Milk-derived functional peptides LQPEVMGVSKVKE, their preparation methods and applications

Abstract

This invention relates to the milk-derived functional peptide LQPEVMGVSKVKE, its preparation method, and its application. Specifically, this invention provides a milk-derived functional peptide comprising a polypeptide with the sequence LQPEVMGVSKVKE as shown in SEQ ID NO:1. The milk-derived functional peptide has anti-wrinkle, soothing, and immunomodulatory functions.

Description

Technical Field

[0001] This invention relates to the field of milk-derived functional peptides, specifically to the milk-derived functional peptide LQPEVMGVSKVKE, its preparation method, and its applications. Background Technology

[0002] Milk-derived bioactive peptides refer to bioactive peptides that are identical or similar to certain segments of the peptide chains of certain proteins in milk, and are inherent in milk or produced during the degradation of milk proteins. With the discovery of milk-derived bioactive peptides with different functions, research on milk protein bioactive peptides has become a new research hotspot in the fields of physiology and nutrition. The discovery of milk-derived bioactive peptides has changed the past simple evaluation of the nutritional functions of milk proteins and has great prospects for application and development. Cow's milk contains a variety of bioactive peptides with different functions, possessing important physiological functions.

[0003] Most current anti-wrinkle or soothing products rely on complex ingredient formulations, often resulting in diffused efficacy signals and decreased skin tolerance due to redundant components. They primarily act on the epidermis, merely reducing external irritation through physical isolation, without linking product efficacy to the core mechanisms of skin aging. This application's active peptides overcome the limitations of traditional products' epidermal action by inhibiting TRPV1 gene expression and simultaneously promoting SIRT1 gene expression, achieving a dual effect of soothing and anti-wrinkle.

[0004] Furthermore, immunomodulatory peptides refer to a collective term for peptide molecules with immunomodulatory activity. These polypeptides are generally present in low amounts in the body, have diverse structures, and act as cell signaling substances. They exert their biological functions through various mechanisms such as endocrine, paracrine, and neurosecretion, facilitating communication between different cell types. Immunomodulatory peptides can stimulate lymphocyte proliferation, enhance macrophage phagocytosis, induce the production of anti-inflammatory mediators and cytokines, and inhibit lipopolysaccharide-induced inflammatory responses in host cells, thereby improving the body's immunity. Bioactive peptides with immunomodulatory functions include oligopeptides, polypeptides, and some protease hydrolysate mixtures of peptides. These immunomodulatory peptides have shown good immunomodulatory effects in animal or cell experiments. Enzymatic hydrolysates or partial peptide mixtures of milk proteins have broad immunomodulatory potential. However, these activities are often attributed to the combined effects of multiple components, and the key single peptide sequence that plays the core function remains unclear. There is an urgent market demand for naturally derived ingredients with clear immunomodulatory functions and relatively well-defined mechanisms of action. Existing products often rely on complex plant extracts or unlabeled peptide mixtures, resulting in unstable effects and complex compositions. The purpose of this invention is to overcome the limitations of existing research and provide a novel, well-defined milk-derived immunomodulatory peptide, addressing the problem of unclear active ingredients in existing technologies. Through MTT assay for cell activity detection and determination of expression levels of specific inflammatory factors (such as IL-1β, IL-6, and TNF-α), the regulatory effect of this synthetic peptide on immune cell function is systematically and quantitatively verified, thus providing solid experimental evidence for its immunomodulatory activity. This invention elucidates the potential application value of this peptide in immunomodulation, providing a new key material basis and theoretical foundation for the development of functional foods, special medical purpose formula foods, or related health products based on a single active peptide. Summary of the Invention

[0005] This invention provides a milk-derived functional peptide or its derivative thereof, wherein the milk-derived functional peptide is a polypeptide with the sequence LQPEVMGVSKVKE shown in SEQ ID NO:1, and the milk-derived functional peptide has immunomodulatory function.

[0006] The present invention provides a milk-derived functional peptide or a derivative thereof, the sequence of which is shown in SEQ ID NO:1.

[0007] In one or more embodiments, the derivative is a polypeptide derivative obtained by methylation or acetylation of the amino or carboxyl terminus of the amino acid sequence shown in SEQ ID NO:1, wherein the derivative retains the bioactivity of the milk-derived functional peptide.

[0008] The present invention also provides a method for preparing the milk-derived functional peptides or their derivatives described herein, the method comprising any one of the following: (1) preparing the milk-derived functional peptides or their derivatives by microbial fermentation; (2) preparing the milk-derived functional peptides or their derivatives by genetic engineering (which can be obtained directly from cells by separation and purification); (3) preparing the milk-derived functional peptides or their derivatives by enzymatic hydrolysis; or (4) synthesizing the milk-derived functional peptides or their derivatives by chemical methods.

[0009] The present invention also provides a formulation comprising the milk-derived functional peptides or derivatives thereof described herein.

[0010] In one or more embodiments, the formulation is a powder, pill, oil drop, capsule, granule, tablet, liquid formulation, or gel.

[0011] The present invention also provides a product comprising the milk-derived functional peptides or derivatives thereof as described in any embodiment herein, or the formulations described in any embodiment herein.

[0012] In one or more embodiments, the product is a food, cosmetic, or pharmaceutical composition.

[0013] In one or more embodiments, the types of food include: plant-based foods, animal-based foods, microbially fermented foods, processed foods, and food additives.

[0014] In one or more embodiments, the food also includes additives and / or nutritional fortifiers.

[0015] In one or more embodiments, the additive includes one or more of the following: flavorings, stabilizers, thickeners, preservatives, antioxidants, emulsifiers, and / or the nutritional fortifier includes one or more of the following: vitamins, minerals, amino acids, fatty acids, dietary fiber.

[0016] In one or more embodiments, the food includes one or more of the following: dairy products, soy products, probiotic powder, probiotic oil droplets, dietary fiber supplements, nutrition bars, rice cereal, cheese, protein powder, fruit puree, fruit and vegetable juice, solid food beverages, fruit juice, ice cream, candy, biscuits, and foods for special medical purposes.

[0017] In one or more embodiments, the food includes health food.

[0018] In one or more embodiments, the health food also includes additives and / or nutritional fortifiers.

[0019] In one or more embodiments, the additive includes one or more of the following: flavorings, stabilizers, thickeners, preservatives, antioxidants, emulsifiers, and / or the nutritional fortifier includes one or more of the following: vitamins, minerals, amino acids, fatty acids, dietary fiber.

[0020] In one or more embodiments, the health food is a powder, pill, capsule, granule, tablet, oil drop, liquid preparation or gel.

[0021] In one or more embodiments, the dosage form of the cosmetic includes liquid dosage form, semi-solid dosage form, solid dosage form, and special dosage form.

[0022] In one or more embodiments, the cosmetic product further includes cosmetic excipients or formulation aids.

[0023] In one or more embodiments, the cosmetic excipients include one or more selected from the following: emulsifiers, humectants, thickeners, preservatives, solvents, pH adjusters, antioxidants, fragrances, flavorings, and skin feel modifiers.

[0024] In one or more embodiments, the cosmetic product includes one or more of the following: toner, serum, makeup remover, moisturizing spray, sunscreen spray, liquid essence oil, moisturizing lotion, sunscreen lotion, essence lotion, facial cleanser, shower gel, eye cream gel, lipstick, lip mask, eyebrow wax, scrub, loose powder, pressed powder, face mask, eye mask, neck mask, eye patch, acne patch, neck wrinkle patch, freeze-dried essence.

[0025] In one or more embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0026] The present invention also provides the use of the milk-derived functional peptides or their derivatives described in any embodiment herein, or the formulations described in any embodiment herein, in the preparation of food.

[0027] The present invention also provides the use of the milk-derived functional peptides or their derivatives described in any embodiment herein, or the formulations described in any embodiment herein, in the preparation of health products with immunomodulatory functions.

[0028] The present invention also provides the use of the milk-derived functional peptides or their derivatives described in any embodiment herein, or the formulations described in any embodiment herein, in the preparation of cosmetics having anti-wrinkle or soothing effects.

[0029] The present invention also provides the use of the milk-derived functional peptides or their derivatives described in any embodiment herein, or the formulations described in any embodiment herein, in the preparation of pharmaceutical compositions having analgesic, skin cell aging-improving and / or immunomodulatory functions.

[0030] In one or more embodiments, the pharmaceutical composition achieves an analgesic effect by inhibiting the expression of TRPV1.

[0031] In one or more embodiments, the pharmaceutical composition improves skin cell aging by enhancing the epidermal barrier structure through promoting SIRT1 expression.

[0032] In one or more embodiments, the cell is a human immortalized keratinocyte (HaCaT).

[0033] In one or more embodiments, the milk-derived functional peptides or their derivatives described in any of the embodiments herein, or the formulations described in any of the embodiments herein, enhance immunomodulatory function by promoting the secretion of inflammatory factors.

[0034] In one or more embodiments, the inflammatory factor is selected from one or more of IL-6, TNF-α, and IL-1β.

[0035] The present invention also provides the use of the milk-derived functional peptides or derivatives thereof described in any embodiment herein, or the formulations described in any embodiment herein, in the preparation of soothing or anti-wrinkle pharmaceutical compositions.

[0036] In one or more embodiments, the pharmaceutical composition achieves a soothing effect by inhibiting TRPV1 expression and blocking pain signal transduction.

[0037] In one or more embodiments, the pharmaceutical composition reduces wrinkles caused by barrier damage by promoting SIRT1 expression, thereby promoting keratinocyte differentiation and strengthening the epidermal barrier structure.

[0038] In one or more embodiments, the cell is a human immortalized keratinocyte (HaCaT). Attached Figure Description

[0039] Figure 1 Relative fluorescence intensity and inhibition rate of TRPV1 in different groups ( This indicates that the difference is statistically significant compared to the model group. P < 0.05 P < 0.01).

[0040] Figure 2 Relative fluorescence intensity of TRPV1 in different groups.

[0041] Figure 3 Relative expression levels of SIRT1 gene in different groups ( This indicates that the difference is statistically significant compared to the control group. P < 0.05 P < 0.01).

[0042] Figure 4 Mass spectrum of active polypeptide LQPEVMGVSKVKE.

[0043] Figure 5 High-performance liquid chromatogram of active peptide LQPEVMGVSKVKE.

[0044] Figure 6 Cell viability test results.

[0045] Figure 7 Results of IL-1β content enhancement rate test ( This indicates that the difference is statistically significant compared to the control group. P < 0.05 P < 0.01).

[0046] Figure 8 : IL-6 content enhancement rate test results ( This indicates that the difference is statistically significant compared to the control group. P < 0.05 P < 0.01).

[0047] Figure 9 Results of TNF-α content enhancement rate test ( This indicates that the difference is statistically significant compared to the control group. P < 0.05 P < 0.01). Detailed Implementation

[0048] Unless otherwise specified, the technical terms in this specification have the same meaning as those generally understood by those skilled in the art; however, in case of any conflict, the definitions in this specification shall prevail.

[0049] The term "anti-wrinkle" refers to the intervention of skin aging-related pathophysiological processes (including dermal collagen loss, elastic fiber degeneration and breakage, decreased skin moisture content, free radical damage, and ultraviolet-induced extrinsic aging) through the milk-derived functional peptide LQPEVMGVSKVKE or its derivatives, as well as preparations or products containing it, to prevent the formation of skin wrinkles, reduce the depth and width of existing wrinkles, improve the appearance of skin folds, or delay the aggravation of wrinkles.

[0050] The term "soothing" refers to the relief of skin discomfort such as redness, itching, burning, and pain caused by irritation, inflammation, or sensitivity through the milk-derived functional peptide LQPEVMGVSKVKE or its derivatives described in this application, as well as preparations or products containing them.

[0051] The term "TRPV1" is a non-selective cation channel protein encoded by the TRPV1 gene. It belongs to the vanillin receptor subfamily of the transient receptor potential (TRP) channel superfamily. It is mainly located on the surface of sensory neurons (such as dorsal root ganglion and trigeminal ganglion neurons) and non-neuronal cells such as keratinocytes, fibroblasts, and vascular endothelial cells in the skin. It mainly mediates physiological or pathological processes such as pain, inflammation, temperature perception, and skin barrier regulation.

[0052] The term "SIRT1" refers to a class of highly conserved NAD+-dependent histone deacetylases that regulate numerous key signaling pathways in prokaryotes and eukaryotes. Belonging to the Sirtuin protein family (7 members in total, SIRT1-SIRT7), SIRT1 is primarily located in the cell nucleus (but can shuttle into the cytoplasm). It regulates key physiological processes such as gene expression, cell metabolism, and oxidative stress response by deacetyling histones and non-histone proteins (e.g., p53, FOXO, NF-κB). SIRT1 is a core regulatory target for aging, inflammation, and metabolic homeostasis. Nuclear-localized SIRT1 promotes keratinocyte differentiation, strengthens the epidermal barrier structure, provides basic support for the skin, and reduces the formation of fine lines caused by barrier damage.

[0053] The term "Foods for Special Medical Purposes (FSMP)" refers to foods specially processed and formulated to meet the specific nutritional or dietary needs of individuals with restricted food intake, digestive and absorptive disorders, metabolic disorders, or specific disease states. These include: complete nutritional formula foods (such as liquid diets for individuals with swallowing difficulties), specific complete nutritional formula foods (such as formulas for diabetes or liver disease), and incomplete nutritional formula foods (such as electrolyte formulas or amino acid component formulas).

[0054] The term "milk-derived functional peptides" refers to small molecule peptides formed by breaking peptide bonds within protein molecules through controlled processes such as enzymatic hydrolysis (preferably food-grade proteases, such as trypsin, alkaline proteases, and lactase), microbial fermentation (such as lactic acid bacteria and yeast fermentation), or mild chemical hydrolysis, using whey proteins (such as whey protein isolate and lactoferrin), caseins (such as α-casein and β-casein), and lactoglobulins from the milk of mammals such as cows, sheeps, and camels as raw materials.

[0055] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0056] As used herein, “improvement” includes any beneficial or desired effect on the symptoms or lesions of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition (e.g., skin aging). Improvement may optionally include a reduction or relief of symptoms of the disease or condition, or a delay in the progression of the disease or condition. “Improvement” does not necessarily mean the complete eradication or cure of the disease or condition or its associated symptoms.

[0057] The terms “milk-derived functional peptides,” “milk-derived bioactive peptides,” and “milk-derived bioactive peptides” used in this article can be used interchangeably.

[0058] Milk-derived functional peptides or their derivatives

[0059] This invention provides a milk-derived functional peptide or its derivative thereof, comprising a polypeptide with the sequence shown in SEQ ID NO:1, wherein the derivative of the milk-derived functional peptide herein comprises a polypeptide derivative obtained by methylation or acetylation modification of the amino acid side chain group, amino terminus or carboxyl terminus of the sequence shown in SEQ ID NO:1.

[0060] The polypeptide derivative of this application retains the biological activity of the parent peptide LQPEVMGVSKVKE in inhibiting TRPV1 expression and promoting SIRT1 expression.

[0061] Preparation method

[0062] The present invention also provides a method for preparing the milk-derived functional peptides or their derivatives described herein, the method comprising: (1) preparing the milk-derived functional peptides or their derivatives by microbial fermentation; (2) preparing the milk-derived functional peptides or their derivatives by genetic engineering (which can be obtained directly from cells by separation and purification); (3) preparing the milk-derived functional peptides or their derivatives by enzymatic hydrolysis; or (4) synthesizing the milk-derived functional peptides or their derivatives by chemical methods.

[0063] Microbial fermentation uses milk protein as a substrate and selects microorganisms with strong protease production capabilities. The microorganisms themselves produce enzymes to directionally hydrolyze the protein, generating the target functional peptide. When preparing derivatives, modification precursors can be introduced during fermentation, and the modification is completed through microbial metabolism. Currently, the main strains used to prepare bioactive peptides include Aspergillus, lactic acid bacteria, Bacillus licheniformis, and Bacillus subtilis.

[0064] Genetic engineering methods include optimizing and synthesizing a target gene against a target peptide sequence, constructing a recombinant expression vector, and transforming it into host cells to achieve heterologous expression. The expression product undergoes cell disruption, chromatography purification, and other steps to obtain a functional peptide or a derivative containing a modified site. An expression vector is a suitable vector for expressing exogenous genes, such as nucleic acid molecules encoding the polypeptide described in this invention, in host cells. These vectors include prokaryotic and eukaryotic expression vectors. Eukaryotic expression systems include yeast, mammalian cell, and insect cell expression systems. After obtaining the expression vector, it is transformed into a host cell to produce the polypeptide LQPEVMGVSKVKE or its derivatives described herein. This transfer process can be performed using conventional techniques well-known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules; it is the site of recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. Specifically, host cells may include bacterial cells such as *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*; fungal cells such as yeast; plant cells; insect cells such as *Drosophila S2* or *Sf9*; animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, including but not limited to the host cells mentioned above. The host cells are preferably various cells that are conducive to gene product expression or fermentation production, and such cells are well-known and commonly used in the art.

[0065] Based on the number of enzyme preparations used and the differences in hydrolysis implementation methods, enzymatic hydrolysis methods include three types: First, single-enzyme hydrolysis, which uses only one protease to complete the hydrolysis, resulting in a simple process and low implementation difficulty; second, compound enzymatic hydrolysis, which combines two or more proteases for synergistic hydrolysis, improving the degree of protein hydrolysis while optimizing the product's bioactivity and sensory flavor; and third, stepwise enzymatic hydrolysis, which involves changing enzyme preparations or adjusting reaction conditions at different reaction stages to perform gradient hydrolysis, allowing for the targeted enrichment of functional peptides within specific molecular weight ranges. The general process of enzymatic hydrolysis is as follows: Milk protein raw materials are taken, water is added to prepare a protein solution of a set concentration, and the solution is stirred until completely dissolved. The system temperature and pH are adjusted to the optimal reaction conditions for the corresponding protease. A quantitative amount of protease is added, and hydrolysis is carried out under constant temperature stirring. After hydrolysis, the protease is inactivated at high temperature to terminate the reaction. The solution is then centrifuged and filtered to remove insoluble solid residues, and the filtrate is collected. The filtrate undergoes further purification processes such as decolorization, membrane separation and fractionation, concentration, and drying to finally obtain the milk-derived functional peptide product.

[0066] This application employs a chemical synthesis method, specifically including the following steps: coupling the first amino acid: adding the amino acid solution and coupling reagent to the resin for reaction; removing the Fmoc protecting group: adding Pip / DMF solution and then vacuum filtering; washing: washing the resin with DMF and filtering; resin detection: detecting whether the Fmoc group has been removed using a reagent; amino acid condensation: adding the amino acid solution and coupling reagent, shaking well and then filtering; repeating the above steps until the synthesis of the last amino acid is completed; reverse chromatography salt conversion: removing trifluoroacetic acid (TFA) residue by reverse chromatography and using an acetic acid system as the mobile phase for salt conversion.

[0067] product

[0068] The present invention also provides a product comprising the milk-derived functional peptides or derivatives thereof described in any embodiment herein, or the formulations described in any embodiment herein, wherein the product may be a food, cosmetic, or pharmaceutical composition.

[0069] In one or more embodiments, the types of food include: plant-based foods, animal-based foods, microbially fermented foods, processed foods, and food additives.

[0070] In one or more embodiments, the food also includes additives and / or nutritional fortifiers.

[0071] In one or more embodiments, the additive includes one or more of the following: flavorings, stabilizers, thickeners, preservatives, antioxidants, emulsifiers, and / or the nutritional fortifier includes one or more of the following: vitamins, minerals, amino acids, fatty acids, dietary fiber.

[0072] In one or more embodiments, the food includes one or more of the following: dairy products, soy products, dietary fiber supplements, nutrition bars, rice cereal, cheese, protein powder, fruit puree, fruit and vegetable juice, solid food beverages, fruit juice, ice cream, candy, biscuits, and foods for special medical purposes.

[0073] The foods applicable to this invention include ordinary foods, health foods, and foods for special medical purposes. None of these foods are intended for disease prevention or treatment. Ordinary foods include, but are not limited to, fermented dairy products, non-fermented dairy products (including fermented vegetable products, fermented soy products, fermented tea, fermented herbs, fermented fruits, and probiotic beverages) or formula milk, such as yogurt, kefir, cheese (e.g., Swiss cheese, Gouda cheese, Cheddar cheese, mozzarella cheese), apple cider vinegar, fermented soy milk, fermented apple juice, fermented carrot juice, bread, steamed buns, lactic acid bacteria beverages, cereal flakes, and chocolate. Health foods include, but are not limited to, vitamin C tablets, calcium and magnesium tablets, probiotic powder, protein powder, fish oil soft capsules, and ginseng lozenges. Foods for special medical purposes include, but are not limited to, complete nutritional formula powders, nutritional powders for diabetes, nutritional solutions for cancer patients, and protein components.

[0074] Solid beverages, as described in this article, refer to solid products with a moisture content of no more than 5 grams per 100 grams of finished product, such as instant coffee, fruit juice powder, and milk tea powder. They are characterized by their ease of preservation and portability. Probiotic oil drops are products that allow probiotics to be ingested orally. The oil drop components can effectively protect the probiotics, allowing them to survive and exert their effects in the digestive system. Candy refers to solid candy made primarily from sugar or syrup (powder) through processes such as mixing, granulation, and compression molding. Dairy products refer to various foods made primarily from fresh cow (sheep) milk and its products. Modified milk powder is a product based on milk powder with the addition of various nutritional fortifiers (such as vitamins, minerals, probiotics, DHA, ARA, etc.), such as milk powder for pregnant women, milk powder for the middle-aged and elderly, and milk powder for children's growth.

[0075] The cosmetics of this invention can be in liquid, semi-solid, solid, or special formulations. Liquid formulations include solutions, emulsions, and suspensions. In one or more embodiments, solutions include toners, serums, makeup removers, moisturizing sprays, sunscreen sprays, and liquid essential oils. In one or more embodiments, emulsions include moisturizing lotions, sunscreen lotions, and essence lotions. In one or more embodiments, suspensions include facial cleansers containing exfoliating particles, exfoliating suspensions, soothing and repairing lotions containing plant particles, and anti-wrinkle serums with retinol encapsulated in microcapsules. Semi-solid formulations include creams, gels, wax-based products, and pastes. Creams include face creams and lotion creams; gels include aloe vera gel, Pro-Xylane gel, eye cream gel, and gel-based sunscreen; wax-based products include lipsticks, lip masks, eyebrow wax, and hair wax; and pastes include face masks, scrubs, and repair pastes. Solid dosage forms include powders, tablets, and films. Powders include loose powder, pressed powder, mineral powder, and cleansing powder; tablets include facial cleansing sheets, solid essence sheets (such as collagen freeze-dried sheets and peptide repair freeze-dried sheets), and soaps; films include sheet masks, eye masks, and neck masks. Special dosage forms include patches, capsules, and freeze-dried preparations. Patches include eye patches, acne patches, and neck wrinkle patches; capsules include essence capsules; freeze-dried preparations include freeze-dried masks and freeze-dried essences.

[0076] The present invention also provides a pharmaceutical composition comprising the milk-derived functional peptide or a derivative thereof as described in any embodiment of the present invention and pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0077] Examples of pharmaceutically acceptable excipients include binders (syrups, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), fillers (lactose, sucrose, starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, etc.), disintegrants (starch, microcrystalline cellulose, etc.), humectants (sodium lauryl sulfate, etc.), and suspending agents (sorbitol, syrups, methylcellulose, glucose syrup). Hydrates, gelatin, hydrogenated edible fats, emulsifiers (lecithin, sorbitan monooleate, gum arabic, etc.), non-aqueous carriers (almond oil, fractionated coconut oil or hydrophobic esters such as glycerin, propylene glycol, and ethanol), preservatives (methylparaben or propylparaben, sorbic acid, etc.), flavorings (synthetic fragrances, natural fragrances, etc.), sweeteners (sucrose, stevia, xylitol, etc.), pH adjusters (sodium bicarbonate, potassium carbonate, etc.), powders (pigments, dyes, resins, etc.), thickeners (gum arabic, methylcellulose, etc.), antioxidants (vitamin C, vitamin E, etc.), etc.

[0078] application

[0079] Uses of the milk-derived functional peptides or their derivatives, formulations or products described in any of the embodiments herein: (1) Uses in food preparation (2) Uses in the preparation of health products with immunomodulatory functions. (3) Use in the preparation of cosmetics with anti-wrinkle, soothing and / or immunomodulatory functions, (4) Use in the preparation of pharmaceutical compositions for analgesia or to improve skin cell aging, (5) Applications for non-therapeutic purposes such as analgesia, improvement of skin cell aging and / or immune regulation.

[0080] In one or more embodiments, the milk-derived functional peptide or its derivatives or formulations achieve an analgesic effect by inhibiting the expression of TRPV1 and blocking pain signal transduction.

[0081] In one or more embodiments, the milk-derived functional peptide or its derivatives or formulations improve skin cell aging by promoting the expression of SIRT1, thereby promoting keratinocyte differentiation and strengthening the epidermal barrier structure.

[0082] In one or more embodiments, the cell is a human immortalized keratinocyte (HaCaT).

[0083] In one or more embodiments, the pharmaceutical composition enhances immunomodulatory function by promoting the secretion of inflammatory factors.

[0084] In one or more embodiments, the inflammatory factor is selected from one or more of IL-6, TNF-α, and IL-1β.

[0085] In one or more embodiments, the analgesia includes relief from mosquito bites, minor physical irritation, and temporary stinging caused by environmental factors.

[0086] In one or more embodiments, the improvement of skin cell aging includes daily anti-aging, photoaging protection and cell damage repair, and gentle anti-aging suitable for sensitive skin.

[0087] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention. The methods and reagents used in the embodiments, unless otherwise stated, are conventional methods and reagents in the art. The compounds of the invention are available from commercial sources or can be synthesized according to or with reference to the disclosed methods. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0088] Example

[0089] Example 1: Peptide Synthesis

[0090] Reagents: Industrial grade N,N-dimethylformamide (DMF), analytical grade N,N-dimethylformamide (DMF), piperidine, N,N-diisopropylethylamine (DIEA), acetonitrile (ACN), methanol. Resin: Fmoc-Glu-2-chloro resin. Instruments: 24-channel fully automated microwave synthesizer, mass spectrometer, high-performance liquid chromatograph (HPLC), preparative HPLC, analytical HPLC, freeze dryer.

[0091] Peptide synthesis: Solid-phase peptide synthesis (SPPS) steps: Coupling the first amino acid: Add the amino acid solution and coupling reagent to the resin for reaction.

[0092] Remove Fmoc protecting groups: Add Pip / DMF solution and then filter under vacuum.

[0093] Washing: Wash the resin with DMF and filter.

[0094] Resin testing: The Fmoc groups are removed by testing with reagents.

[0095] Amino acid condensation: Add amino acid solution and coupling reagent, shake well and filter.

[0096] Repeat the above steps until the synthesis of the last amino acid is complete.

[0097] Salt transfer by reverse chromatography: Trifluoroacetic acid (TFA) residues are removed by reverse chromatography, and salt transfer is performed using an acetic acid system as the mobile phase.

[0098] High-performance liquid chromatography (HPLC) analysis: Column: C18 reversed-phase silica gel column Column temperature: N / A Detection wavelength: 220 nm Flow rate: 1 mL / min Injection volume: 40 μl Mobile phase A: 0.065% aqueous solution of TFA Mobile phase B: 0.05% TFA in acetonitrile solution Crude product purification Crude peptide dissolution: Take crude peptide, add appropriate amount of pure water and acetonitrile mixed reagent and sonicate to dissolve. After the sample is clear and transparent, filter it with a 0.45μm filter membrane and set aside the filtrate.

[0099] Equipment preparation for operation: Use mobile phase A: 0.1% TFA / water solution (v / v); mobile phase B: 0.1% TFA / acetonitrile solution (v / v). Equilibrate with 0.1% TFA / water solution (v / v) for 5 min to reach baseline level before loading the sample.

[0100] Gradient elution: Perform gradient elution according to the settings, and judge the peak shape at a wavelength of 254 nm. If there are obvious impurities, change the tube to collect the liquid.

[0101] Determination of fraction purity: After cleaning the 1ml syringe twice with methanol, take 0.1ml of sample from the estimated target fraction, mix well, and perform pure sample analysis using high performance liquid chromatography.

[0102] High-performance liquid chromatography (HPLC) analysis: Column: C18 reversed-phase silica gel column Column temperature: 40℃ Detection wavelengths: 220 and 254 nm Flow rate: 1 mL / min Injection volume: 20 μl Mobile phase A: 0.065% aqueous solution of TFA Mobile phase B: 0.05% TFA in acetonitrile solution Preservation of qualified fractions: After the qualified fractions are combined and transferred to a clean glass container, the container is sealed with a lint-free cloth and stored in a refrigerator at 0~10℃ until freeze-drying.

[0103] The process of converting freeze-dried food to salt includes the following steps: 1. Pure product weighing record; 2. To dissolve; 3. Trifluoroacetic acid (TFA) residues were removed by reverse chromatography, and an acetic acid-based resin was added to utilize the acetic acid system as the mobile phase for salt transfer; 4. Stir for 2 hours; 5. Filter and freeze-dry.

[0104] Mass spectrometry conditions

[0105] Electrospray ionization (ESI) was used for detection in positive ion mode, with the interface voltage set to +4.5 kV. The nebulizer gas flow rate was 1.5 L / min, and the drying gas flow rate was 5 L / min. The ion transfer tube temperature (CDL temperature) was set to 250 °C, and the heating module temperature (Block temperature) was set to 200 °C. Under these mass spectrometry conditions, the characteristic ion peaks of the target compound were scanned and monitored.

[0106] The synthesized peptide was qualitatively analyzed using mass spectrometry to determine its amino acid sequence. The mass spectrum and high-performance liquid chromatography (HPLC) chromatogram of the synthesized peptide are shown below. Figure 4 and Figure 5 As shown, Figure 4 and Figure 5 If the results match the theoretical values, the primary amino acid sequence of the synthesized polypeptide is the target polypeptide.

[0107] Example 2 TRPV1 Test

[0108] 1. Experimental apparatus

[0109] Table 1 Main Experimental Instruments

[0110] 2. Experimental Materials

[0111] Table 2 Experimental Materials

[0112] 3. Experimental Principle

[0113] As a non-selective cation channel and multimodal receptor, the TRPV1 (transient receptor potential vanillin 1) channel can be activated not only by capsaicin, but also by harmful heat, low extracellular pH, animal toxins, and divalent cations such as Mg. 2+ and Ba2+ Activation. Because the TRPV1 channel has a high affinity and selectivity for capsaicin, it is also known as the capsaicin receptor.

[0114] Immunofluorescence assay: Based on the principle of antigen-antibody reaction, fluorescent dyes that do not affect the activity of antigens and antibodies are labeled on antibodies or secondary antibodies against antibodies. After binding with their corresponding antigens or antibodies, the location and intensity of the fluorescence signal are observed under a fluorescence microscope, thereby determining the nature and location of the antigen or antibody, and performing a preliminary analysis of the expression level of the antigen or antibody.

[0115] When cells are stimulated, they can cause a stinging and burning sensation in the skin by activating the capsaicin receptor (TRPV1). Keratinocytes (HaCaT) are the direct target of capsaicin. The soothing effect can be evaluated by testing the effect of samples on TRPV1 expression using immunofluorescence.

[0116] 4. Experimental Grouping

[0117] Table 3 Experimental Groups

[0118] 5. Experimental Procedure

[0119] 5.1. Cell passage culture

[0120] Resuscitate and culture the cells.

[0121] 5.2. Determination of sample cytotoxicity

[0122] Perform cytotoxicity experiments according to the following procedures, and select appropriate sample concentrations for subsequent efficacy experiments based on the cytotoxicity results.

[0123] Cells in the logarithmic growth phase were digested and centrifuged, and a cell suspension of appropriate density was prepared with complete culture medium. The suspension was then seeded into 96-well plates according to the experimental design and incubated at 37°C with 5% CO2 for 24 h ± 2 h. The culture medium in the wells was discarded, samples were added, and the plates were incubated again at 37°C with 5% CO2 for 24 h ± 2 h. After incubation, the culture medium was discarded, and complete culture medium containing MTT working solution was added to each well. The plates were incubated at 37°C for 2 h ± 30 min. The culture medium was carefully aspirated from the wells, and DMSO was added to dissolve the formazan particles. The absorbance of each well was then read using a microplate reader. The formula for calculating cytotoxicity is: Cell viability (%) = (OD of sample wells - OD of zeroing wells) / (OD of blank control wells - OD of zeroing wells) 100%. Criteria for determining the validity of the test: The coefficient of variation (CV) of the OD values ​​of each set of parallel replicates is ≤20%.

[0124] The test results are as follows: Table 4 Cytotoxicity assay

[0125] 5.3. Cell Climbing and Seeding Plates

[0126] (1) After digesting and centrifuging HaCaT cells in the logarithmic growth phase with 0.25% trypsin, they were prepared into 2×10⁻⁶ cells using complete culture medium. 5 Appropriate density cell suspension; (2) Place one cell crawling sheet in each well of a 12-well plate and inoculate 1 mL into each well of the 12-well plate according to experimental requirements. Incubate at 37°C and 5% CO2 for 24±2 h.

[0127] 5.4. Drug administration

[0128] Remove the original culture medium from the 12-well plates. Add samples according to the experimental groups in Table 3. Add capsaicin to each well of the sample group at a final concentration of 50 μg / mL. Add capsaicin to the model group (M) at a final concentration of 50 μg / mL. Add capsaicin to the positive control group (PC) at a final concentration of 50 μg / mL. Add only culture medium to the blank control group (BC). Add 1 mL of culture medium to each well of the 12-well plate and incubate at 37℃ in a 5% CO2 incubator for 24 ± 2 h. The experimental results are shown in Table 5. Figure 1 , Figure 2 As shown.

[0129] 5.5. Immunofluorescence staining of cells

[0130] After incubation, discard the original culture medium, wash 2-3 times with PBS, add fixative to each well and fix for 20 min, then block. Add primary antibody and incubate overnight at 2-8 ℃ or incubate at 37 ℃ for 1.5 h; wash 2-3 times with PBS, add secondary antibody and incubate at room temperature for 1 h or incubate at 37 ℃ for 40 min, and finally stain the cell nuclei with Hoechst 33342 live cell staining solution for 10 min.

[0131] 5.6. Fluorescent photography

[0132] Cell slides were transferred to a dark room and photographed using a fluorescence microscope. The acquired images were analyzed for fluorescence intensity using ImageJ software.

[0133] 6. Data Analysis

[0134] Data are expressed as mean ± standard deviation. One-way ANOVA was performed, and the p-value was used to determine statistical significance. P < 0.05 was considered statistically significant.

[0135] The formula for calculating cell viability is: Cell viability (%) = (OD of sample wells - OD of zeroing wells) / (OD of blank control wells - OD of zeroing wells) 100%.

[0136] TRPV1 absolute fluorescence intensity calculation formula: TRPV1 absolute fluorescence intensity value = Average TRPV1 fluorescence intensity / Average nuclear fluorescence intensity TRPV1 relative content calculation formula: TRPV1 relative content (%) = T / C × 100% T—Absolute fluorescence intensity value of TRPV1 in the test sample; C—The average absolute fluorescence intensity of TRPV1 in the model group.

[0137] Formula for calculating TRPV1 expression inhibition rate: Inhibition rate (%) = (1 - T / C) × 100% T—The average relative content of TRPV1 in the tested sample; C—The average relative content of TRPV1 in the model group.

[0138] 7. Verification of Experimental Validity

[0139] Determination of the effectiveness of cytotoxicity testing: The coefficient of variation (CV) of absorbance OD values ​​for each of the three biological replicates is ≤ 20%.

[0140] TRPV1 fluorescence intensity analysis

[0141] The experiment requires statistical analysis of fluorescence intensity in images from four different fields of view for each experimental group, and calculation of the standard deviation (SD) of fluorescence intensity in the four different fields of view. After removing the image with the greatest difference, the coefficient of variation (CV) of fluorescence intensity among the remaining three images from different fields of view is calculated. If the CV value is ≤20%, the experiment is considered to be parallel and valid.

[0142] For each test, a model group must be set up. The experimental system is considered effective if the fluorescence intensity of TRPV1 in the blank control group is significantly reduced (P<0.05) compared with the model group.

[0143] 8. Validation of Results

[0144] Compared with the model group, the fluorescence intensity of TRPV1 in the specific sample group was significantly reduced (P < 0.05), which indicates that the sample group has a soothing effect under the test conditions.

[0145] 9. Experimental Results

[0146] Under the experimental conditions, all results met the parallelism requirements, and the experiment was valid. The relative fluorescence intensity of the blank control group was significantly lower than that of the model group, indicating the effectiveness of the experimental system. TRPV1, a key receptor for pain perception and regulation, showed a significant decrease in relative expression level after the addition of LQPEVMGVSKVKE following stimulation of cells with capsaicin at a final concentration of 50 μg / mL to activate TRPV1 (P < 0.05). At test concentrations of 400 μg / mL, 300 μg / mL, and 200 μg / mL, the relative fluorescence intensity of TRPV1 was 72.10%, 84.55%, and 77.83%, respectively, significantly lower than that of the model group by 27.90%, 15.45%, and 22.17% (P < 0.05). The relative fluorescence intensity of TRPV1 in the positive control group was 79.40%, significantly lower than that of the model group by 20.60% (P < 0.05). Specific data are shown in Table 5, Figure 1, and Figure 2.

[0147] Table 5. Relative fluorescence intensity and inhibition rate of TRPV1 in different groups ( This indicates that the difference is statistically significant compared to the model group. P < 0.05 (P < 0.01)

[0148] Example 3 SIRT1 Test

[0149] 1. Instruments

[0150] Table 6 Main Experimental Instruments

[0151] 2. Experimental Materials

[0152] Table 7 Experimental Materials

[0153] 3. Experimental Principle

[0154] Sirtuins (SIRTs) are a class of evolutionarily conserved NAD+-dependent histone deacylases that regulate numerous key signaling pathways and participate in many biological processes in prokaryotes and eukaryotes. There are seven recognized members of the human SIRT family: SIRT1 through SIRT7. The SIRT protein family can interact with proteins such as p53, FOXO / PGC-1α, NF-κB, and Ku70, regulating important processes such as cellular stress response, metabolism, aging, and apoptosis. In the skin's anti-aging process, SIRT family proteins are closely related to anti-wrinkle and firming functions, exhibiting multi-layered regulatory roles. Nuclear-localized SIRT1 promotes keratinocyte differentiation, strengthens the epidermal barrier structure, provides basic support for the skin, and reduces the formation of fine lines caused by barrier damage.

[0155] 4. Experimental Grouping

[0156] Table 8 Experimental Groups

[0157] 5. Experimental Procedure

[0158] 5.1. Cell passage culture

[0159] Resuscitate and culture the cells.

[0160] 5.2. Determination of sample cytotoxicity

[0161] The cytotoxicity test results are the same as those in Example 2, section 5.2.

[0162] 5.3. Cell Seeding Plate

[0163] Cells in the logarithmic growth phase were selected, digested with trypsin, and collected to form a cell suspension for counting. Cells were diluted with cell culture medium to a suitable seeding density (45%–60% confluence 24 h post-seeding) and seeded into 12-well plates, 1 mL per well. After seeding, the plates were incubated in a carbon dioxide (CO2) incubator for 24 h ± 2 h.

[0164] 5.4. Drug Treatment

[0165] Discard the culture medium in the 12-well plate and proceed with the drug administration according to the experimental design. Add 1 mL of complete culture medium containing different concentrations of the sample to the sample group wells, 1 mL of complete culture medium containing the positive control to the positive control wells, and 1 mL of complete culture medium to the blank control group wells. After adding the samples, place the 12-well plate in a CO2 incubator and incubate for 24 h ± 2 h.

[0166] 5.5. Sample Collection

[0167] After incubation, discard the original culture medium in the cell culture plate, wash once with DuPont phosphate-buffered saline (DPBS), and transfer the cell plate to the RNA sample preparation room for later use.

[0168] 5.6. Determination of gene expression levels in cells

[0169] Gene expression was detected using quantitative real-time PCR. RNA was extracted and cDNA was synthesized from the collected cell samples. The synthesized cDNA product was diluted with ultrapure water according to the specified ratio before qRT-PCR was performed to detect the expression level of the SIRT1 gene in the cells.

[0170] 6. Data Analysis

[0171] Quantitative data are expressed as mean ± standard deviation (Mean ± SD). Data are analyzed using one-way ANOVA, and statistical differences are determined by p-value. P < 0.05 indicates statistical significance.

[0172] The formula for calculating cell viability is: Cell viability (%) = (OD of sample wells - OD of zeroing wells) / (OD of blank control wells - OD of zeroing wells) 100%.

[0173] The SIRT1 gene expression level is calculated as follows: Use 2 - △△CT The data obtained from the qRT-PCR experiment were processed. The changes in SIRT1 mRNA expression levels after different sample treatments were compared with the blank control group to calculate the relative SIRT1 expression levels in the sample groups.

[0174] 7. Verification of Experimental Validity

[0175] 7.1. Determination of the validity of cytotoxicity assays: The coefficient of variation (CV) of absorbance OD values ​​for each of the three biological replicates is ≤ 20%.

[0176] 7.2. Validation of qRT-PCR experimental effectiveness

[0177] Each batch of experiments must include a positive control. The positive control must show an upregulation of the relative expression level of the SIRT1 gene in each batch of experiments. If the relative expression level of the SIRT1 gene in the positive control is increased by 0.2 times compared with the blank control, the experimental system is considered effective.

[0178] For qRT-PCR experiments, each sample concentration must be performed in 3 biological replicates, and each biological replicate must be performed in 3 technical replicates. Under the condition that at most one technical replicate value is discarded from each group, the standard deviation (SD) between technical replicates must be ≤ 0.5 and the CV value between the 3 biological replicates must be < 20%. Alternatively, if the experiment is performed in 2 biological replicates, the standard deviation (SD) between technical replicates must be ≤ 0.5 and the CV value between the 2 biological replicates must be < 20% for the experiment to be considered valid for parallelism.

[0179] 8. Validity of Results

[0180] If the relative content and relative expression of SIRT1 in the sample are greater than those in the blank control and show a significant difference (P<0.05) compared with the blank control group, then the sample at this concentration is considered to have anti-wrinkle and firming effects.

[0181] 9. Experimental Results: Under the experimental conditions, all results met the parallelism requirements, and the experiment was valid. The relative expression level of the SIRT1 gene in the positive control group was significantly higher than that in the blank group, indicating that the experimental system was effective.

[0182] The addition of LQPEVMGVSKVKE significantly increased the relative upregulation of SIRT1 gene expression (P < 0.05). In the positive control group, the upregulation rate of SIRT1 was 150.98%, while in the model group, at 400 μg / mL, the upregulation rate was 176.01%. At 300 μg / mL, the upregulation rate was 164.90%, and at 200 μg / mL, it was 241.79%, unexpectedly showing the best effect at lower concentrations. Specific data are shown in Table 9 and Figure 3.

[0183] Table 9. Relative expression levels and increase rates of SIRT1 gene in different groups ( This indicates that the difference is statistically significant compared to the control group. P < 0.05 (P < 0.01)

[0184] Example 4

[0185] Peptide synthesis: Solid-phase peptide synthesis (SPPS): 1) Coupling the first amino acid: Add the amino acid solution and coupling reagent to the resin for reaction.

[0186] 2) Removal of Fmoc protecting groups: Vacuum filtration after adding Pip / DMF solution.

[0187] 3) Washing: Wash the resin with DMF and filter.

[0188] 4) Resin testing: The Fmoc groups are tested using reagents to determine whether they have been removed.

[0189] 5) Amino acid condensation: Add amino acid solution and coupling reagent, shake well and filter.

[0190] 6) Repeat the above steps until the synthesis of the last amino acid is complete.

[0191] 7) Reverse chromatography for salt transfer: Trifluoroacetic acid (TFA) residues are removed by reverse chromatography, and salt transfer is performed using an acetic acid mobile phase.

[0192] MTT assay: RAW 264.7 cells were cultured in DMEM containing 10% FBS and 1% P / S at a 5% CO2 atmosphere and a temperature of 37°C. The cells were then subjected to a reaction at a rate of 3 × 10⁻⁶ cells / mL. 4 Cells were seeded into 96-well plates at a density of cells / mL and cultured overnight, followed by peptide treatment for 24 h. A positive control group was treated with 1 μg / mL LPS. MTT solution (5 mg / mL, 20 μL) was added to each well, and cells were incubated for 4 h. After removing the supernatant, 150 μL of DMSO was added to dissolve the purple dinoflagellate, and the plate was gently shaken for 10 min. Cell viability was determined by measuring the absorbance of the solution in each well at 490 nm using a microplate reader (PerkinElmer, Waltham, Massachusetts).

[0193] MTT cell function verification

[0194] 1. Instruments

[0195] Biological microscopes, centrifuges, carbon dioxide incubators, multi-functional enzyme-linked immunosorbent assay (ELISA) readers, cell counters, and biosafety cabinets.

[0196] 2. Experimental Materials

[0197] Table 10 Experimental Materials

[0198] 3. Experimental Principle

[0199] Succinate dehydrogenase in living cells reduces exogenous thiazolyl blue (MTT) to water-insoluble blue-violet formazan crystals, which are then deposited in the cells. Dead cells lack this function; therefore, the amount of MTT crystals formed is directly proportional to the number of living cells. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its optical density (OD) value is measured at a wavelength of 490 nm using a microplate reader. This OD value reflects cell viability.

[0200] 4. Experimental Procedure

[0201] 4.1. Cell passage culture

[0202] To revive and culture the cells.

[0203] 4.2. Cell viability assay

[0204] RAW 264.7 cells were cultured in DMEM containing 10% FBS and 1% penicillin antibiotics in a 5% CO2 incubator at 37°C. Cells were seeded at an appropriate density into 96-well plates and cultured overnight. Then, the cells were treated with different concentrations of samples for 24 h, with the positive control group treated with 1 μg / mL LPS. MTT solution (1 mg / mL, 100 μL) was added to each well, and the cells were incubated for 2 h. After removing the supernatant, 200 μL of DMSO was added to dissolve the purple formazan, and the plate was gently shaken to ensure complete dissolution. The absorbance of the solution in each well was measured at 490 nm using a microplate reader to determine cell viability.

[0205] 5. Data Analysis

[0206] 5.1. Cytotoxicity Calculation

[0207] Cell viability (%) = (OD of sample wells - OD of zeroing wells) / (OD of blank control wells - OD of zeroing wells) 100%

[0208] 6. Criteria for Determining Test Validity

[0209] The standard deviation (SD) of the optical density between each group of replicates was statistically analyzed, and the coefficient of variation (CV) was calculated. If the CV value is ≤ 20%, the parallelism of the experiment is considered valid.

[0210] Test results: Table 11 Cell viability test results

[0211] As shown in Table 11 and Figure 6 As shown, the coefficient of variation (CV) of absorbance OD values ​​of the sample group, blank control group (BC), and positive control group (PC) are all less than or equal to 20%, indicating that the experiment is valid.

[0212] Example 5: Promoting the secretion of inflammatory factors (IL-6, TNF-α, IL-1β)

[0213] 1. Instruments

[0214] Table 12 Main Experimental Instruments

[0215] 2. Experimental Materials

[0216] Table 13 Experimental Materials

[0217] 3. Experimental Principle

[0218] RAW 264.7 cells are a mouse macrophage cell line commonly used to study immunostimulatory activity. Macrophages play a crucial role in the immune system and are activated upon exposure to external stimuli. Immunostimulants in the body bind to specific receptors on the cell surface, such as Toll-like receptors (TLRs), initiating intracellular signal transduction pathways and activating the cells. Activated RAW 264.7 cells secrete various cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The levels of these cytokines in the cell culture supernatant, measured using methods such as enzyme-linked immunosorbent assay (ELISA), reflect the immune activation status of the cells. Generally, the stronger the immunostimulatory activity of the sample, the higher the level of cytokines secreted by the cells. Activation of RAW 264.7 cells increases the expression of inducible nitric oxide synthase (iNOS), catalyzing the production of nitrogen (NO). The NO content in the cell culture supernatant, commonly measured using the Griess reagent method, can also reflect the degree of cell activation and the immunostimulatory activity of the sample. The higher the NO production, the stronger the substance's potential immune-stimulating effect on cells. UV-VIS spectroscopy (ultraviolet-visible spectroscopy) is an analytical technique based on the principle of light absorption. Its basic principle is to utilize the absorption characteristics of a sample to specific wavelengths of ultraviolet or visible light, analyzing the sample's composition and concentration by measuring the absorbance at different wavelengths. ELISA (enzyme-linked immunosorbent assay) is an immunoassay technique based on the specific binding of antigen-antibody pairs. Its basic principle is to utilize the specific binding of antigens or antibodies on the surface of a solid-phase carrier with the corresponding antibodies or antigens in the sample. The signal is further amplified by enzyme-labeled secondary or tertiary antibodies, and finally, the concentration of the target molecule is quantitatively or qualitatively analyzed through a substrate colorimetric reaction.

[0219] 4. Experimental Grouping

[0220] Table 14 Experimental Groups

[0221] 5. Experimental Procedure

[0222] 5.1. Measurement of inflammatory factors TNF-α and IL-6 levels

[0223] 5.1.1. Cell passage

[0224] The cells are revived, cultured, and passaged.

[0225] 5.1.2. Cell Seeding Plate

[0226] Cells in the logarithmic growth phase were digested and centrifuged, and then prepared into a cell suspension of appropriate density with complete culture medium. The suspension was seeded into 96-well plates according to the experimental design and cultured at 37°C in a 5% CO2 incubator for 24 h ± 2 h.

[0227] 5.1.3. Sample Addition Processing

[0228] Discard the culture medium in the well plate, perform the sample addition operation according to Table 3, and then incubate at 37℃ and 5% CO2 for 24h±2h.

[0229] 5.1.4. Sample Collection

[0230] After the culture is completed, the cell culture medium is collected, and the supernatant is collected by centrifugation for subsequent tests.

[0231] 5.1.5. Measurement of inflammatory factors TNF-α and IL-6

[0232] Follow the instructions for the TNF-α and IL-6 assay kits. Use the human interleukin-6 (IL-6) ELISA kit and the human tumor necrosis factor-α (TNF-α) ELISA kit from Sangon Biotech.

[0233] 5.2. Measurement of IL-1β levels (an inflammatory cytokine)

[0234] 5.2.1. Cell passage

[0235] Cells are revived and cultured.

[0236] 5.2.2. Cell Seeding Plate

[0237] Cells in the logarithmic growth phase were digested and centrifuged, and then prepared into a cell suspension of appropriate density with complete culture medium. The suspension was seeded into 96-well plates according to the experimental design and cultured at 37°C in a 5% CO2 incubator for 24 h ± 2 h.

[0238] 5.2.3. Sample Addition Processing

[0239] Discard the culture medium in the well plate, perform the sample addition procedure according to Table 3, and then incubate at 37℃ in a 5% CO2 incubator for 6h ± 1h. Discard the culture medium in the well plate, add 5μM ATP solution to each well, and incubate at 37℃ in a 5% CO2 incubator for 1h ± 0.5h.

[0240] 5.2.4. Sample Collection

[0241] After the culture is completed, the cell culture medium is collected, and the supernatant is collected by centrifugation for subsequent tests.

[0242] 5.2.5. Determination of IL-1β levels (an inflammatory cytokine)

[0243] Follow the instructions for the IL-1β test kit.

[0244] 6. Data Analysis

[0245] Quantitative data are expressed as mean ± standard deviation (Mean ± SD). Data are analyzed using one-way ANOVA, and statistical differences are determined by p-value, with p < 0.05 indicating statistical significance.

[0246] 6.1. The formula for calculating cell viability is: Cell viability (%) = (OD of sample wells - OD of zeroing wells) / (OD of blank control wells - OD of zeroing wells) 100% 6.2. Calculation method of inflammatory factors: 6.2.1. Inflammatory factor content The obtained OD values ​​were substituted into the standard curves of each kit to calculate the content of inflammatory factors. 6.2.2. Upregulation rate of inflammatory factor levels Upregulation rate of inflammatory factor levels = (TC) / C 100 Where: T— content of inflammatory factors in the sample group; C—The average content of inflammatory factors in the blank control group.

[0247] 7. Criteria for Determining the Validity of the Detection

[0248] 7.1. A positive control group must be set up for each batch of tests. The test system is considered effective if the content of inflammatory factors in the positive control group is significantly increased compared with the blank control group.

[0249] 7.2. If at most one data point is removed, calculate the standard deviation (SD) of the absorbance OD values ​​for each group and the coefficient of variation (CV). If the CV value is ≤20%, the parallelism of the experiment is considered valid.

[0250] The test results are shown in Tables 15-17 and 16. Figure 7-9 As shown, compared with the blank control group, the levels of IL-1β, IL-6, and TNF-α were significantly upregulated after the addition of LQPEVMGVSKVKE (P < 0.05).

[0251] Table 15 Average IL-1β content

[0252] Table 16 Average IL-6 Content

[0253] Table 17 Average TNF-α content .

Claims

1. A milk-derived functional peptide, characterized in that, The amino acid sequence of the milk-derived functional peptide is shown in SEQ ID NO:

1.

2. A method for preparing the milk-derived functional peptides of claim 1, characterized in that, The method synthesizes the milk-derived functional peptides through chemical means.

3. A formulation, characterized in that, The formulation comprises the milk-derived functional peptides of claim 1.

4. The formulation according to claim 3, characterized in that, The preparation is a powder, pill, oil drop, capsule, granule, tablet, liquid preparation or gel.

5. A product comprising the milk-derived functional peptide of claim 1 or the formulation of claim 3.

6. The product as described in claim 5, characterized in that, The product is a food, cosmetic, or pharmaceutical composition.

7. The product as described in claim 6, characterized in that, The cosmetics include one or more of the following: toner, serum, makeup remover, moisturizing spray, sunscreen spray, liquid essence oil, moisturizing lotion, sunscreen lotion, essence lotion, facial cleanser, shower gel, eye cream gel, lipstick, lip mask, eyebrow wax, scrub, loose powder, pressed powder, face mask, eye mask, neck mask, eye patch, acne patch, neck wrinkle patch, and freeze-dried essence.

8. Use of the milk-derived functional peptide of claim 1 or the formulation of claim 3 in the preparation of food.

9. Use of the milk-derived functional peptide of claim 1 or the formulation of claim 3 in the preparation of health products with immunomodulatory functions.

10. Use of the milk-derived functional peptide of claim 1 or the formulation of claim 3 in the preparation of cosmetics having anti-wrinkle, soothing and / or immunomodulatory functions.

11. Use of the milk-derived functional peptide of claim 1 or the formulation of claim 3 in the preparation of a pharmaceutical composition having analgesic, skin cell aging-improving and / or immunomodulatory functions.

12. The use as described in claim 11, characterized in that, The pharmaceutical composition achieves its analgesic effect by inhibiting the expression of TRPV1. The pharmaceutical composition improves skin cell aging by promoting the expression of SIRT1. The pharmaceutical composition enhances immune regulation by promoting the secretion of inflammatory factors.

13. The use as described in claim 12, characterized in that, The cells are human immortalized keratinocytes, and / or the inflammatory factors are selected from one or more of IL-6, TNF-α, and IL-1β.

Images