Use of milk-derived extracellular vesicles in improving brain health and / or improving a nerve-related discomfort condition

Abstract

The present application belongs to the field of biological medicine and food technology, and particularly relates to application of milk-derived extracellular vesicles in improving brain health and / or improving nerve-related discomfort conditions. Compared with the prior art, the present application constructs a chronic restraint stress (CRS) induced anxiety model in adolescent mice, and uses open field test, buried bead test, tail suspension test and other behavioral experiments for functional evaluation. The results show that the milk-derived extracellular vesicles significantly improve the anxiety-like behavior of mice, indicating that the milk-derived extracellular vesicles can significantly improve the anxiety-like behavior of adolescent mice induced by chronic restraint stress (CRS) by remodeling the gut-brain axis function, and that the supplementation of milk-derived extracellular vesicles in adolescents can significantly reduce the susceptibility to anxiety in adulthood. At the same time, the present application also constructs a D-gal induced cognitive impairment model in mice, and the results show that it can reduce the production of pro-inflammatory cytokines, inhibit mucosal inflammation, thereby maintaining intestinal homeostasis and alleviating cognitive impairment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of a milk-derived extracellular vesicle in improving brain health and / or alleviating neurological discomfort. Background Technology

[0002] Anxiety disorders are chronic debilitating illnesses characterized by intense and persistent feelings of fear and distress. As the most prevalent category of mental disorders worldwide, they contribute significantly to global health losses, functional impairments, and adverse consequences throughout the lifespan. These disorders typically manifest early in life and follow a recurring or fluctuating course, making them among the most disabling mental illnesses. While traditional therapies such as psychotherapy and medication have proven effective, their effectiveness is often limited by slow onset of action, adverse reactions, and patient adherence issues, highlighting the need for safer, more sustainable, and acceptable prevention and treatment options. With the emergence of the concept of "precision nutrition" to improve national health, alleviating brain health issues related to anxiety through dietary interventions is of great significance.

[0003] Furthermore, a major limitation of current medications for neurological disorders such as anxiety and cognitive impairment is that they can only alleviate symptoms and cannot cure the disease. In addition, long-term use often leads to serious side effects. Therefore, exploring natural compounds with potential preventative or allergic effects is crucial. Natural bioactive compounds extracted from the diet have attracted considerable attention due to their potential neuroprotective effects. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an application of milk-derived extracellular vesicles in improving brain health and / or improving neurological-related discomfort.

[0005] This invention provides the application of milk-derived extracellular vesicles in the preparation of products that improve brain health and / or alleviate neurological discomfort.

[0006] Preferably, the improvement of brain health and / or improvement of neurological discomfort includes one or more of the following: relieving anxiety, improving memory, and improving cognitive impairment;

[0007] And / or, the milk-derived extracellular vesicles improve brain health and / or alleviate neurological discomfort by remodeling gut-brain axis function;

[0008] And / or, the milk-derived extracellular vesicles improve brain health and / or alleviate neurological discomfort by modulating inflammatory pathways.

[0009] Preferably, the milk-derived extracellular vesicles improve brain health and / or alleviate neurological discomfort by relieving one or more of the following: chronic stress-anxiety-induced growth retardation, reducing stress-related hormone levels, reducing inflammatory cytokine expression levels, and improving gut microbiota metabolism.

[0010] Preferably, the milk-derived extracellular vesicles are selected from milk-derived extracellular vesicles derived from cow's milk and / or milk-derived extracellular vesicles derived from sheep's milk;

[0011] And / or, the average particle size of the extracellular vesicles of the milk source is 100~150 nm.

[0012] Preferably, the milk-derived extracellular vesicles are prepared according to the following method:

[0013] S1) The milk raw material is centrifuged for the first time at low temperature, and the intermediate whey fraction is collected;

[0014] S2) Mix the intermediate whey fraction, rennet and calcium salt and incubate. Then perform a second centrifugation, take the supernatant, and filter it through a filter membrane and then centrifuge it at high speed to obtain milk-derived extracellular vesicles.

[0015] Preferably, the ratio of the rennet to the milk raw material is 0.01~0.1 g: 100 mL;

[0016] And / or, the calcium salt is selected from calcium chloride;

[0017] And / or, the ratio of the calcium salt to the milk raw material is 0.1~0.5 g: 100 mL;

[0018] And / or, the low temperature is 2℃~6℃;

[0019] And / or, the centrifugal force of the first centrifugation is 2000~5000 g; the time of the first centrifugation is 10~20 min;

[0020] And / or, the centrifugal force of the second centrifugation is 10,000 to 20,000 g; the time of the second centrifugation is 10 to 60 min;

[0021] And / or, the ultracentrifugation includes: first centrifuging for a first set time under a first set centrifugal force, and then centrifuging for a second set time under a second set centrifugal force; the first set centrifugal force is 50,000~100,000 g; the first set time is 30~90 min; the second set centrifugal force is 100,000~150,000 g; and the second set time is 30~90 min.

[0022] Preferably, the dosage of the milk-derived extracellular vesicles is 0.3~30 mg / d for a person weighing 60 kg.

[0023] The present invention also provides a composition comprising extracellular vesicles of milk cells for improving brain health and / or improving neurological discomfort.

[0024] The present invention also provides the use of milk-derived extracellular vesicles in the preparation of products for the prevention, treatment and / or improvement of chronic stress-anxiety-like behaviors.

[0025] The present invention also provides a composition comprising milk-derived extracellular vesicles for the prevention, treatment and / or improvement of chronic stress-anxiety-like behaviors.

[0026] This invention also provides the application of milk-derived extracellular vesicles in the preparation of products for the prevention, treatment and / or improvement of nervous system diseases.

[0027] Preferably, the neurological disease is selected from cognitive impairment and / or mental disorders; preferably, the mental disorder includes one or more of stress, anxiety, panic disorder and depression.

[0028] The present invention also provides a composition for the prevention, treatment and / or improvement of nervous system diseases, comprising extracellular vesicles derived from milk cells.

[0029] This invention provides an application of milk-derived extracellular vesicles in improving brain health and / or alleviating neurological-related discomfort. Compared with existing technologies, this invention constructs a chronic restraint stress (CRS)-induced anxiety model in adolescent mice and uses behavioral experiments such as the open field test, bead embedding test, and tail suspension test for functional evaluation. The results show that milk-derived extracellular vesicles significantly improve anxiety-like behavior in mice, indicating that milk-derived extracellular vesicles can significantly improve CRS-induced anxiety-like behavior in adolescent mice by remodeling gut-brain axis function, and that supplementing adolescents with milk-derived extracellular vesicles can significantly reduce anxiety susceptibility in adulthood. Simultaneously, this invention also constructs a D-gal-induced cognitive impairment model in mice, showing that it can maintain intestinal homeostasis and alleviate cognitive impairment by reducing the production of pro-inflammatory cytokines and inhibiting mucosal inflammation. Attached Figure Description

[0030] Figure 1 The graph shows the performance analysis results of gEVs prepared in Example 1 of this invention.

[0031] Figure 2 This is a graph showing the average body weight distribution of mice in different groups at different times in Example 1 of the present invention;

[0032] Figure 3 This is a graph showing the average serum corticosterone levels in each group of mice in Example 1 of the present invention;

[0033] Figure 4This is a route distribution diagram of each group of mice in the open field experiment in Example 1 of the present invention;

[0034] Figure 5 This is a graph showing the central zone dwell time results of each group of mice in the open field experiment of Example 1 of the present invention;

[0035] Figure 6 This is a graph showing the percentage of beads buried in each group of mice in Example 1 of the present invention;

[0036] Figure 7 This is a graph showing the resting time of different groups of mice in the tail suspension test in Example 1 of the present invention;

[0037] Figure 8 These are images showing the histological analysis results of mice in each group in Example 1 of this invention;

[0038] Figure 9 This is a graph showing the effect of extracellular vesicles from milk cells on short-chain fatty acids in the intestines of anxious mice in Example 1 of this invention.

[0039] Figure 10 This is a graph showing the behavioral test results of adult mice after supplementing with milk-derived exocapsids during adolescence, as described in Example 1 of this invention.

[0040] Figure 11 This is a graph showing the behavioral test results of the effect of extracellular vesicles from breast cells on D-gal-induced cognitive impairment in Example 2 of the present invention;

[0041] Figure 12 This is a graph showing the relative mRNA expression levels of inflammatory factors IL-β, IL-6, TNF-α, and NLRP3 in the hippocampus tissue of mice in each group in Example 2 of this invention.

[0042] Figure 13 The image shows the results of colon tissue analysis in each group of mice in Example 2 of this invention. Detailed Implementation

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Milk-derived extracellular vesicles are rich in unique immunomodulatory proteins and possess biological properties such as antioxidation, anti-inflammation, and regulation of immune responses. However, there is a lack of research reports on their effects on improving brain health problems such as mood disorders and their underlying mechanisms. This invention, using a cholecystokinin (CRS) model in adolescent mice, is the first to discover that oral administration of milk-derived extracellular vesicles significantly improves anxiety-like behavior in adolescent mice by remodeling gut-brain axis function and can significantly reduce anxiety susceptibility in adulthood.

[0045] Based on this, the present invention provides an application of milk-derived extracellular vesicles in the preparation of products for the prevention, treatment and / or improvement of nervous system diseases.

[0046] In this invention, the product may be a drug; specifically, the drug may be an oral drug.

[0047] In one specific embodiment of the present invention, the milk-derived extracellular vesicles are preferably milk-derived extracellular vesicles derived from cow's milk and / or milk-derived extracellular vesicles derived from sheep's milk.

[0048] In one specific embodiment of the present invention, the milk-derived extracellular vesicles are preferably milk-derived extracellular vesicles derived from mature cow's milk and / or milk-derived extracellular vesicles derived from mature sheep's milk.

[0049] In one specific embodiment of the present invention, the average particle size of the extracellular vesicles derived from milk cells is preferably 100-150 nm; optionally, the average particle size of the extracellular vesicles derived from milk cells is 100 nm, 110 nm, 114 nm, 120 nm, 125 nm, 129 nm, 130 nm, 140 nm, 150 nm or any two of the above values.

[0050] This invention does not impose any particular limitations on the preparation method of milk-derived extracellular vesicles; any method well-known to those skilled in the art can be used. In a specific embodiment provided by this invention, the milk-derived extracellular vesicles are preferably prepared by a combination of rennet processing and membrane treatment. The milk-derived extracellular vesicles prepared by this method have good uniformity and intact appearance.

[0051] In a specific embodiment of the present invention, the milk-derived extracellular vesicles are prepared by the following method: S1) The milk raw material is centrifuged for the first time at low temperature to collect the intermediate whey portion; S2) The intermediate whey portion, rennet and calcium salt are mixed and incubated, and then centrifuged for the second time. The supernatant is taken and filtered through a filter membrane and then ultracentrifuged in sequence to obtain milk-derived extracellular vesicles.

[0052] In one specific embodiment of the present invention, the milk raw material is preferably cow's milk and / or sheep's milk, more preferably derived from mature cow's milk and / or mature sheep's milk.

[0053] The milk raw material is centrifuged for the first time at a low temperature; the preferred temperature is 2℃~6℃; optionally, the low temperature is 2℃, 3℃, 4℃, 5℃, 6℃ or any two of the above values; the preferred centrifugal force for the first centrifugation is 2000~5000 g; optionally, the preferred centrifugal force for the first centrifugation is 2000 g, 2500 g, 3000 g, 3500 g, 4000 g, 4500 g, 5000 g or any two of the above values; the preferred centrifugation time for the first centrifugation is 10~20 min; optionally, the preferred centrifugation time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min or any two of the above values. By centrifuging the milk raw material at a low temperature for the first time, the upper layer of fat and sediment are discarded, and the central whey fraction is collected.

[0054] The intermediate whey fraction, rennet, and calcium salt are mixed and incubated to precipitate casein. The ratio of rennet to milk raw material is preferably 0.01-0.1 g:100 mL; optionally, the ratio is 0.01 g:100 mL, 0.02 g:100 mL, 0.03 g:100 mL, 0.04 g:100 mL, 0.05 g:100 mL, 0.06 g:100 mL, 0.07 g:100 mL, 0.08 g:100 mL, 0.09 g:100 mL, 0.1 g:100 mL, or any two of the above ratios. The calcium salt is preferably calcium chloride; the ratio of calcium salt to milk raw material is preferably 0.1-0.5 g:100 mL; optionally, the ratio is 0.1 g:100 mL, 0.2 g:100 mL, 0.3 g:100 mL, or any two of the above ratios. The ratios are 0.4 g:100 mL, 0.5 g:100 mL, or any two of the above ratios; the incubation temperature is preferably 35℃~38℃, more preferably 36℃~37℃; the incubation time is preferably 20~40 min; optionally, the incubation time is 20 min, 25 min, 30 min, 35 min, 40 min, or any two of the above values.

[0055] After incubation, a second centrifugation is performed to remove casein, residual fat, and rennet, yielding a supernatant. The centrifugal force for the second centrifugation is preferably 10,000–20,000 g; alternatively, the centrifugal force for the second centrifugation may be 10,000 g, 11,000 g, 12,000 g, 13,000 g, 14,000 g, 15,000 g, 15,500 g, 16,000 g, 16,500 g, 17,000 g, 18,000 g, 19,000 g, 20,000 g, or any two of the above values. The preferred time for the second centrifugation is 10–60 min; alternatively, the time for the second centrifugation may be 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, or any two of the above values.

[0056] The supernatant is filtered through a filter membrane to remove residual large molecular fragments; the pore size of the filter membrane is preferably 0.2~0.6 μm; optionally, the pore size of the filter membrane is 0.2 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm or any two of the above values.

[0057] After filtration, ultracentrifugation is performed, and the resulting precipitate is the extracellular vesicle of milk cells. The ultracentrifugation temperature is preferably [specifically, the ultracentrifugation is performed by first centrifuging at a first set centrifugal force for a first set time, and then centrifuging at a second set centrifugal force for a second set time]. The first set centrifugal force is 50,000~100,000 g; optionally, the first set centrifugal force is 50,000 g, 60,000 g, 70,000 g, 80,000 g, 90,000 g, 100,000 g, or any two of the above values. The first set time is 30~90 min; optionally, the first set time is 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, or any two of the above values. The second set centrifugal force is 100,000~150,000 g; optionally, the second set centrifugal force is 100,000 g, 110,000 g, 115,000 g, 120,000 g. The set time is 30 to 90 minutes; or 125,000 g, 130,000 g, 135,000 g, 140,000 g, 145,000 g, 150,000 g, or any two of the above values.

[0058] In one specific embodiment of the present invention, the obtained extracellular vesicles of milk cells are preferably suspended in a buffer solution and stored at -80°C; the buffer solution can be any buffer solution known to those skilled in the art and there are no special restrictions, but PBS buffer solution is preferred in the present invention.

[0059] In one specific embodiment of the present invention, the neurological disease is specifically cognitive impairment and / or mental disorder.

[0060] In one specific embodiment of this invention, the cognitive impairment is specifically D-galactose (D-gal)-induced cognitive impairment. This invention constructs a D-gal-induced mouse model of cognitive impairment, and the results show that it can maintain intestinal homeostasis and alleviate cognitive impairment by reducing the production of pro-inflammatory cytokines and inhibiting mucosal inflammation.

[0061] In one specific embodiment of the present invention, the mental disorder includes one or more of stress, anxiety, panic disorder, and depression.

[0062] In one specific embodiment of the present invention, the mental disorder is specifically chronic stress-anxiety-like behavior.

[0063] In one specific embodiment of the present invention, the milk-derived extracellular vesicles prevent, treat and / or improve neurological diseases by remodeling gut-brain axis function.

[0064] In one specific embodiment of the present invention, the milk-derived extracellular vesicles prevent, treat and / or improve neurological diseases by alleviating one or more of the following: chronic stress-induced growth retardation, reducing stress-related hormone levels, reducing inflammatory cytokine expression, and improving gut microbiota metabolism.

[0065] In one specific embodiment of the present invention, the stress-related hormone is specifically corticosterone.

[0066] In one specific embodiment of the present invention, the inflammatory cytokines include, but are not limited to, one or more of NLRP3, IL-1β, IL-6 and TNF-α.

[0067] In one specific embodiment of the present invention, the extracellular vesicles derived from milk cells prevent, treat and / or improve neurological diseases by regulating inflammatory pathways.

[0068] In one specific embodiment of the present invention, the daily dosage of the milk-derived extracellular vesicles for the prevention, treatment and / or improvement of nervous system diseases is preferably 0.6 mg / kg, based on animal weight, specifically mouse weight.

[0069] In one specific embodiment of the present invention, based on mouse dosage conversion, for a human weighing 60 kg, the preferred dosage of the milk-derived extracellular vesicles is 0.3~30 mg / d; optionally, based on mouse dosage conversion, for a human weighing 60 kg, the dosage of the milk-derived extracellular vesicles is 0.3 mg / d, 0.5 mg / d, 1 mg / d, 2 mg / d, 2.88 mg / d, 3 mg / d, 4 mg / d, 8 mg / d, 10 mg / d, 12 mg / d, 13 mg / d, 14 mg / d, 18 mg / d, 20 mg / d, 22 mg / d, 23 mg / d, 24 mg / d, 28 mg / d, 30 mg / d, or any two of the above values.

[0070] The present invention also provides a composition comprising extracellular vesicles of milk for the prevention, treatment and / or improvement of nervous system diseases.

[0071] In this invention, the active ingredient in the composition is extracellular vesicles derived from milk cells, and it can be used appropriately according to conventional methods.

[0072] Besides containing the active ingredient as an essential component, the compositions provided by this invention have no limitations on other components and may contain various flavoring agents or natural carbohydrates (such as those in conventional beverages). Examples of such natural carbohydrates include conventional sugars, such as monosaccharides like glucose and fructose; disaccharides like maltose and sucrose; and polysaccharides like dextrin and cyclodextrin, as well as sugar alcohols like xylitol, sorbitol, and erythritol. As sweeteners, both natural and synthetic sweeteners may be used. Natural sweeteners include kiwifruit protein and stevia extract (such as rebaudioside A and glycyrrhizin); synthetic sweeteners include saccharin and aspartame. The proportion of natural carbohydrates can be appropriately determined by those skilled in the art.

[0073] In this invention, the composition may also contain various nutrients, vitamins, minerals (electrolytes), flavoring agents (including synthetic and / or natural flavoring agents), coloring agents, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, or carbonizing agents used in carbonated beverages, and these components may be used alone or in combination. The proportions of these substances may also be selected as needed.

[0074] The present invention also provides the use of milk-derived extracellular vesicles in the preparation of products that improve brain health and / or improve neurological-related discomfort.

[0075] In one specific embodiment of the present invention, the improvement of brain health and / or improvement of neurological discomfort includes one or more of the following: relieving anxiety, improving memory, and improving cognitive impairment.

[0076] In one specific embodiment of the present invention, the product may be a drug, and there are no special limitations.

[0077] In one specific embodiment of the present invention, the milk-derived extracellular vesicles are preferably derived from cow's milk and / or from sheep's milk. The specific preparation method is the same as described above and will not be repeated here.

[0078] In one specific embodiment of the present invention, the milk-derived extracellular vesicles improve brain health and / or alleviate neurological discomfort by remodeling gut-brain axis function.

[0079] In one specific embodiment of the present invention, the milk-derived extracellular vesicles improve brain health and / or improve neurological discomfort by alleviating one or more of the following: chronic stress-anxiety-induced growth retardation, reducing stress-related hormone levels, reducing inflammatory cytokine expression, and improving gut microbiota metabolism.

[0080] In one specific embodiment of the present invention, the stress-related hormone is specifically corticosterone.

[0081] In one specific embodiment of the present invention, the inflammatory cytokines include, but are not limited to, one or more of NLRP3, IL-1β, IL-6 and TNF-α.

[0082] In one specific embodiment of the present invention, the preferred dosage of the milk-derived extracellular vesicles for improving brain health and / or improving neurological-related discomfort is 0.6 mg / kg, based on animal weight, specifically mouse weight.

[0083] In a specific embodiment of the present invention, based on mouse dosage conversion, for a human weighing 60 kg, the preferred dosage of the milk-derived extracellular vesicles for improving brain health and / or improving neurological-related discomfort is 0.3~30 mg / d; optionally, based on mouse dosage conversion, for a human weighing 60 kg, the dosage of the milk-derived extracellular vesicles is 0.3 mg / d, 0.5 mg / d, 1 mg / d, 2 mg / d, 2.88 mg / d, 3 mg / d, 4 mg / d, 8 mg / d, 10 mg / d, 10 mg / d, 12 mg / d, 13 mg / d, 14 mg / d, 18 mg / d, 20 mg / d, 22 mg / d, 23 mg / d, 24 mg / d, 28 mg / d, 30 mg / d, or any two of the above values.

[0084] The present invention also provides a composition comprising extracellular vesicles derived from milk for improving brain health and / or improving neurological discomfort.

[0085] In this invention, the active ingredient in the composition is extracellular vesicles derived from milk cells, and it can be used appropriately according to conventional methods.

[0086] Besides containing the active ingredient as an essential component, the compositions provided by this invention have no limitations on other components and may contain various flavoring agents or natural carbohydrates (such as those in conventional beverages). Examples of such natural carbohydrates include conventional sugars, such as monosaccharides like glucose and fructose; disaccharides like maltose and sucrose; and polysaccharides like dextrin and cyclodextrin, as well as sugar alcohols like xylitol, sorbitol, and erythritol. As sweeteners, both natural and synthetic sweeteners may be used. Natural sweeteners include kiwifruit protein and stevia extract (such as rebaudioside A and glycyrrhizin); synthetic sweeteners include saccharin and aspartame. The proportion of natural carbohydrates can be appropriately determined by those skilled in the art.

[0087] In this invention, the composition may also contain various nutrients, vitamins, minerals (electrolytes), flavoring agents (including synthetic and / or natural flavoring agents), coloring agents, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, or carbonizing agents used in carbonated beverages, and these components may be used alone or in combination. The proportions of these substances may also be selected as needed.

[0088] The present invention also provides the use of the above-mentioned milk-derived extracellular vesicles in the preparation of products for the prevention, treatment and / or improvement of chronic stress-anxiety-like behaviors.

[0089] In one specific embodiment of the present invention, the product may be a drug, and there are no special limitations.

[0090] In one specific embodiment of the present invention, the milk-derived extracellular vesicles are preferably derived from cow's milk and / or from sheep's milk. The specific preparation method is the same as described above and will not be repeated here.

[0091] In one specific embodiment of the present invention, the milk-derived extracellular vesicles prevent and / or treat chronic stress-anxiety-like behaviors by remodeling gut-brain axis function.

[0092] In one specific embodiment of the present invention, the milk-derived extracellular vesicles treat and / or prevent chronic stress-anxiety-like behaviors by alleviating one or more of the following: reducing growth retardation induced by chronic stress and anxiety, reducing stress-related hormone levels, reducing the expression of inflammatory cytokines, and improving gut microbiota metabolism.

[0093] In one specific embodiment of the present invention, the stress-related hormone is specifically corticosterone.

[0094] In one specific embodiment of the present invention, the inflammatory cytokines include, but are not limited to, one or more of IL-1β, IL-6, and TNF-α.

[0095] In one specific embodiment of the present invention, the preferred dosage of the milk-derived extracellular vesicles for improving brain health and / or improving neurological-related discomfort is 0.6 mg / kg, based on animal weight, specifically mouse weight.

[0096] In one specific embodiment of the present invention, based on mouse dosage conversion, for a human weighing 60 kg, the preferred dosage of the milk-derived extracellular vesicles for treating and / or preventing chronic stress-induced anxiety-like behavior is 0.3~30 mg / d; optionally, based on mouse dosage conversion, for a human weighing 60 kg, the dosage of the milk-derived extracellular vesicles is 0.3 mg / d, 0.5 mg / d, 1 mg / d, 2 mg / d, 2.88 mg / d, 3 mg / d, 4 mg / d, 8 mg / d, 10 mg / d, 10 mg / d, 12 mg / d, 13 mg / d, 14 mg / d, 18 mg / d, 20 mg / d, 22 mg / d, 23 mg / d, 24 mg / d, 28 mg / d, 30 mg / d, or any two of the above values.

[0097] The present invention also provides a composition comprising milk-derived extracellular vesicles for the prevention, treatment and / or improvement of chronic stress-anxiety-like behaviors.

[0098] In this invention, the active ingredient in the composition is extracellular vesicles derived from milk cells, and it can be used appropriately according to conventional methods.

[0099] Besides containing the active ingredient as an essential component, the compositions provided by this invention have no limitations on other components and may contain various flavoring agents or natural carbohydrates (such as those in conventional beverages). Examples of such natural carbohydrates include conventional sugars, such as monosaccharides like glucose and fructose; disaccharides like maltose and sucrose; and polysaccharides like dextrin and cyclodextrin, as well as sugar alcohols like xylitol, sorbitol, and erythritol. As sweeteners, both natural and synthetic sweeteners may be used. Natural sweeteners include kiwifruit protein and stevia extract (such as rebaudioside A and glycyrrhizin); synthetic sweeteners include saccharin and aspartame. The proportion of natural carbohydrates can be appropriately determined by those skilled in the art.

[0100] In this invention, the composition may also contain various nutrients, vitamins, minerals (electrolytes), flavoring agents (including synthetic and / or natural flavoring agents), coloring agents, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, or carbonizing agents used in carbonated beverages, and these components may be used alone or in combination. The proportions of these substances may also be selected as needed.

[0101] This invention constructs a chronic restraint stress (CRS)-induced anxiety model in adolescent mice and uses behavioral experiments such as the open field test, bead embedding test, and tail suspension test for functional evaluation. The results show that milk-derived extracellular vesicles significantly improve anxiety-like behavior in mice, indicating that milk-derived extracellular vesicles can significantly improve CRS-induced anxiety-like behavior in adolescent mice by remodeling gut-brain axis function. Furthermore, supplementing adolescents with milk-derived extracellular vesicles can significantly reduce anxiety susceptibility in adulthood.

[0102] To further illustrate the present invention, the following describes in detail, with reference to embodiments, the application of a milk-derived extracellular vesicle provided by the present invention in improving brain health and / or improving neurological-related discomfort.

[0103] All reagents used in the following examples are commercially available.

[0104] Example 1

[0105] 1.1 Preparation of extracellular vesicles from milk cells

[0106] Fresh milk was centrifuged at 3000×g for 15 min at 4°C, and the supernatant fat and precipitate were discarded. The intermediate whey fraction was collected, and 0.05% rennet (w / v) and 0.3% CaCl2 were added and incubated at 37°C for 30 min to precipitate casein. The whey was then centrifuged at 16500×g for 30 min to remove casein, residual fat, and rennet. The resulting whey was filtered through a 0.45 μm filter membrane to remove residual macromolecular debris, and then ultracentrifuged at 4°C to obtain milk-derived extracellular vesicles. Specifically, the ultracentrifugation was performed by centrifuging the filtered solution at 70000×g for 60 min, discarding the precipitate, and then centrifuging the supernatant at 135000×g for 60 min. The precipitate was mEVs, which were resuspended in an appropriate amount of PBS and stored at -80°C.

[0107] Fresh goat milk was centrifuged at 3000×g for 15 min at 4°C, and the supernatant fat and precipitate were discarded. The intermediate whey fraction was collected, and 0.05% rennet (w / v) and 0.3% CaCl2 were added and incubated at 37°C for 30 min to precipitate casein. The whey was then centrifuged at 16500×g for 30 min to remove casein, residual fat, and rennet. The resulting whey was filtered through a 0.45 μm filter membrane to remove residual macromolecular debris, and then ultracentrifuged at 4°C to obtain milk-derived extracellular vesicles. Specifically, the ultracentrifugation was performed by centrifuging the filtered solution at 70000×g for 60 min, discarding the precipitate, and then centrifuging the supernatant at 135000×g for 60 min. The precipitate was gEVs, which were resuspended in an appropriate amount of PBS and stored at -80°C.

[0108] The gEVs obtained in Example 1 were analyzed, and the results are as follows: Figure 1 As shown in Figures AB, the morphology of exosomes was observed using transmission electron microscopy (TEM), and the concentration and particle size distribution of exosomes were determined by nanoparticle tracking analysis (NTA). Figure C shows the results of Western blotting. From Figure 1A, it can be seen that the average particle size of extracellular vesicles obtained from milk-derived cells using the rennet method combined with membrane treatment was 121.37 ± 6.84 nm, with good uniformity, polydispersity < 1%, and a concentration of 4.73 × 10¹² ± 2.13 × 10¹⁰ particles / mL. Figure 1 As can be seen from B, the extracellular vesicles of milk cells obtained by the extraction process of this invention have a complete appearance and structure, appearing as spherical, monodisperse nanoparticles with a complete lipid bilayer; from Figure 1 Western blot results showed significant positive signals for three proteins: CD63, TSG101, and Alix, while the negative protein Calnexin was not expressed, indicating the absence of endoplasmic reticulum protein contamination.

[0109] 1.2 Animal Experiment Design

[0110] SPF-grade male C57BL / 6 adolescent mice (3 weeks old, 13-15 g, Spefair) were placed under standard conditions (22±1℃, 50±10% humidity, 12 h light-dark cycle) with free access to food and water. After 1 week of acclimatization, they were randomly divided into 6 groups (n=10 / group).

[0111] The specific groupings are as follows: Control (unrestrained stress + gavage with PBS solution), gEVs-Control (unrestrained stress + 0.6 mg / kg gEVs), mEVs-Control (unrestrained stress + 0.6 mg / kg mEVs), Model (restrained stress + gavage with PBS solution), gEVs-Model (restrained stress + 0.6 mg / kg gEVs), mEVs-Model (restrained stress + 0.6 mg / kg mEVs).

[0112] The anxiety susceptibility experiment used the above-mentioned 3-week-old SPF-grade C57BL / 6 male adolescent mice. After 1 week of acclimatization, they were randomly divided into groups and intervened until 8 weeks of age (gavage once a day according to the group requirements). Restraint stress modeling (establishing an anxiety model) was carried out for 4 weeks.

[0113] The anxiety model was established using a chronic restraint stress (CRS) protocol: mice were placed in 50 mL conical centrifuge tubes (with sidewall perforations to ensure ventilation); restraint was applied at a fixed time each day for 6 hours. Mice were administered 30 minutes before the restraint stress was applied, and this intervention lasted for 28 days. Normally fed mice were placed in new empty cages (unrestrained) at the same time, and were administered PBS solution by gavage 30 minutes before being placed in the new empty cages.

[0114] 1. Physiological testing

[0115] During the establishment of the anxiety model, the weight of mice was recorded, and the average weight distribution of mice in each group at different time points was obtained as shown in the figure. Figure 2 As shown.

[0116] After establishing the anxiety model, the serum corticosterone levels in the blood of mice were measured, and the average serum corticosterone levels in each group of mice were obtained as follows: Figure 3 As shown.

[0117] 2. Behavioral experiments

[0118] Behavioral tests were conducted within 3 days after the restraint stress period ended. The test sequence was open field test, bead burial test, and tail suspension test, with each test 24 hours apart. The experimental environment was kept quiet, and mice were allowed 30 minutes to acclimatize before each test. The test area was cleaned with 75% ethanol between tests to eliminate odor cues.

[0119] Open Field Test (OFT): The open field device is a 40×40×35 cm open box divided into a central area (25 cm×25 cm) and a peripheral area. A camera is installed on top and connected to a behavior analysis system. Mice are placed in the center of the device, and after acclimatization for 1 minute, their activity is recorded over 6 minutes to obtain a mouse path distribution map. Figure 4 As shown in the figure. Analysis of indicators such as the time mice spent in the central region and the percentage of time spent in the central region relative to the total time yielded the following results: [Figure showing central region dwell time]. Figure 5 As shown, anxiety-like behaviors are assessed.

[0120] Bead Burial Test (MBT): A 5 cm thick bedding layer was placed in a 40 cm × 30 cm × 22 cm rearing box, and 20 glass beads with a diameter of 14–15 mm were evenly placed on it. Mice were placed in the box, and after 30 minutes, the number of buried beads was recorded (more than 2 / 3 covered was considered buried). The percentage of buried beads in different groups of mice is shown in the graph below. Figure 6 As shown.

[0121] Tail Suspension Test (TST): Mice were secured to a tail suspension frame 1 cm from the tip of their tails with tape, suspended at a height of 50 cm for 6 minutes. The immobility time (defined as passive suspension without escape-oriented movement) within the last 4 minutes was recorded using a camera connected to a behavior analysis system, reflecting despair-like behavior. The immobility time of each group of mice was obtained as follows: Figure 7 As shown.

[0122] 3. Histological analysis

[0123] After behavioral testing, the levels of inflammatory factors in the colon of mice were detected using RT-qPCR. The results are shown in the graph below. Figure 8 As shown in B.

[0124] Mice were euthanized after behavioral testing. Proximal colon tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histopathological changes were observed under a light microscope, and the stained tissue images are shown below. Figure 8 As shown in Figure A.

[0125] Depend on Figure 8 It is known that extracellular vesicles derived from milk cells have a protective effect on the intestinal barrier in anxious mice.

[0126] 4. Short-chain fatty acid analysis

[0127] Gas chromatography (GC) was used to quantitatively analyze short-chain fatty acids (acetic acid, propionic acid, and butyric acid) in feces. The results of the effect of milk-derived extracellular vesicles on short-chain fatty acids in the intestines of anxious mice are shown in the figure below. Figure 9 As shown.

[0128] 1.3 Experimental Results

[0129] 1. Physiological Results

[0130] Weight indirectly reflects health status and can serve as a proxy for the severity of anxiety. Figure 2 and Figure 3 It was found that CRS significantly inhibited weight gain and slowed growth rate in adolescent mice (p < 0.05), while intervention with milk-derived extracellular vesicles improved this condition (p < 0.05). Figure 2 Furthermore, serum corticosterone levels were elevated in CRS mice, but significantly decreased after administration of milk-derived extracellular vesicles. Figure 3 These results indicate that long-term supplementation with milk-derived extracellular vesicles can effectively alleviate CRS-induced growth retardation and elevated levels of stress-related hormones.

[0131] 2. Behavioral Results

[0132] Depend on Figures 4-7 The effects of modern expiratory volume (mEVs) and gEVs on chronic restraint stress (CRS)-induced anxiety were assessed using the open field test (OFT), the root-in-the-pillar test (MBT), and the tail-suspended test (TST). CRS significantly reduced central zone dwell time and total movement distance. Figure 4 and Figure 5 ), increased the number of embedded beads ( Figure 6 ), and extended the stationary time ( Figure 7 Intervention with milk-derived extracellular vesicles significantly reversed these behavioral abnormalities (p < 0.05), restoring most parameters to near-control levels. These results indicate that milk-derived extracellular vesicles can alleviate anxiety-like behavior induced by chronic stress in mice.

[0133] 2. Mechanism Indicator Results

[0134] In the colon, CRS causes severe mucosal damage, including crypt destruction, goblet cell loss, and inflammatory infiltration, while intervention with milk-derived extracellular vesicles preserves epithelial structure and reduces inflammation. Figure 8 (A). CRS significantly increased the expression of inflammatory mediators, including IL-1β, IL-6, and TNF-α, while intervention with extracellular vesicles from breast cells significantly reduced the levels of these indicators. Figure 8 (B)

[0135] Compared with the control group, continuous CRS treatment significantly reduced the total content of SCFAs in mouse feces, and all major SCFA components showed varying degrees of downregulation. This suggests that the anxiety-like phenotype may be accompanied by disruption of gut microbiota metabolic function. After intervention with milk-derived extracellular vesicles, the total SCFA content in mouse feces significantly increased (…). Figure 9The levels of acetic acid were close to those of the control group, with the most significant increase observed in acetic acid. These findings suggest that milk-derived extracellular vesicles can improve CRS-induced gut microbiota metabolic abnormalities by selectively upregulating fecal SCFA levels, which may be one of the important mechanisms by which they alleviate anxiety-like behavior in mice.

[0136] 1.4 Supplementing with mEVs or gEVs during adolescence can reduce susceptibility to anxiety in adulthood.

[0137] To investigate the effects of mEVs and gEVs on anxiety susceptibility, mice were treated with extracellular milk vesicles during adolescence, and a CRS model was established in adulthood (model establishment and behavioral testing are as shown above).

[0138] Anxiety susceptibility experiment: Three-week-old SPF-grade male C57BL / 6 adolescent mice were used and, after 1 week of acclimatization, were randomly divided into four groups: Control (no restraint stress + gavage with PBS solution), Model (restraint stress + gavage with PBS solution), gEVs-Model (restraint stress + 0.6 mg / kg gEVs), and mEVs-Model (restraint stress + 0.6 mg / kg mEVs). The intervention continued until 8 weeks of age. Restraint stress modeling (establishing an anxiety model, the establishment method is the same as described above) was carried out for 4 weeks. During the modeling period, gavage intervention was continued.

[0139] Physiological and behavioral tests were performed on adult mice using the methods described above. The results of these tests on adult mice after supplementation with milk-derived exoskeletons during adolescence are shown in the figure below. Figure 10 As shown.

[0140] The study found that mice receiving PBS intervention during adolescence exhibited significantly reduced anxiety-like behaviors when faced with the challenges of chronic traumatic encephalopathy in adulthood, compared to mice that received PBS intervention during adolescence. Figure 10 This indicates that supplementing adolescents with milk-derived extracellular vesicles can reduce anxiety susceptibility in adult mice.

[0141] Example 2

[0142] 2.1 Animal Experiment Design

[0143] Sixty male C57BL / 6 mice (7 weeks old, 20-23 g) were housed in cages of five, with constant temperature (24 ± 2℃), constant humidity (50 ± 10%) and a 12-hour light-dark cycle, and with free access to water and food.

[0144] After one week of acclimatization, mice were randomly divided into four groups: a control group (0.9% saline), a model group (0.9% saline), a mEVs group (0.6 mg / kg bw daily), and a positive control group (VC, 200 mg / d). Cognitive impairment was induced by intraperitoneal injection of D-galactose (D-gal) for six consecutive weeks. mEVs were administered orally via gavage daily, while the control group received an equal volume of PBS. Specifically, control mice were subcutaneously injected with 0.9% saline daily, while other groups were subcutaneously injected with D-gal (200 mg / kg bw) daily. The mEVs group received 0.6 mg / kg bw of milk-derived extracellular vesicles (prepared in Example 1) via gavage daily, while the positive control group received 200 mg of VC via gavage daily.

[0145] 2.2 Behavioral Testing

[0146] 2.2.1 Open Field Experiment

[0147] This method was used to assess the voluntary movement ability of mice. Mice were placed in the center of a 50 cm × 50 cm × 40 cm cube and allowed to move freely for 5 minutes. Figure 11 Figure A shows a schematic diagram of the open field experiment. The trajectory, total distance traveled, time spent in the central area, and number of times the mice entered the central area were recorded using a video tracking system (Shanghai Xinruan). The total distance traveled by each group of mice in the open field experiment was obtained as shown below. Figure 11 As shown in Figure B, the movement trajectories of each group of mice in the open field experiment are obtained as follows. Figure 11 As shown in Figure F. Clean the work area during breaks to avoid odor interference.

[0148] 2.2.2 New Object Recognition Index

[0149] This experiment was used to assess the learning and memory abilities of mice. The experiment consisted of an adaptation period, a familiarization period, and a testing period. On day one (adaptation period), mice were allowed to freely explore an open area for 10 minutes. After 24 hours (familiarization period), two identical objects were placed in the area, and the mice were allowed to explore them for 10 minutes. After 24 hours (testing period), one of the objects was replaced with a new object, and the mice were allowed to explore it again for 5 minutes. Figure 11 Figure C shows a schematic diagram of the novel object recognition experiment. The exploration time of mice for the two objects was recorded during the familiarization and testing periods, and the Novel Object Recognition Index (NOR index) results for each group of mice are shown in the figure below. Figure 11 As shown in D. The formula for calculating the New Object Recognition Index (NORI) is: NORI = (Time to explore new objects / (Time to explore new objects + Time to explore old objects)) × 100%.

[0150] 2.2.3 Barnes Maze

[0151] This was used to assess the spatial learning and reference memory abilities of mice. The maze was a circular platform, 1.2 meters in diameter, with 20 holes around its perimeter, one of which connected to an escape box. Figure 11 Figure E shows a schematic diagram of the Barnes maze experimental setup. The experiment consisted of a 4-day training period and a 5-day exploration period. Each training session lasted a maximum of 3 minutes, guiding the mice to find and enter the escape box. The latency to find the escape box and the number of errors (number of incorrectly explored holes) were recorded. Twenty-four hours after the final training session, the exploration experiment was conducted. The escape box was removed, and the time the mice spent in the target quadrant (the quadrant where the escape box was originally located) and the latency to first reach the target hole were recorded. The average time for each group of mice to find the target hole in the Barnes maze was obtained, as shown in the figure below. Figure 11 As shown in G; example diagrams of the movement paths of each group of mice in the Barnes maze are obtained as follows. Figure 11 As shown in H. Figure 11 In the mean squares, *P < 0.05, **P < 0.01, and ***P < 0.001.

[0152] 2.3 Histological analysis

[0153] 2.3.1 Hippocampal tissue examination

[0154] After behavioral testing, the expression levels of inflammatory cytokines mRNA in the hippocampus of mice were detected using RT-qPCR. The relative expression levels of inflammatory cytokines IL-β, IL-6, TNF-α, and NLRP3 mRNA in the hippocampus of each group of mice are shown in the figure below. Figure 12 As shown. Figure 12 * p < 0.05, ** p < 0.01, *** p < 0.001.

[0155] 2.3.2 Colon tissue examination

[0156] Mice were euthanized after behavioral testing. Proximal colon tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histopathological changes were observed under a light microscope, and the stained tissue images are shown below. Figure 13 As shown in Figure A.

[0157] RNA was extracted from mouse colon tissue, and pro-inflammatory cytokines in the colon tissue were detected using quantitative real-time PCR (qRT-PCR) to assess colonic inflammation. The results of expression levels of inflammatory factors in the colon tissue of each group of mice are shown in the figure below. Figure 13 As shown in B~E.

[0158] 2.4 Experimental Results

[0159] 2.4.1 mEVs improve D-gal-induced cognitive impairment

[0160] To assess the impact of mEVs on cognitive function in model mice, spontaneous motor ability and anxiety-like behavior were first measured using an open field test. Figure 11 (A and B). Compared with the control group, the total movement distance of the model group mice was significantly reduced (P < 0.05), indicating that spontaneous activity was suppressed. Figure 11 (B) Treatment with mEVs significantly restored this indicator to near-normal levels, while the positive control group (VC) also showed a similar trend of improvement. This indicates that mEVs did not cause motor impairment, and their impact on cognitive function was not affected by motor ability.

[0161] Subsequently, a novel object recognition experiment was used to evaluate short-term memory and recognition learning ability. Figure 11 (C) Compared with the control group, the recognition index (NOR index) of the model group was significantly reduced (P < 0.001), showing a decline in novelty recognition ability; while mEVs intervention significantly improved the recognition index (P < 0.05, D in Figure 11), suggesting that it can effectively improve short-term memory deficits.

[0162] Further utilize the Barnes maze to test working memory and spatial learning abilities. Figure 11 (E). The trajectory diagram shows that the model group exhibits obvious disorientation and repeated entry (E). Figure 11 In the F and H sections, the average time to find the target hole was significantly prolonged (P < 0.001). Figure 11 The mEVs treatment significantly shortened the escape latency (P < 0.001), showing a similar improvement effect to the VC group.

[0163] The above results indicate that mEVs can improve the learning and memory abilities of mice.

[0164] 2.4.2 Mechanism Indicators

[0165] Studies have shown that cognitive impairment and neuroinflammation often coexist. PCR results showed that the levels of tumor necrosis factor-α (TNF-α), NLRP3, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the hippocampus of model group mice were significantly increased (P < 0.05 ~ 0.001). Figure 12 (A~D). mEVs significantly downregulated four inflammatory cytokines, particularly IL-1β (P < 0.001), indicating a potent anti-inflammatory effect. These results suggest that mEVs alleviate cognitive impairment by reducing the production of pro-inflammatory cytokines.

[0166] Given the crucial role of the gut-brain axis in regulating neurobehavioral homeostasis, this study investigated whether modified endothelial cells (mEVs) protect the intestinal epithelial barrier in cognitively impaired mice. Histological examination of colonic tissue stained with hematoxylin and eosin (H&E) revealed significant epithelial damage, irregular crypt structures, and increased inflammatory cell infiltration in the model group compared to the control group (P < 0.001). Figure 13 (A). In contrast, mEVs can significantly alleviate these pathological changes and restore epithelial continuity and crypt tissue.

[0167] Furthermore, colonic inflammation was assessed by measuring pro-inflammatory cytokines in colonic tissue. Compared with the control group, the levels of interleukin-1β (IL-1β), NLRP3, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were increased in model mice (P < 0.01). Figure 13 (B~E). Adding mEVs significantly reduced cytokines, with the most significant reduction in IL-6 (P < 0.01). In summary, these data indicate that mEVs exert an effective protective effect on the intestinal barrier by inhibiting mucosal inflammation, thereby maintaining intestinal homeostasis and supporting the gut-brain regulatory network.

[0168] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of milk-derived extracellular vesicles in the preparation of products that improve brain health and / or alleviate neurological discomfort; The improvement of brain health and / or the improvement of neurological discomfort are intended to alleviate anxiety; The milk-derived extracellular vesicles are selected from milk-derived extracellular vesicles derived from cow's milk and / or milk-derived extracellular vesicles derived from sheep's milk. The average particle size of the extracellular vesicles from the milk source is 100-150 nm.

2. The application according to claim 1, characterized in that... The milk-derived extracellular vesicles improve brain health and / or alleviate neurological discomfort by remodeling gut-brain axis function; And / or, the milk-derived extracellular vesicles improve brain health and / or alleviate neurological discomfort by modulating inflammatory pathways.

3. The application according to claim 1, characterized in that, The milk-derived extracellular vesicles improve brain health and / or alleviate neurological discomfort by relieving one or more of the following: chronic stress-anxiety-induced growth retardation, reducing stress-related hormone levels, reducing the expression levels of inflammatory cytokines, and improving gut microbiota metabolism.

4. The application according to claim 1, characterized in that, The extracellular vesicles derived from milk cells were prepared according to the following method: S1) The milk raw material is centrifuged for the first time at low temperature, and the intermediate whey fraction is collected; S2) Mix the intermediate whey fraction, rennet and calcium salt and incubate. Then perform a second centrifugation, take the supernatant, and filter it through a filter membrane and then centrifuge it at high speed to obtain milk-derived extracellular vesicles.

5. The application according to any one of claims 1 to 4, characterized in that, For a person weighing 60 kg, the dosage of the said extracellular vesicles derived from milk is 0.3~30 mg / d.

6. Application of milk-derived extracellular vesicles in the preparation of products for the prevention, treatment and / or improvement of chronic stress-induced anxiety-like behaviors; The milk-derived extracellular vesicles are selected from milk-derived extracellular vesicles derived from cow's milk and / or milk-derived extracellular vesicles derived from sheep's milk. The average particle size of the extracellular vesicles from the milk source is 100-150 nm.

7. Application of milk-derived extracellular vesicles in the preparation of products for the prevention, treatment and / or improvement of nervous system diseases; The neurological disorders are selected from mental disorders; the mental disorders include one or more of stress, anxiety, and depression. The milk-derived extracellular vesicles are selected from milk-derived extracellular vesicles derived from cow's milk and / or milk-derived extracellular vesicles derived from sheep's milk. The average particle size of the extracellular vesicles from the milk source is 100-150 nm.

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