Milk-derived polypeptide VSL for promoting skeletal muscle cell proliferation and use thereof

Abstract

Provided are a milk-derived polypeptide VSL for promoting skeletal muscle cell proliferation and a use thereof. The milk-derived bioactive polypeptide VSL has the effect of promoting muscle cell proliferation, and can be used for preparing a drug for promoting muscle cell proliferation and preventing sarcopenia. The milk-derived bioactive polypeptide VSL is easy to synthesize, can be industrially produced, and has good application prospects in fields such as food, pharmaceuticals, and cosmetics.

Description

A milk-derived polypeptide VSL that promotes skeletal muscle cell proliferation and its applications Technical Field

[0001] This invention relates to a milk-derived polypeptide VSL that promotes skeletal muscle cell proliferation and its applications, in the field of bioactive peptides. Background Technology

[0002] Sarcopenia, also known as sarcopenia, is a progressive, systemic skeletal muscle disorder associated with aging, characterized by a decrease in muscle mass, muscle strength, and muscle function.

[0003] Sarcopenia is characterized by loss of muscle mass and impaired muscle function, including a decrease in muscle cross-sectional area and strength due to aging, without any underlying medical condition. Sarcopenia is also often accompanied by metabolic abnormalities, including impaired insulin sensitivity, oxidative defense, and decreased mitochondrial function. Various treatments have emerged to alleviate sarcopenia. A common treatment for sarcopenia is regulating hormone levels. It has been shown that administering growth hormone and / or testosterone can alleviate sarcopenia by improving certain muscle functions in older men. However, testosterone administration is associated with a greater risk of polycythemia, venous thromboembolism, prostate cancer, and cardiovascular events. Therefore, the search for safer, food-sourced, natural alternatives that can promote muscle proliferation and alleviate muscle damage is receiving increasing attention.

[0004] In recent years, bioactive peptides and proteins have shown promise for further advancements in the treatment of muscle abnormalities. For example, Kitakaze et al. revealed that lactoferrin promotes C2C12 proliferation and differentiation, as well as myotube hypertrophy. Furthermore, Iwasa et al. determined that casein hydrolysis-derived peptides promote glucose uptake in skeletal muscle cells and optimize human metabolism. However, compared to the abundance of milk-derived peptides, research reports are still relatively limited. Therefore, exploring milk-derived bioactive peptides that promote skeletal muscle health and prevent sarcopenia is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a novel use of bioactive peptides in promoting skeletal muscle cell proliferation and / or preventing sarcopenia.

[0006] This invention provides a bioactive peptide: VSL (3 peptide, whose amino acid sequence is Val-Ser-Leu).

[0007] In one embodiment of the present invention, the bioactive polypeptide is a milk-derived polypeptide, and VSL (3-peptide) is derived from α-lactalbumin and located at positions 5-7.

[0008] The amino acid sequences of the κ-casein and α-lactalbumin are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0009] α-lactalbumin (SEQ ID NO.2)

[0010] In one embodiment of the present invention, the bioactive peptide is used as food, medicine or health product; it is used to promote skeletal muscle cell proliferation and muscle cell growth.

[0011] Preferably, the food, pharmaceutical, health product, or nutritional product may also contain derivatives of the polypeptide; the polypeptide derivatives refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation of the amino acid side chain groups, amino terminus, or carbonyl terminus of the polypeptide.

[0012] The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0013] Preferably, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0014] Preferably, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0015] Preferably, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0016] Preferably, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0017] Preferably, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0018] Preferably, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0019] The health products also contain acceptable excipients.

[0020] The present invention also provides an expression vector or recombinant microorganism containing the VSL peptide.

[0021] Preferably, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors; preferably, the recombinant microorganism is bacteria or fungi.

[0022] The present invention also provides a food, medicine, health product or nutritional product containing an effective dose of the above-mentioned polypeptide.

[0023] Preferably, the food, medicine, health product, or nutritional product may also contain derivatives of the polypeptide; the derivatives of the polypeptide refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation of the amino acid side chain groups, amino terminus, or carbonyl terminus of the polypeptide.

[0024] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field;

[0025] Preferably, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0026] Preferably, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0027] Preferably, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0028] Preferably, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0029] Preferably, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0030] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0031] The health products also contain acceptable excipients.

[0032] This invention also provides the application of the above-mentioned VSL peptide in the preparation of food, pharmaceuticals, health products or nutritional products; the food, pharmaceuticals, health products or nutritional products are used to promote skeletal muscle cell proliferation and muscle cell growth.

[0033] In one embodiment of the invention, the drug further comprises a pharmaceutically acceptable carrier.

[0034] In one embodiment of the present invention, the application includes, but is not limited to, promoting muscle cell proliferation.

[0035] The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0036] Preferably, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0037] Preferably, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0038] Preferably, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0039] Preferably, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0040] Preferably, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0041] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0042] The health products also contain acceptable excipients.

[0043] This invention also provides the application of the bioactive peptide VSL peptide in the preparation of health products that help improve muscle quality.

[0044] In one embodiment of the present invention, the health product includes, but is not limited to, functional fermented dairy products.

[0045] The present invention also provides the use of the bioactive peptide VSL peptide in the preparation of drugs that promote skeletal muscle cell proliferation and / or prevent sarcopenia.

[0046] In one embodiment of the invention, the drug further comprises a pharmaceutically acceptable carrier.

[0047] In one embodiment of the present invention, the application includes, but is not limited to, promoting muscle cell proliferation.

[0048] The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0049] Preferably, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0050] Preferably, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0051] Preferably, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0052] Preferably, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0053] Preferably, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0054] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0055] The health products also contain acceptable excipients.

[0056] The present invention also provides a product for promoting myocyte proliferation, the product comprising a derivative of the bioactive peptide VSL;

[0057] In one embodiment of the present invention, the product promoting muscle cell proliferation includes health products that promote muscle mass improvement and drugs that promote muscle cell proliferation; the derivative of the bioactive polypeptide VSL refers to a polypeptide derivative obtained by modifying the amino acid side chain groups, amino terminus or carbonyl terminus of the bioactive polypeptide VSL with hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation. Beneficial effects

[0058] The milk-derived bioactive polypeptide VSL of this invention has a significant effect on promoting skeletal muscle cell proliferation and is expected to be used in the preparation of drugs or functional foods that promote skeletal muscle cell proliferation and prevent sarcopenia. The VSL of this invention is easy to synthesize and can be industrially produced, showing good application prospects in the fields of food, medicine, and cosmetics. Attached Figure Description

[0059] Figure 1: Schematic diagram of the docking of whey peptides with the FGFR1 receptor molecule. Pymol 1.5 and LigPlot were used to represent the specific binding sites of the peptides to FGFR1. The interaction forces include hydrogen bonding and hydrophobic interactions.

[0060] Figure 2: Influence of peptides on skeletal muscle cell proliferation. The effect of 0.01 μM bioactive peptide VSL on skeletal muscle cell proliferation was tested; CCK-8 assay was quantified by measuring absorbance at 450 nm using a microplate reader. n=3, data are expressed as mean ± SEM, and were analyzed by one-way ANOVA followed by Tukey multiple comparison test (p<0.05).

[0061] Figure 3: EdU staining results for peptide proliferation. (a) Cells were treated with a modified DMEM medium and 0.01 μM of bioactive peptide VSL. Cells were then stained with EdU staining solution and images were taken using an inverted fluorescence microscope; (b) The number of positive cells was counted using Image software. Data are the mean ± SEM values ​​from 9 independent experiments. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 compared with the control group.

[0062] Figure 4: Effect of peptides on muscle cell cycle. The effect of 0.01 μM bioactive peptide VSL on skeletal muscle cell cycle was tested. Cell numbers at each stage were collected and analyzed by flow cytometry, plotted using Modefit5, and the data were analyzed. n=3, data are expressed as mean ± SEM, and analyzed by one-way ANOVA followed by Tukey multiple comparison test (p<0.05).

[0063] Figure 5: Effect of peptide on reactive oxygen species (ROS) content in muscle cells. The effects of 0.01 μM bioactive peptide VSL on ROS damage in skeletal muscle cells were tested; the relative ROS content in cells was measured by absorbance at 532 nm using a microplate reader. n=3, data are expressed as mean ± SEM, and were analyzed by one-way ANOVA followed by Tukey multiple comparisons (p<0.05). ****p<0.0001, compared with the control group.

[0064] Figure 6: Effect of peptides on MDA content in muscle cells. The oxidative damage of 0.01 μM bioactive peptide VSL to skeletal muscle cells was tested. MDA content in cells was measured at 532 nm using a microplate reader and normalized to BCA protein content. n=3, data are expressed as mean ± SEM, and analyzed by one-way ANOVA followed by Tukey multiple comparisons (p<0.05). *p<0.05, **p<0.01, compared with the control group.

[0065] Figure 7: Liquid phase spectrum.

[0066] Figure 8: Effects of Lebentope milk mineral salt glucosamine chondroitin powder on muscle function in mice with sarcopenia. Muscle function was assessed using a limb grip strength tester and a wheel fatigue rotundus tester. n=3, data are expressed as mean ± SEM, and analyzed by one-way ANOVA followed by Tukey multiple comparison test (p<0.05). *p<0.05, **p<0.01, ***p<0.001 compared with the control group. # p<0.05, ## p<0.01, ### p<0.001 compared with the model group.

[0067] Figure 9: Effect of Lebentop Milk Mineral Salt Glucosamine Chondroitin Powder on Muscle Coefficient in Mice with Sarcopenia Syndrome. Mouse muscle coefficient = muscle wet weight / body weight × 100%. n = 3. Data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey multiple comparison test (p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group. # p<0.05, ##p<0.01, ### p<0.001 compared with the model group.

[0068] Figure 10: Effects of Lebentope emulsion mineral salt glucosamine chondroitin powder on gastrocnemius muscle fibers in mice with sarcopenia. Three 15× local muscle fiber images (avoiding blood vessels, lymph nodes, and connective tissue) were selected from the original slice images of the mouse gastrocnemius muscle. ImageJ was used to count the muscle fiber area and cell nucleus number. n=3, data are expressed as mean ± SEM, and were analyzed by one-way ANOVA, followed by Tukey multiple comparison test (p<0.05). *p<0.05, **p<0.01, ***p<0.001 compared with the control group. # p<0.05, ## p<0.01, ### p<0.001 compared with the model group.

[0069] Figure 11: Cross-section of mouse gastrocnemius muscle fibers (×150). Paraffin sections of mouse gastrocnemius muscle were prepared and stained with hematoxylin and eosin (HE). Detailed Implementation

[0070] The EdU working solution and cell cycle assay kits used in the following examples were purchased from Wuhan Saive Biotechnology Co., Ltd. and Shanghai Beyotime Biotechnology Co., Ltd., respectively.

[0071] The LC-MS / MS analysis method involved in the following embodiments is as follows:

[0072] LC-MS / MS analysis was performed using an EASY nLC 1200 system equipped with an Acclaim PepMap™ RSLC column (50 μm × 15 cm, 2 μm, 100 μm). The moving phase A consisted of an aqueous solution containing 0.1% (v / v) formic acid and 2% (v / v) acetonitrile, while the moving phase B consisted of an aqueous solution containing 0.1% (v / v) formic acid and 90% (v / v) acetonitrile. The gradient elution program was: 6%–20% B, 0–40 min; 20%–32% B, 40–52 min; 32%–80% B, 52–56 min, at a flow rate of 0.2 μL / min. The MS spectral acquisition range was 150–2000 m / z, with a resolution of 60,000. The MS / MS data collection range was 180–2000 m / z, with a scan time of 0.1 s.

[0073] The method for identifying the active peptide sequence involved in the following examples is as follows:

[0074] Peptide identification was performed using bovine proteins from the UniProt database (https: / / www.uniprot.org / ) as a reference database. Raw data were retrieved using Maxquant (v.1.5.2.8) in non-specific enzymatic cleavage mode with a mass tolerance of 0.02 Da, a tolerance of 10 ppm for de novo synthesis, and a tolerance of 7 ppm for isotope removal in MS / MS. The protein-peptide match false detection rate (FDR) threshold was set at 1%. To identify bioactive peptides, each sample was analyzed in triplicate; a peptide was considered significant if it appeared at least twice.

[0075] The following examples illustrate the methods for screening the docking ability of whey peptides with FGFR1 receptor molecules:

[0076] For virtual screening and molecular docking, the molecular structure of the FGFR1 receptor (PDB ID: 1EVT) was downloaded from the RCSB Protein Database (https: / / www.rcsb.org / ). Water molecules and irrelevant atoms were removed before docking. Peptide structures were constructed using Discovery Studio 2019 with energy minimized. Virtual screening was performed using AutoDock Wiener 1.2. Following docking method references, the box center position was set to -10.083, 2.000, -12.444, with a size of 120×120×120, and other parameters remained at their default values. The conformation with the highest affinity was selected as the result. Under the same conditions, the binding mode and orientation of the FGFR1 receptor were analyzed and selected. The first three conformations of each peptide docked with FGFR1 were generated, and the optimal conformation was selected for further analysis based on the docking results. Discovery Studio (DS) 2019, Pymol 1.5, and LigPlot were used.

[0077] The culture media involved in the following examples are as follows:

[0078] DMEM complete culture medium: purchased from Thermo Fisher Scientific (China) Co., Ltd., Shanghai, China.

[0079] The ARHPHPHLSF (10-peptide) involved in the following examples is derived from κ-casein, located at positions 96-105.

[0080] κ-casein (SEQ ID NO.1)

[0081] Example 1: Preparation of bioactive peptides

[0082] 1. Isolation and preparation of peptides

[0083] (1) Preparation of fermented whey

[0084] The purified strain CCFM1263 (described in Chinese invention patent application publication number CN117305158A) was passaged in first-generation MRS solid plates and second-generation MRS liquid tubes. After centrifugation at 6000×g for 5 min, the culture was resuspended in 11% (w / w) skim milk and inoculated into 11% (w / w) skim milk at a 2% (v / v) inoculation rate. After thorough mixing, the culture was incubated in an anaerobic incubator at 37℃ for 18 h. Then, the culture was passaged in skim milk again using the same method, incubated in an anaerobic incubator at 37℃ for 18 h, and then the protein was removed by Tris acid washing, and the viable count was adjusted to 1×10⁻⁶. 6 CFU / mL was inoculated into 11% (w / w) skim milk at an inoculum of 2% (v / v) and fermented in a constant temperature incubator at 37°C for 48 h.

[0085] Different fermented milk samples were taken, and the pH of the samples was adjusted to 4.6 with 1 mol / L sodium hydroxide solution. The samples were then pasteurized in an autoclave at 95°C for 10 min. The sterilized fermented milk samples were centrifuged at 10000×g, 4°C for 10 min. The supernatant was collected and filtered through a 0.45 μm organic filter membrane to obtain the *Lactobacillus helveticus* fermented milk whey sample. The collected *Lactobacillus helveticus* fermented milk whey was freeze-dried into powder and stored in freeze-drying bottles at 4°C for later use.

[0086] (2) Fermented whey was desalted and purified using a C18 solid-phase extraction column. The sample was desalted and purified using a C18 solid-phase extraction desalting column with a loading volume of 3 mg. The process consisted of five steps: moistening, leveling, loading, desalting, and washing, with the flow rate controlled by a constant pressure pump.

[0087] (3) After purification, the peptide solution was dried using a cold centrifugal vacuum concentrator and reconstituted in an aqueous solution consisting of 0.1% (v / v) formic acid and 2% (v / v) acetonitrile to achieve a peptide concentration of 0.5 mg / mL. The solution was stirred at 12000 g and 4 °C for 15 min, and the whey was collected and stored in sample vials for LC-MS / MS analysis. The sequences of the peptides in the mixed solution were obtained.

[0088] 2. Identification of active peptide sequences and screening of the docking ability of whey peptides with FGFR1 receptor molecules.

[0089] The sequences of the peptides obtained in step 1 were identified, and the docking ability of whey peptides with FGFR1 receptor molecules was screened.

[0090] The results are shown in Table 1.

[0091] Table 1: Molecular docking results

[0092] The results showed that a lower binding energy indicated a better binding effect with the receptor. Ultimately, a group of peptides, ARHPHPHLSF and VSL, were obtained that exhibited good virtual binding to the receptor. Figure 1 shows a schematic diagram of the docking of the two peptides with the FGFR1 molecule.

[0093] Example 2: Effects of bioactive peptides on muscle cells

[0094] 1. Effects on muscle cell proliferation

[0095] The specific method is as follows:

[0096] (1) ARHPHPHLSF polypeptide powder and VSL polypeptide powder were prepared by chemical synthesis by Shanghai Jier Polypeptide Co., Ltd.

[0097] (2) The ARHPHPHLSF and VSL peptide powders obtained in step (1) were added to the DMEM complete medium to make the final peptide concentrations in the medium 0.001 μmol / L, 0.01 μmol / L, and 0.1 μmol / L, respectively. The following were finally prepared: DMEM complete medium containing 0.001 μmol / L, 0.01 μmol / L, and 0.1 μmol / L bioactive peptide ARHPHPHLSF; and DMEM complete medium containing 0.001 μmol / L, 0.01 μmol / L, and 0.1 μmol / L bioactive peptide VSL.

[0098] (3) Mouse C2C12 myoblasts were seeded in 96-well plates at a seeding density of 2 × 10⁶ cells. 4 Cells / well were cultured at 37℃ for 24 h, and then DMEM complete medium containing 0 μmol / L, 0.001 μmol / L, 0.01 μmol / L, and 0.1 μmol / L bioactive peptides ARHPHPHLSF (named ARH10), and VSL were added to each well. After culturing at 37℃ for 24 h, 10 μL of CCK-8 solution was added to each well under aseptic conditions, and the cells were incubated at 37℃ for 30 min. After mixing, the absorbance was measured at 450 nm using a microplate reader. The results are shown in Table 2 and Figure 2.

[0099] Table 2: Effects of different concentrations of peptides on muscle cell proliferation

[0100] The results showed that the milk-derived peptide VSL could promote myoblast proliferation, with the best effect achieved at a concentration of 0.01 μmol / L, and the proliferation rate reaching 128% at a concentration of 0.01 μmol / L. ARHPHPHLSF could also promote myoblast proliferation, and the effect increased with increasing dosage.

[0101] 2. Effects on EdU proliferation staining of muscle cells

[0102] The specific method is as follows:

[0103] (1) ARHPHPHLSF polypeptide powder and VSL polypeptide powder were prepared by chemical synthesis by Shanghai Jier Polypeptide Co., Ltd.

[0104] (2) The ARHPHPHLSF peptide and VSL peptide powder obtained in step (1) were added to DMEM complete medium to make the final concentration of ARHPHPHLSF peptide in the medium 0.1 μmol / L and the final concentration of VSL peptide 0.01 μmol / L. DMEM complete medium containing ARHPHPHLSF peptide with a final concentration of 0.1 μmol / L and DMEM complete medium containing VSL peptide with a final concentration of 0.01 μmol / L were prepared.

[0105] (3) Mouse C2C12 myoblasts were seeded in 96-well plates at a seeding density of 2 × 10⁶ cells. 4 Cells / well were cultured at 37°C for 24 h, and then DMEM complete medium containing 0 μmol / L and 0.1 μmol / L bioactive peptide ARHPHPHLSF (named: ARH10) and 0.01 μmol / L bioactive peptide VSL were added, and cultured at 37°C for 24 h.

[0106] (4) Add 500 μL of preheated EdU working solution (37℃) to the treated cells and incubate for 2 h. Discard the EdU working solution, add 1 mL of fixative, and fix at room temperature for 15 min. Discard the fixative and wash 3 times with PBS containing 5% BSA. Add 1 mL of 0.3% Triton X-100 to each well and incubate at room temperature for 15 min. Discard the Triton X-100 and wash 3 times with wash buffer. Add 500 μL of Click reaction solution and incubate at room temperature in the dark for 30 min. Discard the Click reaction solution and wash 3 times with wash buffer. Add 1 mL of Hoechsty solution and incubate at room temperature in the dark for 10 min. Discard the Hoechsty solution and wash 3 times with wash buffer. Acquire images using a fluorescence microscope. The results are shown in Table 3 and Figure 3:

[0107] Table 3: Effects of different concentrations of peptides on the proportion of positive cells in muscle cells

[0108] As shown in Figure 3, the milk-derived peptide VSL significantly increased the proportion of positive cells in myoblasts, and its proliferation effect was significantly higher than that of ARHPHPHLSF on C2C12 cell proliferation. When the concentration of milk-derived peptide VSL was 0.01 μmol / L, the proportion of positive cells increased by 20.11% compared to the normal group.

[0109] 3. Effects on the muscle cell growth cycle

[0110] The specific method is as follows:

[0111] (1) ARHPHPHLSF polypeptide powder and VSL polypeptide powder were prepared by chemical synthesis by Shanghai Jier Polypeptide Co., Ltd.

[0112] (2) The ARHPHPHLSF peptide and VSL peptide powder obtained in step (1) were added to DMEM complete medium to make the final concentration of ARHPHPHLSF peptide in the medium 0.1 μmol / L and the final concentration of VSL peptide 0.01 μmol / L. DMEM complete medium containing ARHPHPHLSF peptide with a final concentration of 0.1 μmol / L and DMEM complete medium containing VSL peptide with a final concentration of 0.01 μmol / L were prepared.

[0113] (3) Mouse C2C12 myoblasts were seeded into 12-well plates at a seeding density of 1×10⁶ cells. 5 Cells / well were cultured at 37°C for 24 h, and then DMEM complete medium containing 0 μmol / L, 0.1 μmol / L bioactive peptide ARHPHPHLSF (named: ARH10), and 0.01 μmol / L bioactive peptide VSL were added and cultured at 37°C for 24 h.

[0114] (4) Discard the culture medium in the culture plate, lyse the cells with lysis buffer, collect the cells into 1.5 mL centrifuge tubes, discard the supernatant after centrifugation, add 0.5 mL of pre-cooled PBS buffer to dissolve the precipitate, discard the supernatant after centrifugation, and wash 2-3 times with PBS solution. Add propidium iodide (PI) staining solution to disperse the cells, and then stain overnight at 4°C in the dark (more than 24 h). Finally, analyze the cell fluorescence using flow cytometry. The DNA signal, represented by the red fluorescence intensity after PI staining, was collected by computer, while forward scattering (FSC) and side scattering (SSC) data were collected in real time. All data were analyzed using Modefit5. The results are shown in Table 4 and Figure 4:

[0115] Table 4: Effects of different peptides on muscle cells and the muscle cell cycle

[0116] The results are shown in Figure 4. Milk-derived VSL can accelerate the progression of the muscle cell cycle. When the concentration of milk-derived peptide VSL was 0.01 μmol / L, compared with the normal group, VSL significantly reduced the number of cells in the G0 / G1 phase by 10.9%, while significantly increasing the number of cells in the S phase by 9.15%, indicating that VSL treatment accelerated the cell cycle and promoted cell growth.

[0117] 4. Determination of ROS content in muscle cells

[0118] The specific method is as follows:

[0119] (1) ARHPHPHLSF polypeptide powder and VSL polypeptide powder were prepared by chemical synthesis by Shanghai Jier Polypeptide Co., Ltd.

[0120] (2) The ARHPHPHLSF and VSL peptide powders obtained in step (1) were added to DMEM complete medium to make the final peptide concentrations in the medium 0.1 μmol / L and 0.01 μmol / L, respectively. At the same time, the TNF-α powder synthesized by Shanghai Sangon Biotech was dissolved in DMEM medium to make the final concentration of TNF-α in the medium 20 ng / mL. Finally, the following DMEM complete mediums were prepared: DMEM complete medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, DMEM complete medium containing 0.01 μmol / L bioactive peptide VSL, and DMEM complete medium containing 20 ng / mL TNF-α.

[0121] (3) Mouse C2C12 myoblasts were seeded into six-well plates, 1 × 10⁶ cells per well. 5 Cells were cultured for 24 hours and then divided into groups for intervention. DMEM complete medium containing 0 μmol / L and 0.1 μmol / L bioactive peptide ARHPHPHLSF (named ARH10), 0.01 μmol / L bioactive peptide VSL, and 20 ng / mL TNF-α were added respectively, and the cells were cultured at 37°C for another 24 hours.

[0122] (4) Discard the old culture medium and add 500 μL of DCFH-DA solution to a final concentration of 10 μmol / L. Incubate at 37°C for 20-40 min. Wash three times with serum-free culture medium (purchased from Thermo Fisher Scientific) to remove DCFH-DA that has not entered the cells. Finally, measure the absorbance at a wavelength of 532 nm. The results are shown in Table 5 and Figure 5:

[0123] Table 5: Effects of different concentrations of peptides on ROS levels in muscle cells

[0124] As shown in Figure 5, compared with the model group, the content of reactive oxygen species (ROS) in the milk-derived peptide VSL treatment group was reduced by 1.90 times compared with the TNF-α group.

[0125] 5. Determination of MDA content in muscle cells

[0126] The specific method is as follows:

[0127] (1) ARHPHPHLSF polypeptide powder and VSL polypeptide powder were prepared by chemical synthesis by Shanghai Jier Polypeptide Co., Ltd.

[0128] (2) The ARHPHPHLSF and VSL peptide powders obtained in step (1) were added to DMEM complete medium to make the final peptide concentrations in the medium 0.1 μmol / L and 0.01 μmol / L, respectively. Simultaneously, the TNF-α powder synthesized by Shanghai Sangon Biotech was dissolved in DMEM medium to make the final TNF-α concentration in the medium 20 ng / mL. The final preparations included: DMEM complete medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, 0.01 μmol / L bioactive peptide VSL, and 20 ng / mL TNF-α.

[0129] (3) Mouse C2C12 myoblasts were seeded into six-well plates, 1 × 10⁶ cells per well. 5 Cells were cultured for 24 hours and then divided into groups for intervention. DMEM complete medium containing 0 μmol / L and 0.1 μmol / L bioactive peptide ARHPHPHLSF (named ARH10), 0.01 μmol / L bioactive peptide VSL, and 20 ng / mL TNF-α were added respectively, and the cells were cultured at 37°C for another 24 hours.

[0130] (4) The treated cells were lysed using lysis buffer and collected in 1.5 mL centrifuge tubes. After centrifugation at 12000 g for 10 minutes, the supernatant was collected for subsequent assays. In a new centrifuge tube, 0.1 mL of homogenate and lysis buffer were added as a blank control, 0.1 mL of standards at different concentrations were added to prepare a standard curve, and 0.1 mL of sample was added for assays. Then, 0.2 mL of MDA detection working solution was added. After mixing, the mixture was heated at 100 °C or in a boiling water bath for 15 minutes. After cooling to room temperature in a water bath, the cells were centrifuged at 1000 g for 10 minutes at room temperature. 200 μL of the supernatant was added to a 96-well plate, and the absorbance was measured at 532 nm using a microplate reader.

[0131] The results are shown in Table 6 and Figure 6:

[0132] Table 6: Effects of different concentrations of peptides on MDA content in muscle cells

[0133] As shown in Figure 6, compared with the model group, the MDA content in the milk peptide VSL treatment group was reduced by 2.05 times compared with the TNF-α group.

[0134] Example 3: The effect of Lebentop Milk Mineral Salt Glucosamine Chondroitin Powder containing VSL polypeptide from Shanghai Lebentop Health Technology Co., Ltd. on muscle repair in mice.

[0135] 1. Detection of VSL (Vitamin Sludge) from the in vitro digestion products of Lebentop milk mineral salt glucosamine chondroitin powder

[0136] The specific method is as follows:

[0137] (1) Experimental materials and equipment

[0138] a) Sample: a) Milk mineral salt glucosamine chondroitin powder; b) Simulated gastric juice (SGF): Accurately weigh 6.9mM KCl, 72.2mM NaCl, 0.9mM KH2PO4, 0.1mM MgCl2, and 0.5mM (NH4)2CO3, dissolve in ultrapure water and bring to a final volume of 1L, adjust pH to 3.0 with 1M HCl, and prepare fresh before use; c) Simulated intestinal juice (SIF): Weigh 6.8mM KCl, 123.4mM NaCl, 0.8mM KH2PO4, and 0.33mM MgCl2, dissolve in ultrapure water and bring to a final volume of 1L, adjust pH to 7.0 with 1M NaOH, and prepare fresh before use; d) Digestive enzymes and reagents: pepsin (4000U / mL), trypsin (200U / mL), bile salts (20mM), 1M NaOH solution, 1M HCl solution, methanol (chromatographic grade), acetonitrile (chromatographic grade), VSL peptide standard (purity ≥98%); e) Experimental instruments: constant temperature circulating bath, magnetic stirrer, pH meter, electronic balance, low temperature centrifuge, ice-water bath device, LC-MS / MS (equipped with Waters BEH C18 column, 1.7μm, 2.1×100mm).

[0139] (2) Sample pretreatment

[0140] Weigh out 30g according to the product instructions. Place the milk mineral salt glucosamine chondroitin powder in a 200 mL enzyme reactor, add 180 mL of 40 °C ultrapure water, dissolve thoroughly, and prepare a product solution for later use;

[0141] (3) In vitro static digestion experiment

[0142] a) In vitro simulated gastric digestion

[0143] Add the corresponding volume of simulated gastric juice to the reactor at a product solution:SGF (simulated gastric juice) ratio of 1:1 (v / v), wherein the final concentration of pepsin in SGF is 4000 U / mL; adjust the pH of the reaction system to 3.0, and stir at a constant temperature of 37℃ to simulate the gastric digestion stage; after digestion for 2 hours, add 1M NaOH solution dropwise to adjust the pH of the reaction system to 7.0, terminate the pepsin reaction, and complete the gastric digestion stage.

[0144] b) In vitro simulated intestinal digestion

[0145] A corresponding volume of simulated intestinal fluid was added to the reactor at a ratio of gastric digestive fluid to SIF (simulated intestinal fluid) of 1:1 (v / v), wherein the final concentration of trypsin in the SIF was 200 U / mL and the final concentration of bile salts was 20 mM. The reaction system was kept at pH 7.0 and stirred at 37°C to simulate the intestinal digestion stage. After 2 hours of digestion, the reactor was placed in a 95°C water bath for 10 minutes to terminate the trypsin reaction, and then immediately transferred to an ice-water bath for 10 minutes to complete the digestion and obtain the intestinal digestion products.

[0146] c) Detection of VSL peptides in intestinal digestive products

[0147] ① Sample pretreatment

[0148] Add chromatographically pure methanol rapidly to the digestion products at a ratio of 1:4 (v / v) of "enteral digestion products: methanol", vortex mix for 5 min to ensure full contact between methanol and sample; place the mixed solution in a 4°C refrigerator for 30 min to allow incompletely digested large molecular proteins, enzymes (pepsin, trypsin), and salts in the digestion solution to denature and aggregate; place the settled solution in a pre-cooled centrifuge, set the parameters to 4°C, 12000 rpm, and centrifuge for 20 min to completely separate the denatured protein precipitate to the bottom of the tube; collect the supernatant after centrifugation, filter it through a 0.22 μm organic phase filter membrane, and place the filtered solution in a sample vial for LC-MS / MS analysis.

[0149] ② LC-MS / MS detection of VSL peptide parameters settings

[0150] Chromatographic conditions

[0151] Chromatographic column: Waters BEH C18 column (1.7 μm, 2.1 × 100 mm); Mobile phase: Phase A (water, containing 0.1% FA), Phase B (acetonitrile); Gradient elution program: 0 min, 95% Phase A, 5% Phase B; 1 min, 95% Phase A, 5% Phase B; 6 min, 5% Phase A, 95% Phase B; 8 min, 5% Phase A, 95% Phase B; 8.1 min, 95% Phase A, 5% Phase B; Flow rate: 0.3 L / min; Column temperature: 40 °C; Injection volume: 2 μL.

[0152] Mass spectrometry conditions

[0153] Ion source: Electrospray ionization source (ESI+, positive ion mode); characteristic ion information is shown in the table below:

[0154] Table 7: Characteristic Ion Information under Mass Spectrometry Conditions

[0155] ③ Plotting the standard curve

[0156] VSL standard solutions of 5 μg / L, 10 μg / L, 50 μg / L, 100 μg / L, and 1000 μg / L were prepared and detected under the chromatographic and mass spectrometric conditions described above. A standard curve was plotted with peak area as the ordinate and concentration as the abscissa (R2≥0.99).

[0157] ④ Qualitative and quantitative analysis of samples

[0158] The filtered sample solution was injected into LC-MS / MS, and the VSL peptides were identified based on retention time (compared with VSL standards) and characteristic ions. The mass concentration of VSL peptides in the sample was calculated using the standard curve. Combined with the relative molecular mass of VSL (317.38 g / mol), the molar concentration of VSL peptides was calculated using the formula "molar concentration (μmol / L) = mass concentration (μg / L) ÷ molar mass (g / mol)".

[0159] ⑤ Detection Results - VSL Peptide Quantification Results

[0160] The LC-MS / MS detection spectrum is shown in Figure 7. After in vitro simulated gastrointestinal digestion, the Lebento milk mineral salt chondroitin sulfate product can produce VSL peptide. The calculated molar concentration is 0.048 μmol / L, which is within the effective activity concentration range (0.001-0.1 μmol / L) confirmed by this invention, and is higher than the optimal concentration for muscle cell proliferation (0.01 μmol / L).

[0161] 2. Effect of Lebentop Milk Mineral Salt Glucosamine Chondroitin Powder on Muscle Repair in Mice with Sarcopenia Syndrome.

[0162] The specific method is as follows:

[0163] (1) Animal experimental design

[0164] Forty-eight 6-week-old SPF-grade healthy male C57BL / 6 mice with a body weight of 21.63±0.76 g were selected for the experiment. They were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. and raised in the Animal Experiment Center of Jiangnan University. The license number is SYXK (Su) 2021-0056, and the approval number (IACUC Issue No.) is JN.No20250415c0880710

[0195] . According to the national standard for rodent experimental animal feeding, the experimental animals had free access to water and food under a constant room temperature of 23±2°C, a humidity of 40-45%, and a 12-hour light-dark cycle. After one week of adaptive feeding, the mice were numbered and weighed, and then randomly divided into two groups. One group was the control group (n = 8), and the other group was the model group (n = 32). The mice in the model group were further divided into a model group (n = 8), a low-dose intervention group of the Lebeintuo product (n = 8), a medium-dose intervention group of the Lebeintuo product (n = 8), and a high-dose intervention group of the Lebeintuo product (n = 8).

[0165] (2) Modeling stage

[0166] All mice were administered by subcutaneous injection. The mice in the model group were given dexamethasone sodium phosphate solution for intervention at a dose of 10 mg / kg / d for 21 consecutive days; the control group was given 0.9% normal saline and injected continuously for 21 days. The administration volume was 0.1 ml / 10 g for both groups. After 21 days of modeling, a four-limb pull force meter and a wheel-type rotarod were used to detect the muscle function of the mice to evaluate the modeling effect. If, compared with the control group, the mice in the model group showed slow movement, decreased daily activity ability, significantly decreased four-limb pull force, and a significantly increased number of times of falling off the wheel-type rotarod per unit time, it indicated that the muscle atrophy model was successfully established.

[0167] (3) Intervention stage

[0168] After the mice were successfully modeled, they were intervened by oral gavage for 21 consecutive days. The control group and the model group were gavaged with 0.9% normal saline; the sample intervention group was gavaged with Lebeintuo milk mineral glucosamine chondroitin powder orally. The low, medium, and high-dose groups were prepared according to the dose concentrations of 6.17 g / kg / d, 12.33 g / kg / d, and 18.5 g / kg / d respectively. The gavage volume was 0.2 ml / 10 g of mouse body weight.

[0169] (4) Behavioral testing

[0170] The pulling force of mice's limbs was assessed using a gripping device. Before the test, the mice were placed on the device for 5 minutes to acclimatize. The device was then set to measurement mode, and after ensuring that all four paws were firmly gripping the device, the mice were gently pulled backward by the tail end, and the gripping device reading was recorded. The pulling force was measured three times for each mouse, and the average value in grams was used as the indicator for assessing limb gripping force. The endurance and coordination of mice were assessed using a rotundus apparatus. Before the experiment, mice underwent acclimatization training: the rotation speed was set to a constant 5 rpm / min, and mice were placed one by one in the rotundus for 5 minutes of acclimatization exercise. After training, the mice were placed in cages to rest for at least 30 minutes. In the formal experiment, the rotation speed was gradually increased from 5 rpm / min to 35 rpm / min, and the time (s) it took for the mouse to fall from the rotundus was recorded. The experiment was repeated three times.

[0171] (5) Organize collection and testing

[0172] The experiment was completed after mice were treated with samples by gavage for 3 weeks. The mice were then anesthetized by inhalation with 3% isoflurane, and blood was collected from the ocular venous plexus. After blood collection, the mice were dissected, and gastrocnemius muscle tissue was collected. Pathological sections were prepared and stained to observe the morphology of gastrocnemius muscle fiber tissue, cross-sectional area of ​​muscle fibers, and number of cell nuclei.

[0173] (6) The results of the analysis of mouse behavioral outcomes are shown in Table 8 and Figure 8:

[0174] Table 8: Effects of different concentrations of lebento milk mineral salt glucosamine chondroitin powder on muscle function in mice with sarcopenia syndrome

[0175] The results showed that, compared with the model group, the limb pulling force of mice in all Lebento intervention groups was significantly increased (25.05% increase in the low-dose group, p < 0.001; 18.87% increase in the medium-dose group, p < 0.01; 21.79% increase in the high-dose group, p < 0.001), indicating an improvement in the maximum muscle contraction force of mice. Compared with the model group, the dwell time on the rotarod was significantly prolonged in all Lebento intervention groups (114.99% prolonged in the low-dose group, p < 0.01; 116.26% prolonged in the medium-dose group, p < 0.05; 124.59% prolonged in the high-dose group, p < 0.01), indicating an improvement in muscle endurance and coordination of mice. The results indicate that Lebento milk mineral salt glucosamine chondroitin powder has a good effect on improving muscle strength and muscle endurance in mice with sarcopenia syndrome.

[0176] (7) Analysis of mouse muscle mass results, as shown in Table 9 and Figure 9:

[0177] Table 9: Effects of different concentrations of Lebento milk mineral salt glucosamine chondroitin powder on muscle in mice with sarcopenia syndrome

[0178] The results showed that, compared with the model group, the gastrocnemius muscle coefficient of mice in the medium- and high-dose intervention groups of Lebento was significantly increased (23.14% increase in the medium-dose group, p < 0.001; 14.88% increase in the high-dose group, p < 0.01); the soleus muscle coefficient of mice in all Lebento intervention groups was significantly increased (26.13% increase in the low-dose group, p < 0.01; 40.02% increase in the medium-dose group, p < 0.001; 29.67% increase in the high-dose group, p < 0.05). The results indicate that the intervention of Lebento milk mineral salt glucosamine chondroitin powder has a good effect on increasing the gastrocnemius and soleus muscle coefficients and muscle mass in mice with sarcopenia syndrome.

[0179] (8) Analysis of the results of the gastrocnemius muscle fibers in mice. The results are shown in Table 10, Figure 10 and Figure 11.

[0180] Table 10: Effects of different concentrations of lebento milk mineral salt glucosamine chondroitin powder on gastrocnemius muscle fibers in mice with sarcopenia

[0181] The results showed that, compared with the model group, the cross-sectional area of ​​gastrocnemius muscle fibers in all Lebento intervention groups was significantly increased (15.34% increase in the low-dose group, p < 0.001; 15.70% increase in the medium-dose group, p < 0.001; 15.79% increase in the high-dose group, p < 0.001); the number of gastrocnemius muscle nuclei in both the low-dose and high-dose Lebento intervention groups was significantly increased (15.64% increase in the low-dose group, p < 0.01; 24.20% increase in the high-dose group, p < 0.05). As shown in Figure 11, the gastrocnemius muscle fibers in the model group were disordered and accompanied by widened interstitium and loose arrangement, while the gastrocnemius muscle fibers in all Lebento intervention groups were compact and dense, and the cell structure tended to be normal. The results indicate that Lebento milk mineral salt glucosamine chondroitin powder intervention has a good effect on the recovery of muscle fiber status in mice with sarcopenia syndrome.

[0182] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of VSL peptides derived from α-lactalbumin in the preparation of food, pharmaceuticals, health products or nutritional products; wherein the food, pharmaceuticals, health products or nutritional products are used to promote skeletal muscle cell proliferation and muscle cell growth.

2. The application according to claim 1, characterized in that, The VSL peptide is derived from α-lactalbumin and is located at positions 5-7; the amino acid sequence of the α-lactalbumin is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The VSL peptide is prepared as follows: (1) Preparation of fermented whey The purified strain CCFM1263 was passaged in first-generation MRS solid medium and second-generation MRS liquid medium, centrifuged at 6000×g for 5 min, resuspended in 11% (w / w) skim milk, and inoculated into 11% (w / w) skim milk at a 2% (v / v) inoculation rate. After thorough mixing, the culture was incubated in an anaerobic incubator at 37℃ for 18 h. Subculturing in skim milk was then repeated using the same method, followed by incubation in an anaerobic incubator at 37℃ for 18 h. Protein was removed by Tris acid washing, and the viable count was adjusted to 1×10⁻⁶. 6 CFU / mL, inoculated into 11% (w / w) skim milk at an inoculum of 2% (v / v), and fermented in a constant temperature incubator at 37°C for 48 h; Different fermented milk samples were taken, and the pH of the samples was adjusted to 4.6 by adding 1 mol / L sodium hydroxide solution. The samples were then pasteurized in a high-pressure steam sterilizer at 95°C for 10 min. The sterilized fermented milk samples were centrifuged at 10000×g at 4°C for 10 min. The supernatant was collected and filtered through a 0.45 μm organic filter membrane to obtain the whey sample of Lactobacillus helveticus fermented milk. (2) The fermented whey sample obtained in step (1) was desalted and purified using a C18 solid phase extraction column; (3) After purification, the peptide solution was dried using a cold centrifugal vacuum concentrator and reconstituted in an aqueous solution consisting of 0.1% (v / v) formic acid and 2% (v / v) acetonitrile to achieve a peptide concentration of 0.5 mg / mL. After stirring the solution at 12000 g and 4 °C for 15 min, the whey was collected and stored in a sample vial, and then analyzed by LC-MS / MS. The sequence of the peptide in the mixed solution was obtained by analysis. (4) The sequence of the peptide obtained in step (3) is identified, and the docking ability of whey peptide with FGFR1 receptor molecule is screened to finally obtain VSL peptide derived from α-lactalbumin.

4. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

5. The application according to claim 4, characterized in that, The excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; Other excipients may also be added to the composition: flavoring agents and sweeteners.

6. The application according to claim 5, characterized in that, The dosage forms of the medicine are oral, injectable, and inhaled.

7. The application according to claim 6, characterized in that, The oral dosage forms are tablets, capsules, granules, oral liquids, and oral suspensions.

8. The application according to claim 6, characterized in that, The injectable dosage forms are injection solutions and injection powders for injection.

9. The application according to claim 6, characterized in that, The inhalation dosage form is an aerosol or powder inhaler.

10. The application according to claim 1, characterized in that, The food products include grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food products also include special dietary foods. The health products also contain acceptable excipients.

11. The application according to claim 1, characterized in that, The food product is whey protein powder or food products for which whey protein powder has been added.

12. The use of VSL peptides in the preparation of health products that help improve muscle quality or in the preparation of drugs that promote skeletal muscle cell proliferation and / or prevent sarcopenia.

Images