Nutritional composition containing bovine colostrum and application thereof

Abstract

The invention discloses a nutritional composition containing bovine colostrum and application of the nutritional composition. The present invention provides a nutritional composition, in which the nutritional composition comprises the following essential components: colostrum, a peptide from collagen, and a peptide from casein; furthermore, in the nutritional composition, the mass ratio of the colostrum (in terms of solid), the collagen-derived peptide, and the casein-derived peptide is (3-150): (1-100): (0.5-50). It is found that the composition containing colostrum, peptide from casein and peptide from collagen can synergistically relieve body fatigue and reduce lactic acid accumulation. A zebra fish model and a mouse model are further adopted for anti-oxidation function verification, and experimental results show that the composition containing the colostrum, the peptide from the casein and the peptide from the collagen can synergistically reduce oxidative stress, remove free radicals in the body, slow down aging and enhance immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of functional nutrient research, and in particular relates to a nutrient composition containing bovine colostrum and application thereof. Background Art

[0002] Fatigue is a physiological or psychological phenomenon caused by excessive mental activity, physical activity and mental stimulation. It is related to factors such as imbalance in the supply and demand of energy substances in the body, accumulation of metabolites and oxidative stress response. Fatigue, as a sub-health state, usually manifests as fatigue, weakness, lethargy, weakness and other discomforts. When the body is in a fatigued state for a long time, it will lead to a decline in physical function and even cause diseases, seriously affecting work efficiency and quality of life.

[0003] If fatigue cannot be recovered in time, it will easily lead to overwork, endocrine disorders, decreased immunity, and even organic lesions in the body, posing a threat to health. In addition, excess free radicals in the body and insufficient antioxidant capacity will lead to premature appearance of skin aging symptoms, accompanied by adverse effects such as insomnia, fatigue, dizziness, endocrine disorders, and low immunity. Therefore, anti-fatigue and anti-oxidation are important physiological functions of body regulation. It is of great practical significance to develop products with high safety, anti-fatigue and significant oxidation effects.

[0004] Colostrum is the milk secreted by healthy animals within 72 hours after giving birth. It contains precious active immunoglobulins (IgG, IgA, IgM, IgE, IgD) and rich milk calcium, protein, various trace elements and other nutrients. Studies have found that colostrum contains rich immunoglobulins, which can directly improve immunity, supplement the immune system, reduce the chance of respiratory and gastrointestinal infections, and ensure the healthy growth of the next generation. Colostrum includes cows, sheep, horses, camels, etc.

[0005] Collagen peptides are collagen peptides obtained from enzymatic hydrolysis of collagen-rich animal tissues, including collagen tripeptides, with a molecular weight of less than 10,000 Da accounting for no less than 90%. The absorption rate of collagen peptides is as high as over 95%, which is dozens of times higher than that of ordinary collagen. Scientific research has shown that the absorption rate of ordinary collagen by the human body is only 0%-2.5%, and only small molecule peptides that have been decomposed by enzymes can be truly absorbed by the human body. After being absorbed by the human body, collagen peptides provide the amino acids necessary for the body's synthesis, reconstitute the collagen required by the human body, supplement the lack of human collagen, and promote other functional ingredients to enter human cells to improve the nutritional status and microenvironment of cells and repair damaged cells. At present, collagen peptides can be used as food additives in ordinary foods, such as oral beauty functional foods, anti-aging functional foods, calcium supplement functional foods, body shaping functional foods and foods for special populations.

[0006] Casein is the protein with the highest content in milk, accounting for about 80% of milk protein. Casein peptides produced by protease hydrolysis technology have many physiological effects, such as preventing mineral loss, preventing tooth decay, preventing and treating osteoporosis and rickets, promoting in vitro fertilization of animals, regulating blood pressure, treating iron deficiency anemia, magnesium deficiency neuritis, etc., especially its functional characteristics of promoting the efficient absorption of macroelements (Ca, Mg) and trace elements (Fe, Zn, Cu, Cr, Ni, Co, Mn, Se).

[0007] Reference 1 discloses a skin care composition with calming, sleep-inducing and anti-fatigue effects, which includes collagen peptides, elastin peptides, fish maw oligopeptides, sea cucumber oligopeptides and casein hydrolyzed peptides. However, it does not disclose whether bovine colostrum, collagen tripeptides and casein peptides have anti-fatigue and antioxidant effects. Reference 2 discloses a collagen peptide solid drink, which includes collagen peptide powder, milk mineral salts, casein phosphopeptides and colostrum alkaline protein, which can effectively avoid problems such as lack of sleep, palpitations, and lack of energy caused by tension and stress. However, it also does not disclose whether bovine colostrum, collagen tripeptides and casein peptides have anti-fatigue, antioxidant and immunity-enhancing effects.

[0008] There are no reports on the anti-fatigue, anti-oxidation and immune-enhancing effects of bovine colostrum, collagen tripeptide and casein peptide. In addition, the biological empirical research on the combination of the three is not sufficient and lacks experimental data support. Therefore, there is still room for further exploration to provide a functional combination with a clearer mechanism and simpler ingredients for anti-fatigue, oxidation and immune-enhancing effects.

[0009] Citations

[0010] Reference 1: CN114868928A

[0011] Reference 2: CN115363162A Summary of the invention

[0012] Problem that the invention aims to solve

[0013] Although there have been studies on nutritional compositions such as anti-fatigue or anti-oxidation in the prior art, such research is still not perfect, and there is still room for development of compositions for anti-fatigue, anti-oxidation and immunity enhancement.

[0014] The nutritional composition provided by the present invention can relieve body fatigue, oxidative stress and enhance immunity. By scientifically combining colostrum with a variety of peptides to prepare a corresponding nutritional composition, through nutritional intervention, it can significantly relieve body fatigue and remove free radicals in the human body, delay body aging, and enhance immunity.

[0015] Solutions for solving problems

[0016] [1]. A nutritional composition, wherein the nutritional composition comprises the following essential components:

[0017] colostrum, peptides from collagen, and peptides from casein;

[0018] Furthermore, in the nutritional composition, the mass ratio of the colostrum (in terms of solid content), the collagen-derived peptide, and the casein-derived peptide is (3 to 150):(1 to 100):(0.5 to 50).

[0019] [2] The nutritional composition according to [1], wherein

[0020] The colostrum includes colostrum and / or colostrum powder, and optionally, the colostrum is derived from at least one of cattle, sheep, horses and camels;

[0021] The collagen-derived peptide is a hydrolyzate of collagen-containing tissue; and / or

[0022] The casein-derived peptides are hydrolyzates of casein and / or milk protein.

[0023] [3] The nutritional composition according to [2], wherein

[0024] In the casein-derived peptide, the protein component in the casein-derived peptide has the following molecular weight distribution:

[0025] (i) the content of protein components with a molecular weight of less than 5000 Da is 10% by mass or more;

[0026] The content is the content of the protein component in condition (i) based on dry weight relative to the total protein component in the casein-derived peptides; and or

[0027] In the collagen-derived peptide, the protein component in the collagen-derived peptide has the following molecular weight distribution:

[0028] (ii) the content of protein components with a molecular weight of less than 10,000 Da is 80% by mass or more;

[0029] The content is the content of the protein component in condition (ii) relative to the total protein component in the collagen-derived peptides on a dry weight basis.

[0030] [4] A food comprising or using the nutritional composition according to any one of [1] to [3] as a raw material.

[0031] [5] The food according to [4], wherein the food is a nutritional food or a health food.

[0032] [6] The food according to [4] or [5], wherein the food further contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives and any acceptable excipients.

[0033] [7] Use of the nutritional composition described in any one of [1] to [3] in the preparation of food for anti-fatigue, anti-oxidation and / or immunity enhancement.

[0034] [8]. The use according to [7], wherein the anti-fatigue effect includes at least one of improving hypoxic fatigue, improving exercise behavior, promoting adenosine triphosphate production, promoting lactic acid metabolism, and promoting active oxygen scavenging.

[0035] [9]. The use according to [7], wherein the antioxidant activity includes promoting the removal of active oxygen;

[0036] Optionally, the anti-oxidation comprises reducing malondialdehyde; increasing total superoxide dismutase; increasing reduced glutathione; and / or reducing protein carbonyls.

[0037]

[10] . The use according to [7], wherein the enhancing immunity comprises promoting the secretion of lysozyme, promoting the secretion of immunoglobulins and / or promoting the secretion of cytokines.

[0038] Effects of the Invention

[0039] The present invention found that a composition comprising colostrum, peptides from casein and peptides from collagen can synergistically relieve body fatigue and reduce lactic acid accumulation. The present invention also uses a zebrafish model and a mouse model to verify the antioxidant function. The experimental results show that the composition comprising colostrum, peptides from casein and peptides from collagen can synergistically reduce oxidative stress, remove free radicals in the body, slow down aging, and enhance immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Typical diagram of the movement trajectory of zebrafish after different sample treatments in Example 1. In the figure, the black line represents the slow movement trajectory, the green line represents the medium speed movement trajectory, and the red line represents the fast movement trajectory. DETAILED DESCRIPTION

[0041] I. Definition of Terms

[0042] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0043] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0044] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0045] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0046] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0047] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0048] In the present invention, the term "about" is used to define the numerical range and parameters of the present invention are approximate values, and the specific relevant values ​​are presented as accurately as possible. Unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" usually means that the actual value is within ±3%, ±2%, ±1% or ±0.5% of a particular value or range.

[0049] In the present invention, "infant" is used to refer to a human group of 0 to 6 months old.

[0050] In the present invention, "older infants" are used to refer to a human group of 6 to 12 months of age.

[0051] In the present invention, the term "infant" is used to refer to a human group of 12 to 36 months old.

[0052] In the present invention, "infants" are used to refer to a human group under the age of 3.

[0053] In the present invention, the term "children" used herein refers to a human living group that is older than 3 years old and younger than 12 years old and is in the growth and development stage.

[0054] In this specification, the term "adult" refers to a person over the age of 18 years.

[0055] In this specification, the term "youth" refers to people aged 7-40 years old.

[0056] In this specification, the term "middle-aged people" refers to people aged 41-65 years old.

[0057] In this specification, the term "elderly" or "elderly person" refers to a person over the age of 65 years.

[0058] II. Nutritional Composition

[0059] The present invention provides a nutritional composition, which comprises the following essential components: colostrum, peptides from collagen and peptides from casein; and in the nutritional composition, the mass ratio of the colostrum (in terms of solids), the peptides from collagen and the peptides from casein is (3-150):(1-100):(0.5-50).

[0060] Compared with colostrum, peptides from collagen or peptides from casein alone, the nutritional composition provided by the present invention can be more effective in anti-fatigue (relieving physical fatigue), anti-oxidation (helping to resist oxidation, anti-aging, and delaying aging) and improving immunity (helping to enhance immunity). In terms of anti-fatigue, anti-oxidation and improving immunity, colostrum, peptides from collagen and peptides from casein have a synergistic effect, especially when the content ratio of the three is within the above range, the relevant effect is particularly prominent.

[0061] In order to obtain better synergistic anti-fatigue, antioxidant and immunity-enhancing effects, in some embodiments, in the nutritional composition, the mass ratio of the colostrum, the peptides from collagen and the peptides from casein is (3-130):(1-95):(0.5-45).

[0062] In some preferred embodiments, in the nutritional composition, the mass ratio of the colostrum, the peptides derived from collagen, and the peptides derived from casein is (5-125):(2-90):(1-40).

[0063] In some exemplary embodiments, in the nutritional composition, the mass ratio of the colostrum, the peptides derived from collagen, and the peptides derived from casein is 25:10:10; 5:10:10; 125:90:40; 25:90:40; or 125:2:1.

[0064] In order to obtain further better synergistic anti-fatigue, anti-oxidation and immunity-enhancing effects, in some more preferred embodiments, in the nutritional composition, the mass ratio of the colostrum, the peptides from collagen and the peptides from casein is (5-100): (5-50): (5-30). In some further preferred embodiments, in the nutritional composition, the mass ratio of the colostrum, the peptides from collagen and the peptides from casein is (5-50): (5-20): (5-20), and the more preferred mass ratio is (5-30): (5-15): (10-20), for example 25:10:10; 5:10:10. When the content ratio of colostrum, the peptides from collagen and the peptides from casein is within the above range, the nutritional composition further obtains better synergistic anti-fatigue, anti-oxidation and immunity-enhancing effects.

[0065] (Colostrum components)

[0066] In the present invention, the colostrum refers to the milk secreted within 72 hours after the birth of a healthy animal or prepared into a powder. There is no particular limitation on the source of the colostrum of the present invention in principle. In some embodiments, the colostrum can be bovine colostrum, sheep colostrum, camel colostrum, horse colostrum, etc.

[0067] In some embodiments, the content of immunoglobulin IgG in the colostrum component is 10% by mass or more, based on the weight of the solid matter in the colostrum (ie, when the colostrum is in the form of powder, based on the total mass (dry weight) of the colostrum).

[0068] The present invention finds that controlling the content of immunoglobulin in colostrum to above 10% by mass can better cooperate with peptides from casein and peptides from collagen to produce anti-fatigue, anti-oxidation and immunity-enhancing effects.

[0069] In some specific embodiments, the content of immunoglobulin IgG in the colostrum is 10% by mass or more, preferably 15% by mass or more, and more preferably no more than 30% by mass, based on the weight of the solids in the colostrum (that is, when the colostrum is in the form of powder, based on the total mass (dry weight) of the colostrum), for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, etc. If the content of IgG is too low, the synergistic effect with the peptides from casein and collagen is not obvious, while if the content of IgG is too high, there are usually certain difficulties in industrial preparation, and in some cases, the loss of other nutrients caused by component concentration is too serious.

[0070] In some embodiments, the colostrum may preferably be bovine colostrum. The bovine colostrum is a raw material made by spray drying or freeze drying the milk of a cow within about 3 days after calving. When bovine colostrum is used in combination with peptides from collagen and peptides from casein, the nutritional composition obtains better synergistic anti-fatigue, anti-oxidation and immunity-enhancing effects.

[0071] In some exemplary embodiments, the bovine colostrum may be commercially available bovine colostrum, such as bovine colostrum powder from Feihe (Tailai) Dairy Co., Ltd., but is not limited thereto.

[0072] (Peptide fraction from casein)

[0073] In this specification, casein is a calcium-phosphorus-containing binding protein that is acid-sensitive and precipitates at low pH. Casein is the main protein in the milk of mammals including cows, sheep and humans, and is also known as casein, casein and lactocasein.

[0074] In the present specification, casein-derived peptides (also referred to simply as casein peptides in the present invention) refer to hydrolyzates obtained from casein and / or milk protein (optionally separated and purified) by biotechnological means such as enzymatic hydrolysis.

[0075] In some preferred embodiments, the peptides derived from casein include casein peptides hydrolyzed from casein and / or milk protein peptides hydrolyzed from milk protein.

[0076] In some embodiments, in the peptide fraction from casein, the protein components in the peptide fraction from casein have the following molecular weight distribution:

[0077] (i) the content of protein components with a molecular weight of less than 5000 Da is 10% by mass or more;

[0078] The content is the content of the protein component in condition (i) based on dry weight relative to the total protein component in the casein-derived peptide component.

[0079] In some specific embodiments, in the peptide component derived from casein, the protein component in the peptide component derived from casein has the following molecular weight distribution:

[0080] (i) the content of protein components with a molecular weight of less than 5000 Da is 10% by mass or more, preferably 15% by mass or more, more preferably not more than 40% by mass, for example 15% by mass, 16% by mass, 17% by mass, 18% by mass, 19% by mass, 20% by mass, 25% by mass, 30% by mass, 35% by mass, 40% by mass, etc.;

[0081] The content is the content of the protein component in condition (i) based on dry weight relative to the total protein component in the casein-derived peptide component.

[0082] In this composition (degree of hydrolysis), the peptide component derived from casein can better synergize with the colostrum component and the peptide component derived from collagen in terms of anti-fatigue, anti-oxidation and improving immunity.

[0083] In some exemplary embodiments, the peptide component derived from casein can be commercially available casein peptides, such as casein peptides from Hunan Feile Biotechnology Co., Ltd., derived from bovine milk casein, with a molecular weight of less than 5000 Da accounting for 20%.

[0084] (Peptide fraction from collagen)

[0085] In this specification, collagen is a helical, fibrous protein that is the main component of animal connective tissue.

[0086] In the present specification, peptides derived from collagen (also referred to as collagen peptides or collagen peptides in the present invention) refer to hydrolyzates prepared by protease hydrolysis (optionally separation and purification) of animal tissues rich in collagen.

[0087] In some embodiments, in the peptide component derived from collagen, the protein component in the peptide component derived from collagen has the following molecular weight distribution:

[0088] (ii) the content of protein components with a molecular weight of less than 10,000 Da is 80% by mass or more;

[0089] The content is the content of the protein component in condition (ii) based on dry weight relative to the total protein component in the collagen-derived peptide component.

[0090] In some specific embodiments, in the peptide component derived from collagen, the protein component in the peptide component derived from collagen has the following molecular weight distribution:

[0091] (ii) the content of protein components having a molecular weight of less than 10,000 Da is 80% by mass or more, preferably 90% by mass or more, for example, 90% by mass, 91% by mass, 92% by mass, 93% by mass, 94% by mass, 95% by mass, 96% by mass, 97% by mass, 98% by mass, 99% by mass, 100% by mass, etc.;

[0092] The content is the content of the protein component in condition (ii) based on dry weight relative to the total protein component in the collagen-derived peptide component.

[0093] Under this composition (degree of hydrolysis), the peptide component from collagen can better synergize with the colostrum component and the peptide component from casein in terms of anti-fatigue, anti-oxidation and improving immunity, and has a better absorption rate of more than 95%.

[0094] In some preferred embodiments, the peptide derived from collagen comprises collagen tripeptide.

[0095] In this specification, collagen tripeptide is a collagen fragment composed of three amino acid molecules, and is the collagen essence obtained after enzymatic hydrolysis of collagen. Compared with macromolecular collagen, collagen tripeptide has a smaller molecular weight and is easier to be absorbed by the human body.

[0096] In some exemplary embodiments, the peptide component derived from collagen can be commercially available self-collagen peptides, such as collagen peptides from Hunan Feile Biotechnology Co., Ltd., which are derived from fish skin and contain 92% collagen peptides with a relative molecular weight of less than 10,000 Da.

[0097] Surprisingly, in the present invention, a zebrafish model is used to evaluate the anti-fatigue of a composition comprising colostrum, peptides from casein and peptides from collagen. The model is made by sodium sulfite, which consumes oxygen in the water through chemical reactions, causing zebrafish in the water to be hypoxic, and after a period of time, hypoxic fatigue is induced. Fatigued zebrafish show weakened motor ability, decreased ATP content in the body, increased lactic acid accumulation and increased ROS levels. Zebrafish hypoxic fatigue and physical fatigue that occurs in the human body after high-intensity exercise or long-term work are highly similar in both phenotype and mechanism. The present invention finds that a composition comprising colostrum, peptides from casein and peptides from collagen can synergistically relieve body fatigue and reduce lactic acid accumulation. The present invention also uses a zebrafish model and a mouse model to verify the antioxidant function. The experimental results show that a composition comprising colostrum, peptides from casein and peptides from collagen can synergistically reduce oxidative stress, remove free radicals in the body, and slow down aging. In addition, the present invention has also verified in mice that a composition comprising colostrum, peptides from casein and peptides from collagen promotes the secretion of lysozyme, enhances the body's defense function, and improves the body's immune status; promotes the secretion of immunoglobulins (such as IgA, IgM), enhances the body's humoral immunity; and promotes the secretion of cytokines (such as IL-4, TNF-α), thereby improving the body's immunity.

[0098] The present invention does not particularly limit the form of the nutritional composition, and typically, the nutritional composition may be liquid or solid.

[0099] In some embodiments, in the nutritional composition of the present invention, the content of colostrum is greater than or equal to 10 μg / mL, preferably greater than or equal to 20 μg / mL, more preferably 20-1500 μg / mL, and further preferably 25-1250 μg / mL.

[0100] In some embodiments, in the nutritional composition of the present invention, the content of the peptides derived from casein is greater than or equal to 5 μg / mL, preferably greater than or equal to 10 μg / mL, more preferably 10-1000 μg / mL, and further preferably 10-900 μg / mL.

[0101] In some embodiments, in the nutritional composition of the present invention, the content of the peptides derived from casein is greater than or equal to 3 μg / mL, preferably greater than or equal to 5 μg / mL, more preferably 5-500 μg / mL, and further preferably 5-400 μg / mL.

[0102] The nutritional composition of the present invention is usually an artificially synthesized or compounded composition, that is, it is not a natural composition.

[0103] III. Anti-fatigue and / or anti-oxidative uses

[0104] The present invention proposes to compound colostrum, peptides from casein and peptides from collagen in a specific ratio, which can resist fatigue (relieve physical fatigue), resist oxidation (help resist oxidation and anti-aging) and / or improve immunity (help enhance immunity), and the three substances have a synergistic effect. Therefore, the present invention provides the use of the above-mentioned nutritional composition in preparing food for resisting fatigue, resisting oxidation and / or improving immunity.

[0105] In some embodiments, the anti-fatigue, anti-oxidation and / or immunity enhancement described in the present invention is not intended to treat or prevent diseases.

[0106] In some embodiments, the anti-fatigue comprises improving hypoxic fatigue.

[0107] In some embodiments, the anti-fatigue comprises improving motor performance, such as increasing the distance a subject can move.

[0108] In some embodiments, the anti-fatigue comprises promoting the generation of adenosine triphosphate (ATP), that is, promoting the cellular oxidative phosphorylation process to generate energy.

[0109] In some embodiments, the anti-fatigue comprises promoting lactate metabolism.

[0110] In some embodiments, the anti-fatigue comprises promoting the scavenging of reactive oxygen species (ROS), such as the scavenging of oxygen free radicals.

[0111] In some embodiments, the anti-oxidation includes promoting the scavenging of reactive oxygen species (ROS), such as the scavenging of oxygen free radicals.

[0112] In some specific embodiments, the anti-oxidation includes reducing malondialdehyde (MDA), increasing total superoxide dismutase (T-SOD), increasing reduced glutathione (GSH), and / or reducing protein carbonyl groups to eliminate free radicals, thereby increasing the body's antioxidant capacity.

[0113] In some embodiments, the improving immunity includes promoting the secretion of lysozyme, enhancing the body's defense function, and improving the body's immune status.

[0114] In some embodiments, the improving immunity includes promoting the secretion of immunoglobulins (eg, IgA, IgM), thereby enhancing the body's humoral immunity.

[0115] In some embodiments, the improving immunity includes promoting the secretion of cytokines (eg, IL-4, TNF-α) to improve the body's immunity.

[0116] IV. Nutritional / Health Food

[0117] Furthermore, the present invention also provides a food comprising the above-mentioned nutritional composition as a raw material, which can be a nutritional or health-care food. The nutritional / health-care food has the effects of anti-fatigue (relieving physical fatigue), anti-oxidation (helping to resist oxidation and anti-aging) and / or improving immunity (helping to enhance immunity) through the addition or use of the above-mentioned nutritional composition. And the above-mentioned nutritional / health-care food of the present invention also includes any nutritional / health-care food obtained by further food processing technology of the food precursor obtained by adding the above-mentioned nutritional composition as a raw material.

[0118] In some exemplary embodiments, in the food, based on the weight of the food, colostrum (in powder form) is 0.04%-2% by mass, peptides from collagen are 0.04%-2% by mass, and peptides from casein are 0.04%-2% by mass, and the three satisfy the requirement of “ II. Nutritional Composition The proportion of the three recorded in the "Part.

[0119] Furthermore, the above-mentioned nutritional / health food may contain, in addition to the limited nutritional composition, other ingredients that may be added or used as required.

[0120] Containing milk ingredients or protein ingredients, the milk ingredients include dairy products including fresh milk from fresh cow (sheep) milk, milk powder, whey protein or cheese, etc.; the protein ingredients can come from plant proteins, such as soy protein, peanut protein, etc.

[0121] Plants or plant extracts, including fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, rubber, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblica chinensis and bilberry or their extracts; fruit and vegetable substances such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow rice, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts or their extracts; coffee or its extracts.

[0122] Animal ingredients include meat products from beef, lamb, fish or poultry.

[0123] The fat component may include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, DHA, EPA, ARA, and phospholipids. More specifically, the fat includes safflower seed oil, walnut oil, peanut oil, corn oil, soybean oil, argan oil, olive oil, tea oil, sacha inchi oil, olive oil, coconut oil, perilla oil, deep-sea fish oil, cocoa butter, palm oil, butter, cream, lard, medium-chain triglycerides or lecithin, etc.

[0124] Functional added components include vitamins (one or more of vitamin A, β-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, biotin), starch, modified starch, amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine or L-leucine, etc.), traditional Chinese medicine or traditional Chinese medicine extracts, dietary fiber (inulin, konjac flour, galacto-oligosaccharides, fructo-oligosaccharides, isomalto-oligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin or soybean fiber, etc.).

[0125] Trace element supplements may include metal ion salts of organic acids, such as one or more of calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate and magnesium gluconate.

[0126] Any food-acceptable excipients, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0127] The specific form of the nutritional / health food of the present invention is not particularly limited, and may be solid (powder or block, etc.), semi-solid (soft, paste or thick, etc.) or liquid.

[0128] In the food, the specific contents of colostrum, collagen-derived peptides, and casein-derived peptides are not particularly limited as long as the requirements of food-related laws and regulations are met.

[0129] The present invention does not specifically limit the food with anti-fatigue, anti-oxidation and / or immunity-enhancing effects that can be prepared using the above nutritional composition. In some embodiments, the food is a solid dairy product or a liquid dairy product; for example, milk powder, cheese, yogurt, liquid milk, etc.

[0130] In some specific embodiments, the nutritional or health food can be a powdered reconstituted food (solid beverages, milk powder, instant coffee, cereal powder, nut powder or lotus root powder, etc.), a liquid beverage (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.).

[0131] Typically, the above-mentioned foods include: infant formula milk powder, baby complementary food, children's formula milk powder, maternal milk powder, middle-aged and elderly milk powder or nutritional or dietary supplements, etc.

[0132] Example

[0133] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0134] Example 1: Anti-fatigue-animal experiment example

[0135] The zebrafish fatigue model is created by sodium sulfite, which consumes oxygen in the water through chemical reactions, causing the zebrafish in the water to be hypoxic, and after a period of time, hypoxic fatigue is induced. Fatigued zebrafish show weakened athletic ability, decreased ATP content in the body, increased lactic acid accumulation, and increased ROS levels. Zebrafish hypoxic fatigue is highly similar to the physical fatigue that occurs in the human body after high-intensity exercise or long-term work in both phenotype and mechanism. In addition, zebrafish are small in size; have a short development cycle; have a high number of single spawning times; have a large number of offspring born from the same parent and small individual differences; have obvious behavioral characteristics and are easy to observe; these advantages make zebrafish an ideal model for studying fatigue mechanisms and developing anti-fatigue products.

[0136] 1. Test materials

[0137] 1.1. Experimental animals

[0138] Zebrafish (purchased from Hangzhou Huante Biotechnology Co., Ltd.) were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), and were bred and provided by the applicant's fish farming center. The experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0139] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 4 days post fertilization (4 dpf) were used for the evaluation of the anti-fatigue efficacy of the samples.

[0140] 1.2. Instruments, consumables and reagents

[0141] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); behavior analyzer (Zebra Lab 3.22.3.31, Viewpoint, France); multifunctional microplate reader (SPARK, TECAN, Austria); fully automatic sample rapid grinding instrument (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); 96-well microplate (NestBiotech, China); 96-well plate (Nest Biotech, China).

[0142] Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); anhydrous sodium sulfite (batch number I1428128, Shanghai Aladdin Biochemical Technology Co., Ltd., China); CM-H2DCFDA (batch number 2652734, Invitrogen, USA); BCA protein concentration assay kit (batch number 18C08A89, Boster Biotechnology, China); ATP content assay kit (batch number 0000593109, Promega, USA); lactate (LD) test kit (batch number 20230811, Nanjing Jiancheng Bioengineering Institute, China); sodium chloride injection (batch number S21091609, Hunan Kelun Pharmaceutical Co., Ltd., China).

[0143] 2. Detection method

[0144] 2.1. Safety evaluation of the composition

[0145] 4dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble, and a normal control group and a model control group were set up at the same time, with a capacity of 3mL per well. After 1 day of treatment at 28℃, all experimental groups except the normal control group were given anhydrous sodium sulfite in water-soluble form to establish a zebrafish fatigue model. After 1 hour at 28℃, the toxic phenotype and number of deaths of zebrafish were observed and counted, the safety of the composition was evaluated, and the maximum detection concentration (maximum toxic concentration, MTC) of each substance was determined.

[0146] 2.2. Improvement of motor behavior of the composition

[0147] 4dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The sample was given water-soluble, and the positive control was Red Bull (Red Bull Vitamin Beverage Co., Ltd.). At the same time, a normal control group and a model control group were set up, and the capacity of each well was 3mL. After 1 day of treatment at 28℃, except for the normal control group, all other experimental groups were given anhydrous sodium sulfite in water to establish a zebrafish fatigue model. Subsequently, 10 zebrafish were randomly selected from each experimental group and the total movement distance of the zebrafish for 1h was detected by a behavior analyzer. The statistical analysis results of this indicator were used to evaluate the improvement of the sample's motor behavior. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0148] 2.3. Efficacy of the composition in increasing ATP content

[0149] 4dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The sample was given water-soluble, and the positive control was Red Bull. At the same time, a normal control group and a model control group were set up, and the capacity of each well was 3mL. After 1 day of treatment at 28℃, all experimental groups except the normal control group were given anhydrous sodium sulfite in water to establish a zebrafish fatigue model. After 1h of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and treated according to the ATP content determination kit. The data were collected using a multifunctional microplate reader to detect the ATP content in the zebrafish of each experimental group. The statistical analysis results of this indicator were used to evaluate the efficacy of the sample in increasing the ATP content. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0150] 2.4. ROS scavenging efficacy of the composition

[0151] 4dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble, and the positive control Red Bull was set up at the same time. The normal control group and the model control group were set up at the same time, and the capacity of each well was 3mL. After 1 day of treatment at 28℃, all experimental groups except the normal control group were given anhydrous sodium sulfite in water to establish the zebrafish fatigue model. After 1h of treatment at 28℃, CM-H2DCFDA was added to each experimental group and incubated for 20min. The data were collected using a multifunctional microplate reader, and the ROS fluorescence value of zebrafish in each experimental group was detected. The statistical analysis of this indicator was used to evaluate the ROS scavenging efficacy of the sample. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0152] 2.5. Effect of the composition on reducing lactic acid content

[0153] 4dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble, and the positive control Red Bull was set up at the same time. The normal control group and the model control group were set up at the same time. The volume of each well was 3mL. Three experiments were set up in parallel. After 1 day of treatment at 28℃, all experimental groups except the normal control group were given anhydrous sodium sulfite in water to establish a zebrafish fatigue model. After 1h of treatment at 28℃, zebrafish samples were collected according to the instructions of the lactate determination kit, and data were collected using a multifunctional microplate reader to detect the lactate content in zebrafish in each experimental group. The statistical analysis results of this indicator were used to evaluate the efficacy of the sample in reducing lactate content. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0154] 2.6. Sources of bovine colostrum, collagen peptides, and casein peptides

[0155] Bovine colostrum powder, purchased from Feihe (Tailai) Dairy Co., Ltd., sourced from bovine colostrum, IgG content 20%;

[0156] Collagen peptides (including collagen tripeptide) were purchased from Hunan Feile Biotechnology Co., Ltd., sourced from fish skin, and the proportion of collagen peptides with a relative molecular weight of less than 10,000 Da accounted for 92%;

[0157] Casein peptides were purchased from Hunan Feller Biotechnology Co., Ltd. and were derived from bovine milk casein, with a molecular weight of less than 5000 Da accounting for 20%.

[0158] 3. Test results

[0159] 3.1. Safety evaluation of the composition

[0160] Under the experimental conditions, the maximum detection concentration (MTC) of bovine colostrum, collagen peptide, and casein peptide on zebrafish for fatigue relief was 2000 μg / mL. See Table 1 for details.

[0161] Table 1. Experimental results of the maximum detection concentration of the sample's fatigue relief effect (n=30)

[0162]

[0163] 3.2. Evaluation of the Composition for Improvement of Motor Behavior

[0164] Under the experimental conditions, the improvement effects of the samples and compositions on motor behavior are shown in Table 2.

[0165] Table 2. Efficacy of samples in improving motor behavior (n=10)

[0166]

[0167] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0168] This experiment used zebrafish model animals, and exposed 4-day-old (4dpf) wild-type AB strain zebrafish to different concentrations of bovine colostrum (50, 250, 1250μg / mL) and collagen peptides (20, 100, 900μg / mL) and casein peptides (10, 100, 400μg / mL) monomers and compositions, 20.8μL / mL Red Bull (positive control), and set up a normal control group (normal zebrafish treated with fish farming water) and a model control group (both water-soluble and given anhydrous sodium sulfite to establish a zebrafish fatigue model). After 1 day of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and the total movement distance of the zebrafish in 1h was detected using a behavior analyzer. The statistical analysis results of this indicator were used to evaluate the improvement effect of the sample's movement behavior.

[0169] The experimental results showed that compared with the blank control group, the total movement distance of the model group decreased, indicating that the modeling was successful. Sodium sulfite consumed oxygen in the water through chemical reactions, causing hypoxia in the zebrafish in the water. After a period of time, hypoxic fatigue was induced. After fatigue, the zebrafish's movement ability weakened and the movement distance decreased. The results showed that compared with the model group, 1250μg / mL bovine colostrum (Comparative Example 3) and 100, 900μg / mL collagen peptides (Comparative Examples 5 and 6), 100, 400μg / mL (Comparative Examples 8 and 9) casein peptides were treated to increase the movement distance of zebrafish and improve hypoxic fatigue, with significant differences.

[0170] From the absolute value, the total movement distance of zebrafish in Experimental Example 1 is 5022mm, which is much higher than the total movement distances of 3198mm, 3722mm, and 3697mm in Comparative Examples 2, 5, and 8, and also higher than Comparative Examples 3 and 6 with the same or higher total dosage. The longer the movement distance, the higher the ability to improve hypoxic fatigue. It is suggested that the combination of bovine colostrum 250μg / mL, collagen peptide 100μg / mL, and casein peptide 100μg / mL (Experimental Example 1) has a synergistic effect compared with the single use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0171] The total movement distance of zebrafish in Experimental Example 3 was 4879 mm, which was higher than the total movement distances of 3618 mm, 3840 mm, and 3658 mm in Comparative Examples 3, 6, and 9, indicating that the combined use of 1250 μg / mL bovine colostrum, 900 μg / mL collagen peptide, and 400 μg / mL casein peptide (Experimental Example 3) had a synergistic effect compared with the respective bovine colostrum (Comparative Example 3), collagen peptide (Comparative Example 6), and casein peptide (Comparative Example 9) groups alone.

[0172] The total movement distance of zebrafish in Experimental Example 5 was 3796 mm, which was higher than the total movement distances of 3145 mm, 3722 mm, and 3697 mm in Comparative Example 1, Comparative Example 5, and Comparative Example 8, indicating that the combined use of 50 μg / mL bovine colostrum, 100 μg / mL collagen peptide, and 100 μg / mL casein peptide (Experimental Example 5) had a synergistic effect compared with the respective bovine colostrum (Comparative Example 1), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8) groups alone.

[0173] 3.3. Evaluation of the efficacy of the composition in increasing ATP content

[0174] Under the experimental conditions, the samples and compositions promoted ATP production in fatigued zebrafish. See Table 3 for details.

[0175] Table 3. Efficacy of the composition in increasing ATP content (n=10)

[0176]

[0177] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0178] This experiment used zebrafish model animals, and exposed 4-day-old (4dpf) wild-type AB strain zebrafish to different concentrations of bovine colostrum (50, 250, 1250μg / mL) and collagen peptides (20, 100, 900μg / mL) and casein peptides (10, 100, 400μg / mL) monomers and compositions, 20.8μL / mL Red Bull (positive control), and set up a normal control group (normal zebrafish treated with fish farming water) and a model control group (both water-soluble and given anhydrous sodium sulfite to establish a zebrafish fatigue model). After 1 day of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group, treated according to the ATP content determination kit, and data were collected using a multifunctional microplate reader to detect the ATP content in the zebrafish of each experimental group. The statistical analysis results of this indicator were used to evaluate the efficacy of the sample in increasing ATP content.

[0179] The experimental results showed that compared with the model group, treatment with 1250μg / mL bovine colostrum (Comparative Example 3), 900μg / mL collagen peptide (Comparative Example 6), and 400μg / mL casein peptide (Comparative Example 9) can promote ATP production in zebrafish, generate energy, promote cellular oxidative phosphorylation process, and increase spontaneous activity of the body, with significant differences.

[0180] From the absolute value, the ATP content of zebrafish in Experimental Example 1 is 107446RLU, which is much higher than the ATP content of zebrafish in Comparative Example 2, Comparative Example 5, and Comparative Example 8, which is 84212RLU, 85727RLU, and 86658RLU, and is also higher than Comparative Examples 3 and 6 with the same or higher total dosage. The higher the ATP content, the higher the energy generated by the sample and the composition. The sample and the composition promote the oxidative phosphorylation process of cells and increase the spontaneous activity of the body. It is suggested that the combination of bovine colostrum 250μg / mL, collagen peptide 100μg / mL, and casein peptide 100μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0181] The ATP content of zebrafish in Experimental Example 2 is 94931RLU, which is much higher than the ATP content of zebrafish in Comparative Example 1, Comparative Example 6, and Comparative Example 9, which are 83486RLU, 92890RLU, and 93383RLU. It is suggested that the combination of bovine colostrum 250μg / mL, collagen peptide 900μg / mL, and casein peptide 400μg / mL (Experimental Example 2) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 1), collagen peptide (Comparative Example 6), and casein peptide (Comparative Example 9).

[0182] The ATP content of zebrafish in Experimental Example 3 is 107446RLU, which is much higher than the ATP content of zebrafish in Comparative Example 3, Comparative Example 6, and Comparative Example 9, which are 93028RLU, 92890RLU, and 93383RLU. It is suggested that the combination of bovine colostrum 1250μg / mL, collagen peptide 900μg / mL, and casein peptide 400μg / mL (Experimental Example 3) has a synergistic effect compared with the single use groups of bovine colostrum (Comparative Example 3), collagen peptide (Comparative Example 6), and casein peptide (Comparative Example 9).

[0183] Corresponding conclusions can also be drawn from Experimental Examples 4 and 5. In particular, Experimental Example 5 is much higher than Comparative Examples 2, 3, 6, and 9 with the same or higher total dosage.

[0184] 3.4. Evaluation of ROS scavenging efficacy of the composition

[0185] The effects of samples and compositions on free radical scavenging in fatigued zebrafish under the experimental conditions are shown in Table 4.

[0186] Table 4. Efficacy of ROS scavenging by samples and compositions (n=10)

[0187]

[0188] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0189] This experiment used zebrafish model animals, and 4-day-old (4dpf) wild-type AB strain zebrafish were exposed to different concentrations of bovine colostrum (50, 250, 1250μg / mL) and collagen peptides (20, 100, 900μg / mL) and casein peptides (10, 100, 400μg / mL) monomers and compositions, 20.8μL / mL Red Bull (positive control), and a normal control group (normal zebrafish treated with fish farming water) and a model control group (both water-soluble and given anhydrous sodium sulfite to establish a zebrafish fatigue model) were set up at the same time. After continuing to treat at 28℃ for 1h, CM-H2DCFDA was added to each experimental group and incubated for 20min. The data was collected using a multifunctional microplate reader, and the ROS fluorescence value of the zebrafish in each experimental group was detected. The statistical analysis of this indicator was used to evaluate the ROS scavenging efficacy of the sample.

[0190] The experimental results showed that compared with the model group, 1250 μg / mL bovine colostrum (Comparative Example 3), 900 μg / mL collagen peptide (Comparative Example 6), and 400 μg / mL casein peptide (Comparative Example 9) treatment can eliminate the excessive accumulation of oxygen free radicals in the body of fatigue zebrafish and reduce the body damage caused by fatigue.

[0191] From the absolute value, the ROS content of zebrafish in Experimental Example 1 is 16161, which is much lower than the ROS content of fatigue zebrafish in Comparative Example 2, Comparative Example 5, and Comparative Example 8 (18582, 18406, and 18486), and is also lower than Comparative Examples 3 and 6 with the same or higher total dosage. The lower the value, the higher the ability of the sample and composition to remove free radicals from the fatigue body, and the stronger the ability to reduce the body damage caused by fatigue. It is suggested that the combination of bovine colostrum 250μg / mL, collagen peptide 100μg / mL, and casein peptide 100μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0192] The ROS content of zebrafish in Experimental Example 3 was 15651, which was much lower than the ROS content of fatigue zebrafish in Comparative Example 3, Comparative Example 6, and Comparative Example 9, which were 17944, 17976, and 17378, respectively. This indicates that the combination of bovine colostrum 1250 μg / mL, collagen peptide 900 μg / mL, and casein peptide 400 μg / mL (Experimental Example 3) has a synergistic effect compared with the respective bovine colostrum (Comparative Example 3), collagen peptide (Comparative Example 6), and casein peptide (Comparative Example 9) groups used alone.

[0193] Corresponding conclusions can also be drawn from Experimental Examples 4 and 5. In particular, Experimental Example 5 is lower than Comparative Examples 2, 3, and 6 with the same or higher total dosage.

[0194] 3.5. Effect of the composition on reducing lactic acid content

[0195] Under the experimental conditions, the samples and compositions promote lactic acid metabolism in fatigued zebrafish. See Table 5 for details.

[0196] Table 5. Efficacy of samples and compositions in reducing lactic acid content (n=10)

[0197]

[0198] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0199] This experiment used zebrafish model animals, and exposed 4-day-old (4dpf) wild-type AB strain zebrafish to different concentrations of bovine colostrum (50, 250, 1250μg / mL) and collagen peptides (20, 100, 900μg / mL) and casein peptides (10, 100, 400μg / mL) monomers and compositions, 20.8μL / mL Red Bull (positive control), and set up a normal control group (normal zebrafish treated with fish farming water) and a model control group (both water-soluble and given anhydrous sodium sulfite to establish a zebrafish fatigue model). After continuing to treat at 28℃ for 1h, zebrafish samples were collected according to the instructions of the lactate determination kit, and data were collected using a multifunctional microplate reader to detect the lactate content in the zebrafish of each experimental group. The statistical analysis results of this indicator were used to evaluate the efficacy of the sample in reducing lactate content.

[0200] The experimental results showed that compared with the model group, 1250μg / mL bovine colostrum, 900μg / mL collagen peptide, and 400μg / mL casein peptide treatment could reduce the excessive lactic acid in the body of fatigue zebrafish and promote the metabolism of lactic acid. There was a significant difference.

[0201] From the absolute value, the lactic acid content of zebrafish in Experimental Example 1 is 0.296, which is much lower than the lactic acid content of fatigue zebrafish in Comparative Example 2, Comparative Example 5, and Comparative Example 8, which is 0.411, 0.397, and 0.394, and is also lower than Comparative Examples 3 and 6 with the same or higher total dosage. The lower the value, the higher the ability of the sample and composition to promote lactic acid metabolism. It is suggested that the combination of bovine colostrum 250 μg / mL, collagen peptide 100 μg / mL, and casein peptide 100 μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0202] The lactic acid content of zebrafish in Experimental Example 3 was 0.287, which was much lower than the lactic acid content of fatigue zebrafish in Comparative Example 3, Comparative Example 6, and Comparative Example 9, which were 0.339, 0.367, and 0.353, respectively. This indicates that the combined use of 1250 μg / mL bovine colostrum, 900 μg / mL collagen peptide, and 400 μg / mL casein peptide (Experimental Example 3) has a synergistic effect compared with the respective bovine colostrum (Comparative Example 3), collagen peptide (Comparative Example 6), and casein peptide (Comparative Example 9) groups used alone.

[0203] The corresponding conclusion can also be drawn from Experimental Example 5. Experimental Example 5 is lower than Comparative Example 2 with the same or higher total dosage.

[0204] From the above experimental results, it can be seen that the highest concentration (2000 μg / mL) of bovine colostrum, collagen peptides and casein peptides in the composition of the present invention did not cause any abnormality in zebrafish, and its state was similar to that of normal zebrafish, indicating that the sample and the composition are highly safe. Under this condition, the composition can significantly improve the motor ability of hypoxic fatigue zebrafish, reduce lactic acid content, promote ATP production, and reduce free radicals generated by fatigue.

[0205] Example 2: Antioxidant-Animal Experiment Example

[0206] Free radicals are highly active molecules or atoms produced in human life activities. The production and removal of free radicals are in a dynamic equilibrium state under normal circumstances. However, when the free radicals in the body are produced too much or removed too slowly, this dynamic equilibrium will be destroyed, causing the free radicals to accumulate in the body. These accumulated free radicals will cause damage to the body and cause a series of reactions such as oxidative stress and inflammation. This study refers to the "Health Food Function Test and Evaluation Method" (2023 Edition) and uses the ethanol oxidative damage mouse animal model to study the antioxidant function of the nutritional composition.

[0207] 1. Instruments and kits

[0208] 1.1. Instruments and Equipment

[0209] Multifunctional microplate reader (BioTek Synergy LX), HH-6 constant temperature water bath (Guohua Electric Co., Ltd.), desktop centrifuge (LABGIC CF1524R), vortex mixer (DLAB MX-S), various specifications of pipettes (Thermo Fisher Scientific), 37°C constant temperature incubator (Shanghai Bowen Instrument Co., Ltd., WGP-400)

[0210] 1.2. Kit

[0211] The trace reduced glutathione (GSH) assay kit (catalog number: A006-2-1), malondialdehyde (MDA) test kit (catalog number: A003-1) and total superoxide dismutase (T-SOD) assay kit (catalog number: A001-1) were purchased from Nanjing Jiancheng Bioengineering Institute, the protein carbonyl content colorimetric test kit (catalog number: E-BC-K117-M) was purchased from Wuhan Elaruite Biotechnology Co., Ltd., and the BCA protein concentration assay kit (catalog number: PC0020) was purchased from Beijing Solebow Technology Co., Ltd.

[0212] 2. Experimental Methods

[0213] 2.1. Mouse model construction

[0214] 200 25g healthy adult Balb / c female mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were purchased and randomly divided into 20 groups, 1 model control group, 1 blank control group, 1 positive control group and 17 test sample groups. The 17 sample groups were given different concentrations of test samples (see Table 6), the model control group and the blank control group were given the same volume of deionized water, and the positive control group was given the same volume of vitamin C (100 mg / kg) by continuous intragastric gavage for 30 days. After the last intragastric gavage, the model control group, the positive control group and the 35 sample groups were fasted for 16 hours (overnight), and then 50% ethanol 12mL / kg BW was given by intragastric gavage once. Blood was collected after 6 hours, and the whole blood of the 20 groups of mice was left to stand at 4°C overnight. The next day, it was centrifuged at 4000rpm / min for 10 minutes at 4°C, and the mouse serum was drawn for subsequent experiments.

[0215] 2.2. Determination of malondialdehyde content in mouse blood lipid oxidation products

[0216] Take the malondialdehyde (MDA) test kit (item number: A003-1) and let it stand at room temperature for 20 minutes. Add 0.1mL of anhydrous ethanol, 0.1mL of reagent one, 3mL of reagent two and 1mL of reagent three to the blank tube, add 0.1mL of 10nmol / mL standard, 0.1mL of reagent one, 3mL of reagent two and 1mL of reagent three to the standard tube, add 0.1mL of mouse serum to be tested, 0.1mL of reagent one, 3mL of reagent two and 1mL of reagent three to the measurement tube, cover the centrifuge tube, poke a small hole in the cover with a needle, mix with a vortex mixer, place in a 95℃ water bath for 40 minutes, take out and cool with running water, then centrifuge at 4000rpm / min for 10 minutes, then take 200μL of supernatant from each tube and add it to the 96-well ELISA plate, use an ELISA reader to measure the absorbance value of each well at 532nm for calculation. The formula is as follows:

[0217]

[0218] 2.3. Determination of total superoxide dismutase activity in mouse serum

[0219] Take the total superoxide dismutase (T-SOD) assay kit (Cat. No.: A001-1) and let it stand at room temperature for 20 minutes. Add 1.0mL of reagent 1, 0.05mL of distilled water, 0.1mL of reagent 2, 0.1mL of reagent 3, 0.1mL of reagent 4 and 2mL of color developer to the control tube, add 1.0mL of reagent 1, 0.05mL of 2.5-fold diluted mouse serum to be tested, 0.1mL of reagent 2, 0.1mL of reagent 3, 0.1mL of reagent 4 and 2mL of color developer to the assay tube, mix well, let stand at room temperature for 10 minutes, then take 200μL of supernatant from each tube and add it to the 96-well ELISA plate, use an ELISA reader to measure the absorbance value of each well at 550nm for calculation. The formula is as follows:

[0220]

[0221] 2.4. Determination of reduced glutathione content in mouse serum

[0222] Take the trace reduced glutathione (GSH) assay kit (Cat. No.: A006-2-1) and let it stand at room temperature for 20 minutes. Take 0.05 mL of mouse serum, add 0.2 mL of reagent 1 and mix well, centrifuge at 3500 rpm / min for 10 minutes, and take the supernatant as the sample to be tested. Prepare the sample and reagent as follows and add them to the 96-well plate. Add 100 μL of supernatant, 100 μL of reagent 2 and 25 μL of reagent 3 to the blank well, 100 μL of reagent 1, 100 μL of reagent 2 and 25 μL of reagent 3 to the assay well, 100 μL of 20 μmol / LGHS standard solution, 100 μL of reagent 2 and 25 μL of reagent 3 to the standard well, gently shake the plate to mix, let stand for 5 minutes, and measure the absorbance value A of each well at 405 nm using an enzyme reader. The formula is as follows:

[0223]

[0224] 2.5. Determination of protein carbonyl content in mouse serum

[0225] Take the protein carbonyl content colorimetric test kit (Cat. No.: E-BC-K117-M) and the BCA protein concentration assay kit (Cat. No.: PC0020) and let it stand at room temperature for 20 minutes. Test tube: Take 0.1mL of the sample to be tested and 0.4mL of reagent three and add them to a 2mL EP tube; control tube: Take 0.1mL of the sample to be tested and 0.4mL of reagent four and add them to a 2mL EP tube. Vortex mix for 1min and react at 37℃ for 30 minutes in the dark. Add 0.5mL of reagent five to each tube, vortex mix for 1min, centrifuge at 4℃, 13780×g for 10 minutes. Remove the supernatant with a pipette and discard it, leaving the precipitate. Add 1mL of anhydrous ethanol and ethyl acetate mixed application solution to each tube, vortex mix for 1 minute, centrifuge at 4℃, 13780×g for 10 minutes, remove the supernatant with a pipette and discard it, leaving the precipitate. After washing three times, add 1.25 mL of reagent VI to each tube, vortex to mix, and react at 37°C for 15 minutes. Vortex to dissolve the precipitate, and centrifuge at 4°C, 13780×g for 15 minutes. Use a micropipette to take 0.3 mL of the supernatant to the ELISA plate, and measure the OD value at 370 nm on the ELISA instrument; at the same time, take the supernatant from the assay tube and measure its protein concentration using the BCA protein concentration kit. The results are calculated as follows:

[0226]

[0227] Data analysis

[0228] SPSS25.0 software was used to statistically analyze the serum oxidation factor data of each group of mice, calculate the mean value and standard deviation, and analyze the significant differences.

[0229] 3. Test results

[0230] 3.1. Evaluation of the antioxidant function of the composition

[0231] Under the experimental conditions, the effects of the samples and compositions on the contents of malondialdehyde (MDA), total superoxide dismutase (T-SOD), reduced glutathione (GSH) and protein carbonyl (PCO) in blood are shown in Table 6.

[0232] Table 6. Effects of samples on malondialdehyde in mouse blood

[0233]

[0234] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0235] The experimental results showed that compared with the blank control group, the MDA content in the model group increased, the SOD and GSH contents decreased, and the protein carbonyl increased, indicating that the modeling was successful. Excessive ethanol intake can cause an imbalance in the free radical reaction in the body and increase peroxidation products. The content of MDA, the end product of lipid peroxidation, can reflect the degree of peroxidation damage and cell damage; the oxidation of carbohydrates and lipids can produce reactive carbonyls, and the combination of reactive oxygen and reactive carbonyls with proteins leads to the formation of carbonyl dysfunction proteins in plasma and tissues. The increase in PCO content indicates oxidative stress-dependent cell damage. Oxidative damage is one of the important mechanisms of alcoholic liver damage. When ethanol is metabolized by the liver, ROS is produced in the cells, consuming a large amount of reducing substances SOD, GSH-Px, and GSH, resulting in a decrease in antioxidant capacity and an inability to remove excessive free radicals, thereby causing oxidative stress and a rapid increase in cellular lipid peroxidation products.

[0236] The results showed that compared with the model group, 125, 625 μg / mL bovine colostrum (Comparative Examples 2 and 3) and 10, 50, 450 μg / mL collagen peptide (Comparative Examples 4, 5 and 6) and 50, 200 μg / mL (Comparative Examples 8 and 9) casein peptide treatment can reduce the content of MDA, increase the content of SOD and GSH, and reduce the content of protein carbonyl, with significant differences. It suggests that it can eliminate excessive free radicals and increase the body's antioxidant capacity.

[0237] From the absolute value, the malondialdehyde content in the serum of mice in Experimental Example 1 is 8.99nmoL / mL, which is much lower than the MDA content of 10.06nmoL / mL, 9.43nmoL / mL, and 9.85nmoL / mL in Comparative Examples 2, 5, and 8, and also lower than Comparative Example 3. The lower the MDA content, the stronger the ability to improve oxidative damage. It is suggested that the combination of bovine colostrum 125μg / mL, collagen peptide 50μg / mL, and casein peptide 50μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0238] The content of SOD in serum of Experimental Example 1 is 165.00U / mL, which is much higher than the content of SOD in Comparative Examples 2, 5, and 8 (157.70U / mL, 161.37U / mL, and 158.87U / mL), and also higher than Comparative Example 3. The higher the SOD content, the stronger the ability to scavenge free radicals. It is suggested that the combination of bovine colostrum 125μg / mL, collagen peptide 50μg / mL, and casein peptide 50μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0239] The content of GSH in serum of Experimental Example 1 is 76.53 μmoL / mL, which is much higher than the content of GSH in Comparative Examples 2, 5, and 8 (70.97 μmoL / mL, 72.73 μmoL / mL, and 72.67 μmoL / mL), and also higher than Comparative Example 3. The higher the GSH content, the stronger the ability to scavenge free radicals. It is suggested that the combination of bovine colostrum 125 μg / mL, collagen peptide 50 μg / mL, and casein peptide 50 μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0240] The content of protein carbonyl in serum of Experimental Example 1 is 1.57nmoL / mg, which is much lower than the content of protein carbonyl in Comparative Examples 2, 5, and 8, 1.74nmoL / mg, 1.72nmoL / mg, and 1.73nmoL / mg. The lower the protein carbonyl content, the lower it is than Comparative Examples 3 and 6, indicating that the ability to improve oxidative stress is stronger. It is suggested that the combination of bovine colostrum 125μg / mL, collagen peptide 50μg / mL, and casein peptide 50μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8).

[0241] Corresponding conclusions can also be drawn from Experimental Examples 2, 3, 4, and 5. Among them, the MDA content of Experimental Example 5 is lower than that of Comparative Examples 2, 3, and 9; the SOD content of Experimental Example 5 is higher than that of Comparative Examples 2, 3, and 9; the GSH content of Experimental Example 5 is higher than that of Comparative Examples 2 and 3; and the protein carbonyl content of Experimental Example 5 is lower than that of Comparative Examples 2, 3, and 9.

[0242] Example 3: Immunization-Animal Experiment Example

[0243] 1. Instruments and kits

[0244] 1.1. Instruments and Equipment

[0245] Multifunctional microplate reader (BioTek Synergy LX), vortex mixer (DLAB MX-S), various pipettes (Thermo Fisher Scientific), 37°C constant temperature incubator (Shanghai Platinum Instrument Co., Ltd. WGP-400)

[0246] 1.2. Kit

[0247] Mouse immunoglobulin A (IgA) ELISA kit (Cat. No.: E-EL-M0690), mouse lysozyme (LZM) ELISA kit (Cat. No.: E-EL-M0764), mouse interleukin 4 (IL-4) ELISA kit (Cat. No.: E-EL-M0043), and mouse tumor necrosis factor α (TNF-α) ELISA kit (Cat. No.: E-EL-M3063) were purchased from Wuhan Elaruite Biotechnology Co., Ltd.; mouse immunoglobulin M (IgM) ELISA kit (Cat. No.: RE3086M) was purchased from Wuhan Redi Biotechnology Co., Ltd.

[0248] 2. Experimental Methods

[0249] 2.1. Mouse model construction

[0250] 200 25g healthy adult Balb / c female mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were purchased and randomly divided into 20 groups, 1 model control group, 1 blank control group, 1 positive control group and 17 test sample groups. The 20 sample groups were given different concentrations of test samples (see Table 1), the model control group and the blank control group were given the same volume of deionized water, and the positive control group was given the same volume of vitamin C (100 mg / kg) by continuous intragastric administration for 30 days. After the last intragastric administration, they were fasted for 16 hours (overnight), and then the whole blood of 20 groups of mice was collected and left to stand at 4°C overnight. The next day, the blood was centrifuged at 4000 rpm / min for 10 minutes at 4°C to obtain mouse serum.

[0251] 2.2. Determination of immunoglobulin A content in mouse serum

[0252] Take the mouse immunoglobulin A (IgA) ELISA kit (Cat. No.: E-EL-M0690) and let it stand at room temperature for 20 minutes. Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of the diluted standard to the standard wells, 100 μL of the standard or mouse serum to the blank wells, and 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 minutes. Then shake off the liquid in the wells, add 100 μL of the biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Then add 100 μL of HRP enzyme conjugate working solution to each well, cover the plate with a film, incubate at 37°C for 30 minutes, shake off the liquid in the well, and repeat the above washing operation 5 times. Add 90 μL of substrate solution to each well, cover the plate with a film, and incubate at 37°C in the dark for 15 minutes. Then add 50 μL of stop solution to each well to stop the reaction, and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader. Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, fit the standard curve of the four-parameter logistic function on the double logarithmic coordinate axis. Substitute the measured OD value into the curve to calculate the IgA content in the sample.

[0253] 2.3. Determination of lysozyme (LZM) content in mouse serum

[0254] Take the mouse lysozyme (LZM) ELISA kit (Cat. No.: E-EL-M0764) and let it stand at room temperature for 20 minutes. Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of the diluted standard to the standard wells, 100 μL of the standard or mouse serum to the blank wells, and 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 minutes. Then shake off the liquid in the wells, add 100 μL of the biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this plate washing step 3 times. Then add 100 μL of HRP enzyme conjugate working solution to each well, cover the plate with a film, incubate at 37°C for 30 minutes, shake off the liquid in the well, and repeat the above plate washing operation 5 times. Add 90 μL of substrate solution to each well, cover the plate with a film, and incubate at 37°C in the dark for 15 minutes. Then add 50 μL of stop solution to each well to stop the reaction, and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader. Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, fit the standard curve of the four-parameter logistic function on the double logarithmic coordinate axis. Substitute the measured OD value into the curve to calculate the lysozyme content in the sample.

[0255] 2.4. Determination of interleukin-4 content in mouse serum

[0256] Take the mouse interleukin 4 (IL-4) enzyme-linked immunosorbent assay kit (Cat. No.: E-EL-M0043) and let it stand at room temperature for 20 minutes. Set standard wells, blank wells, and sample wells respectively. Add 100 μL of the diluted standard to the standard wells, 100 μL of the standard or mouse serum to the blank wells, and 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 minutes. Then shake off the liquid in the wells, add 100 μL of the biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Then add 100 μL of HRP enzyme conjugate working solution to each well, cover the plate with a film, incubate at 37°C for 30 minutes, shake off the liquid in the well, and repeat the above washing operation 5 times. Add 90 μL of substrate solution to each well, cover the plate with a film, and incubate at 37°C in the dark for 15 minutes. Then add 50 μL of stop solution to each well to stop the reaction, and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader. Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, fit the standard curve of the four-parameter logistic function on the double logarithmic coordinate axis. Substitute the measured OD value into the curve to calculate the IL-4 content in the sample.

[0257] 2.5. Determination of immunoglobulin M content in mouse serum

[0258] Mouse Immunoglobulin M (IgM) ELISA Kit (Cat. No.: RE3086M), let stand at room temperature for 20 minutes. Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of the diluted standard to the standard wells, 100 μL of the standard or mouse serum to the blank wells, and 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 minutes. Then shake off the liquid in the wells, add 100 μL of the biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Then add 100 μL of HRP enzyme conjugate working solution to each well, cover the plate with a film, incubate at 37°C for 30 minutes, shake off the liquid in the well, and repeat the above washing operation 5 times. Add 90 μL of substrate solution to each well, cover the plate with a film, and incubate at 37°C in the dark for 15 minutes. Then add 50 μL of stop solution to each well to stop the reaction, and immediately measure the OD value of each well at a wavelength of 450 nm using an ELISA reader. Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, fit the standard curve of the four-parameter logistic function on the double logarithmic coordinate axis. Substitute the measured OD value into the curve to calculate the IgM content in the sample.

[0259] 2.6. Determination of tumor necrosis factor-α content in mouse serum

[0260] Take the mouse tumor necrosis factor α (TNF-α) ELISA kit (Cat. No.: E-EL-M3063) and let it stand at room temperature for 20 minutes. Set standard wells, blank wells and sample wells respectively. Add 100 μL of the diluted standard to the standard wells, 100 μL of the standard or mouse serum to the blank wells, and 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 minutes. Then shake off the liquid in the wells, add 100 μL of the biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Then add 100 μL of HRP enzyme conjugate working solution to each well, cover the plate with a film, incubate at 37°C for 30 minutes, shake off the liquid in the well, and repeat the above washing operation 5 times. Add 90 μL of substrate solution to each well, cover the plate with a film, and incubate at 37°C in the dark for 15 minutes. Then add 50 μL of stop solution to each well to stop the reaction, and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader. Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, fit the standard curve of the four-parameter logistic function on the double logarithmic coordinate axis. Substitute the measured OD value into the curve to calculate the TNF-α content in the sample.

[0261] Data analysis

[0262] SPSS25.0 software was used to statistically analyze the serum immune factor data of each group of mice, calculate the mean value and standard deviation, and analyze the significant differences.

[0263] 3. Experimental results

[0264] 3.1. Effects of samples and compositions on lysozyme in mouse serum

[0265] The effects of the samples and compositions on the lysozyme content in blood under the experimental conditions are shown in Table 7.

[0266] Table 7. Effects of samples on lysozyme in mouse serum

[0267]

[0268] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0269] Lysozyme in serum is present in the body's tissues and body fluids, and plays an important role in the body's repair and regeneration and resistance to foreign infections. Its secretion level can reflect the active state of phagocytes and the ability to transmit antigens, and largely reflects the body's nonspecific immune state. The experimental results show that compared with the blank control group, 25, 125, 625 μg / mL bovine colostrum (Comparative Example 1, Comparative Example 2 and Comparative Example 3) and 50, 450 μg / mL collagen peptide (Comparative Example 5 and Comparative Example 6) and 50, 200 μg / mL casein peptide (Comparative Example 8 and Comparative Example 9) can promote the secretion of lysozyme in serum, enhance the body's defense function, and improve the body's immune state.

[0270] From the absolute value, the content of lysozyme in the serum of mice in Experimental Example 1 is 1971.80ng / mL, which is much higher than the content of lysozyme in Comparative Examples 2, 5, and 8 (1780.25ng / mL, 1788.97ng / mL, and 1749.40ng / mL), and also higher than Comparative Examples 3 and 6. The higher the lysozyme content, the stronger the ability to enhance the body's defense function. It is suggested that the combination of bovine colostrum 125μg / mL, collagen peptide 50μg / mL, and casein peptide 50μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8). The content of lysozyme in the serum of mice in Experimental Example 2 was 2088.11 ng / mL, which was much higher than the content of lysozyme in Comparative Examples 2, 6, and 9, which was 1780.25 ng / mL, 1849.67 ng / mL, and 1849.60 ng / mL. The higher the content of lysozyme, the stronger the ability to enhance the body's defense function. It is suggested that the combination of bovine colostrum 125 μg / mL, collagen peptide 450 μg / mL, and casein peptide 200 μg / mL (Experimental Example 1) has a synergistic effect compared with the single-use groups of bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 6), and casein peptide (Comparative Example 9).

[0271] This conclusion can also be drawn from Experimental Examples 3, 4, and 5. Among them, Experimental Example 5 is even higher than Comparative Examples 2, 3, 6, and 9.

[0272] 3.2. Effects of samples and compositions on immunoglobulins in mouse serum

[0273] The effects of the samples and compositions on the immunoglobulin content in serum under the experimental conditions are shown in Table 8.

[0274] Table 8. Effects of samples on immunoglobulins in mouse serum

[0275]

[0276] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0277] Immunoglobulin is a protein with antibody activity, present in blood or body fluids, can kill pathogenic microorganisms under the synergistic action of complement, is an important part of the body's humoral immunity, mediates humoral immunity, and is also the main immunoglobulin for serological diagnosis and detection. The level of body immunity can be reflected by the immunoglobulin content in serum. The experimental results show that compared with the blank control group, 25, 125, 625 μg / mL bovine colostrum (Comparative Example 1, Comparative Example 2 and Comparative Example 3) and 10, 50, 450 μg / mL collagen peptide (Comparative Example 4, Comparative Example 5 and Comparative Example 6) and 5, 50, 200 μg / mL casein peptide (Comparative Example 7, Comparative Example 8 and Comparative Example 9) can promote the secretion of immunoglobulin in serum after treatment, and enhance the ability of body humoral immunity.

[0278] From the absolute value point of view, the content of IgA in the serum of mice in Experimental Example 1 is 13.07 mg / mL, and the content of IgM is 131.31 μg / mL, which is much higher than the content of IgA in Comparative Examples 2, 5, and 8 of 11.75 mg / mL, 11.83 mg / mL, and 11.57 mg / mL, and the content of IgM is 117.61 μg / mL, 119.74 μg / mL, and 115.78 μg / mL. The higher the immunoglobulin content, the higher it is than Comparative Examples 3 and 6, indicating that the body's immune ability is stronger, suggesting that the combination of 125 μg / mL of bovine colostrum, 50 μg / mL of collagen peptide, and 50 μg / mL of casein peptide (Experimental Example 1) has a synergistic effect compared with the respective bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5) and casein peptide (Comparative Example 8) groups alone.

[0279] This conclusion can also be drawn from Experimental Examples 2, 3, 4, and 5. Example 5 is even higher than Comparative Examples 2, 3, 6, and 9.

[0280] 3.3. Effects of samples and compositions on cytokines in mouse serum

[0281] Under the experimental conditions, the effects of the samples and compositions on the secretion of cytokines in serum are shown in Table 9.

[0282] Table 9. Effects of samples on cytokines in mouse serum

[0283]

[0284] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0285] Cytokines are small molecule proteins secreted by immune cells and non-immune cells after stimulation. They can be divided into interleukin interferon, tumor necrosis factor, etc. Among them, interleukin refers to cytokines that regulate each other between self cells or immune cells. In the immune response process, it has an important regulatory effect on promoting cell-to-cell interaction, cell proliferation and differentiation. It can maintain the physiological balance of the body and resist the infection of pathogens. Through specific target cells, in combination with corresponding receptors, information is transmitted to cells to cause immune response reactions, thereby improving the body's immune capacity. The experimental results show that compared with the blank control group, 25, 125, 625 μg / mL colostrum (Comparative Example 1, Comparative Example 2 and Comparative Example 3) and 10, 50, 450 μg / mL collagen peptides (Comparative Example 4, Comparative Example 5 and Comparative Example 6) and 5, 50, 200 μg / mL casein peptides (Comparative Example 7, Comparative Example 8 and Comparative Example 9) can promote the secretion of cytokines in serum after treatment, and improve the body's immunity.

[0286] From the absolute value point of view, the content of IL-4 in the serum of mice in Experimental Example 1 was 28.90pg / mL, and the content of TNF-α was 52.02pg / mL, which were much higher than the contents of IL-4 in Comparative Examples 2, 5, and 8 (25.85pg / mL, 26.02pg / mL, and 25.45pg / mL), and the contents of TNF-α were 46.53pg / mL, 46.83pg / mL, and 45.81pg / mL, which were also higher than those in Comparative Examples 3 and 6. The higher the secretion of cytokines, the stronger the body's immune ability, suggesting that the combination of 125μg / mL of bovine colostrum, 50μg / mL of collagen peptide, and 50μg / mL of casein peptide (Experimental Example 1) has a synergistic effect compared with the respective bovine colostrum (Comparative Example 2), collagen peptide (Comparative Example 5), and casein peptide (Comparative Example 8) groups used alone.

[0287] This conclusion can also be drawn from Experimental Examples 2, 3, 4, and 5. Example 5 is also higher than Comparative Examples 2, 3, 6, and 9.

[0288] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0289] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A nutritional composition, wherein: The nutritional composition comprises the following essential components: colostrum, peptides from collagen, and peptides from casein; Furthermore, in the nutritional composition, the mass ratio of the colostrum (in terms of solid content), the collagen-derived peptide, and the casein-derived peptide is (3 to 150):(1 to 100):(0.5 to 50).

2. The nutritional composition according to claim 1, wherein The colostrum includes colostrum and / or colostrum powder, and optionally, the colostrum is derived from at least one of cattle, sheep, horses and camels; The collagen-derived peptide is a hydrolyzate of collagen-containing tissue; and / or The casein-derived peptides are hydrolyzates of casein and / or milk protein.

3. The nutritional composition according to claim 2, wherein In the casein-derived peptide, the protein component in the casein-derived peptide has the following molecular weight distribution: (i) the content of protein components with a molecular weight of less than 5000 Da is 10% by mass or more; The content is the content of the protein component in condition (i) based on dry weight relative to the total protein component in the casein-derived peptides; and / or In the collagen-derived peptide, the protein component in the collagen-derived peptide has the following molecular weight distribution: (ii) the content of protein components with a molecular weight of less than 10,000 Da is 80% by mass or more; The content is the content of the protein component in condition (ii) relative to the total protein component in the collagen-derived peptides on a dry weight basis.

4. A food, wherein The food comprises or uses the nutritional composition according to any one of claims 1 to 3 as a raw material.

5. The food according to claim 4, wherein The food is a nutritional food or a health food.

6. The food according to claim 4 or 5, wherein The food further contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional added ingredients and any acceptable auxiliary materials.

7. Use of the nutritional composition according to any one of claims 1 to 3 in the preparation of food for anti-fatigue, anti-oxidation and / or immunity enhancement.

8. The use according to claim 7, wherein The anti-fatigue comprises at least one of improving hypoxic fatigue, improving exercise behavior, promoting adenosine triphosphate generation, promoting lactic acid metabolism and promoting active oxygen removal.

9. The use according to claim 7, wherein: The anti-oxidation includes promoting the removal of active oxygen; Optionally, the anti-oxidation comprises reducing malondialdehyde; increasing total superoxide dismutase; increasing reduced glutathione; and / or reducing protein carbonyls.

10. The use according to claim 7, wherein: The enhancement of immunity includes promoting the secretion of lysozyme, promoting the secretion of immunoglobulin and / or promoting the secretion of cytokines.

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