Costrum peptide composition and application thereof

Abstract

The invention mainly provides a novel colostrum peptide composition (composite peptide composition) and application thereof, the colostrum peptide composition takes colostrum hydrolyzed peptide, collagen peptide and casein peptide as necessary components, the three components have synergistic effect to achieve the effects of assisting in resisting fatigue, assisting in resisting oxidation (delaying senescence) and improving immunity, and in addition, the colostrum peptide composition has the effects of resisting fatigue and resisting oxidation (delaying senescence) and improving immunity. The synergistic effect can be embodied in the form of synergistic reduction and synergism.

Description

Technical Field

[0001] The invention belongs to the field of food, and in particular relates to a nutritional composition based on colostrum hydrolysate and its application in anti-fatigue and anti-aging. 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] 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.

[0005] 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).

[0006] 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.

[0007] In addition, in the field of food, the combined application of multiple nutrients has also been paid attention to for a long time. For example, reference 1 provides a functional solid beverage, which includes collagen tripeptide, casein phosphopeptide and many other nutrients.

[0008] Nevertheless, in the field of food, there is still a broad space for research to provide a complex nutritional composition that helps to resist fatigue and oxidation.

[0009] References:

[0010] Reference 1: CN108967800A Summary of the invention

[0011] Problem that the invention aims to solve

[0012] At present, there is no technical report on the combined anti-fatigue, anti-oxidation and immunity enhancement of collagen peptides, casein peptides and colostrum hydrolyzed peptides. Although some results show that colostrum hydrolyzed peptides, collagen tripeptides and casein peptides each seem to have anti-fatigue and oxidation effects, biological empirical research on the three is not obvious. The most common use of a variety of substances with health benefits is currently lacking in experimental data support. Therefore, there is still room for further exploration in providing a functional composition with a clearer mechanism and simpler ingredients for anti-fatigue, oxidation and immunity enhancement.

[0013] Based on the existing situation in the art, the applicant unexpectedly discovered that collagen peptides (also called "collagen peptides", the same below), casein peptides and colostrum hydrolyzed peptides show a synergistic effect in assisting anti-fatigue, assisting in slowing down aging and increasing immunity, and that this effect can be reflected in the form of synergistic reduction and synergy.

[0014] Specifically, it is surprising that in the study of the scheme of the present invention, the applicant used a zebrafish model to evaluate the anti-fatigue effect of the composition. The model was created by sodium sulfite, which consumed oxygen in the water through chemical reactions, causing the zebrafish in the water to be hypoxic, and after a period of time, hypoxic fatigue was induced. The fatigued zebrafish showed weakened motor 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.

[0015] Based on the above biology, the applicant has found that a composition based on colostrum hydrolyzed peptides, collagen peptides, and casein peptides can simultaneously show the effects of improving anti-fatigue, anti-oxidation / anti-aging, and immune capacity. And through the synergistic effect of the three, the above effects can be achieved with a smaller dosage.

[0016] Therefore, the present invention mainly provides a new composite peptide composition and its application, which uses colostrum hydrolyzed peptide, collagen peptide and casein peptide as essential components. The synergistic effect of the three can play the role of assisting anti-fatigue and anti-oxidation (slowing down aging) as well as improving immunity.

[0017] Furthermore, the present invention also provides a new application, that is, using the composite peptide composition to simultaneously help improve anti-fatigue, anti-oxidation and immune capabilities.

[0018] Solutions for solving problems

[0019] It has been found that the above technical problems can be solved by implementing the following technical solutions:

[0020] [1] The present invention first provides an edible composition that helps to improve anti-fatigue, anti-oxidation and immune capabilities at the same time, wherein the edible composition includes as essential ingredients:

[0021] Colostrum hydrolyzed peptide ingredients, collagen peptide ingredients and casein peptide ingredients.

[0022] [2] The edible composition according to [1], characterized in that the colostrum hydrolyzed peptides and casein peptides are derived from animal milk; and the collagen peptides include collagen tripeptides.

[0023] Preferably, the colostrum hydrolyzed peptides are prepared by protease hydrolysis of colostrum and / or colostrum powder, and the proportion of molecular weight less than 5000Da is not less than 50%; the casein peptides are obtained by hydrolysis of casein or milk protein, and the proportion of molecular weight less than 5000Da is not less than 10%; the collagen peptides are prepared by protease hydrolysis of collagen-rich animal tissues, and the proportion of molecular weight less than 10000Da is not less than 90%.

[0024] [3]. Furthermore, the present invention also provides a food that helps to improve anti-fatigue, anti-oxidation and immune capabilities at the same time, wherein the food contains or uses the edible composition according to [1] or [2].

[0025] [4] The food according to [3], wherein the food is liquid, semi-solid or solid at room temperature.

[0026] [5]. Furthermore, the present invention also provides a new application:

[0027] One is the use of a composition containing three essential ingredients, namely, colostrum hydrolyzed peptide component, collagen peptide component and casein peptide component, in the preparation of an edible composition that helps to improve anti-fatigue, anti-oxidation and immune capabilities.

[0028] Another aspect is the use of a composition containing three essential ingredients, namely, colostrum hydrolyzed peptide component, collagen peptide component and casein peptide component, in the preparation of food that helps to improve anti-fatigue, anti-oxidation and immune capabilities.

[0029] [6]. The use according to [5], wherein the food comprises a solid beverage, a dairy product, a food for special dietary uses or a nutritional food, and the dairy product comprises a children's formula powder product or a middle-aged and elderly formula powder product.

[0030] Effects of the Invention

[0031] By implementing the above technical solution, the present invention can achieve the following technical effects:

[0032] 1) By using colostrum hydrolyzed peptides, collagen peptides and casein peptides as essential ingredients, it can simultaneously assist in anti-fatigue and anti-oxidation (slowing down aging) as well as improving immunity;

[0033] 2) Through the synergistic effect of the three ingredients of colostrum hydrolyzed peptide, collagen peptide and casein peptide, a smaller total amount of the three substances can be used to obtain the same or similar effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the swimming trajectory of zebrafish in 3.2 evaluation of motor behavior improvement in (I) Anti-fatigue-animal experiment example; Note: 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

[0035] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. 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.

[0036] 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.

[0037] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0038] 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.

[0039] In the present invention, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0040] In the present invention, the use of "optional" or "optionally" indicates that certain substances, components, execution steps, applied conditions and other factors are used or not used.

[0041] In the present invention, "Da" is used to represent the unit of molecular weight "Dalton", that is, "Dalton".

[0042] In the present invention, unless otherwise specified, the "normal temperature" used generally refers to a temperature of 23±2°C.

[0043] In the present invention, unless otherwise stated, "%" used herein means weight or mass percentage.

[0044] In this specification, the term "about" or "substantially" or "essentially" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value essentially inevitably contains the standard deviation caused by the aforementioned testing device or method. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" generally means that the standard deviation of the actual value and the theoretical model or theoretical data is within 3%, preferably 2%, and more preferably 1%.

[0045] In the present invention, 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.

[0046] The present invention mainly provides a nutritional composition that can simultaneously assist in improving or enhancing anti-fatigue ability, antioxidant ability and immune ability. The composition is obtained with colostrum hydrolyzed peptide components, collagen peptide components and casein peptide components as essential components. Through biological verification, it has been found that the combined use of the three components can significantly obtain the above three effects. In particular, when obtaining the same or similar effects, the amount of the complex of the three components used is significantly lower than the case of using one substance alone.

[0047] (Colostrum Hydrolyzed Peptides)

[0048] The colostrum hydrolyzed peptide of the present invention may also be referred to as "colostrum peptide". In principle, there is no particular limitation on its source. For example, it may be colostrum (liquid colostrum or colostrum powder) derived from various animal milks, such as bovine colostrum, sheep colostrum, horse colostrum, camel colostrum, etc. Preferably, it may be derived from bovine colostrum.

[0049] Furthermore, there is no particular limitation on the preparation method of colostrum hydrolyzed peptides. In some specific embodiments, colostrum raw materials are obtained and then the colostrum hydrolyzed peptides are obtained by defatting, enzymolysis, filtration, concentration, drying, etc. In addition, colostrum hydrolyzed peptides can also be directly obtained through various commercial channels.

[0050] In addition, with regard to the molecular weight of the colostrum hydrolyzed peptides of the present invention, from the perspective of both nutritional properties and digestibility, the proportion of colostrum hydrolyzed peptides with a molecular weight of less than 5000 Da is not less than 50%.

[0051] (Collagen Peptide)

[0052] Collagen peptides, also known as "collagen peptides", are a type of product that is extracted, hydrolyzed and refined from fresh animal tissues rich in collagen (including skin, bones, tendons, tendons, scales, etc.), with a relative molecular mass of less than 10,000 Da. In addition, the number of amino acids contained in these peptides ranges from 2 to 100, including collagen tripeptides.

[0053] In some preferred embodiments, the content of components smaller than 10,000 Da in the collagen peptide of the present invention is not less than 90% by mass.

[0054] Generally speaking, after a series of physical and chemical treatments or biological enzymes, the collagen raw materials undergo depolymerization and cleavage of their molecular chains, ultimately yielding low molecular weight collagen polypeptides. The molecular weight and distribution of collagen polypeptides vary with the extraction method. Commonly used extraction methods include chemical hydrolysis, enzymatic hydrolysis, and subcritical water hydrolysis.

[0055] In some preferred embodiments of the present invention, the collagen peptide refers to a collagen peptide mainly comprising collagen tripeptide (CTP) or substantially consisting of it. The so-called collagen tripeptide is usually the smallest and most stable structural unit of collagen prepared from pig skin and fish skin using bioengineering technology. It is a tripeptide containing glycine, proline (or hydroxyproline) plus one other amino acid. Simply put, the structure of collagen tripeptide can be simply expressed as Gly-xy. Its average molecular weight is 280 to 600 Daltons. Since the molecular weight of collagen tripeptide is very small, it can be fully absorbed by the human body.

[0056] (Casein Peptide)

[0057] The source of the casein peptide of the present invention is not particularly limited, and may be, for example, a hydrolyzate derived from various animal milks, such as cow's milk, goat's milk, horse's milk, camel's milk, etc. Preferably, the casein peptide may be derived from cow's milk.

[0058] By defatting and separating the components of animal milk, an enriched casein component can be obtained, and further, casein peptides can be obtained by enzymolysis with one or more enzymes, preferably, the proportion of casein peptides with a molecular weight less than 5000 Da is not less than 10%. Similarly, the casein peptides of the present invention can be self-made or commercially available.

[0059] (Nutritional composition)

[0060] The nutritional composition of the present invention is mainly a composition containing the above three components as essential components.

[0061] In principle, there is no particular restriction on the ratio of the three components in the composition, and any ratio or combination can be used, for example, the ratio of colostrum hydrolyzed peptide component: collagen peptide component: casein peptide is (0.2-30): 1: 1, (0.3-10): 1: 1, (0.4-3): 1: 1, (0.5-2.5): 1: 1, etc. The nutritional composition of the present invention can simultaneously provide the effect of assisting in improving or enhancing anti-fatigue ability, antioxidant ability and immune ability.

[0062] (food)

[0063] The food of the present invention uses or contains the above-mentioned nutritional composition.

[0064] In principle, there is no particular limitation on the form of the food of the present invention, and the food may be in liquid, semi-solid or solid form at room temperature.

[0065] Furthermore, the food of the present invention may generally be pasta, beverages, reconstituted foods, baked goods, sauces or functional nutritional supplements.

[0066] Wheaten food may include staple foods processed or made from flour-based raw materials, coarse grain staple foods, etc., such as steamed bread, cakes, noodles, stuffed staple foods, etc.

[0067] For baked pastries, it can be baked cakes or biscuits mainly based on cream, eggs and baking powder.

[0068] There are no particular restrictions on beverages, which may be solid protein beverages, fruit beverages, vegetable beverages, milk tea beverages, tea beverages, milk, yogurt, vitamin beverages, etc. For fruit or vegetable beverages, in addition to the three functional components of the present invention, they may also include fruit or vegetable juice, or their solid components, etc. For vitamin beverages, in addition to the three functional components of the present invention, they may also mainly include various functional vitamins and other functional ingredients, such as white sugar, sodium cyclamate, acesulfame potassium, taurine, potassium sorbate, lysine, inositol, vitamin pp, vitamin B6, vitamin B12, citric acid, etc.

[0069] Reconstituted foods may typically be reconstituted milk powder products, such as adult milk powder, middle-aged and elderly milk powder, and the like.

[0070] In addition, there are no special restrictions on functional nutritional supplement foods, which can be used as nutritional supplements or meal replacements. Such foods, in addition to the three functional components of the present invention, may also include one or more of the following components: protein, fat, essential carbohydrates, dietary fiber, supplementary elements, vitamins, plants or dietary ingredients, etc.

[0071] Wherein, the protein is selected from at least one of milk protein powder, whey protein powder, soy protein isolate, whole milk powder, whole egg powder, lactoferrin, bovine colostrum, amino acids, and other protein peptides; and the amino acids are selected from at least one of L-lysine-L-glutamate, L-glutamate, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, and L-leucine; the other protein peptides are selected from one or more of soybean oligopeptides, wheat protein peptides, silkworm pupa protein peptides, marine fish oligopeptide powder, kola peptides, amino peptides, and ovalbumin peptides.

[0072] The fat 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, 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, and lecithin.

[0073] The carbohydrates include starch or modified starch. The dietary fiber includes one or more of inulin, konjac flour, galacto-oligosaccharide, fructo-oligosaccharide, isomalto-oligosaccharide, soybean polysaccharide, cyclodextrin, resistant dextrin and soybean fiber.

[0074] The supplementary elements are selected from metal ion salts of citric organic acids, such as one or more of calcium citrate, calcium L-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.

[0075] The vitamins are selected from 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, and biotin.

[0076] The plants or dietary ingredients include one or more of cereals, beans, potatoes, vegetables, fungi and algae, nuts and seeds, fish and shrimp, poultry and livestock meat, and fruits.

[0077] In addition, there is no particular restriction on the content of the three essential components of the present invention in food in principle, which is related to the relevant laws and regulations of different countries or regions. As long as the above regulations are met, the content of the three components can be arbitrarily configured.

[0078] In some preferred embodiments, the content of colostrum hydrolyzed peptides in the food can be greater than 0.04 mass%, for example, 0.04 mass% to 1 mass%; the collagen peptide can be greater than 0.04 mass%, for example, 0.04 mass% to 2 mass%; the casein peptide can be greater than 0.04 mass%, for example, 0.04 mass% to 2 mass%.

[0079] (New Application)

[0080] The present invention also provides new uses of a compound of three components, namely, colostrum hydrolyzed peptide component, collagen peptide component and casein peptide component, or the above nutritional composition.

[0081] The use is in the preparation of edible compositions or foods that are simultaneously helpful in improving anti-fatigue, anti-oxidation and immune capabilities.

[0082] Example

[0083] 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.

[0084] (Source of raw materials)

[0085] Colostrum hydrolyzed peptides, homemade, sourced from bovine colostrum, 65% of which are less than 5000Da in molecular weight;

[0086] 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%;

[0087] 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%.

[0088] (I) Anti-fatigue-animal experiment example

[0089] 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.

[0090] 1. Test materials

[0091] 1.1. Experimental animals

[0092] 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), bred and provided by the company'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).

[0093] 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.

[0094] 1.2. Instruments, consumables and reagents

[0095] 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); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); 96-well plate (NestBiotech, China); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); multi-function microplate reader (SPARK, TECAN, Austria).

[0096] ATP content determination kit (Batch No. 0000511614, Promega, USA); lactate determination kit (Cat. No. A019-2-1, Batch No. 20230105, Nanjing Jiancheng Bioengineering Institute, China); anhydrous sodium sulfite (Batch No. I1428128, Shanghai Aladdin Biochemical Technology Co., Ltd., China); CM-H2DCFDA (Batch No. 2456929, Invitrogen, USA); BCA protein concentration determination kit (Batch No. AR1189, Boster Bioengineering Co., Ltd., China).

[0097] 2. Detection Method

[0098] 2.1. Safety evaluation of the composition

[0099] 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.

[0100] 2.2. Improvement of motor behavior of the composition

[0101] 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.

[0102] 2.3. Efficacy of the composition in increasing ATP content

[0103] 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.

[0104] 2.4. ROS scavenging efficacy of the composition

[0105] 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.

[0106] 2.5. Effect of the composition on reducing lactic acid content

[0107] 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.

[0108] 3. Test results

[0109] 3.1. Safety evaluation of the composition

[0110] Under the experimental conditions, the maximum detection concentration (MTC) of colostrum hydrolyzed peptides, collagen peptides, and casein peptides for fatigue relief in zebrafish is 2000 μg / mL. See Table 1-1 for details.

[0111] Table 1-1. Results of the maximum detection concentration test for the fatigue relief effect of the samples (n=30)

[0112]

[0113] 3.2. Evaluation of the Composition for Improvement of Motor Behavior

[0114] Under the experimental conditions, the improvement effects of the samples and compositions on motor behavior are shown in Table 1-2.

[0115] Table 1-2. Efficacy of samples in improving motor behavior (n=10)

[0116]

[0117] Note: Compared with the model group, *p<0.05, **p<0.01, ***p<0.001

[0118] This experiment used zebrafish model animals. Wild-type AB strain zebrafish of 4 days old (4 dpf) were exposed to different concentrations of colostrum hydrolyzed peptides (50, 250, 1250 μg / mL), collagen peptides (20, 100, 900 μg / mL), and casein peptides (10, 100, 400 μg / mL) monomers and compositions, and 20.8 μL / mL Red Bull (positive control). A blank 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 also set up. After 1 day of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and the total movement distance of the zebrafish in 1 hour was detected using a behavior analyzer. The statistical analysis results of this indicator were used to evaluate the efficacy of the sample in improving movement behavior. The swimming trajectory of zebrafish can be seen in Figure 1 .

[0119] The experimental results show:

[0120] i. When the three components are used separately, the total movement distance changes unilaterally with the increase in the dosage of the three components;

[0121] ii. In Example 1, the total concentration of the three components is 250+100+100=450 μg / mL, and the corresponding effect exceeds that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL), indicating that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone;

[0122] ii. The total concentration of the three components in Example 2 is 50+100+100=250 μg / mL, and the corresponding effect is significantly higher than that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL), indicating that when the same or similar effects are obtained, the total amount of the three components used is much smaller than the amount used when the three components are used alone.

[0123] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0124] 3.3. Evaluation of the efficacy of the composition in increasing ATP content

[0125] Under the experimental conditions, the samples and compositions promote ATP production in fatigued zebrafish. See Tables 1-3 for details.

[0126] Table 1-3. Efficacy of the composition in increasing ATP content (n=10)

[0127]

[0128] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0129] This experiment used zebrafish model animals, and 4-day-old (4dpf) wild-type AB strain zebrafish were exposed to different concentrations of colostrum hydrolyzed peptides (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 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.

[0130] The experimental results show:

[0131] i. When the three components are used separately, the ATP content changes unilaterally with the increase in the dosage of the three components;

[0132] ii. In Example 1, the total concentration of the three components is 250+100+100=450 μg / mL, and the corresponding effect exceeds that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL), indicating that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone;

[0133] ii. The total concentration of the three components in Example 2 is 50+100+100=250 μg / mL, and the corresponding effect is significantly higher than that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL), indicating that when the same or similar effects are obtained, the total amount of the three components used is much smaller than the amount used when the three components are used alone.

[0134] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0135] 3.4. Evaluation of ROS scavenging efficacy of the composition

[0136] Under the experimental conditions, the effects of the samples and compositions on the clearance of free oxygen species (ROS) in fatigued zebrafish are shown in Tables 1-4.

[0137] Table 1-4. Efficacy of ROS scavenging by samples and compositions (n=10)

[0138]

[0139] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0140] This experiment used zebrafish model animals, and 4-day (4dpf) wild-type AB strain zebrafish were exposed to different concentrations of colostrum hydrolyzed peptides (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 zebrafish in each experimental group was detected. The statistical analysis of this indicator was used to evaluate the ROS scavenging efficacy of the sample.

[0141] The experimental results show:

[0142] i. When the three components are used alone, the ROS scavenging effect changes unilaterally with the increase in the dosage of the three components;

[0143] ii. The total concentration of the three components in Example 1 is 250+100+100=450 μg / mL, and the corresponding effect is comparable to that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone;

[0144] ii. The total concentration of the three components in Example 2 is 50+100+100=250 μg / mL, and the corresponding effect is comparable to that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone.

[0145] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0146] 3.5. Effect of the composition on reducing lactic acid content

[0147] Under the experimental conditions, the samples and compositions promote lactic acid metabolism in fatigued zebrafish. See Tables 1-5 for details.

[0148] Table 1-5. Efficacy of samples and compositions in reducing lactic acid content (n=10)

[0149]

[0150] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0151] This experiment used zebrafish model animals, and 4-day-old (4dpf) wild-type AB strain zebrafish were exposed to different concentrations of colostrum hydrolyzed peptides (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, 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.

[0152] The experimental results show:

[0153] i. When the three components are used alone, the lactic acid content changes unilaterally with the increase in the dosage of the three components;

[0154] ii. The total concentration of the three components in Example 1 is 250+100+100=450 μg / mL, and the corresponding effect is comparable to that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone;

[0155] ii. The total concentration of the three components in Example 2 is 50+100+100=250 μg / mL, and the corresponding effect is comparable to that of Comparative Example 3 (1250 μg / mL), Comparative Example 6 (900 μg / mL) and Comparative Example 9 (400 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone.

[0156] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0157] (II) Antioxidant-Animal Experiment Example

[0158] 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 functional composition.

[0159] 1. Instruments and kits

[0160] 1.1 Instruments and Equipment

[0161] 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 Platinum Instrument Co., Ltd. WGP-400)

[0162] 1.2 Kit

[0163] Malondialdehyde (MDA) test kit (Cat. No.: A003-1) and total superoxide dismutase (T-SOD) assay kit (Cat. No.: A001-1) were purchased from Nanjing Jiancheng Bioengineering Institute.

[0164] 2. Experimental Methods

[0165] 2.1 Mouse model construction

[0166] 140 25g healthy adult Balb / c female mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were purchased and randomly divided into 14 groups, including 1 model control group, 1 blank control group, 1 positive control group and 11 test sample groups. The 11 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 gavage for 30 days. After the last intragastric gavage, the model control group, the positive control group and the 11 sample groups were fasted for 16 hours (overnight), and then 50% ethanol 12mL / kg BW was given by intragastric gavage once. Blood was collected 6 hours later, and the whole blood of the 14 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 collected for subsequent experiments.

[0167] 2.2 Determination of malondialdehyde content in mouse blood lipid oxidation products

[0168] 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:

[0169]

[0170] 2.3 Determination of total superoxide dismutase activity in mouse serum

[0171] 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:

[0172]

[0173] 2.4 Data Analysis

[0174] 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.

[0175] 3. Evaluation of the antioxidant function of the composition

[0176] Under the experimental conditions, the effects of the samples and compositions on the contents of various substances in the blood are shown in Tables 1-6.

[0177] Table 1-6. Data on the effect of samples on mouse blood

[0178]

[0179] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0180] The experimental results show:

[0181] i. When the three components are used separately, the test parameters change unilaterally with the increase of the dosage of the three components;

[0182] ii. The total concentration of the three components in Example 1 is 125+50+50=225 μg / mL, and the corresponding effect is comparable to or better than that of Comparative Example 3 (625 μg / mL), Comparative Example 6 (450 μg / mL) and Comparative Example 9 (200 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone;

[0183] ii. The total concentration of the three components in Example 2 is 25+50+50=125 μg / mL, and the corresponding effect is comparable to or better than that of Comparative Example 3 (625 μg / mL), Comparative Example 6 (450 μg / mL) and Comparative Example 9 (200 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone.

[0184] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0185] (III) Immunization-Animal Experiment Example

[0186] 1. Instruments and kits

[0187] 1.1 Instruments and Equipment

[0188] 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)

[0189] 1.2 Kit

[0190] Mouse lysozyme (LZM) ELISA kit (Cat. No.: E-EL-M0764) 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.

[0191] 2. Experimental Methods

[0192] 2.1 Mouse model construction

[0193] 140 25g healthy adult Balb / c female mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were purchased and randomly divided into 14 groups, including 1 model control group, 1 blank control group, 1 positive control group and 11 test sample groups. The 11 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 oral gavage for 30 consecutive days. After the last oral gavage, they were fasted for 16 hours (overnight), and then whole blood from the 14 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.

[0194] 2.2 Determination of lysozyme (LZM) content in mouse serum

[0195] 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.

[0196] 2.3 Determination of immunoglobulin M content in mouse serum

[0197] 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.

[0198] 2.4 Determination of tumor necrosis factor-α content in mouse serum

[0199] 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.

[0200] 2.5 Data Analysis

[0201] 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.

[0202] 3. Experimental results

[0203] 3.1. Effects of samples and compositions on lysozyme in mouse serum

[0204] Under the experimental conditions, the effects of the samples and compositions on the lysozyme content in the blood are shown in Tables 1-7.

[0205] Table 1-7. Effects of samples on lysozyme in mouse serum

[0206]

[0207] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0208] Lysozyme in serum exists in the body's tissues and body fluids, and plays an important role in the body's repair and regeneration and in fighting against foreign infections. Its secretion level can reflect the active state of phagocytes and their ability to transmit antigens, and to a large extent reflects the body's nonspecific immune state.

[0209] The experimental results show:

[0210] i. When the three components are used alone, the lysozyme content changes unilaterally with the increase in the dosage of the three components;

[0211] ii. The total concentration of the three components in Example 1 is 125+50+50=225 μg / mL, and the corresponding effect is significantly better than that of Comparative Example 3 (625 μg / mL), Comparative Example 6 (450 μg / mL) and Comparative Example 9 (200 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone;

[0212] ii. The total concentration of the three components in Example 2 is 25+50+50=125 μg / mL, and the corresponding effect is comparable to that of Comparative Example 3 (625 μg / mL), Comparative Example 6 (450 μg / mL) and Comparative Example 9 (200 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components used is less than the amount used when the three components are used alone.

[0213] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0214] 3.2. Effects of samples and compositions on immunoglobulins in mouse serum

[0215] Under the experimental conditions, the effects of the samples and compositions on the content of immune protein IgM are shown in Table 1-8.

[0216] Table 1-8. Effects of samples on IgM in mouse serum

[0217]

[0218] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0219] Immunoglobulin is a protein with antibody activity that exists in the blood or body fluids. It can kill pathogenic microorganisms under the synergistic action of complement. It is an important part of the body's humoral immunity and mediates humoral immunity. It is also the main immunoglobulin for serological diagnostic testing. The body's immune level can be reflected by the immunoglobulin content in the serum.

[0220] The experimental results show:

[0221] i. When the three components are used alone, the IgM content changes unilaterally with the increase in the dosage of the three components;

[0222] ii. The total concentration of the three components in Example 1 is 125+50+50=225 μg / mL, and the corresponding effect is significantly better than that of Comparative Example 3 (625 μg / mL), Comparative Example 6 (450 μg / mL) and Comparative Example 9 (200 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone;

[0223] ii. The total concentration of the three components in Example 2 is 25+50+50=125 μg / mL, and the corresponding effect is comparable to that of Comparative Example 3 (625 μg / mL), Comparative Example 6 (450 μg / mL) and Comparative Example 9 (200 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components used is less than the amount used when the three components are used alone.

[0224] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0225] 3.3. Effects of samples and compositions on cytokines in mouse serum

[0226] Under the experimental conditions, the effects of the samples and compositions on the secretion of cytokines in serum are shown in Tables 1-9.

[0227] Table 1-9. Effects of samples on cytokines in mouse serum

[0228]

[0229] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0230] Cytokines are small molecule proteins secreted by immune cells and non-immune cells after stimulation. They can be divided into interferon, tumor necrosis factor, etc. Among them, interferon refers to cytokines that regulate each other between cells or immune cells. In the immune response process, they play an important regulatory role in promoting cell-to-cell interaction, cell proliferation and differentiation. They can maintain the physiological balance of the body and resist the infection of pathogens. Through specific target cells, they bind to corresponding receptors, transmit information to cells to generate immune response reactions, and improve the body's immune ability.

[0231] The experimental results show:

[0232] i. When the three components are used alone, the TNF-α content changes unilaterally with the increase in the dosage of the three components;

[0233] ii. The total concentration of the three components in Example 1 is 125+50+50=225 μg / mL, and the corresponding effect is comparable to or better than that of Comparative Example 3 (625 μg / mL), Comparative Example 6 (450 μg / mL) and Comparative Example 9 (200 μg / mL). This shows that when the same or similar effect is obtained, the total amount of the three components can be less than the amount of the three components used alone.

[0234] Therefore, it can be seen that when the three components of the present invention are used in combination, compared with using a single component alone to obtain the same or similar effect, the former allows the use of a smaller total dosage, which shows the synergistic effect of the three components.

[0235] 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.

[0236] 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. An edible composition that helps to improve anti-fatigue, anti-oxidation and immune capabilities at the same time, characterized in that: The edible composition is an edible composition comprising a composite peptide, wherein the composite peptide includes as an essential ingredient: Colostrum hydrolyzed peptide ingredients, collagen peptide ingredients and casein peptide ingredients.

2. The edible composition according to claim 1, characterized in that The colostrum hydrolyzed peptide and casein peptide are derived from animal milk; and the collagen peptide includes collagen tripeptide.

3. The edible composition according to claim 1 or 2, characterized in that The colostrum hydrolyzed peptides are prepared by hydrolyzing colostrum and / or colostrum powder with protease, and the proportion of peptides with a molecular weight less than 5000 Da is not less than 50%.

4. The edible composition according to any one of claims 1 to 3, characterized in that The casein peptides are obtained by hydrolysis of casein or milk protein, and the proportion of peptides with a molecular weight less than 5000Da is not less than 10%.

5. The edible composition according to any one of claims 1 to 4, characterized in that The collagen peptides are prepared by protease hydrolysis of collagen-rich animal tissues, and the proportion of peptides with a molecular weight less than 10,000 Da is not less than 90%.

6. A food that helps to improve anti-fatigue, anti-oxidation and immunity at the same time, characterized in that: The food comprises or uses the edible composition according to claims 1 to 5.

7. The food according to claim 6, characterized in that The food is in liquid, semi-solid or solid state at room temperature.

8. Use of a composition containing three essential ingredients, namely, colostrum hydrolyzed peptide component, collagen peptide component and casein peptide component, in the preparation of an edible composition that helps to improve anti-fatigue, anti-oxidation and immune capabilities.

9. Use of a composition containing three essential ingredients, namely, colostrum hydrolyzed peptide component, collagen peptide component and casein peptide component, in the preparation of food that helps to improve anti-fatigue, anti-oxidation and immune capabilities at the same time.

10. The use according to claim 9, wherein the food comprises a solid beverage, a dairy product, a food for special dietary uses or a nutritional food, and the dairy product comprises a children's formula powder product or a middle-aged and elderly formula powder product.

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