Polypeptide as well as preparation method and application thereof in delaying muscle attenuation

Abstract

The invention discloses a polypeptide, a preparation method thereof and application of the polypeptide in delaying muscle attenuation. The amino acid sequence of the polypeptide is Ile-Arg-Leu-Ser-Phe-Asn-Pro-Thr. The polypeptide with the amino acid sequence can effectively relieve senescence-related markers, improve abnormal lipid metabolism and improve muscle functions, has the effect of improving muscle attenuation, has the advantages of being safe, free of side effects and the like, and can be applied to products for improving muscle attenuation and related symptoms thereof.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to a polypeptide, its preparation method, and its application in delaying muscle loss. Background Technology

[0002] Sarcopenia, or muscle loss syndrome, is a syndrome characterized by a progressive and systemic loss of muscle mass, strength, or function associated with aging. Its disease code is ICD-10-CM (M62.84). Skeletal muscle is a vital tissue in vertebrates, playing a crucial role in metabolism, daily physical activity, and maintaining posture and balance. Muscle loss can lead to a range of health problems, such as falls, functional decline, dyslipidemia, chronic diseases, weakness, and even death. The incidence of sarcopenia increases with age.

[0003] Currently, there are no approved drugs for treating muscle loss. The main recognized interventions are resistance training and nutritional supplementation. The reduction in muscle protein mass results from an imbalance between protein synthesis and protein breakdown. Protein synthesis requires sufficient amino acids from the diet. For patients with sarcopenia over 65 years of age, the recommended daily protein intake is 1.0 to 1.2 grams per kilogram of body weight, compared to 0.8 grams per kilogram of body weight for non-standard adults. However, meeting higher dietary protein requirements is challenging. With aging, kidney function declines, and high protein intake may further burden the kidneys. Simultaneously, efficient digestion and absorption of protein are essential for maintaining muscle protein synthesis, but the generally diminished digestive function in the elderly further limits the actual utilization of protein.

[0004] Bioactive peptides are derived from specific amino acid sequences of proteins and exhibit different biological activities due to factors such as molecular weight, amino acid composition and sequence, net charge, and hydrophobicity. Numerous studies have shown that peptides possess various nutritional and health-promoting functions. Currently, patent document CN119060126A discloses an oligopeptide that can delay muscle loss, containing a tetrapeptide with the amino acid sequence LWPF and / or LPGF, and a tripeptide with the amino acid sequence FEP. Experiments have confirmed that these oligopeptides can improve muscle loss, but their efficacy is limited. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a polypeptide that can better delay muscle loss, a method for preparing the polypeptide, and its application in delaying muscle loss.

[0006] A polypeptide with the amino acid sequence shown in SEQ ID No:1, namely Ile-Arg-Leu-Ser-Phe-Asn-Pro-Thr, abbreviated as IRLSFNPT; Wherein, Ile is the corresponding amino acid residue of isoleucine, Arg is the corresponding amino acid residue of arginine, Leu is the corresponding amino acid residue of leucine, Ser is the corresponding amino acid residue of serine, Phe is the corresponding amino acid residue of phenylalanine, Asn is the corresponding amino acid residue of asparagine, Pro is the corresponding amino acid residue of proline, and Thr is the corresponding amino acid residue of threonine.

[0007] The polypeptide with the above amino acid sequence is an octapeptide with a molecular weight of 947.12 Da, and its chemical formula is shown in Formula I below; Formula I: .

[0008] The above-mentioned polypeptide is prepared by chemical solid-phase synthesis or enzymatic hydrolysis.

[0009] Furthermore, the chemical solid-phase synthesis method includes: coupling the corresponding amino acids one by one on a solid-phase support according to the amino acid sequence shown in SEQ ID NO:1 to obtain a peptide resin, and then cleaving it to obtain the target polypeptide.

[0010] Furthermore, the solid support is resin, and the process of obtaining peptide resin is as follows: a solid support preloaded with the first amino acid and a protected amino acid are obtained respectively, and the protected amino acid and the resin are coupled one by one according to the amino acid sequence from the C-terminus to the N-terminus of the peptide.

[0011] The protected amino acid is a single amino acid whose N-terminus is protected by Fmoc, including: Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Ile-OH; The solid support preloaded with the first amino acid is Fmoc-Thr(tBu)-Wang Resin, where WangResin is the solid support Wang resin.

[0012] Specifically, when the polypeptide of the present invention is synthesized by solid-phase synthesis, the standard Fmoc scheme is adopted, the solid-phase support is resin, and the resin selected is Wang resin (CAS:1365700-43-1) pre-loaded with the first amino acid, namely Fmoc-Thr(tBu)-Wang. Resin; Fmoc protects the N-terminus of amino acids, with the protectant amino acids being Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Ile-OH. The protectant amino acids are coupled one by one to the resin. Conventional coupling reagents in the field of solid-phase synthesis can be used for coupling, such as tetrafluoroborate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea (TBTU) as a condensing agent, and N,N-diisopropylethylamine (DIEA) as a base, to couple the amino acids. The protectant amino acids are coupled one by one to the resin according to the amino acid sequence from the C-terminus to the N-terminus of the peptide. Then, the protecting groups of the resin and the side chains of the protectant amino acids are removed using a lysis buffer to obtain the crude product. After purification of the crude product, a peptide with the effect of slowing muscle attenuation is obtained.

[0013] The enzymatic hydrolysis process includes: mixing pretreated milk protein-containing raw materials with distilled water and adjusting the pH; heating to the enzymatic hydrolysis temperature; adding a first protease and a second protease in two steps for enzymatic hydrolysis; inactivating the enzymes after hydrolysis to obtain the hydrolysate; centrifuging and membrane filtration of the hydrolysate to obtain a preliminary hydrolysate containing a polypeptide with the amino acid sequence shown in SEQ ID No:1. The milk protein-containing raw materials can be sourced from one or more of cow's milk, goat's milk, camel's milk, donkey's milk, and mare's milk. The distilled water and the milk protein in the raw material containing milk protein are mixed at a ratio of (10-20):1; the pH is adjusted to 6.0-8.5; the enzymatic hydrolysis temperature is 52-58℃; during the enzymatic hydrolysis process, the total enzyme-to-protein ratio is 1:(20-100), and the total enzymatic hydrolysis time is 4-6 hours; the first protease includes one or more of serine protease, plant protease, animal protein hydrolase, Bacillus licheniformis protease, and Bacillus thermophilus protease; and / or, the second protease includes one or more of Bacillus subtilis protease, Bacillus licheniformis protease, plant protease, animal protein hydrolase, alkaline protease, Streptomyces protease, and Aspergillus niger fermentation protease.

[0014] This invention also provides the application of the above-mentioned peptides in the preparation of products that delay muscle loss.

[0015] Furthermore, the product slows down muscle loss by reducing the accumulation of age-related biomarkers, improving lipid metabolism abnormalities, and significantly enhancing the body's motor function; age-related biomarkers include lipofuscin and lipid droplets.

[0016] As an optional configuration, the dosage form of the product includes solutions, oral liquids, capsules, microcapsule powders, tablets, granules, emulsions, and tablets.

[0017] The technical solution of this invention has the following advantages: 1. This invention provides a polypeptide with the amino acid sequence shown in SEQ ID No:1, which is Ile-Arg-Leu-Ser-Phe-Asn-Pro-Thr. Experiments have confirmed that this polypeptide has a better effect on improving muscle loss than existing disclosed polypeptides, and has advantages such as safety, long-term use, and no side effects. It can be applied to products that improve muscle loss and related symptoms.

[0018] 2. Compared with an equal amount of protein, the polypeptides provided by this invention exhibit a faster absorption and digestion rate, can rapidly increase plasma amino acid concentration, thereby initiating muscle protein synthesis (MPS) and maintaining net muscle protein balance, and have a more significant impact on muscle health.

[0019] 3. The polypeptide preparation method provided by this invention uses chemical synthesis, which overcomes the shortcomings of enzymatic hydrolysis, the main method for preparing bioactive peptides, compared with the enzymatic hydrolysis method. Enzymatic hydrolysis easily generates a large number of non-target peptides, and subsequent separation and purification is time-consuming, costly, and complex. At the same time, it is difficult to control the stability of the preparation. It has many advantages such as high controllability and the ability to accurately synthesize bioactive peptides with specific sequences and structures. The prepared polypeptides have the advantages of high purity and safety. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The mass spectrum of the polypeptide synthesized in Example 1 that has the function of delaying muscle loss; Figure 2 This is a schematic diagram illustrating the effects of different treatments on the accumulation level of lipofuscin in the adult cristatum 'Seventh Antennae' in Example 2 of the present invention. Figure 3 This is a schematic diagram illustrating the effects of different treatments on lipid droplet accumulation in the adult seventh antennal worm in Example 3 of the present invention; Figure 4 This is a schematic diagram illustrating the effects of different treatments on the swimming ability of adult Antennae Septoria in Embodiment 4 of the present invention, including, for example... Figure 4 The activity index shown in (a) is as follows: Figure 4 The moving speed shown in (b) is as follows: Figure 4 The oscillation intensity shown in (c) is as follows: Figure 4 The four indicators of wave initiation frequency are shown in (d). Figure 5 This is a schematic diagram illustrating the effects of different treatments on the morphology of adult Antennae occulta during swimming, as shown in Example 4 of the present invention. Figure 5 The moving speed shown in (a) is as follows: Figure 5 The asymmetry shown in (b) is as follows: Figure 5 The three indicators of body wave quantity are shown in (c). Detailed Implementation

[0022] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0023] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0024] Example 1 - Preparation of Polypeptides A polypeptide, the preparation process of which is as follows: S1. Weigh out Fmoc-Thr(tBu)-Wang Resin containing 0.1 mol of amino acid active groups, place it in a glass reaction column, add DCM at a rate of 10 mL per gram of resin to swell for 30 min, and remove DCM under reduced pressure. S2. Wash the resin three times with DMF to remove residual solvent, and add 20% piperidine / DMF solution at a rate of 15 mL per gram of resin; this 20% piperidine / DMF solution means that the solution contains piperidine and DMF, and the volume ratio of piperidine in the solution is 20%; react at room temperature for 20 min to remove the protecting group Fmoc, remove the solution under reduced pressure, and wash with DMF six times; S3. Weigh out 0.4 mol of the second amino acid Fmoc-Pro-OH (corresponding to 4 times the excess of 0.1 mol of resin), 0.4 mol of TBTU, and 0.4 mol of HOBT, and add them to the resin. Dissolve them in DMF and add 0.8 mol of DIEA. Stir at room temperature for 5-10 min to activate the amino acids. Add the activated solution to the resin and react for 30 min. Take a small amount of resin and test it with ninhydrin. Observe the color of the solution and the color of the resin. A bright yellow solution and a yellow resin indicate that the reaction is complete. Remove the solvent under reduced pressure. S4. Repeat steps S2 and S3, and connect amino acid residues Asn, Phe, Ser, Leu and Arg in sequence according to the same steps and amounts, until the last amino acid Fmoc-Ile-OH is connected. Then wash three times each with DMF, DCM and methanol, and dry the resin. S5. After the peptide side chain is synthesized, the peptide chain containing the resin is added to a mixture of dichloromethane and trifluoroacetic acid in a volume ratio of 99:1 to cleave the peptide chain from the resin; the peptide is then added to a mixture of trifluoroacetic acid, ethylenediamine tartrate, distilled water, and trypsin inhibitor (TIS) in a volume ratio of 94.5:2.5:2:1 and reacted for 2 hours to remove the side chain protecting groups.

[0025] The mass spectrum of the polypeptide synthesized in this embodiment that has the function of delaying muscle atrophy is shown below. Figure 1 As shown, the amino acid sequence analysis of the mass spectrum reveals that the primary amino acid sequence of the synthesized polypeptide is Ile-Arg-Leu-Ser-Phe-Asn-Pro-Thr.

[0026] Example 2 - Evaluation of the effect of peptides on alleviating aging using Caenorhabditis elegans N2 1. Solution preparation: Nematode growth medium (NGM): 2.5 g peptone, 3 g NaCl, 20 g agar, 25 mL 1 M phosphate buffer, 1 mL 1 M CaCl2 solution, 1 mL 1 M MgSO4 solution, 973 mL H2O. Autoclave at 121℃ for 20 min. Cool to approximately 55℃, add 1 mL 5 mg / mL cholesterol solution, mix well, and pour into sterile petri dishes.

[0027] M9 buffer: 5g NaCl, 3g KH2PO4, 6g Na2HPO4, 1ml 1M MgSO4, bring to a final volume of 1L, autoclave at 121℃ for 20 min.

[0028] Nematode lysis buffer: 5 M NaOH and 5% NaClO (1:1 mixture, store at 4°C). Mix with M9 buffer at a 1:2 volume ratio before use. Prepare fresh before use.

[0029] Levomisole hydrochloride solution: Prepare 25 mM levamisole using M9 buffer for live nematode observation. Aliquot into 1.5 mL centrifuge tubes and store at -20°C for later use.

[0030] 2. Specific experimental methods: When there are enough eggs and reproductive nematodes on the NGM culture dish, wash them into a 1.5 mL centrifuge tube with M9 buffer. Repeat the washing twice, discard the supernatant, add 1 mL of nematode washing buffer, and mix thoroughly in a rotary mixer for 10 min. Then, observe whether the nematode bodies are fully lysed using a stereomicroscope. Centrifuge at 1000×g for 1 min, wash 3 times with M9 buffer, add an appropriate volume of M9 buffer to mix the eggs evenly, and drop them onto an NGM culture plate coated with OP50. Once the eggs hatch, L1 stage larvae with consistent development stages can be obtained. When the larvae grow to the L4 stage, plate transfer intervention can be performed.

[0031] A 50 μL M9 buffer solution was mixed with 50 μL of *E. coli* OP50 (OD600>1.0) to serve as the blank control group. The pure peptide from Example 1 was prepared into a 400 μM pure peptide solution using M9 buffer, and 50 μL of this pure peptide solution was mixed with 50 μL of *E. coli* OP50 (OD600>1.0) to serve as the IRLSFNPT intervention group. Both groups were coated onto nematode growth medium (NGM) to create NGM plates with different compositions. Synchronized L4 stage *C. elegans* N2 cells were picked and cultured in the NGM plates with different compositions. The plates were incubated at 20°C, transferred every other day, and cultured for 7 days. This yielded nematodes in the aged control group (corresponding to the blank control group) and nematodes in the peptide intervention group (IRLSFNPT) after intervention in the IRLSFNPT intervention group.

[0032] After intervention, each group of nematodes was washed three times with M9 buffer and then fixed onto a 2% agarose pad with 25 mM levamisole. Fluorescence imaging was performed using an inverted biofluorescence microscope (Guangzhou Daoyi Science & Technology Co., Ltd., B80iF) with blue excitation light (405-488 nm), DAPI, and 10x magnification. The lipofuscin accumulation level of each group of nematodes was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Results are shown in [Figure number missing]. Figure 2 .

[0033] like Figure 2As shown, compared with the elderly control group, the synthetic polypeptide IRLSFNPT of this invention significantly reduced the accumulation of lipofuscin in nematodes after intervention, indicating that the polypeptide can significantly reduce the accumulation of aging markers in the body.

[0034] Example 3 - Evaluation of the effects of peptides on improving lipid metabolism and protecting muscle function using Caenorhabditis elegans N2 1. Solution preparation: Oil Red O dye: Add 0.5g of Oil Red O powder to 100ml of isopropanol, stir magnetically at 300rpm overnight at 30℃, and filter using a 0.22μm organic filter membrane to prepare a saturated Oil Red O solution. It can be stored long-term at 4℃ under light-protected conditions. Before use, dilute the saturated Oil Red O solution to 60% with ddH2O, and filter the precipitate again to prepare the Oil Red O working solution. Prepare fresh before use.

[0035] 2. Specific experimental methods: Obtain the nematodes after intervention in Example 2.

[0036] After each intervention, the nematodes were washed three times with M9 buffer, the supernatant was removed, and 150 μL of 1% paraformaldehyde was added for fixation for 15 min. The fixed nematodes were placed in a -80℃ freezer for 2 min, and then placed in room temperature water until thawed. This operation was repeated three times. Afterward, the nematodes were washed three times with M9 buffer and the supernatant was removed.

[0037] Add 200 μL of Oil Red O working solution to each nematode suspension and stain in the dark for 1 h. Wash three times with M9 buffer containing 0.01% Triton-X100, centrifuge and discard the supernatant. Transfer the nematodes to a 2% agarose pad and take bright-field photographs at 10x magnification using an inverted microscope. Quantify the lipid droplet accumulation in each group of nematodes using ImageJ software. Results are shown in [Figure number missing]. Figure 3 .

[0038] like Figure 3 As shown, compared with the elderly control group, the synthetic peptide IRLSFNPT of this invention can significantly reduce the accumulation of lipid droplets in nematodes, indicating that the peptide can significantly regulate abnormal lipid metabolism and thus delay muscle aging.

[0039] Example 4 - Evaluation of the effect of peptides on improving muscle motor function using Caenorhabditis elegans N2 Obtain the nematodes after intervention in Example 2; 100 μL of M9 buffer was added to a grooved glass slide. The treated nematodes were then picked up and placed in the M9 buffer, positioned under a 1X microscope, and allowed to stand for 10 seconds before recording a 30-second motion video. The motion videos were analyzed using CeleST software to obtain kinematic parameters for each nematode during swimming, including activity index, movement speed, oscillation intensity, and wave initiation frequency, as well as morphological parameters including extension, asymmetry, and number of body waves. The results are shown in [Table missing]. Figure 4 , Figure 5 As shown; where, the activity index refers to the total number of pixels covered by the body / min, that is, the activity level of the nematode's bending during swimming; the movement speed refers to the distance the nematode moves per unit time, in mm / min; the oscillation intensity refers to the total number of pixels covered by the body / time, that is, the total number of pixels covered by the nematode's body in one complete flexed stroke, that is, the depth of the nematode's movement and the degree of bending in a given stroke; the wave initiation frequency refers to the number of body oscillations initiated by the head per unit time, in times / min; the extension refers to the maximum difference between the two extreme curvature values ​​of any part of the animal's body during each stroke, in curvature range / mm; asymmetry is used to assess the balance of swimming posture during each stroke; and the number of body waves refers to the number of repetitions of waves present in the body at a certain moment, in times.

[0040] like Figure 4 As shown, the synthetic polypeptide IRLSFNPT of this invention has a significant promoting effect on the motility of nematodes. Specifically, compared with the aged control group, the overall motility of adult nematodes on the seventh day after IRLSFNPT intervention was significantly improved in swimming, specifically reflected in a significant increase in their activity index, movement speed, oscillation intensity, and wave initiation frequency.

[0041] like Figure 5 As shown, the motility of nematodes in the peptide intervention group was significantly higher than that in the aged control group, but their motility patterns underwent characteristic changes. The nematodes in the peptide intervention group exhibited higher oscillation intensity; this enhanced muscle contraction resulted in deeper body folding during movement, leading to lower extension observed morphologically. Simultaneously, the powerful contractile oscillation replaced the ineffective, high-frequency shallow waves of the aged nematodes, resulting in a reduction in the number of body waves. This indicates that peptides primarily improve the motility of aged nematodes by enhancing muscle strength, rather than completely reversing age-related changes in motility patterns.

[0042] Comparing the wave initiation frequency index of nematodes, the nematodes treated with the polypeptide (IT8) with the amino acid sequence shown in SEQ ID No:1 in this application showed a 266.67% increase compared to the control group. However, according to patent document CN119060126A, the LWPF, LPGF, and FEP disclosed therein only showed increases of 42.06%, 35.71%, and 35.71% respectively in the corresponding indexes compared to the control group described in that document, indicating a significantly weaker improvement than the polypeptide with the amino acid sequence shown in SEQ ID No:1 in this application.

[0043] Meanwhile, in a nematode model, the effect of the IT8 characteristic peptide was compared with that of an equal dose of hydrolysate containing the IT8 characteristic peptide (i.e., replacing the IT8 characteristic peptide with the same mass of hydrolysate containing the IT8 characteristic peptide). The effect of the IT8 characteristic peptide was 30.18 times that of the hydrolysate containing the IT8 characteristic peptide. In a zebrafish model, the hydrolysate containing the IT8 characteristic peptide showed a 19.3% improvement compared to conventional whey protein isolate (WPI). The preparation process of the hydrolysate containing the IT8 characteristic peptide was as follows: milk protein powder (skimmed) was weighed and dissolved in distilled water at a ratio of 1g:15mL. The pH was adjusted to 7.0 using sodium carbonate solution (0.1 mol / L). A 50 mg / mL solution of the first and second proteases was prepared and activated at 37°C for 15 min before use. The enzymatic hydrolysis employed a two-step scheme with a total enzyme-to-protein ratio (w / w) of 1:25. First, the dissolved milk protein solution was preheated to 55°C. In the first step, the first protease was added at a w / w ratio of 1:50, and the hydrolysis time was 4 hours. In the second step, the second protease was added and activated at 37°C before use, with a w / w ratio of 1:50, and the hydrolysis time was 2 hours. After hydrolysis, the protein was inactivated at 85°C for 30 minutes, cooled, and centrifuged at 25°C, 4000 rpm for 30 minutes. The hydrolysate obtained after centrifugation was stored at -80°C for later analysis. Specifically, the first protease was a food-grade serine protease with an activity ≥10 U / mg produced by Shanghai Zhongfeng Biotechnology, and the second protease was a food-grade Bacillus subtilis protease with an activity 20 U / mg produced by Jiangsu Jiujia Biotechnology.

[0044] As can be seen from the above, the characteristic peptides of this invention are superior to the peptides disclosed in comparative patent literature, as well as traditional whey protein isolate (WPI) with muscle-building effects. In summary, the peptides prepared by the method of this invention can improve the accumulation of aging markers, improve abnormal lipid metabolism, and enhance muscle performance, thereby delaying muscle loss.

[0045] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence is shown in SEQ ID No:1, which is Ile-Arg-Leu-Ser-Phe-Asn-Pro-Thr; Wherein, Ile is the corresponding amino acid residue of isoleucine, Arg is the corresponding amino acid residue of arginine, Leu is the corresponding amino acid residue of leucine, Ser is the corresponding amino acid residue of serine, Phe is the corresponding amino acid residue of phenylalanine, Asn is the corresponding amino acid residue of asparagine, Pro is the corresponding amino acid residue of proline, and Thr is the corresponding amino acid residue of threonine.

2. The method for preparing the polypeptide according to claim 1, characterized in that, The polypeptide was prepared by chemical solid-phase synthesis or enzymatic hydrolysis.

3. The preparation method according to claim 2, characterized in that, The chemical solid-phase synthesis method includes: sequentially coupling the corresponding amino acids on a solid support to obtain a peptide resin, which is then cleaved to obtain the target polypeptide. The enzymatic hydrolysis process includes: mixing the pretreated milk protein-containing raw material with distilled water and adjusting the pH; heating to the enzymatic hydrolysis temperature; adding the first protease and the second protease in two steps for enzymatic hydrolysis; inactivating the enzyme after enzymatic hydrolysis to obtain the enzymatic hydrolysate; centrifuging and filtration of the enzymatic hydrolysate to obtain the preliminary enzymatic hydrolysis product, which contains a polypeptide with the amino acid sequence shown in SEQ ID No:

1.

4. The preparation method according to claim 3, characterized in that, The solid support is resin. The process of obtaining peptide resin is as follows: a solid support preloaded with the first amino acid and a protected amino acid are obtained respectively. The protected amino acid and the resin are coupled one by one according to the amino acid sequence from the C-terminus to the N-terminus of the peptide. In the enzymatic hydrolysis process, the distilled water and the milk protein in the raw material containing milk protein are mixed at a ratio of (10-20):1; the pH is adjusted to 6.0-8.5; the enzymatic hydrolysis temperature is 52-58℃; during the enzymatic hydrolysis process, the total enzyme-to-protein ratio is 1:(20-100), and the total enzymatic hydrolysis time is 4-6 hours; the first protease includes one or more of serine protease, plant protease, animal protein hydrolase, Bacillus licheniformis protease, and Bacillus thermophilus protease; the second protease includes one or more of Bacillus subtilis protease, Bacillus licheniformis protease, plant protease, animal protein hydrolase, alkaline protease, Streptomyces protease, and Aspergillus niger fermentation protease.

5. The preparation method according to claim 4, characterized in that, The protected amino acid is a single amino acid whose N-terminus is protected by Fmoc, including: Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Ile-OH; The solid support preloaded with the first amino acid is Fmoc-Thr(tBu)-Wang Resin, where Wang Resin is the solid support Wang resin.

6. The preparation method according to claim 4, characterized in that, The coupling reagents used in the coupling process include condensing agents and bases.

7. The preparation method according to claim 6, characterized in that, The condensing agent includes tetrafluoroboronic acid-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea; The base includes N,N-diisopropylethylamine.

8. The application of a polypeptide in the preparation of products that delay muscle loss, characterized in that, The polypeptide is the polypeptide of claim 1 or the polypeptide prepared by any one of the preparation methods of claims 2-7.

9. The application according to claim 8, characterized in that, The product slows down muscle loss by reducing the accumulation of aging-related markers, improving lipid metabolism abnormalities, and significantly enhancing the body's motor function; aging-related markers include lipofuscin and lipid droplets.

10. The application according to claim 8, characterized in that, The dosage forms of the products include solutions, oral liquids, capsules, microcapsule powders, tablets, granules, and emulsions.

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