Application of milk-derived extracellular vesicles to improvement of brain health conditions and / or improvement of nerve-related discomfort conditions

Abstract

The invention belongs to the technical field of biological medicine and food, and particularly relates to application of milk-derived extracellular vesicles to improvement of brain health conditions and / or improvement of nerve-related discomfort conditions. Compared with the prior art, a chronic constraint stress (CRS)-induced puberty mouse anxiety model is constructed, functional evaluation is performed by behavioral experiments such as an open field experiment, a bead embedding experiment and a tail suspension experiment, and results show that the milk-derived extracellular vesicles remarkably improve mouse anxiety-like behaviors; it is shown that the milk-derived extracellular vesicles can significantly improve the chronic bound stress (CRS) induced adolescent mouse anxiety-like behavior by reshaping the intestinal-brain axis function, and the anxiety susceptibility of adolescents can be significantly reduced by supplementing the milk-derived extracellular vesicles to teenagers; meanwhile, a D-gal induced mouse cognitive impairment model is constructed, and the result shows that mucosa inflammation can be inhibited by reducing generation of proinflammatory cytokines, so that intestinal homeostasis is maintained, and cognitive impairment is relieved.

Description

TECHNICAL FIELD

[0001] 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. BACKGROUND

[0002] Anxiety disorders are chronic debilitating illnesses characterized by intense and persistent fear and distress. As the most prevalent category of mental disorders worldwide, they contribute significantly to global health loss, impairment of functioning, and adverse outcomes throughout the life cycle. These disorders often manifest in early life and follow a course of recurrence or fluctuation, making them one of the most disabling mental illnesses. Although traditional therapies such as psychotherapy and medication have been proven effective, their effects are often limited by slow onset, adverse reactions, and patient compliance issues, highlighting the need for safer, sustainable, and acceptable prevention and treatment options. With the rise of the concept of "precision nutrition" to enhance national health, dietary interventions to alleviate anxiety and brain health issues are of great significance.

[0003] In addition, one of the major limitations of current drugs for neurological diseases such as anxiety and cognitive impairment is that they can only alleviate symptoms and cannot cure the disease. In addition, long-term use often causes serious side effects. Therefore, it is crucial to explore natural compounds with potential preventive or alleviating effects. Natural bioactive compounds extracted from diet have attracted considerable attention due to their potential neuroprotective effects. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide application of milk-derived extracellular vesicles in improving brain health and / or improving nerve-related discomfort conditions.

[0005] The present application provides application of milk-derived extracellular vesicles in preparing products for improving brain health and / or improving nerve-related discomfort conditions.

[0006] Preferably, the improvement of brain health and / or the improvement of nerve-related discomfort conditions includes one or more of relieving anxiety, improving memory, and improving cognitive impairment;

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

[0008] And / or, the milk-derived extracellular vesicles improve brain health and / or improve nerve-related discomfort conditions by regulating inflammatory pathways.

[0009] Preferably, the milk-derived extracellular vesicles improve brain health and / or improve neuro-related discomfort by one or more of alleviating chronic stress-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 the group consisting of milk-derived extracellular vesicles derived from bovine milk and / or milk-derived extracellular vesicles derived from goat milk.

[0011] Preferably, the milk-derived extracellular vesicles have an average particle size of 100-150 nm.

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

[0013] S1) centrifuging the milk raw material at low temperature to obtain an intermediate whey fraction;

[0014] S2) mixing the intermediate whey fraction, rennet and calcium salt, incubating, then centrifuging to obtain a supernatant, and then filtering through a filter membrane and ultracentrifuging to obtain the milk-derived extracellular vesicles.

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

[0016] Preferably, the calcium salt is selected from the group consisting of calcium chloride.

[0017] Preferably, the ratio of calcium salt to milk raw material is 0.1-0.5 g: 100 mL.

[0018] Preferably, the low temperature is 2-6°C.

[0019] Preferably, the centrifugal force of the first centrifugation is 2000-5000 g, and the time of the first centrifugation is 10-20 min.

[0020] Preferably, the centrifugal force of the second centrifugation is 10000-20000 g, and the time of the second centrifugation is 10-60 min.

[0021] Preferably, the ultracentrifugation comprises: 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 50000-100000 g; the first set time is 30-90 min; the second set centrifugal force is 100000-150000 g; and the second set time is 30-90 min.

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

[0023] The present application also provides a composition for improving brain health and / or improving a neurological related discomfort, comprising milk-derived extracellular vesicles.

[0024] The present application also provides a use of milk-derived extracellular vesicles in the preparation of a product for preventing, treating and / or improving chronic stress anxiety-like behavior.

[0025] The present application also provides a composition for preventing, treating and / or improving chronic stress anxiety-like behavior, comprising milk-derived extracellular vesicles.

[0026] The present application also provides a use of milk-derived extracellular vesicles in the preparation of a product for preventing, treating and / or improving a neurological disease.

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

[0028] The present application also provides a composition for preventing, treating and / or improving a neurological disease, comprising milk-derived extracellular vesicles.

[0029] The present application provides a use of milk-derived extracellular vesicles in improving brain health and / or improving a neurological related discomfort. 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) through 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. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure is the performance analysis result graph of gEVs prepared in Example 1 of the present application;

[0031] Figure 2 Figure is the average weight distribution graph of mice in each group at different times in Example 1 of the present application;

[0032] Figure 3 Figure is the average content graph of serum corticosterone of mice in each group in Example 1 of the present application;

[0033] Figure 4Route distribution diagram for open field test of mice in each group in Example 1 of the present application;

[0034] Figure 5 Result diagram of central area residence time of mice in each group in Example 1 of the present application;

[0035] Figure 6 Result diagram of percentage of buried beads of mice in each group in Example 1 of the present application;

[0036] Figure 7 Result diagram of resting time of mice in different groups in tail suspension test in Example 1 of the present application;

[0037] Figure 8 Result diagram of histological analysis of mice in each group in Example 1 of the present application;

[0038] Figure 9 Result diagram of influence of milk-derived extracellular vesicles on short-chain fatty acids in the intestines of anxious mice in Example 1 of the present application;

[0039] Figure 10 Result diagram of behavioral detection of mice supplemented with milk-derived extracellular vesicles during adolescence in adulthood in Example 1 of the present application;

[0040] Figure 11 Result diagram of behavioral detection of the influence of milk-derived extracellular vesicles on D-gal-induced cognitive impairment in Example 2 of the present application;

[0041] Figure 12 Result diagram of mRNA relative expression level of inflammatory factors IL-β, IL-6, TNF-α and NLRP3 in hippocampal tissue of mice in each group in Example 2 of the present application;

[0042] Figure 13 Result diagram of colon tissue detection of mice in each group in Example 2 of the present application. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0044] Milk-derived extracellular vesicles are rich in unique immunomodulatory proteins, have biological characteristics such as antioxidant, anti-inflammatory and immune response regulation, but there is a lack of relevant research reports on the improvement effect and mechanism of brain health problems such as emotional disorders. The present application uses the CRS model of adolescent mice for the first time to find that oral administration of milk-derived extracellular vesicles can significantly improve the anxiety-like behavior of adolescent mice by remodeling the function of the gut-brain axis, and can significantly reduce the susceptibility to anxiety in adulthood.

[0045] Based on this, the present application provides a use of milk-derived extracellular vesicles in the preparation of a product for preventing, treating and / or improving nervous system diseases.

[0046] In the present application, the product can be a drug or a functional food; specifically, the drug can be an oral drug.

[0047] In a specific embodiment provided by the present application, 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 a specific embodiment provided by the present application, 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 a specific embodiment provided by the present application, the average particle size of the milk-derived extracellular vesicles is preferably 100-150 nm; optionally, the average particle size of the milk-derived extracellular vesicles is 100 nm, 110 nm, 114 nm, 120 nm, 125 nm, 129 nm, 130 nm, 140 nm, 150 nm or a range between any two of the above values.

[0050] The present application does not have special restrictions on the preparation method of milk-derived extracellular vesicles, which can be prepared according to methods well known to those skilled in the art; in a specific embodiment provided by the present application, the milk-derived extracellular vesicles are preferably prepared by a combination of renneting and membrane treatment. The milk-derived extracellular vesicles prepared by this method have good uniformity and complete appearance structure.

[0051] In a specific embodiment provided by the present application, the milk-derived extracellular vesicles are prepared according to the following method: S1) centrifuging the milk raw material at low temperature for the first time, and collecting the intermediate whey part; S2) mixing the intermediate whey part, renneting enzyme and calcium salt, incubating, then centrifuging for the second time, taking the supernatant, and sequentially filtering through a filter membrane and ultracentrifuging to obtain milk-derived extracellular vesicles.

[0052] In a specific embodiment provided by the present application, the milk raw material is preferably cow milk and / or goat milk, more preferably mature cow milk and / or mature goat milk.

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

[0054] The intermediate whey part, chymosin and calcium salt are mixed and incubated to precipitate casein; the ratio of the chymosin to the milk raw material is preferably 0.01-0.1 g:100 mL; alternatively, the ratio of the chymosin to the milk raw material 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 a range between any two of the above values; the calcium salt is preferably calcium chloride; the ratio of the calcium salt to the milk raw material is preferably 0.1-0.5 g:100 mL; alternatively, the ratio of the calcium salt to the milk raw material is 0.1 g:100 mL, 0.2 g:100 mL, 0.3 g:100 mL, 0.4 g:100 mL, 0.5 g:100 mL or a range between any two of the above values; the temperature of the incubation is preferably 35-38°C, more preferably 36-37°C; the time of the incubation is preferably 20-40 min; alternatively, the time of the incubation is 20 min, 25 min, 30 min, 35 min, 40 min or a range between any two of the above values.

[0055] After the incubation, a second centrifugation is performed to remove the casein, residual fat and chymosin, and to obtain the supernatant; the centrifugal force of the second centrifugation is preferably 10000-20000 g; alternatively, the centrifugal force of the second centrifugation is 10000 g, 11000 g, 12000 g, 13000 g, 14000 g, 15000 g, 15500 g, 16000 g, 16500 g, 17000 g, 18000 g, 19000 g, 20000 g or a range between any two of the aforementioned values; the time of the second centrifugation is preferably 10-60 min; alternatively, the time of the second centrifugation is 10 min, 20 min, 30 min, 40 min, 50 min, 60 min or a range between any two of the aforementioned 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; alternatively, 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 a range between any two of the aforementioned values.

[0057] After the filtration, an ultracentrifugation is performed to obtain the precipitate, which is the milk-derived extracellular vesicle; the temperature of the ultracentrifugation is preferably 4-10°C; alternatively, the temperature of the ultracentrifugation is 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or a range between any two of the aforementioned values; the ultracentrifugation is preferably performed in a stepwise manner, i.e., first under a first centrifugal force for a first time, and then under a second centrifugal force for a second time; the first centrifugal force is 50000-100000 g; alternatively, the first centrifugal force is 50000 g, 60000 g, 70000 g, 80000 g, 90000 g, 100000 g or a range between any two of the aforementioned values; the first time is 30-90 min; alternatively, the first time is 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min or a range between any two of the aforementioned values; the second centrifugal force is 100000-150000 g; alternatively, the second centrifugal force is 100000 g, 110000 g, 115000 g, 120000 g, 125000 g, 130000 g, 135000 g, 140000 g, 145000 g, 150000 g or a range between any two of the aforementioned values; the second time is 30-90 min; alternatively, the second time is 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min or a range between any two of the aforementioned values.

[0058] In a specific embodiment provided by the present application, the obtained milk-derived extracellular vesicles are preferably suspended in a buffer and stored at -80°C; the buffer can be any buffer known to those skilled in the art and is not particularly limited, and in the present application, a PBS buffer is preferred.

[0059] In a specific embodiment provided by the present application, the nervous system disease is specifically cognitive impairment and / or mental disorder.

[0060] In a specific embodiment provided by the present application, the cognitive impairment is specifically D-galactose (D-gal)-induced cognitive impairment. The present application constructs a D-gal-induced mouse cognitive impairment model, 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 a specific embodiment provided by the present application, the mental disorder includes one or more of stress, anxiety, panic, and depression.

[0062] In a specific embodiment provided by the present application, the mental disorder is specifically chronic stress anxiety-like behavior.

[0063] In a specific embodiment provided by the present application, the milk-derived extracellular vesicles prevent, treat, and / or improve the nervous system disease by remodeling the function of the gut-brain axis.

[0064] In a specific embodiment provided by the present application, the milk-derived extracellular vesicles prevent, treat, and / or improve the nervous system disease by one or more of alleviating chronic stress anxiety-induced growth retardation, reducing stress-related hormone levels, reducing inflammatory cytokine expression, and improving intestinal microbial flora metabolism.

[0065] In a specific embodiment provided by the present application, the stress-related hormone is specifically corticosterone.

[0066] In a specific embodiment provided by the present application, the inflammatory cytokine includes, but is not limited to, one or more of NLRP3, IL-1β, IL-6, and TNF-α.

[0067] In a specific embodiment provided by the present application, the milk-derived extracellular vesicles prevent, treat, and / or improve the nervous system disease by regulating the inflammatory pathway.

[0068] In a specific embodiment provided by the present application, the amount of the milk-derived extracellular vesicles used per day in preventing, treating, and / or improving the nervous system disease is preferably 0.6 mg / kg based on the weight of the animal, specifically the weight of a mouse.

[0069] In one specific embodiment provided in the present application, the amount of the milk-derived extracellular vesicles is preferably 0.3-30 mg / d for a 60 kg person, converted from a mouse. Alternatively, the amount 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 a range between any two of the above values for a 60 kg person, converted from a mouse.

[0070] The present application also provides a composition for preventing, treating, and / or improving nervous system diseases, comprising milk-derived extracellular vesicles.

[0071] In the present application, the effective ingredient in the composition is milk-derived extracellular vesicles, which can be directly added to food or used with other food or food ingredients, and can be appropriately used according to conventional methods.

[0072] In addition to containing the effective ingredient as an essential ingredient, the composition provided in the present application is not limited to other components, and can contain various flavorings or natural carbohydrates (such as conventional beverages). Examples of the above-mentioned natural carbohydrates include conventional sugars, such as monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., and sugar alcohols, e.g., xylitol, sorbitol, erythritol, etc. As a sweetener, it can be a natural sweetener or a synthetic sweetener, such as thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), aspartame, etc. The proportion of the natural carbohydrate can be appropriately determined by the selection of one of ordinary skill in the art.

[0073] In the present application, the composition can also contain various nutrients, vitamins, minerals (electrolytes), flavorings (including synthetic flavorings and / or natural flavorings), colorings, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerol, alcohols, or carbonating agents used in carbonated beverages, and these ingredients can be used alone or in combination. The proportions of these substances can also be selected as needed.

[0074] The present application also provides a use of milk-derived extracellular vesicles in the preparation of a product for improving brain health and / or improving the condition of a neurological-related discomfort.

[0075] In one specific embodiment provided in the present application, the improvement of brain health and / or the improvement of neurological related discomforts comprises one or more of relieving anxiety, improving memory, and improving cognitive impairment.

[0076] In one specific embodiment provided in the present application, the product can be a medicine or a functional food, and is not particularly limited.

[0077] In one specific embodiment provided in the present application, the milk-derived extracellular vesicles are preferably milk-derived extracellular vesicles derived from cow milk and / or milk-derived extracellular vesicles derived from goat milk. The specific preparation method is as described above, and will not be repeated here.

[0078] In one specific embodiment provided in the present application, the milk-derived extracellular vesicles improve brain health and / or improve neurological related discomforts by remodeling the function of the gut-brain axis.

[0079] In one specific embodiment provided in the present application, the milk-derived extracellular vesicles improve brain health and / or improve neurological related discomforts by one or more of relieving chronic stress-induced anxiety, reducing stress-related hormone levels, reducing inflammatory cytokine expression, and improving gut microbiota metabolism.

[0080] In one specific embodiment provided in the present application, the stress-related hormone is specifically corticosterone.

[0081] In one specific embodiment provided in the present application, 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 provided in the present application, the amount of milk-derived extracellular vesicles used to improve brain health and / or improve neurological related discomforts is preferably 0.6 mg / kg based on the weight of the animal, specifically the weight of a mouse.

[0083] In one embodiment of the present application, the amount of the milk-derived extracellular vesicles for improving brain health and / or improving a neurological-related discomfort condition is preferably 0.3 to 30 mg / d in terms of a mouse equivalent for a 60 kg human. Alternatively, the amount 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 a range between any two of the above values in terms of a mouse equivalent for a 60 kg human.

[0084] The present application also provides a composition for improving brain health and / or improving a neurological-related discomfort condition, comprising the milk-derived extracellular vesicles.

[0085] In the present application, the effective ingredient in the composition is the milk-derived extracellular vesicles, which can be directly added to food or used with other food or food ingredients, and can be appropriately used according to conventional methods.

[0086] In addition to containing the effective ingredient as an essential ingredient, the composition provided by the present application is not limited to other components, and can contain various flavorings or natural carbohydrates (such as conventional beverages). Examples of the above-mentioned natural carbohydrates include conventional sugars, such as monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., and sugar alcohols, e.g., xylitol, sorbitol, erythritol, etc. As a sweetener, it can be a natural sweetener or a synthetic sweetener, such as thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), aspartame, etc. The proportion of the natural carbohydrate can be appropriately determined by the selection of one of ordinary skill in the art.

[0087] In the present application, the composition can also contain various nutrients, vitamins, minerals (electrolytes), flavorings (including synthetic flavorings and / or natural flavorings), colorings, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerol, alcohols, or carbonating agents used in carbonated beverages, and these ingredients can be used alone or in combination. The proportions of these substances can also be selected as needed.

[0088] The present application also provides the use of the above-mentioned milk-derived extracellular vesicles in the preparation of a product for preventing, treating, and / or improving chronic stress anxiety-like behavior.

[0089] In one specific embodiment provided in the present application, the product can be a medicine or a functional food, and there is no special limitation.

[0090] In one specific embodiment provided in the present application, the milk-derived extracellular vesicles are preferably milk-derived extracellular vesicles derived from cow milk and / or milk-derived extracellular vesicles derived from goat milk. The specific preparation method is as described above, and will not be repeated here.

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

[0092] In one specific embodiment provided in the present application, the milk-derived extracellular vesicles treat and / or prevent chronic stress anxiety-like behavior by one or more of relieving chronic stress anxiety-induced growth retardation, reducing stress-related hormone levels, reducing inflammatory cytokine expression, and improving intestinal microbial flora metabolism.

[0093] In one specific embodiment provided in the present application, the stress-related hormone is specifically corticosterone.

[0094] In one specific embodiment provided in the present application, the inflammatory cytokine includes, but is not limited to, one or more of IL-1β, IL-6, and TNF-α.

[0095] In one specific embodiment provided in the present application, the amount of milk-derived extracellular vesicles used to improve brain health and / or improve neuro-related discomfort is preferably 0.6 mg / kg based on the weight of the animal, specifically the weight of a mouse.

[0096] In one specific embodiment provided in the present application, the amount of milk-derived extracellular vesicles used to treat and / or prevent chronic stress anxiety-like behavior is preferably 0.3-30 mg / d based on a 60 kg person converted from a mouse; alternatively, the amount of milk-derived extracellular vesicles used 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 a range between any two of the above values, based on a 60 kg person converted from a mouse.

[0097] The present application also provides a composition for preventing, treating, and / or improving chronic stress anxiety-like behavior, comprising milk-derived extracellular vesicles.

[0098] In the present application, the effective ingredient in the composition is milk-derived extracellular vesicles, which can be directly added to food or used with other food or food ingredients, and can be appropriately used according to conventional methods.

[0099] In addition to containing the effective ingredient as an essential ingredient, the composition provided by the present application has no limitation on other components, and can contain various flavorings or natural carbohydrates (such as conventional beverages). Examples of the above-mentioned natural carbohydrates include conventional sugars, such as monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., and sugar alcohols, e.g., xylitol, sorbitol, erythritol, etc. As a sweetener, it can be a natural sweetener or a synthetic sweetener, such as thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), aspartame. The proportion of the natural carbohydrate can be appropriately determined by the selection of those skilled in the art.

[0100] In the present application, the composition can also contain various nutrients, vitamins, minerals (electrolytes), flavorings (including synthetic flavorings and / or natural flavorings), colorings, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerol, alcohols, or carbonating agents used in carbonated beverages, and these ingredients can be used alone or in combination. The proportions of these substances can also be selected as needed.

[0101] 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 milk-derived extracellular vesicles significantly improve anxiety-like behavior in mice, indicating that milk-derived extracellular vesicles can significantly improve anxiety-like behavior in adolescent mice induced by chronic restraint stress (CRS) by remodeling the gut-brain axis function, and that adolescent supplementation with milk-derived extracellular vesicles can significantly reduce the susceptibility to anxiety in adulthood.

[0102] To further illustrate the present application, the following embodiments describe in detail the application of a milk-derived extracellular vesicle provided by the present application in improving brain health and / or improving neuro-related discomfort conditions.

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

[0104] Example 1

[0105] 1.1 Preparation of milk-derived extracellular vesicles

[0106] Fresh bovine milk was centrifuged at 3000 x g for 15 min at 4 ℃, and the upper fat and precipitate were discarded. The middle whey fraction was collected, 0.05% rennet (W / V) and 0.3% CaCl2 were added, and incubation was performed at 37 ℃ for 30 min to precipitate casein, centrifugation was performed at 16500 x g for 30 min to remove casein, residual fat and rennet. The obtained whey was removed of residual large molecular fragments through a 0.45 μm filter membrane, and then ultracentrifugation was performed at 4 ℃ to obtain milk-derived extracellular vesicles. The specific ultracentrifugation was as follows: the filtered solution was centrifuged at 70000 x g for 60 min, the precipitate was discarded, and then the supernatant was centrifuged at 135000 x g for 60 min, the precipitate was bEVs, which was resuspended with an appropriate amount of PBS and stored at -80 ℃.

[0107] Fresh bovine milk was centrifuged at 3000 x g for 15 min at 4 ℃, and the upper fat and precipitate were discarded. The middle whey fraction was collected, 0.05% rennet (W / V) and 0.3% CaCl2 were added, and incubation was performed at 37 ℃ for 30 min to precipitate casein, centrifugation was performed at 16500 x g for 30 min to remove casein, residual fat and rennet. The obtained whey was removed of residual large molecular fragments through a 0.45 μm filter membrane, and then ultracentrifugation was performed at 4 ℃ to obtain milk-derived extracellular vesicles. The specific ultracentrifugation was as follows: the filtered solution was centrifuged at 70000 x g for 60 min, the precipitate was discarded, and then the supernatant was centrifuged at 135000 x g for 60 min, the precipitate was bEVs, which was resuspended with an appropriate amount of PBS and stored at -80 ℃.

[0108] The gEVs obtained in Example 1 were analyzed, and the results are shown in Figure 1 Fig. A-B is the observation of exosome morphology by transmission electron microscopy (TEM), the determination of exosome concentration and particle size distribution by nanoparticle tracking analysis (NTA), and C is the Western Blot result figure. As can be seen from A in 1, the average particle size of the milk-derived extracellular vesicles obtained by the combination of rennet method and membrane treatment is 121.37 ± 6.84 nm, the uniformity is good, the polydispersity is < 1%, and the concentration is 4.73 x 1012 ± 2.13 x 1010 particles / mL; as can be seen from B in 1, the milk-derived extracellular vesicles obtained by the extraction process of the application have complete appearance structure, are spherical, monodisperse nanoparticles, and have complete lipid bilayers; as can be seen from Figure 1 the Western Blot result, the signals of three positive proteins CD63, TSG101 and Alix are significant, and the negative protein Calnexin has no expression, indicating that there is no endoplasmic reticulum protein pollution. Figure 1

[0109] 1.2 Animal experiment design

[0110] ​SPF grade C57BL / 6 male pubertal mice (3 weeks old, 13-15 g, Sibeifeng) were placed in standard conditions (22±1℃, 50±10% humidity, 12 h light-dark cycle) and could freely access food and water. After 1 week of adaptive feeding, they were randomly divided into 6 groups (n=10 / group).

[0111] The specific grouping is as follows: Control (no restraint stress + intragastric PBS solution), gEVs-Control (no restraint stress + 0.6 mg / kg gEVs), mEVs-Control (no restraint stress + 0.6 mg / kg mEVs), Model (restraint stress + intragastric PBS solution), gEVs-Model (restraint stress + 0.6 mg / kg gEVs), mEVs-Model (restraint stress + 0.6 mg / kg mEVs).

[0112] The anxiety susceptibility experiment used the above 3-week-old SPF grade C57BL / 6 male pubertal mice, which were randomly divided into groups after 1 week of adaptive feeding and intervened until 8 weeks of age (intragastric administration once a day according to the grouping requirements), and subjected to restraint stress modeling (anxiety model establishment) for 4 weeks.

[0113] The anxiety model was established using the chronic restraint stress (CRS) protocol: the mice were placed in 50 mL conical centrifuge tubes (with holes punched in the side wall to ensure ventilation); restraint was performed at a fixed time every day for 6 hours / day. Intragastric administration was performed 30 min before the restraint stress, and the intervention was continuous for 28 d. Normal feeding mice were placed in new empty cages (without restraint) at the same time period, and were intragastrically administered with PBS solution 30 min before being placed in the new empty cages.

[0114] 1. Physiological detection

[0115] The body weight of the mice was recorded during the establishment of the anxiety model, and the average body weight distribution of the mice in each group at different times is shown in Figure 2 .

[0116] The content of serum corticosterone in the blood of the mice was detected after the establishment of the anxiety model, and the average content of serum corticosterone in each group of mice is shown in Figure 3 .

[0117] 2. Behavioral experiment

[0118] The behavioral test was performed within 3 days after the end of restraint stress, and the test order was open field test, buried bead test, and tail suspension test, with an interval of 24 hours between each test. The test environment was kept quiet, and the mice were adapted for 30 minutes before the test. The test site was cleaned with 75% ethanol between tests to eliminate odor cues.

[0119] Open field test (OFT): The open field device is a 40x40x35 cm uncovered box divided into a central area (25 cm x 25 cm) and a peripheral area, with a camera installed above connected to a behavior analysis system. The mouse is placed in the center of the device, and after 1 min of adaptation, the mouse's activity is recorded for 6 min, and the mouse's route distribution map is obtained as shown in Figure 4 . The mouse's central area residence time, the percentage of central area residence time in total time, and other indicators are analyzed, and the central area residence time result graph is obtained as shown in Figure 5 . The anxiety-like behavior is evaluated.

[0120] Marble burying test (MBT): A 40 cm x 30 cm x 22 cm feeding box is filled with 5 cm thick bedding, and 20 glass beads with a diameter of 14-15 mm are evenly placed. The mouse is placed in the box, and after 30 minutes, the number of buried beads (more than 2 / 3 covered is considered buried) is recorded, and the percentage of buried beads for different groups of mice is obtained as shown in Figure 6 .

[0121] Tail suspension test (TST): The mouse's tail tip 1 cm is fixed to the tail suspension rack with tape, with a suspension height of 50 cm, lasting for 6 minutes. The immobility time (defined as passive suspension without escape-oriented movement) in the last 4 minutes is recorded using a camera connected to a behavior analysis system, reflecting the despair-like behavior, and the resting time of each group of mice is obtained as shown in Figure 7 .

[0122] 3. Histological analysis

[0123] After the behavior test, the mouse's colon is detected for inflammation factor content using RT-qPCR, and the inflammation factor content graph of each group of mice is obtained as shown in Figure 8 B.

[0124] The mouse is euthanized after the behavior test. The proximal colon tissue is fixed with 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). The histopathological changes are observed under a light microscope, and the tissue staining graph is obtained as shown in Figure 8 A.

[0125] As can be seen from Figure 8 , milk-derived extracellular vesicles have a protective effect on the intestinal barrier of anxious mice.

[0126] 4. Short-chain fatty acid analysis

[0127] Gas chromatography (GC) is used to quantitatively analyze short-chain fatty acids (acetic acid, propionic acid, and butyric acid) in feces, and the result graph of the effect of milk-derived extracellular vesicles on the intestinal short-chain fatty acids of anxious mice is obtained as shown in Figure 9 .

[0128] 1.3 Experimental results

[0129] 1. Physiological outcomes

[0130] Body weight indirectly reflects health status and can be used as a surrogate indicator of anxiety severity. From Figure 2 , it is known that CRS significantly inhibited body weight gain in adolescent mice, slowing down the growth rate (p < 0.05), while milk-derived exosomal intervention improved this condition (p < 0.05, Figure 3 Figure 2 ). In addition, the serum corticosterone level of CRS mice was elevated, but significantly decreased after administration of milk-derived exosomes ( Figure 3 ). These results indicate that long-term supplementation of milk-derived exosomes can effectively alleviate CRS-induced growth retardation and stress-related hormone level elevation.

[0131] 2. Behavioral outcomes

[0132] From Figures 4-7 , the effects of mEVs and gEVs on chronic restraint stress (CRS)-induced anxiety were evaluated using the open field test (OFT), the marble burying test (MBT), and the tail suspension test (TST). CRS significantly reduced the central zone residence time and total movement distance ( Figure 4 Figure 5 ), increased the number of buried marbles ( Figure 6 ), and prolonged the immobility time ( Figure 7 ). Milk-derived exosomal intervention significantly reversed these behavioral abnormalities (p < 0.05), restoring most parameters to levels close to the control group. The above results indicate that milk-derived exosomes can alleviate anxiety-like behavior in mice induced by chronic stress.

[0133] 2. Mechanistic indicator outcomes

[0134] In the colon, CRS caused severe mucosal damage, including crypt destruction, goblet cell loss, and inflammatory infiltration, while milk-derived exosomal intervention preserved the epithelial structure and reduced inflammation ( Figure 8 A). CRS significantly increased the expression of inflammatory mediators, including IL-1β, IL-6, and TNF-α, while milk-derived exosomal intervention 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 each major SCFA component was down-regulated to varying degrees. This suggests that anxiety-like phenotypes may be accompanied by the destruction of intestinal microbiota metabolic function. After milk-derived exosomal intervention, the total SCFA content in mouse feces significantly increased ( Figure 9 ​​), approaching control levels, with the most significant increase in acetate content. These findings suggest that milk-derived extracellular vesicles can ameliorate CRS-induced metabolic abnormalities of the intestinal microbiota by selectively upregulating fecal SCFA levels, which can be one of the important mechanisms for its alleviation of anxiety-like behaviors in mice.

[0136] 1.4 Adolescent supplementation of mEVs or gEVs can reduce anxiety susceptibility in adulthood

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

[0138] Anxiety susceptibility experiment: 3-week-old SPF C57BL / 6 male adolescent mice were used, and after 1 week of adaptive feeding, they were randomly divided into 4 groups: Control (no restraint stress + intragastric administration of PBS solution), Model (restraint stress + intragastric administration of PBS solution), gEVs-Model (restraint stress + 0.6 mg / kg gEVs), mEVs-Model (restraint stress + 0.6 mg / kg mEVs) and intervened until 8 weeks of age, then restrained stress modeling (establishment of anxiety model, the establishment method is the same as described above) for 4 weeks, and continuous intragastric intervention during modeling.

[0139] According to the above method, physiological detection and behavioral testing were performed on adult mice, and the physiological detection and behavioral testing results of mice supplemented with milk-derived extracellular vesicles in adolescence were obtained, as shown in Figure 10 .

[0140] It was found that compared with mice receiving PBS intervention in adolescence, mice receiving milk-derived extracellular vesicle intervention exhibited significantly reduced anxiety-like behavior when facing the challenge of chronic traumatic encephalopathy in adulthood ( Figure 10 ). This indicates that adolescent supplementation of milk-derived extracellular vesicles can reduce the anxiety susceptibility of adult mice.

[0141] Example 2

[0142] 2.1 Design of animal experiment scheme

[0143] 60 male C57BL / 6 mice (7 weeks old, 20-23 g) were housed in animal cages at a rate of 5 per cage, maintained at a constant temperature (24 ± 2°C), constant humidity (50 ± 10%), and a 12-hour light-dark cycle, and had free access to water and food.

[0144] After 1 week of adaptation, mice were randomly divided into 4 groups: control group (0.9% saline), Model group (0.9% saline) and mEVs group (0.6 mg / kg bw per day), positive control group (VC, 200 mg / d). D-galactose (D-gal) was used to induce cognitive impairment by intraperitoneal injection (150 mg / kg·d) for 6 weeks, and mEVs were given orally every day, and the control group was given the same volume of PBS. The specific operation is that the control group of mice is injected subcutaneously with 0.9% saline every day, and the other groups are injected subcutaneously with D-gal (200 mg / kg bw) every day. The mEVs group was given 0.6 mg / kg bw of bovine milk-derived milk extracellular vesicles (prepared in Example 1) by gavage every day, and the positive control group was given 200 mg VC by gavage every day.

[0145] 2.2 Behavioral test

[0146] 2.2.1 Open field test

[0147] The open field test was used to evaluate the spontaneous motor ability of mice. The mice were placed in the center of a 50 cm × 50 cm × 40 cm cubic field, and freely moved for 5 min, Figure 11 Figure A is a schematic diagram of the open field test. The trajectory, total travel, center stay time and center entry times were recorded by a video tracking system (Shanghai Xinsoft), and the total movement distance of each group of mice in the open field test was obtained as shown in Figure 11 Figure B, and the movement trajectory diagram of each group of mice in the open field test was obtained as shown in Figure 11 Figure F. The field was cleaned between experiments to avoid odor interference.

[0148] 2.2.2 Novel object recognition index

[0149] The novel object recognition test was used to evaluate the learning and memory ability of mice. The experiment was divided into adaptation period, familiarization period and test period. On the first day (adaptation period), the mice freely explored the empty field for 10 minutes. 24 hours later (familiarization period), two identical objects were placed in the field, and the mice were allowed to explore for 10 minutes. 24 hours later (test period), one of the objects was replaced with a new object, and the mice were allowed to explore again for 5 minutes, Figure 11 Figure C is a schematic diagram of the novel object recognition test procedure. The exploration time of the mice on the familiarization period and the test period was recorded, and the novel object recognition index (NOR index) result diagram of each group of mice was obtained as shown in Figure 11 Figure D. The novel object recognition index (NORI) calculation formula is: NORI = (exploration time of new object / (exploration time of new object + exploration time of old object)) × 100%.

[0150] 2.2.3 Barnes maze

[0151] To evaluate the spatial learning and reference memory ability of mice. The maze is a circular platform with a diameter of 1.2 meters, with 20 holes around the perimeter, one of which is connected to an escape box below, Figure 11 Figure 1 shows the schematic diagram of the Barnes maze experimental device. The experiment includes a 4-day training period and a 5th-day exploration period. Each training lasts a maximum of 3 minutes, guiding the mouse to find and enter the escape box. Record the latency of the mouse to find the escape box and the number of errors (the number of times to explore the wrong hole). 24 hours after the last training, the exploration experiment was conducted, and the escape box was removed. Record the time the mouse spends in the target quadrant (the quadrant where the escape box is located) and the latency of the first time it reaches the target hole. The average time for each group of mice to find the target hole in the Barnes maze is shown in Figure 1G; an example of the movement path of each group of mice in the Barnes maze is shown in Figure 1H. Figure 11 Figure 11 Figure 11 In Figure 1, *P < 0.05, **P < 0.01, ***P < 0.001.

[0152] 2.3 Histological analysis

[0153] 2.3.1 Hippocampal tissue detection

[0154] After behavioral testing, the expression levels of inflammatory factors mRNA in the hippocampus of mice were detected by RT-qPCR. The relative expression levels of inflammatory factors IL-β, IL-6, TNF-α, and NLRP3 mRNA in the hippocampus of each group of mice are shown in Figure 2. Figure 12 Figure 12 In Figure 2, *p < 0.05, **p < 0.01, ***p < 0.001.

[0155] 2.3.2 Colonic tissue detection

[0156] Mice were euthanized after behavioral testing. The proximal colonic tissue was fixed with 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). Histopathological changes were observed under a light microscope, and the tissue staining images are shown in Figure 3A. Figure 13

[0157] RNA was extracted from the colonic tissue of mice, and real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of pro-inflammatory cytokines in the colonic tissue to evaluate colonic inflammation. The results of the expression levels of inflammatory factors in the colonic tissue of each group of mice are shown in Figures 3B~E. Figure 13

[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] In view of the key role of the gut-brain axis in regulating neurobehavioral homeostasis, whether mEVs protect the intestinal epithelial barrier of cognitively impaired mice was investigated. Hematoxylin-eosin (H&E) staining of colon tissue histological examination showed that compared with the control group, the model group had obvious epithelial damage, irregular crypt structure and increased inflammatory cell infiltration (P < 0.001, Figure 13 Fig. 5A). In contrast, mEVs can significantly alleviate these pathological changes and restore the continuity of the epithelium and crypt structure.

[0167] In addition, the inflammation of the colon was evaluated by measuring pro-inflammatory cytokines in the colon 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) in the model mice were increased (P < 0.01, Figure 13 Fig. 5B-E). The addition of mEVs can significantly reduce cytokines, with the most significant reduction in IL-6 (P < 0.01). In summary, these data suggest that mEVs play an effective protective role in the intestinal barrier by inhibiting mucosal inflammation, thereby maintaining intestinal homeostasis and supporting the gut-brain regulatory network.

[0168] The above only is the preferred embodiment of the present application, it should be pointed out that, for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of milk-derived extracellular vesicles in the preparation of a product for improving brain health and / or improving a neurological discomfort condition.

2. Use according to claim 1, characterized in that, The improvement of brain health and / or the improvement of a neurological discomfort condition comprises one or more of relieving anxiety, improving memory, and improving cognitive impairment; And / or, the milk-derived extracellular vesicles improve brain health and / or improve a neurological discomfort condition by remodeling the function of the gut-brain axis; And / or, the milk-derived extracellular vesicles improve brain health and / or improve a neurological discomfort condition by regulating inflammatory pathways.

3. Use according to claim 1, characterized in that, The milk-derived extracellular vesicles improve brain health and / or improve a neurological discomfort condition by one or more of relieving chronic stress anxiety-induced growth retardation, reducing stress-related hormone levels, reducing inflammatory cytokine expression levels, and improving intestinal microbial flora metabolism.

4. Use according to claim 1, characterized in that, The milk-derived extracellular vesicles are selected from milk-derived extracellular vesicles derived from cow milk and / or milk-derived extracellular vesicles derived from goat milk; And / or, the average particle size of the milk-derived extracellular vesicles is 100-150 nm.

5. Use according to claim 4, characterized in that, The milk-derived extracellular vesicles are prepared according to the following method: S1) centrifuging a milk raw material at low temperature to collect an intermediate whey fraction; S2) mixing the intermediate whey fraction, rennet, and calcium salt, incubating, then centrifuging for the second time, taking the supernatant, and sequentially filtering through a filter membrane and ultracentrifuging to obtain milk-derived extracellular vesicles.

6. The use according to any one of claims 1 to 5, characterized in that, The amount of the milk-derived extracellular vesicles is 0.3-30 mg / d for a 60 kg person.

7. A composition for improving brain health and / or for improving a neurological related discomfort condition, characterized in that, The product comprises milk-derived extracellular vesicles.

8. Use of milk-derived extracellular vesicles in the preparation of a product for preventing, treating, and / or improving chronic stress anxiety-like behavior.

9. A composition for preventing, treating and / or ameliorating chronic stress anxiety-like behavior, characterized by, The product comprises milk-derived extracellular vesicles.

10. Use of milk-derived extracellular vesicles in the preparation of a product for preventing, treating, and / or improving a nervous system disease.

11. Use according to claim 10, characterized in that, The nervous system disease is selected from cognitive impairment and / or mental disorder.

12. A composition for preventing, treating and / or ameliorating a nervous system disease, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof. The product comprises milk-derived extracellular vesicles.

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