Collagen peptide composition with function of increasing muscle strength and application of collagen peptide composition

Abstract

The invention discloses a collagen peptide composition with a function of increasing muscle strength and application of the collagen peptide composition. The collagen peptide composition comprises fish collagen peptide and beef tendon collagen peptide. A mouse senescence sarcopenia model is constructed, then a collagen peptide composition is used for intragastric intervention, and the improvement effect is evaluated through behavioral testing, body component analysis and HE staining. The potential target and regulatory pathway of the collagen peptide composition are explored by combining network pharmacology and molecular docking. Results show that the four-limb grip strength, treadmill exhaustion time and movement distance of a model mouse can be remarkably improved, quadriceps femoris muscle and gastrocnemius muscle indexes are recovered, and muscle fiber atrophy and collagen deposition are relieved. And it is found that TNF, IL1B, AKT1, mTOR and MAPK1 may be core targets of the TNF, IL1B, AKT1, mTOR and MAPK1. The polypeptide plays a role by regulating and controlling biological processes such as MAPK cascade, protein processing and inflammatory reaction, cell apoptosis, a cAMP pathway and a calcium signal pathway.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, and in particular to a collagen peptide composition that increases muscle strength and its application. Background Technology

[0002] On the one hand, exercise and balanced nutrition are important for improving athletic abilities such as muscle strength. Among sports enthusiasts and athletes, in order to improve muscle strength more efficiently, they not only train but also try to use nutritional supplements such as growth promoters.

[0003] On the other hand, sarcopenia is a syndrome characterized by progressive and widespread decline in skeletal muscle strength, muscle mass, and muscle function. It not only reduces patients' quality of life but also imposes a heavy economic burden. Based on etiology, it can be divided into two main types: primary sarcopenia and secondary sarcopenia. Age-related sarcopenia is the only subtype of primary sarcopenia. It is a degenerative disease with aging as the core driving factor, characterized by age-related decline in skeletal muscle mass and function. Its development is closely related to chronic inflammation and metabolic disorders. The pathological mechanisms involve excessive degradation of skeletal muscle proteins mediated by the ubiquitin-proteasome system (UPS), oxidative stress imbalance, and mitochondrial dysfunction. Current research on age-related sarcopenia is not in-depth; understanding mainly relies on clinical observation. Clinical interventions primarily focus on exercise therapy and basic nutritional support, which can only improve the overall physiological state of the body and cannot specifically regulate the aforementioned deep pathological targets, leading to significant individual differences in intervention effects. While hormonal interventions (such as exogenous testosterone supplementation and the use of growth hormone analogs) have shown some effectiveness in improving muscle mass and strength, their safety concerns are a key obstacle to their widespread clinical application. Testosterone replacement therapy significantly increases the risk of adverse cardiovascular events and prostate cancer, while growth hormone therapy can easily induce glucose metabolism disorders (such as decreased insulin sensitivity). Furthermore, the abuse of anabolic steroids can lead to endocrine disorders and liver damage. These safety concerns make hormonal interventions unsuitable as a long-term, routine clinical treatment.

[0004] Therefore, given the high demand for muscle-strengthening nutrients and the accelerating global aging process, it is of great significance to explore safe and active substances that can effectively enhance muscle strength and improve age-related sarcopenia. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention proposes a collagen peptide composition that increases muscle strength and its application, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A collagen peptide composition for increasing muscle strength, the collagen peptide composition comprising fish collagen peptides and bovine tendon collagen peptides.

[0007] Preferably, the mass ratio of fish collagen peptides to bovine tendon collagen peptides in the collagen peptide composition is (5~10):(0.5~3).

[0008] Preferably, the mass ratio of fish collagen peptides to bovine tendon collagen peptides in the collagen peptide composition is 9:1.

[0009] Preferably, the fish collagen peptide has a molecular weight of 500-1200 Da, and the beef tendon collagen peptide has a molecular weight of 800-2500 Da, both provided by Hainan Yuantai Biotechnology Co., Ltd. Using tilapia and beef tendon as raw materials, the mixture is washed multiple times with purified water, sterilized by heating, cooled, and then enzymatically hydrolyzed for 5-15 hours. The residue is filtered to obtain a clear enzymatic hydrolysate. Food-grade activated carbon is added to the hydrolysate, and after thorough mixing, the activated carbon is used to decolorize the solution for one hour. The solution is then filtered through a plate and frame filter to obtain a pure hydrolysate. The hydrolysate is sent to a vacuum concentration tower for concentration at an evaporation temperature of 60-70°C. The concentrate is then aseptically filtered through a high-efficiency filter to obtain a high-concentration sterile solution. The high-concentration solution is sprayed into a water mist and then enters a spray drying tower. The hot, dry air in the tower quickly removes the moisture from the solution, and granular collagen peptides are obtained within seconds.

[0010] Preferably, the preparation method of the collagen peptide composition includes the following steps: mixing fish collagen peptides and bovine tendon collagen peptides, and then sieving to obtain the collagen peptide composition.

[0011] Preferably, the mesh size of the sieve is 120-300 mesh.

[0012] Preferably, the collagen peptide composition involved in this application is used in enhancing muscle strength and / or in preparing products for treating age-related sarcopenia.

[0013] Preferably, the dosage form of the product is powder, granules, capsules, or oral liquid.

[0014] Preferably, the collagen peptide composition accounts for 20-80% of the mass of the product.

[0015] Preferably, the collagen peptide composition improves sarcopenia by acting on targets of TNF, IL1B, AKT1, mTOR, and MAPK1, thereby regulating the MAPK cascade, protein processing, inflammatory response, apoptosis, cAMP pathway, and calcium signaling pathway.

[0016] Compared with the prior art, the beneficial effects of the present invention are: The collagen peptide composition (CP) of this application can effectively improve muscle function and structure in age-related sarcopenia model mice. After 6 weeks of gavage intervention with the collagen peptide composition (CP), the grip strength of the limbs, the time to exhaustion on the treadmill, and the distance of movement were all significantly improved (p<0.01), and muscle strength and endurance were effectively restored. The quadriceps and gastrocnemius muscle indices significantly increased (p<0.01), and HE staining confirmed that it can reduce muscle fiber atrophy and collagen deposition, making the muscle fiber morphology closer to normal. The collagen peptide composition (CP) exerts its intervention effect through a multi-target, multi-pathway mechanism, namely, by acting on TNF, IL1B, AKT1, mTOR, and MAPK1 targets, regulating the MAPK cascade, protein processing, inflammatory response, apoptosis, cAMP pathway, and calcium signaling pathway, thereby improving sarcopenia and related symptoms. Attached Figure Description

[0017] Figure 1 Behavioral results for the control group and the D-galactose model group; where (a) is grip force, (b) is time to exhaustion, and (c) is displacement. Figure 2 The effect of collagen peptides on D-galactose-induced mice; where (a) represents grip strength, (b) represents time to exhaustion, and (c) represents displacement. Figure 3 Changes in mouse body weight; Figure 4 The effect of collagen peptides on muscle content in an aging sarcopenia model mouse; Figure 5 The effect of collagen peptides on the muscle index of mice; (a) gastrocnemius, (b) quadriceps femoris, (c) tibialis anterior, and (d) extensor digitorum longus. Figure 6 HE staining results for unilateral gastrocnemius muscle; (a) is the blank group, (b) is the model group, and (c) is the CP gavage group. Figure 7 GO enrichment analysis bar chart for potential targets of collagen peptides; Figure 8 Bubble graph of KEGG enrichment analysis for potential targets of collagen peptides. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0019] Example 1 A collagen peptide composition for increasing muscle strength, wherein the mass ratio of fish collagen peptide to bovine tendon collagen peptide in the collagen peptide composition is 10:1. The molecular weight of the fish collagen peptide is 500~1200 Da, and the molecular weight of the bovine tendon collagen peptide is 800~2500 Da. The preparation method of the collagen peptide composition includes the following steps: mixing fish collagen peptides and bovine tendon collagen peptides, and passing them through a 120-mesh sieve to obtain the collagen peptide composition.

[0020] Example 2 A collagen peptide composition that increases muscle strength, wherein the mass ratio of fish collagen peptide to bovine tendon collagen peptide in the collagen peptide composition is 10:3. The molecular weight of the fish collagen peptide is 500~1200 Da, and the molecular weight of the bovine tendon collagen peptide is 800~2500 Da. The preparation method of the collagen peptide composition includes the following steps: mixing fish collagen peptides and bovine tendon collagen peptides, and passing them through a 200-mesh sieve to obtain the collagen peptide composition.

[0021] Example 3 A collagen peptide composition for increasing muscle strength, wherein the mass ratio of fish collagen peptide to bovine tendon collagen peptide in the collagen peptide composition is 9:1. The molecular weight of the fish collagen peptide is 500~1200 Da, and the molecular weight of the bovine tendon collagen peptide is 800~2500 Da. The preparation method of the collagen peptide composition includes the following steps: mixing fish collagen peptides and bovine tendon collagen peptides, and passing them through a 300-mesh sieve to obtain the collagen peptide composition.

[0022] Experimental Example 1. Experimental Materials and Instruments Experimental materials: SPF-grade male C57 mice were purchased from Zhuhai Baishitong Biotechnology Co., Ltd.; fish collagen peptides and bovine tendon collagen peptides were provided by Hainan Yuantai Biotechnology Co., Ltd. The following experiments used the collagen peptide composition prepared in Example 3; D-galactose was purchased from Maclean Chemical Reagent Co., Ltd. Experimental instruments: ZS-ZL grip strength tester was purchased from Beijing Zhongshidichuang Technology Development Co., Ltd.; XR-PT-10B treadmill was purchased from Shanghai Xinruan Information Technology Co., Ltd.; PARAMETER 3D animal dual-energy X-ray bone densitometer was purchased from Shanghai Bozhuo Biotechnology Co., Ltd.

[0023] 2. Experimental Methods 2.1 Establishment and experimental grouping of a mouse model of age-related sarcopenia SPF-grade male C57 mice weighing 21±1g were randomly divided into a control group, a D-galactose model group (D-gal), and a collagen peptide group (CP) (n=8). The control group received an intraperitoneal injection of 0.1mL / 10g of 0.9% sodium chloride solution once daily. The D-galactose model group and the collagen peptide group received an injection of 500mg / kg of D-gal solution once daily for 14 days. The D-galactose model group and the collagen peptide group continued to receive D-gal injections daily. In addition, the collagen peptide group was administered a collagen peptide composition solution by gavage at a dose of 1000mg / kg (prepared by mixing collagen peptide composition and 0.9% sodium chloride solution at a mass ratio of 1g:20mL, stirring magnetically for 30min until completely dissolved, and then sterilizing through a 0.22μm filter membrane). That is, the modeling reagent D-gal solution was administered in the morning and collagen peptide intervention was administered in the afternoon. The experiment was conducted after 43 days of continuous gavage.

[0024] 2.2 Mouse behavioral tests After the D-gal modeling group mice were modeled, their limb grip strength was tested at weeks 1, 2, 3, 4, 5 and 6 after drug administration. The specific operation method is as follows: The mouse was placed on the grid platform of the grip strength meter, ensuring that its four paws fully grasped the grid; then the mouse was slowly and uniformly dragged backward, keeping the mouse's body horizontal throughout the process, until its four paws left the grid (i.e., it lost grip strength). The grip strength data at this time point was recorded. The above operation was repeated 5 times for each mouse, and the average value of the 3 maximum values ​​was calculated.

[0025] After the D-gal modeling group mice were modeled, running tests were conducted on each group of animals in weeks 1, 2, 3, 4, 5 and 6 after drug administration. The specific operation was as follows: Before the first formal running test, all mice had to complete 3 consecutive days of treadmill adaptation training: the treadmill incline was set to 0° during the training period, and the training time was 15 minutes per day; the adaptation speed was increased in a gradient, namely 10 m / min on the first day, 15 m / min on the second day and 18 m / min on the third day.

[0026] The formal running test parameters were set as follows: treadmill incline 20°, running speed 18 m / min, electric shock tolerance time 3 s, and stimulation current 0.8 mA. The criterion for exhaustion was: the mouse continuously fell onto the electric grid below the treadmill, triggering an electric shock, and remained in the rear 1 / 3 of the track, in a supine resting position with rapid breathing, unable to continue the treadmill exercise task. During the test, the number of electric shocks before exhaustion and the time to exhaustion were recorded.

[0027] 2.3 Mouse body composition analysis and gastrocnemius muscle staining Mice in the blank control group, D-galactose model group, and collagen peptide group had their body weight measured twice a week, with three measurements taken per mouse and the average value recorded. Six weeks after gavage administration of the collagen peptide composition solution, three mice from each group were selected for body composition analysis. Procedure: Mice were placed in an induction chamber connected to isoflurane. After anesthesia, the mice were transferred to a 3D animal dual-energy X-ray bone densitometer, where isoflurane was continued to maintain anesthesia. The instrument was started, with a current of 1000 μA and a voltage of 40 kV. After automatic calibration, body composition analysis was performed. After the experiment, mice were anesthetized, and bilateral tibialis anterior, gastrocnemius, quadriceps femoris, and extensor digitorum longus muscles were dissected and stored at -80°C. Unilateral gastrocnemius muscle was stained with hematoxylin and eosin (HE) to analyze muscle changes.

[0028] 2.4 The known amino acid sequences of collagen peptides were imported into the online tool ExPASy Peptide Cutter (http: / / web.expasy.org / peptide_cutter / ). Three enzymes—pepsin (pH=1.3), highly specific chymotrypsin ([FYWML] is in item C, not before item P), and trypsin—were selected to simulate the gastrointestinal environment for virtual enzymatic digestion, yielding 925 collagen peptides with lengths ranging from 2 to 10 amino acids. Water solubility and toxicity were predicted using Discovery Studio 2019 Client, and 508 peptides with good water solubility and no toxicity were selected for target prediction.

[0029] The 3D structures of these 508 peptides were constructed using the "Macromolecules" module in the DS software. Under the CHARM force field, the "MinimizeLigands" and "PrepareLigands" tools were used for energy optimization of the peptide structures, and the results were saved in MOL2 file format. The MOL2 format files of the peptides were imported into the Novopro platform to obtain the corresponding peptide smile numbers, which were then input into the SwissTargetPrediction database. The species "Homo sapiens" was selected to predict the potential targets of the collagen peptide composition, resulting in a target set for the collagen peptide composition. Gene names were corrected and transformed using the UniProt database. In the GeneCards, CTD, and DisGeNET databases, using "sarcopenia" as the search term, targets related to sarcopenia were searched. The targets from the three databases were merged and duplicate targets were removed to obtain a target set related to sarcopenia. The obtained set of collagen peptide targets was mapped with the set of targets related to sarcopenia using Venny 2.1.0 online software. The intersection targets are the potential targets of the collagen peptide composition for sarcopenia.

[0030] 2.5 Construction of protein interaction network Using the Multiple proteins module of the STRING database, the potential targets of the obtained collagen peptide composition for sarcopenia were imported. "Homo sapiens" was selected for Organizations, and "medium confidence (0.400)" was chosen for the minimum required interaction score. Unconnected nodes in the network were hidden, and the protein interaction network was downloaded as a TSV file. The file was imported into Cytoscape 3.9.1 software for visualization analysis. The Analyze Network function in the "Tools" module was used to calculate the degree, betweenness centrality (BC), and closeness centrality (CC) of each node in the network. In the Style module, the node size and color were adjusted according to the degree value. The Threshold values ​​of Closeness unDir, Betweenness unDir, and Degree unDir were calculated using the "Centiscape 2.2" function in the Apps module. The data was exported using Excel software, and the Threshold and Degree values ​​were used to screen for key targets.

[0031] 2.6 Enrichment Analysis The potential targets of the collagen peptide composition for sarcopenia were imported into the Metascape platform (https: / / metascape.org / gp / index.html). The "Analysis as species" option was set to "H. sapiens", the p-value cutoff to "0.01", and other parameters were left at their default values. GO Molecular Functions, GO Biological Processes, GO Cellular Components, and KEGG Pathway analyses were performed. The data were then imported into Excel for formatting. The processed data were plotted online using the Bioinformatics online platform (https: / / www.bioinformatics.com.cn / ), and the top 10 GO entries and top 20 KEGG pathways with the most significant enrichment results were visualized.

[0032] 2.7 Molecular docking of collagen peptides with key targets Two bioinformatics tools were used in combination to screen collagen peptides: Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) and CPP pred (http: / / distilldeep.ucd.ie / CPPpred / ). Peptide Ranker ranks peptides based on their predicted bioactivity probability, with scores ranging from 0 to 1; higher scores indicate greater bioactivity potential. CPP pred predicts the cell penetration ability of peptides, also using a 0-1 scoring system, with higher scores representing better cell permeability. In this study, both a PeptideRanker score > 0.6 and a CPP pred score > 0.6 were used as screening thresholds to identify peptides with both potential bioactivity and good cell permeability.

[0033] Using the online AutoDock Vina platform Prank Web 4 (https: / / prankweb.cz / ) as the docking tool, the binding conformation of collagen peptides with key targets was analyzed. By inputting the PDB IDs of protein receptors TNF, IL1B, AKT1, mTOR, and MAPK1, the platform automatically processed the protein receptors, removing irrelevant atoms such as water molecules and ligands. The MGL Tools tool was used to add hydrogen atoms and optimize hydrogen bond interactions, generating a PDBQT format file. The SMILES numbers of candidate peptides were input, converted to PDB format using OpenBabel, and further optimized through energy minimization and conformation. Then, MGL Tools scripts were used to add hydrogen atoms and assign charges, finally generating a PDBQT format file. The selected collagen peptides were then molecularly docked with the sites on the protein receptors with the highest scores. A milk-derived peptide, VSL, which promotes skeletal muscle cell proliferation, was used as a positive control, and molecular docking was performed according to the above method.

[0034] 2.8 Data Processing All measurement data were statistically analyzed using SPSS software, and the results are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used to test the statistical significance of differences in mean values ​​between groups, and Duncan's multiple range test was used for pairwise comparisons between groups. A p-value < 0.05 was used as the criterion for statistical significance.

[0035] 3. Experimental Results 3.1 Validation of the effectiveness of the age-related sarcopenia mouse model Six weeks after the intervention, visual analysis of grip strength, time to exhaustion, and motor displacement was performed (see details). Figure 1 The grip strength of the D-galactose model group (1.59±0.01N) was significantly lower than that of the control group (1.96±0.04N) (p<0.001); the time to exhaustion of the model group (757.13±85.97s) was significantly shorter than that of the control group (1809.25±216.54s) (p<0.001); and the displacement of the model group (218.62±26.74m) was also significantly less than that of the control group (531.11±64.34m) (p<0.001). According to the criteria for successful establishment of an age-related sarcopenia model (decreased grip strength, shortened time to exhaustion, and reduced displacement), the D-galactose model group and the control group showed statistically significant differences in all of the above indicators, indicating that the age-related sarcopenia model was successfully established.

[0036] 3.2 Muscle-improving effect of collagen peptide composition on age-related sarcopenia model mice After 6 weeks of intervention with collagen peptide solution, mice in the collagen peptide group showed significantly higher grip strength (p<0.01), treadmill exhaustion time (p<0.01), and range of motion (p<0.05) compared to mice in the D-galactose model group. However, the recovery level was still lower than that of the control group (see details). Figure 2 This indicates that the collagen peptide composition can effectively improve muscle strength, exercise endurance, and exercise capacity in D-galactose-induced mice.

[0037] 3.3 Effects of collagen peptide composition on body weight and body composition regulation in age-related sarcopenia model mice 3.3.1 Effects of body weight like Figure 3 As shown, compared with the blank group, the D-galactose group (D-gal) and the collagen peptide group (CP) were significantly lower than the blank group. The body weight of mice in the collagen peptide group was similar to that in the D-galactose group, but slightly higher than that in the D-galactose group, indicating that the collagen peptide composition prepared in this application has a certain improving effect on the increase of mouse body weight.

[0038] 3.3.2 Muscle content analysis The muscle mass of mice in the blank control group was significantly higher than that of mice in the model group (see details). Figure 4 Behavioral analysis showed that intervention with the collagen peptide composition significantly improved grip strength (strength), time to exhaustion (endurance), and range of motion (overall muscle function). This suggests that intervention with the collagen peptide composition may not improve muscle quality in mice by increasing muscle volume, but rather by targeting and optimizing muscle function.

[0039] 3.3.3 Muscle Index The gastrocnemius, tibialis anterior, quadriceps femoris, and extensor digitorum longus muscles of both sides of mice were harvested and weighed. The muscle index was calculated by dividing muscle weight (mg) by body weight (g). Compared with the control group, the gastrocnemius, quadriceps femoris, tibialis anterior, and extensor digitorum longus muscle indices of the model group mice were significantly decreased, indicating that the muscles of the model group mice had undergone significant atrophy. The gastrocnemius and quadriceps femoris muscle indices of the collagen peptide group mice were significantly increased compared with the model group (see details). Figure 5 However, the muscle recovery in the control group still showed a certain gap compared to the control group; the recovery of the tibialis anterior and extensor digitorum longus muscles was not significant, with no statistically significant difference. This is because the gastrocnemius and quadriceps femoris are large muscle groups that bear the main weight of the body, with active muscle fiber metabolism and high metabolic synthesis requirements. Collagen peptide composition can serve as an important raw material for the synthesis of myofascia and tendons, promoting muscle recovery and functional improvement, and significantly increasing muscle mass index by stimulating collagen synthesis. In contrast, the tibialis anterior and extensor digitorum longus muscles are small muscle groups with low load and lower metabolic requirements, resulting in less significant intervention effects from the collagen peptide composition.

[0040] 3.3.4 Effects of CP intervention on gastrocnemius muscle fibers in age-related sarcopenia mice The dissected unilateral gastrocnemius muscle was stained with hematoxylin and eosin (HE), and the changes in muscle fibers were determined by analyzing the images. Figure 6 As shown in (a), the myofibril cells in the blank group (normal muscle tissue) are regular polygonal in shape, with clear boundaries and uniform morphology; the cells are arranged tightly and orderly without obvious gaps, reflecting the integrity of the inter-muscle fiber connection structure; the interstitial background is a uniform light pink, without any abnormally proliferating white gaps, indicating no obvious fibrosis or inflammatory infiltration.

[0041] The model group of myofibroblasts showed significant atrophy and irregular cell morphology. Figure 6 (b) In this context, the cells are arranged in a disordered manner, and the boundaries between cells are still visible, but the gaps are enlarged, reflecting a decrease or breakage of the number of muscle fibers; the fibrosis is aggravated, and the white gaps are significantly increased, which may be due to abnormal proliferation of the endomysium and perimysium, which is a typical pathological feature of sarcopenia.

[0042] The myofibroblasts in the CP gavage group showed significant repair, with morphology close to that of normal muscle cells in the control group, indicating an improvement in muscle atrophy. Figure 6 (c) The arrangement of muscle cells was significantly more regular than that of the model group, approaching the regularity of the blank group; the gaps were significantly smaller than those of the model group, reflecting a reduced degree of fibrosis, indicating that CP can promote muscle fiber regeneration or functional recovery and improve D-gal-induced age-related sarcopenia.

[0043] 3.4 Analysis of key targets and mechanisms of action of collagen peptide compositions in improving sarcopenia based on network pharmacology 3.4.1 Prediction of potential targets and construction of protein-protein interaction networks A total of 461 collagen peptide targets for human action were predicted from the SwissTargetPrediction database. Using "sarcopenia" as the search term, 8041 targets related to sarcopenia were collected from the database. Through visualization and statistical analysis of these genes to determine the intersection of the two groups, 249 potential collagen peptide targets for sarcopenia were ultimately identified.

[0044] Potential targets extracted from the Venn diagram were imported into the STRING database to generate corresponding protein-protein interaction network diagrams. In this network, nodes are represented by colors, with different colored nodes indicating peptide targets. The more nodes interacting with a gene protein, the more protein-coding nodes there are. Nodes also represent the degree of association between target proteins; larger nodes with darker colors indicate a higher position of the target molecule in protein-protein interactions and a greater likelihood of regulatory function. The results showed that a PPI network with 247 nodes and 2882 edges was obtained. Screening using Threshold and Degree values ​​revealed key targets TNF, IL1β, AKT1, MTOR, and MAPK1. Tumor necrosis factor (TNF) and interleukin-1β (IL1β) are important pro-inflammatory cytokines whose expression levels are closely related to inflammatory responses and play a crucial role in the development of sarcopenia. Studies have shown that low levels of chronic inflammation activate the ubiquitin-proteasome system, promoting muscle protein hydrolysis and inhibiting muscle regeneration, thereby inducing sarcopenia. For example, elevated TNF-α levels have been observed in the skeletal muscle of aged mice, which is associated with muscle atrophy and a decrease in the number of satellite cells and type IIA myofiber. Similar phenomena have been observed in adult mice, suggesting that inhibiting TNF-α-induced proteolysis may be an effective strategy for treating or preventing age-related sarcopenia. IL1B, by activating the nuclear factor-κB (NF-κB) signaling pathway, works synergistically with TNF-α to further promote inflammatory responses and muscle protein degradation.

[0045] The PI3K / AKT / mTOR signaling pathway plays a central role in cell growth, proliferation, survival, protein synthesis, and metabolic regulation. Protein kinase B (AKT1) is a key component of this pathway; its activation significantly promotes protein synthesis and is a crucial signaling node for the stimulation of muscle anabolic metabolism by insulin and insulin-like growth factor-1 (IGF-1). AKT1 can also promote muscle cell growth and survival and inhibit protein degradation by phosphorylating downstream molecules, such as mammalian target of rapamycin (mTOR) and forkhead box transcription factor (FOXO1). mTOR, a key positive regulator of cell growth, proliferation, survival, and protein synthesis, experiences a decline in activity with aging, leading to a weakened muscle response to anabolic stimuli, i.e., anabolic resistance. AKT1 and mTOR, as core components of the PI3K / AKT / mTOR signaling pathway, work synergistically to activate this pathway and effectively promote protein synthesis.

[0046] Mitogen-activated protein kinase 1 (MAPK1) is a key component of the MAPK signaling pathway, which is widely involved in various biological processes such as cell proliferation, differentiation, migration and apoptosis. The MAPK signaling pathway also forms a complex molecular network with the PI3K-AKT signaling pathway and the NF-κB signaling pathway, jointly regulating cell function and the occurrence and development of diseases.

[0047] 3.4.2 GO analysis and KEGG pathway enrichment analysis of potential targets Gene Ontology (GO) analysis is an important tool for understanding the function of biological systems. It categorizes the functional annotation of genes or proteins into three main categories: biological processes (BP), cellular components (CC), and molecular functions (MF). By performing GO enrichment analysis on specific sets of genes or proteins, their potential roles in cellular activities, localization, and molecular interactions can be revealed. The top 10 enriched entries were visualized using GO enrichment functions, as shown in the following figure. Figure 7 As shown, the potential targets of the collagen peptide composition for sarcopenia mainly exert their effects through biological processes such as cellular response to nitrogen compounds, regulation of the MAPK cascade, protein processing, and inflammatory responses; cellular components such as membrane rafts, dendrites, the outer surface of the plasma membrane, the extracellular matrix, focal adhesion, and vesicles also participate in regulation; the main molecular functions involved include endopeptidase activity, protein kinase activity, kinase binding, G protein-coupled peptide receptor activity, hormone binding, protein tyrosine kinase activity, exopeptidase, cysteine-type endopeptidase activity, and protein domain-specific binding.

[0048] Collagen peptide compositions may improve sarcopenia through synergistic effects on biological processes such as the regulation of the MAPK cascade, protein processing, and inflammatory responses. The mechanisms include modulating the MAPK signaling pathway, promoting muscle cell proliferation, differentiation, and survival, and mitigating oxidative stress, thereby promoting muscle repair and regeneration and reducing cell damage. Activation of the PI3K / AKT / mTOR signaling pathway can enhance the efficiency of muscle protein synthesis, effectively combating muscle atrophy. Experimental studies have shown that bovine collagen oligopeptide (BCOP) can alleviate dexamethasone-induced muscle atrophy by activating the PI3K / AKT / mTOR signaling pathway. Furthermore, collagen peptide compositions may also inhibit the production and activity of pro-inflammatory cytokines such as TNF-α and IL-1β, reducing chronic inflammation-induced muscle damage, decreasing protein degradation, and promoting muscle regeneration.

[0049] KEGG pathway enrichment analysis revealed 157 pathways, identifying and visualizing the top 20 pathways by p-value, such as... Figure 8As shown. The top 20 signaling pathways include cancer pathogenesis, neuroactive ligand-receptor interactions, apoptosis, lipids and atherosclerosis, plaque formation, chemical oncogenesis-receptor activation, Alzheimer's disease, microRNAs in cancer, cyclic adenosine monophosphate (cAMP) signaling pathway, diabetic cardiomyopathy, programmed necrosis, calcium signaling pathway, cell clearance, neutrophil extracellular trap formation, regulation of TRP channels by inflammatory mediators, phospholipase D signaling pathway, axonal guidance, neuroactive ligand signal transduction, the renin-angiotensin system, complement and coagulation cascades, etc.

[0050] Apoptosis, the cAMP signaling pathway, and the calcium signaling pathway may play key roles. Apoptosis can lead to the direct loss of muscle cells, thus exacerbating sarcopenia. On the other hand, moderate apoptosis is necessary to clear damaged or dysfunctional muscle cells, creating space for the regeneration of new muscle fibers. The cAMP signaling pathway plays a crucial role in muscle cell growth and metabolism. cAMP activates protein kinase A (PKA), which in turn activates downstream Akt kinase. Akt can directly phosphorylate the Foxo3a transcription factor, causing it to remain in the cytoplasm and prevent it from entering the nucleus. This process specifically inhibits the transcriptional expression of muscle atrophy-related E3 ubiquitin ligases (atrogin-1 / MAFbx and MuRF1), thereby reducing skeletal muscle protein degradation and maintaining muscle mass homeostasis. Calcium ions play a vital role in muscle contraction, cell signaling, and gene expression. Disruptions in the calcium signaling pathway can lead to muscle dysfunction and atrophy.

[0051] In summary, the collagen peptide composition may improve muscle health and increase muscle mass at multiple levels by regulating several key biological processes and signaling pathways. On one hand, it promotes muscle cell growth and metabolism by activating the cAMP signaling pathway, regulating PKA activity, promoting protein synthesis, and inhibiting protein degradation. On the other hand, it regulates intracellular calcium levels by maintaining the stability of the calcium signaling pathway. 2+ Homeostasis may help alleviate muscle dysfunction caused by nerve damage and inhibit pathological cell apoptosis and protein degradation, thereby improving sarcopenia.

[0052] 3.5 Collagen peptide-target interaction mechanism Five collagen peptides, AWMR, MR, CAR, MAR, and ARPR, with both bioactivity and delivery potential were obtained through screening. Their predicted bioactivity values ​​were 0.943, 0.849, 0.648, 0.636, and 0.621, respectively, and their predicted delivery potential values ​​were 0.684, 0.823, 0.639, 0.707, and 0.855, respectively.

[0053] Docking was performed using the PrankWeb platform, and the results are shown in Table 1. In molecular docking studies, -5.0 kcal / mol is often used as the threshold for judging binding stability. Docking energy ≤ -5.0 kcal / mol usually indicates a stable and biologically significant binding between the ligand and the receptor. As shown in Table 1, the docking energy value of the positive control VSL is close to that of the collagen peptide. Some collagen peptides showed higher binding scores with specific targets (such as IL1B and mTOR), indicating that they can stably bind to the active sites of the targets, thereby inhibiting or activating the biological functions of the targets. The pathogenesis of sarcopenia is complex and is often related to chronic inflammation (involving inflammatory factors such as TNF and IL1B) and disorders of multiple signaling pathways. The collagen peptides screened this time can target key targets such as TNF and IL1B related to inflammation, as well as AKT1, mTOR, and MAPK1 related to cell proliferation and protein metabolism, which means that they may have the unique advantage of multi-target and synergistic effects. From the perspective of their mechanism of action, these collagen peptides may not only directly promote muscle synthesis, but also create a more favorable internal environment for muscle cell growth and proliferation by regulating the microinflammatory environment.

[0054] Table 1. Docking results of collagen peptides with key targets and positive controls.

[0055] Studies have shown that collagen peptide composition (CP) effectively improves muscle function and structure in age-related sarcopenia model mice. After 6 weeks of intervention with CP (1000 mg / kg body weight) by gavage, the grip strength of the limbs of the mice (recovered from the model group to near the level of the blank group), the time to exhaustion on the treadmill and the distance of movement were significantly improved (p<0.01), and muscle strength and endurance were effectively restored. The quadriceps and gastrocnemius muscle indices were significantly increased (p<0.01), and HE staining confirmed that it can reduce muscle fiber atrophy and collagen deposition, and make the muscle fiber morphology closer to normal. Collagen peptide composition (CP) exerts its interventional effect through multiple targets and pathways. CP targets 249 intersection points related to sarcopenia, with TNF, IL1B, AKT1, mTOR, and MAPK1 likely being core targets, involved in key physiological processes such as inflammation regulation, protein synthesis, and degradation. Enrichment analysis further reveals that CP primarily improves muscle atrophy through biological processes such as MAPK cascade regulation, protein processing, and inflammatory responses (GO analysis), as well as by regulating apoptosis, cAMP signaling pathway, and calcium signaling pathway (KEGG analysis). Molecular docking validation shows that five highly active collagen peptide fragments can stably bind to core targets such as IL1B and mTOR (docking energy ≤ -5.0 kcal / mol), providing evidence for the target mechanisms of collagen peptides.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A collagen peptide composition having the effect of increasing muscle strength, characterized in that, The collagen peptide composition includes fish collagen peptides and bovine tendon collagen peptides.

2. The collagen peptide composition according to claim 1, characterized in that, The mass ratio of fish collagen peptides to beef tendon collagen peptides in the collagen peptide composition is (5~10):(0.5~3).

3. The collagen peptide composition according to claim 2, characterized in that, The mass ratio of fish collagen peptides to beef tendon collagen peptides in the collagen peptide composition is 9:

1.

4. The collagen peptide composition according to claim 1, characterized in that, The molecular weight of the fish collagen peptide is 500~1200 Da, and the molecular weight of the beef tendon collagen peptide is 800~2500 Da.

5. The method for preparing the collagen peptide composition according to claim 1, characterized in that, The process includes the following steps: mixing fish collagen peptides and beef tendon collagen peptides, and then sieving them to obtain a collagen peptide composition.

6. The preparation method according to claim 5, characterized in that, The sieve mesh size is 120-300 mesh.

7. The use of the collagen peptide composition according to any one of claims 1 to 4 or the collagen peptide composition prepared by the preparation method according to any one of claims 5 to 6 in enhancing muscle strength and / or preparing products for treating age-related sarcopenia.

8. The application according to claim 7, characterized in that, The product is available in the form of powder, granules, capsules, or oral liquid.

9. The application according to claim 7, characterized in that, The collagen peptide composition accounts for 10-80% of the mass of the product.

10. The application according to claim 7, characterized in that, The collagen peptide composition enhances muscle strength and improves sarcopenia by acting on TNF, IL1B, AKT1, mTOR and MAPK1 targets, regulating MAPK cascade, protein processing, inflammatory response, apoptosis, cAMP pathway and calcium signaling pathway.

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