source listed above or any combination of various carbohydrate sources.

[0039] In this specification, the term “fat” refers to lipids, fats, oils, and combinations thereof, unless otherwise specified. In certain embodiments, the nutritional composition constitutes about 0.5 wt% to 20 wt% of the nutritional composition. In more specific embodiments, fat constitutes about 0.5 wt% to 18 wt% of the nutritional composition, which includes about 0.5 wt% to 15 wt%, about 0.5 wt% to 10 wt%, about 0.5 wt% to 5 wt%, about 2 wt% to 8 wt%, about 5 wt% to 10 wt%, about 8 wt% to 12 wt%, or about 12 wt% to 18 wt% of the nutritional composition.

[0040] Suitable fats for use in nutritional compositions include, but are not limited to, algal oil, canola oil, linseed oil, borage oil, safflower oil, high-oleic safflower oil, high-gamma-linolenic acid (GLA) safflower oil, corn oil, soybean oil, sunflower oil, high-oleic sunflower oil, cottonseed oil, coconut oil, fractionated coconut oil, medium-chain triglyceride (MCT) oils, palm oil, palm kernel oil, palm olein, and long-chain polyunsaturated fatty acids such as docosahexanoic acid (DHA), arachidonic acid (ARA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), and combinations thereof.

[0041] The concentrations and relative amounts of protein, carbohydrates, and / or fat in the exemplary nutritional composition will vary considerably depending, for example, on the specific dietary needs of the intended user. In one particular embodiment, the nutritional composition contains, based on the weight of the nutritional composition, about 2 wt% to 15 wt% of protein, about 5 wt% to 25 wt% of carbohydrates, and about 0.5 wt% to 12 wt% of fat.

[0042] In certain embodiments, the nutritional composition has a neutral pH, i.e., a pH of about 6 to 8, or more specifically, a pH of about 6 to 7.5. In more specific embodiments, the nutritional composition has a pH of about 6.5 to 7.2, or more specifically, a pH of about 6.8 to 7.1.

[0043] The nutritional composition may further contain one or more additional components that can alter the physical, chemical, aesthetic, or processing properties of the nutritional composition, or that can function as additional nutrients. Non-limiting examples of additional components include preservatives, emulsifiers (e.g., lecithin), buffers, sweeteners including artificial sweeteners (e.g., saccharin, aspartame, acesulfame K, sucralose), colorants, flavorings, thickeners, and stabilizers.

[0044] Furthermore, the nutritional composition may further contain vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin B12, vitamin C, vitamin D, vitamin K, thiamine, riboflavin, pyridoxine, niacin, folic acid, pantothenic acid, biotin, choline, inositol, its salts and derivatives, and combinations thereof. Water-soluble vitamins may be added in the form of a water-soluble vitamin (WSV) premix, and / or fat-soluble vitamins may be added to one or more oily carriers as needed.

[0045] In another embodiment, the nutritional composition may further contain one or more minerals, non-limiting examples of which include calcium, phosphorus, magnesium, zinc, manganese, sodium, potassium, molybdenum, chromium, chlorine, and combinations thereof.

[0046] The nutritional composition may be prepared using any technique known in the art. In one embodiment, the nutritional composition may be prepared by the following steps: (a) preparing an aqueous solution containing proteins and carbohydrates; (b) preparing a mixed oil containing fats and lipid-soluble components; and (c) mixing the aqueous solution and the mixed oil together to prepare an emulsified liquid nutritional composition. The undamaged exosomes may be added at a desired time in the process, for example, to the aqueous solution or the emulsified mixture. The undamaged exosomes may be dry-mixed with one or more dry components in powder form, for example, for the purpose of adding them in combination to a liquid composition, or if a powdered nutritional product is desired.

[0047] A method for reducing muscle atrophy and / or increasing muscle regeneration includes administering a nutritional composition, such as those described herein, to a subject at risk of muscle atrophy. The nutritional composition may be administered in powder or liquid form, as may be required. In certain embodiments, a nutritional composition comprising bovine milk-isolated exosomes is administered to the subject once or more times per day or week. In certain embodiments, the nutritional composition is administered to the subject about once to about six times per day or week, or about once to about five times per day or week, or about once to about four times per day or week, or about once to about three times per day or week. 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.

[0048] In a particular embodiment, the nutritional composition is administered in an effective amount to reduce muscle atrophy and / or increase muscle regeneration. In another particular embodiment, a sufficient amount of the nutritional composition is administered to result in a dose of approximately 0.01 g to approximately 10 g of isolated bovine milk exosomes. In yet another embodiment, the administration of the nutritional composition targets a dose of approximately 0.1 to approximately 10 g or approximately 1 to approximately 5 g of isolated bovine milk exosomes.

[0049] The following examples illustrate aspects of the method of the present invention, but they are presented solely for the purpose of illustrating specific examples. These examples should not be considered limiting to the general concept of the present invention, for many modifications are possible without departing from the spirit and scope of the general concept of the present invention.

[0050] Example 1 This example describes the preparation of powdered exosomes. First, exosomes were isolated from bovine milk and then dried. Specifically, untreated raw bovine milk was dispensed and immediately frozen at -80°C. The dispensed material was thawed on ice and subjected to a first centrifugation at approximately 4°C and 12,000 G for approximately 15 minutes to obtain the whey layer formed between the uppermost layer of fat (lipids) and the cell debris precipitate. This whey fraction was transferred to a clean tube and subjected to two further centrifugations to remove fat and cell debris; each centrifugation was performed at approximately 4°C, 21,000 G, and 30 minutes, conditions that kept the exosomes in an undamaged form. A substantially clear whey fraction was obtained. The substantially clear whey fraction was microfiltered using a 0.22 μm hydrophilic polyethersulfone filter, and the microfiltered whey was then centrifuged at 100,000 G at 4°C for approximately 60 minutes to obtain a precipitate containing exosomes. The exosome-containing precipitate in the centrifugation tube was carefully suspended in sterile PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4; pH 7.4) or molecular biology-grade sterile water, and then incubated with a swirling shaker at 150 rpm at 4°C for 12–36 hours. Notably, without the swirling shaker step, aggregates appeared and required sufficient pipetting to disintegrate, which resulted in exosome membrane damage.

[0051] First, the exosomes were frozen at -80°C for at least 2 hours, and then the frozen exosomes were freeze-dried under conditions that preserved the exosomes in an undamaged form. Specifically, this freeze-drying process involved placing the frozen exosomes at -80°C and under a vacuum of less than 0.3 mbar for a sufficient time (approximately 24 hours) to achieve a low moisture content. The resulting product consisted of powdered exosomes.

[0052] Importantly, thawing the exosomes before the vacuum stage of freeze-drying damaged the membranes of the milk exosomes. Therefore, embodiments of the method described herein include freezing the milk exosomes and keeping them frozen until freeze-drying is complete and powdered milk exosomes are obtained. Under the isolation and drying conditions described herein, the integrity of the exosomal lipid membrane and the bioactivity of the exosomal contents are preserved.

[0053] Example 2 A portion (the first portion) of the powdered, undamaged bovine milk exosomes prepared in Example 1 was dissolved in water. The second portion of the powdered exosomes prepared in Example 1 was dissolved in water and treated with an Ultrasons P-selecta sonifier for 1 hour. After sonication, the sonicated milk exosomes were incubated at 95°C for 15 minutes to completely destroy the milk exosome membrane.

[0054] The ability of exosomes to influence the mechanisms causing muscle atrophy and muscle regeneration was evaluated using in vitro assays. In vitro experiments were performed using the L6.C11 rat skeletal muscle myoblast cell line (ECACC number: 92102119). The cell line was grown in DMEM medium (Dulbecco's modified Eagle medium) supplemented with 10% (v / v) fetal bovine serum (FBS), 2 mmol / L glutamine, 100 units / ml penicillin, and 0.1 mg / ml streptomycin, under an atmosphere of 5% CO2 and 95% air, and then maintained at a sub-confluent density in growth medium. The cells were differentiated into myotubes by culturing them in DMEM containing 2% FBS (v / v) for 5 days.

[0055] Dexamethasone-Induced Proteolysis: To evaluate the ability of isolated bovine milk exosomes to reduce muscle atrophy / muscle fatigue, proteolysis was first evaluated in the presence or absence of undamaged milk exosomes. Dexamethasone, a glucocorticoid that induces proteolysis in muscle, was used to mimic the pathological state caused by increased catabolism. Myoblasts from L6.C11 rat skeletal muscle were differentiated into myotubes (5 days in differentiation medium), and undamaged isolated bovine milk exosomes or sonicated exosomes were labeled with 1 μCi / ml L-[ring-3,5-3H]-tyrosine for 48 hours in 10% FBS-containing DMEM medium containing 15 μg / ml each. After rinsing the cells once with PBS-Tyr, they were placed in DMEM (dexamethasone, degradation medium) supplemented with 10% FBS, 2 mM L-tyrosine, and 5 μM DEX for 2 hours to degrade the very short-lived proteins. Next, the cells were rinsed twice with PBS-Tyr and fresh degradation medium was added. The cells were incubated in the degradation medium for a further 24 hours. At the end of incubation, the medium was transferred to a microcentrifuge tube containing 100 μl of bovine serum albumin (BSA) (10 mg / ml) to measure the degradation rate. Trichloroacetic acid (TCA) was added to a final concentration of 10% (w / v). After incubation at 4°C for at least 1 hour, the sample was centrifuged for 5 minutes. The precipitate was then dissolved in a tissue lysis agent. Monolayer cells were washed with ice-cold PBS and then solubilized with 0.5 M NaOH containing 0.1% Triton X-100. Radioactivity was measured using a Beckman LS6000 SE scintillation counter. Proteolysis was expressed as "24-hour protein degradation %".

[0056] As can be seen in Figure 1, when muscle cells were incubated in the presence of dexamethasone, the addition of undamaged bovine milk isolate exosomes (Ex) reduced dexamethasone-induced proteolysis, and this reduction was statistically significant, compared to a control without exosomes (C). This is a very reasonable result, assuming that muscle proliferation reflects a balance between protein anabolism and catabolism. Therefore, undamaged bovine milk isolate exosomes are useful for treating or preventing conditions characterized by increased proteolysis rates.

[0057] Molecular markers for protein degradation and expression Further experiments were conducted to demonstrate the effects of undamaged bovine milk exosomes, and in some cases sonicated milk exosomes, on key molecular markers. These key molecular markers were specifically the ubiquitin-proteasome pathway markers Akt, FoxO, ubiquitin, and atrogin-1 as molecular markers related to muscle atrophy, and the marker Mef2 as a molecular marker related to muscle regeneration and muscle formation.

[0058] To test the phosphorylation status of proteins involved in signal transduction events, L6 myotubes prepared as described above were incubated with 15 μg / ml of undamaged bovine milk isolation exosomes for 15, 30, 45, and 60 minutes. To prepare protein extracts, the plates were rapidly frozen in liquid nitrogen. Cells were scraped from 60 mm plates using 400 μl of cold solution per plate; the composition of the solution used was as follows: 30 mM Tris-HCl (pH 7.4), 25 mM NaCl, 1% (v / v) Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM ethylene glycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA), 20 nM okadaic acid, 10 μg / ml aprotinin, 10 μg / ml leupeptin, and 10 μg / ml pepstatin. After being placed on ice for 10 minutes, the extracts were centrifuged at 4°C and 13,000 G for 10 minutes. Protein concentrations were measured using the bicinchonic acid method. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Schleicher & Schull), and then immunoblotted with a selective antibody. The immunoblots were developed using an enhanced chemiluminescence detection system (Amersham Biosciences) according to the manufacturer's instructions.

[0059] To test the expression of proteolytic proteins FoxO, ubiquitin, and atrogin-1, L6 myotubes prepared as described above were pre-incubated for 48 hours with 15 μg / ml of undamaged bovine milk exosomes or sonicated milk exosomes, respectively, and then incubated for 24 hours with 5 μM dexamethasone (DEX) in or without an effector. If 100 nM bafilomycin A1 was used, it was added together with DEX and maintained in the treatment throughout the process. After treatment, cells were lysed with RIPA buffer containing phosphatase inhibitors and protease inhibitors, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM EGTA, 20 nM okadaic acid, 10 μg / ml aprotinin, 10 μg / ml leupeptin, and 10 μg / ml pepstatin. Protein concentration was measured using a method involving bicinconic acid. Protein (40 μg) was separated by SDS-PAGE and transferred to a nitrocellulose membrane, and immunoblotting was performed using a specific antibody; the immunoblot was developed by a sensitized chemiluminescence detection method.

[0060] To test MEF2 expression during differentiation, L6 myoblasts were treated with 15 μg / ml of undamaged bovine milk isolated exosomes or sonicated exosomes for 3 days. As described above, the cells were treated with RIPA and phosphatase inhibitors.

[0061] The protein kinase Akt is one of the major regulators of proteolysis and subsequent muscle atrophy. This process, which includes myofibrils degenerating due to the net loss of proteins, intracellular organelles, and cytoplasm, primarily occurs via the ubiquitin-proteasome pathway. Under anabolic conditions, the ubiquitin-proteasome pathway is inhibited by phosphorylated Akt, which occurs by inactivating the phosphorylation of the forkheadbox O (FoxO) transcription factor. Figure 2 shows the effect of incubation with undamaged bovine milk exosomes on the Akt phosphorylation state. When myotubes were incubated with undamaged bovine milk exosomes, Akt phosphorylation was significantly increased compared to the control (C) in the absence of exosomes.

[0062] Within the ubiquitin-proteasome degradation pathway, two elements are considered to be the main effectors of muscle atrophy. The first is the ubiquitin gene, which is induced under degradation conditions and produces a small regulatory ubiquitin protein. While the degraded protein undergoes ubiquitin tagging, ubiquitin itself is a monomeric 76-amino acid protein that acts as a signal to guide the ubiquitinated protein to the proteasome. This reaction is catalyzed by two ubiquitin ligases, atrogin and Murfl. To induce ubiquitin-proteasome-mediated proteolysis, both ubiquitin and ubiquitin ligase levels must increase. Figure 3 shows that ubiquitin promoter transcriptional activity is induced by incubation with dexamethasone (Dex). Dexamethasone-induced transcriptional activity of the ubiquitin promoter is a prominent feature of proteolysis. When myotubes were incubated with either intact bovine milk exosomes (Ex) alone or sonicated milk exosomes (sEx) alone, the transcriptional activity induction described above was not observed, suggesting that neither intact bovine milk exosomes nor sonicated milk exosomes themselves activate the ubiquitin-proteasome degradation pathway. When cells were treated with dexamethasone and intact bovine milk exosomes (Dex+Ex), the measured level of ubiquitin promoter transcriptional activity was equivalent to the level of the control without dexamethasone. Therefore, lyophilized exosomes acted protectively against dexamethasone-induced ubiquitin promoter transcriptional activity. Notably, when cells were treated with dexamethasone and sonicated exosomes (Dex+sEx) and the exosome membrane was disrupted, the protective effect against protein degradation described above was not observed.

[0063] In addition to increasing ubiquitin gene transcription, activation of the ubiquitin-proteasome pathway requires compartment-specific ubiquitin ligase complexes that target substrates with ubiquitin for proteasome degradation. Therefore, we evaluated the protein levels of atrogin-1, a muscle-specific ubiquitin ligase, in myotubes. As shown in Figure 4, dexamethasone (Dex) significantly increased the expression level of atrogin-1, but neither isolated bovine milk exosomes (Ex) nor sonicated milk exosomes (sEx) alone increased atrogin-1 expression to a statistically significant level; that is, the respective p-values ​​were higher than 0.05, and therefore statistically, neither isolated bovine milk exosomes (Ex) nor sonicated milk exosomes (sEx) were considered different from the control (C). Only Dex was statistically different from the control (C). As can be further seen in Figure 4, incubating myotubes with dexamethasone and isolated bovine milk exosomes (Dex+Ex) suppressed the dexamethasone-induced increase in atrogin-1 levels. However, this protective effect was not obtained when myotubes were incubated with dexamethasone and sonicated exosomes (Dex+sEx). Thus, the increase in atrogin-1 protein levels necessary for proteolysis by the ubiquitin-proteasome system was prevented by using isolated bovine milk exosomes.

[0064] As described above, phosphorylated Akt inhibits the ubiquitin-proteasome pathway by inactivating the phosphorylation of the FoxO transcription factor. Therefore, we also decided to evaluate FoxO gene transcription. FoxO is a master regulator of the ubiquitin-proteasome catabolism pathway, and its phosphorylation is necessary for increased protein degradation. As shown in Figure 5, when myotubes were incubated with dexamethasone (Dex) alone, FoxO promoter transcriptional activity was significantly induced. Incubation with either intact bovine milk isolated exosomes (Ex) alone or sonicated exosomes (sEx) alone did not result in an increase in FoxO promoter transcriptional activity. This is also quite reasonable if we assume that neither intact bovine milk isolated exosomes nor sonicated lyophilized exosomes alone cause protein degradation. When myotubes were incubated with dexamethasone and isolated bovine milk exosomes (Dex+Ex), the transcriptional activity of the FoxO promoter was significantly reduced. Notably, when myotubes were incubated with dexamethasone and sonicated milk exosomes (Dex+sEx), the above protective effect was not observed. Thus, the dexamethasone-induced transcriptional activity of the proteolytic promoter factor FoxO was abolished by isolated bovine milk exosomes, but this did not occur with sonicated milk exosomes.

[0065] The effects of undamaged bovine milk-isolated exosomes on muscle hypertrophy or muscle differentiation were also investigated; specifically, this was done by measuring Mef2 levels. During muscle cell differentiation, Mef2 levels increase, leading to the formation of mature myotubes, which are necessary for muscle proliferation and regeneration. As can be seen in Figure 6, when L6 myoblasts were transferred to differentiation medium (C), Mef2 expression transiently increased from day 0 levels, particularly on days 1 and 2. As can be further seen in Figure 6, when undamaged bovine milk-isolated exosomes were added to differentiation medium (Ex), Mef2 expression significantly increased over all three days. No such induction occurred when sonicated exosomes were added to differentiation medium (sEx).

[0066] In summary, these results indicate the following: (1) Undamaged bovine milk isolate exosomes do not induce proteolysis or muscle fatigue on their own; (2) Under conditions of muscle wasting, undamaged bovine milk isolate exosomes can mitigate proteolysis by inhibiting the ubiquitin-proteasome pathway; (3) Undamaged bovine milk isolate exosomes can significantly increase Mef2 markers for muscle regeneration and muscle differentiation; and (4) these effects conferred by undamaged bovine milk isolate exosomes are not obtained with membrane-ruptured, sonicated bovine milk exosomes. Thus, undamaged bovine milk isolate exosomes do both: protect muscle cells from proteolysis and reduce muscle atrophy, and promote muscle regeneration by significantly increasing Mef2 markers for muscle regeneration and muscle differentiation.

[0067] Therefore, undamaged bovine milk isolated exosomes provide a novel tool for promoting muscle protein anabolism over muscle catabolism, thereby reducing muscle atrophy and promoting muscle regeneration in subjects with or at risk of diseases or conditions including muscle fatigue.

[0068] The present application is specifically described by its embodiments, and these embodiments are described in considerable detail, but these descriptions are not intended in any way to limit or restrict the scope of the accompanying claims to such detail. Further advantages and modifications will be readily apparent to those skilled in the art. Thus, in its broader context, the present invention is not limited to those specific details, representative compositions and methods, or specific examples presented and described. That is, various modifications and variations are possible starting from such details without departing from the spirit or scope of the general concept of the present invention.

Claims

1. A method for reducing muscle atrophy and / or promoting muscle regeneration, comprising orally administering to a subject at risk of muscle atrophy a nutritional composition containing at least one of protein, fat, and carbohydrate, as well as bovine milk-isolated exosomes containing intact exosomes.

2. A method according to claim 1, wherein more than 90% of the isolated bovine milk exosomes are approximately 10 nanometers to approximately 250 nanometers in diameter.

3. A method according to claim 1 or 2, wherein the nutritional composition comprises about 0.001 to about 10 wt% of isolated bovine milk exosomes based on the weight of the nutritional composition.

4. A method according to any one of claims 1 to 3, wherein the subject is an adult over 40 years of age.

5. A method according to any one of claims 1 to 4, wherein the subject is suffering from malnutrition, acquired immunodeficiency syndrome (AIDS), cancer, diabetes, chronic obstructive pulmonary disease (COPD), amyotrophic lateral sclerosis (ALS), non-alcoholic fatty liver disease (NAFLD), or burns, or is receiving clinical treatment with corticosteroids.

6. A method according to any one of claims 1 to 5, for reducing muscle atrophy.

7. The method according to any one of claims 1 to 6, which promotes muscle regeneration.

8. A method according to any one of claims 1 to 7, wherein the nutritional composition comprises protein.

9. A method according to claim 8, wherein the nutritional composition further comprises fat and carbohydrates.

10. A method according to any one of claims 1 to 9, wherein the nutritional composition is: whole egg powder, egg yolk powder, egg white powder, whey protein, whey protein concentrate, whey protein isolate, whey protein hydrolysate, acid casein, casein protein isolate, sodium caseate, calcium caseate, potassium caseate, casein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, skim milk powder, concentrated skim milk powder, whole bovine milk, partially or completely skimmed milk, coconut milk, soy protein concentrate, soy protein isolate, soy protein hydrolysate, endo A method comprising: green bean protein concentrate, pea protein isolate, pea protein hydrolysate, rice protein concentrate, rice protein isolate, rice protein hydrolysate, broad bean protein concentrate, broad bean protein isolate, broad bean protein hydrolysate, collagen protein, collagen protein isolate, meat protein, potato protein, chickpea protein, canola protein, mung bean protein, quinoa protein, amaranth protein, chia protein, hemp protein, flax protein, earthworm protein, insect protein, and at least one protein selected from a combination of two or more of these.

11. A method according to any one of claims 1 to 10, wherein the nutritional composition comprises algal oil, canola oil, linseed oil, borage oil, safflower oil, high-oleic safflower oil, high-gamma-linolenic acid (GLA) safflower oil, corn oil, soybean oil, sunflower oil, high-oleic sunflower oil, cottonseed oil, coconut oil, fractionated coconut oil, medium-chain triglyceride (MCT) oil, palm oil, palm kernel oil, palm olein, long-chain polyunsaturated fatty acids, and at least one fat selected from two or more combinations thereof.

12. A method according to any one of claims 1 to 11, wherein the nutritional composition comprises maltodextrin, hydrolyzed starch, modified starch, hydrolyzed corn starch, modified corn starch, polydextrose, dextrins, corn syrup, corn syrup solids, rice maltodextrin, soft powdery brown rice powder, brown rice syrup, sucrose, glucose, fructose, lactose, high-fructose corn syrup, honey, reduced maltose, erythritol, sorbitol, isomaltulose, sucromalt, pullulan, potato starch, corn starch, fructooligosaccharides, galactooligosaccharides, oat fiber, soy fiber A method comprising fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, gellan gum, locust bean gum, konjac powder, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, acacia gum, chitosan, arabinolactin, glucomannan, xanthan gum, alginic acid, pectin, low methoxypectin, high methoxypectin, cereal β-glucans, carrageenan, psyllium, fiber, fruit puree, vegetable puree, isomaltoligosaccharides, monosaccharides, disaccharides, tapioca-derived carbohydrates, inulin, and artificial sweeteners, as well as at least one carbohydrate selected from combinations of two or more of these.

13. A method according to any one of claims 1 to 12, wherein the nutritional composition comprises, based on the weight of the nutritional composition, about 1 wt% to about 30 wt%, about 1 wt% to about 25 wt%, about 1 to about 20 wt%, about 1 to about 15 wt%, about 1 to about 10 wt%, or about 10 wt% to about 30 wt% of protein.

14. A method according to any one of claims 1 to 13, wherein the nutritional composition contains, based on the weight of the nutritional composition, 0.5 wt% to 20 wt%, about 0.5 to about 15 wt%, about 0.5 to about 10 wt%, about 0.5 to about 5 wt%, or about 5 to about 15 wt% of fat.

15. A method according to any one of claims 1 to 14, wherein the nutritional composition comprises, based on the weight of the nutritional composition, about 5 wt% to about 75 wt%, about 5 wt% to about 70 wt%, 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%, or about 15 wt% to about 25 wt% of carbohydrates.

16. A method according to any one of claims 1 to 15, wherein the nutritional composition is a liquid nutritional composition, and the nutritional composition comprises, based on the weight of the nutritional composition, about 1 to about 15 wt% of protein, about 0.5 to about 10 wt% of fat, and about 5 to about 30 wt% of carbohydrates.

17. A method according to any one of claims 1 to 15, wherein the nutritional composition is a powdered nutritional composition, and the nutritional composition comprises, based on the weight of the nutritional composition, about 10 to about 30 wt% of protein, about 5 to about 15 wt% of fat, and about 30 wt% to about 65 wt% of carbohydrates.

18. A method according to any one of claims 1 to 17, wherein the nutritional composition comprises at least one protein, including milk protein concentrate and / or soy protein isolate; at least one fat, including canola oil, corn oil, coconut oil and / or fish oil; and at least one carbohydrate, including maltodextrin, sucrose and / or short-chain fructooligosaccharides.

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