Cow milk source MFGM micromolecular antioxidant peptide as well as preparation method and application thereof
A two-stage enzymatic hydrolysis process for MFGM peptides addresses inefficiencies in existing purification methods, producing peptides with high antioxidant activity and bioavailability to enhance muscle development and address sarcopenia.
Patent Information
- Application Number
- CN202510253498.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-07-15
AI Technical Summary
The existing MFGM peptide isolation and purification technology has slow separation speed, limited sample load, high protein loss rate, and poor in vitro activity screening effect, making it difficult to effectively solve the problem of degradation in skeletal muscle mass and strength caused by protein nutrition imbalance.
Two-stage enzymatic method was used to hydrolyze the MFGM protein from bovine milk, combined with membrane filtration, ultrafiltration concentration and vacuum freeze-drying, and MFGM small molecule antioxidant peptides with high antioxidant activity and strong bioavailability, including TGIIT and IITQ, and the preparation process was optimized by simulating the human digestion process.
The prepared MFGM small molecule antioxidant peptide has significant antioxidant activity, which can improve cell survival and promote skeletal muscle development. In particular, the DPPH radical scavenging rate of TGIIT and IITQ in the range of 0.5–2 mg/mL is up to 46.7%, which is significantly higher than other peptides, effectively alleviating muscle damage caused by oxidative stress.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of drug preparation for promoting skeletal muscle development, and specifically relates to a class of milk-derived MFGM small molecule antioxidant peptides, their preparation methods and applications. Background Art
[0002] Sarcopenia is a degenerative disease with a high incidence in the global elderly population, mainly accompanied by a decrease in skeletal muscle mass and strength, seriously threatening the health of the elderly. Although there are many causes of sarcopenia, for the elderly population, protein malnutrition is considered to be the main cause of the imbalance between myoprotein synthesis and catabolism in myoblasts. Due to insufficient protein intake or impaired digestion and absorption, abnormal Akt phosphorylation can cause myoblast metabolic disorders and cell apoptosis. Apoptotic cells can accelerate muscle protein breakdown, ultimately leading to the occurrence of sarcopenia. Therefore, promoting myoblast proliferation, differentiation and fusion helps to maintain the normal structure and function of skeletal muscle.
[0003] Since MFGM (milk fat globule membrane) protein has the effect of improving skeletal muscle mass and function; due to the complex composition of membrane proteins, more than 500 kinds have been identified, and the mechanism of its action is not yet clear. Modern nutrition believes that MFGM protein is not only absorbed in the form of amino acids after gastrointestinal digestion, but more is absorbed in the form of small peptides of 2-6 amino acids, and then plays a role in promoting cell proliferation. In addition, small peptides can be absorbed into the circulatory system in a complete form, without any waste products and metabolites being produced and can be fully utilized by the human body, avoiding the burden on the liver caused by the decomposition of excessive protein intake in the body to produce metabolites such as ammonia, keto acids and urea. Therefore, aiming at "small peptide preparation - small molecule absorption", establishing "in vitro enzymatic hydrolysis - screening" of small peptides that can be absorbed into the circulatory system in a complete form can perfectly solve the metabolic disorders caused by excessive protein intake or impaired digestion and absorption. The existing separation and purification techniques of MFGM peptides are mainly: ①, enzymatic hydrolysis of proteins; ②, separation and purification by Sephadex gel and DEAE-52 cellulose anion exchange chromatography; ③, purification and identification by LC-MS / MS. However, it is inevitable that although the MFGM protein obtained by column chromatography separation and purification has a high purity, the separation speed is low, the sample loading capacity is limited, the protein loss rate is high, and a large amount of in vitro activity screening is required. Summary of the Invention
[0004] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a class of milk-derived MFGM small molecule antioxidant peptides, their preparation methods and applications. The class of milk-derived MFGM small molecules has the advantages of high antioxidant activity, strong bioavailability, and green preparation process, and can be used for the development of functional foods (nutrients) and anti-aging products, helping to solve protein malnutrition and health problems, and having important application value.
[0005] To solve the problems of the existing technology, the technical solution adopted by the present invention is as follows:
[0006] A class of milk-derived MFGM small molecule antioxidant peptides, including one or more combinations of TGIIT, TSPLG, IITQ, YIC or DTP.
[0007] As an improvement, the class of milk-derived MFGM small molecule antioxidant peptides includes TGIIT and IITQ, wherein both TGIIT and IITQ have a high binding energy with keap1.
[0008] The preparation method of the above-mentioned milk-derived MFGM small molecule antioxidant peptides uses milk-derived MFGM protein as a raw material, hydrolyzes the MFGM protein by a two-stage enzymatic hydrolysis method, and after membrane filtration, ultrafiltration concentration and vacuum freeze-drying, obtains the MFGM small molecule antioxidant peptides.
[0009] The above preparation method includes the following steps:
[0010] Step 1: Take milk-derived MFGM protein in a beaker, and prepare a 5 mg / mL MFGM protein solution under the condition of a constant temperature water bath at 42-45 °C.
[0011] Step 2: Add an equal volume of simulated gastric juice to the MFGM protein solution, adjust the pH of the solution to 3.0 with 1 mol / L HCl, then sequentially add CaCl2, pepsin and gastric lipase, and hydrolyze in a constant temperature magnetic stirrer at 37 °C for 2 hours, adjust the pH to 6.5 to obtain a mixed solution A.
[0012] Step 3: Add an equal volume of simulated intestinal juice to the mixed solution A, adjust the pH to 7.0 with 1 mol / L NaOH solution, then sequentially add bile salts, CaCl2 solution and pancreatin, and hydrolyze in a constant temperature magnetic stirrer at 37 °C to obtain a mixed solution B.
[0013] Step 4: After the enzymatic hydrolysis is completed, heat-inactivate the enzyme in the mixed solution B at 95 °C to terminate the digestion process of the MFGM protein, and then perform centrifugation to remove pepsin, pancreatin, undigested protein, insoluble peptides and impurities.
[0014] Step 5: Use membrane separation technology to separate the MFGM peptide component with a molecular weight lower than 3000 Da from the supernatant after centrifugation, concentrate by ultrafiltration, and vacuum freeze-dry to obtain milk-derived MFGM small molecule antioxidant peptides.
[0015] As an improvement, the MFGM protein solution described in Step 1 is obtained by mixing pepsin and MFGM protein and dissolving them in 2.0 mg / mL NaCl, and the mass ratio of pepsin to MFGM protein is 13:1.
[0016] As an improvement, the rotation speed of centrifugation in step 4 is 10,000 rpm.
[0017] As an improvement, the temperature of vacuum freeze-drying in step 5 is (-40°C) - (-10°C).
[0018] Application of the milk-derived MFGM small molecule antioxidant peptide prepared by the above preparation method in the preparation of drugs for promoting skeletal muscle development.
[0019] Application of the milk-derived MFGM small molecule antioxidant peptide prepared by the above preparation method in the preparation of nutrients.
[0020] Beneficial effects:
[0021] Compared with the prior art, the milk-derived MFGM small molecule antioxidant peptide of the present invention, its preparation method and application have the following advantages:
[0022] 1. The present invention uses milk-derived MFGM as a raw material, and through an in vitro method simulating human digestion, membrane separation and purification, mass spectrometry identification combined with molecular docking technology to obtain MFGM small molecule antioxidant peptides that promote skeletal muscle development. The preparation method is simple, and the milk-derived MFGM small molecule antioxidant peptides have the advantages of high antioxidant activity, strong bioavailability, and green preparation process.
[0023] 2. The MFGM small molecule antioxidant peptides of the present invention, especially TGIIT and IITQ, can effectively improve cell viability. Among them, TGIIT increased by 2.57% to 8.27%, and IITQ increased by 7.21% to 15.32% (p < 0.01). The DPPH free radical scavenging rates of TGIIT and IITQ increase with the increase of concentration. The scavenging rate of IITQ is up to 46.7% at most in the range of 0.5 - 2 mg / mL, and is significantly higher than that of TGIIT. It provides a broad application prospect for MFGM small molecule antioxidant peptides in the fields of functional foods, cosmetics and pharmaceuticals.
[0024] 3. Experiments of the present invention show that the muscle fibers in the IITQ group and the TGIIT group are arranged tightly, the cell gap is reduced, and the striated muscle edema disappears, indicating that the two groups can effectively relieve lipid deposition, mitochondrial dysfunction and muscle structure damage caused by oxidative stress, further proving the application of the MFGM small molecule antioxidant peptide of the present invention in the preparation of drugs for promoting skeletal muscle development, and providing a new direction for solving sarcopenia. Description of the drawings
[0025] Figure 1 It is a schematic diagram for the preparation and identification of MFGM small molecule peptides.
[0026] Figure 2Schematic diagram of the docking sites and interaction 2D and 3D model results of Keap1 and MFGM peptides.
[0027] Figure 3 Schematic diagram of the results of the regulatory effect of MFGM peptides on rat skeletal muscle.
[0028] Figure 4 Schematic diagram of the digestion stability of MFGM protein by pepsin and trypsin.
[0029] Figure 5 Schematic diagram of the changes in particle size and ζ potential of MFGM protein during gastrointestinal digestion.
[0030] Figure 6 Schematic diagram of the results of the effect of selected MFGM peptides on the activity of L6 cells.
[0031] Figure 7 Schematic diagram of the results of the determination of the scavenging rate of DPPH free radicals by selected MFGM peptides.
[0032] Figure 8 Schematic diagram of the results of the determination of the scavenging rate of ABTS+ free radicals by selected MFGM peptides. Detailed implementation method
[0033] The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.
[0034] Example 1 Preparation of milk-derived MFGM small molecule antioxidant peptides
[0035] Step 1, Take milk-derived MFGM protein in a beaker, and under the condition of a constant temperature water bath at 42 °C, prepare a 5 mg / mL MFGM protein solution (the MFGM protein solution is obtained by mixing pepsin and MFGM protein and dissolving them in 2.0 mg / mL NaCl, and the mass ratio of pepsin to MFGM protein is 13:1);
[0036] Step 2, Add an equal volume of simulated gastric juice to the MFGM protein solution, adjust the pH of the solution to 3.0 with 1 mol / L HCl, refer to the INFOGEST static simulation digestion method, and then sequentially add CaCl2, pepsin and gastric lipase, and hydrolyze in a constant temperature magnetic stirrer at 37 °C for 2 hours, adjust the pH to 6.5 to inactivate pepsin, and obtain mixed solution A;
[0037] Step 3, Add an equal volume of simulated intestinal fluid to mixed solution A, adjust the pH to 7.0 with 1 mol / L NaOH solution, refer to the INFOGEST static simulation digestion method, and then sequentially add bile salts, CaCl2 solution and pancreatin, and hydrolyze in a constant temperature magnetic stirrer at 37 °C to obtain mixed solution B;
[0038] Step 4, after the enzymatic hydrolysis is completed, the mixed solution B is inactivated at 95 °C to terminate the digestion process of MFGM protein, and then centrifuged to remove pepsin, pancreatin, undigested protein, insoluble peptides and impurities;
[0039] Step 5, the supernatant after centrifugation is separated by membrane separation technology to obtain MFGM peptide components with a molecular weight lower than 3000 Da, concentrated by ultrafiltration, and freeze-dried under vacuum to obtain milk-derived MFGM small molecule antioxidant peptides.
[0040] Example 2 Analysis and Identification of Milk-Derived MFGM Small Molecule Antioxidant Peptides
[0041] SDS-PAGE combined with ζ potential and particle size measurement was used to evaluate the digestibility of milk-derived MFGM small molecule antioxidant peptides during the 240-minute digestion process.
[0042] The digestion samples were diluted to 1 mg / mL, mixed with 5× loading buffer, and heated in a boiling water bath for 5 minutes. The samples were separated by SDS-PAGE (75 V, 30 minutes; 125 V, 60 minutes), stained with Coomassie Brilliant Blue R250 for 30 minutes, and then decolorized until the protein bands were clear. Images were captured by a gel imaging system (Bio-Rad, Hercules, USA) and analyzed with Quantity One software for protein digestion stability ( Figure 4 )
[0043] The particle size and ζ potential of MFGM protein samples at different digestion times were measured using a Malvern ZetaSizer Nano (Malvern Instruments Ltd, Malvern, UK). After the samples were diluted with deionized water, each group was measured 3 times to reduce errors caused by voltage fluctuations.
[0044] The changes in particle size and ζ potential of MFGM protein during simulated gastrointestinal digestion are as Figure 5 shown. It can be seen from the figure that the particle size of undigested MFGM protein is 0.20 μm, which increases to 3.50 μm within 60 minutes of gastric digestion and then decreases to 2.44 μm. This is mainly due to aggregation caused by the initial pH approaching the isoelectric point, and subsequent enzymatic hydrolysis decomposes the aggregates and releases active peptides and amino acids. During the intestinal digestion stage, the particle size gradually decreases and stabilizes after 60 minutes, indicating that the digestion process is basically completed. During gastric digestion, the ζ potential changes from negative to positive, and the increase in positive charge generates electrostatic repulsion, which helps to disperse the aggregates; while during intestinal digestion, the ζ potential stabilizes at -16.4 to -29.5 mV, enhancing the stability of the system. After 60 minutes of digestion, both the particle size and ζ potential remain stable, and the system reaches an equilibrium state.
[0045] The research results show that MFGM protein exhibits good digestive stability and biological functional characteristics under digestive conditions and has the potential to develop highly efficient bioactive products.
[0046] Liquid chromatography - electrospray ionization tandem mass spectrometry (LC - ESI - MS / MS) technology was used for determination. The specific operation was as follows: After desalting the sample, 10 μL was injected into a C18 reversed - phase chromatographic column (0.15×150 mm, 5 μm, Column Technology Inc., CA, USA). Chromatographic separation was carried out using mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (84% acetonitrile solution of 0.1% formic acid). The gradient elution program was: The initial ratio was 4% B, linearly increased to 50% B within 50 minutes, then the ratio was increased to 100% B, maintained for 5 minutes and then continued with isocratic elution for 5 minutes. The flow rate was set at 300 nL / min, and the column temperature was controlled at 30 °C. Mass spectrometry data was acquired using the Top 10 data - dependent acquisition mode (DDA). The top 10 most abundant precursor ions in the range of 300–1800 m / z were dynamically selected for high - energy collision dissociation (HCD), the collision energy was set at 27 eV, and the dynamic exclusion time was 20 seconds. The resolution of the full - scan and HCD spectra was set at 70,000 and 17,500 (m / z 200), respectively. The obtained peptide sequences were verified by comparing with bovine sequences in the UniProt and NCBI databases. Among them, the mass spectrometry data was analyzed using the Thermo Scientific Xcalibur 3.2 software and the MASCOT search engine. The database search used the bovine (Bos taurus, 9913) protein sequence file (uniprot_cetartiodactyla.fasta) in the UniProt database.
[0047] In LC - ESI - MS / MS analysis, peptides with smaller molecular weights showed stronger proliferation - promoting effects, which was attributed to their stronger binding ability to target molecules. In particular, the content of small peptides composed of 3 - 5 amino acids was 52.94%, higher than the content of small peptides in MFGM peptides, while MFGM peptides mainly contained peptides with more than 5 amino acids. Generally speaking, small peptides were considered to be the effective components regulating the proliferation of L6 cells, and their biological activity was stronger than that of larger peptides.
[0048] The PepDraw software was used to calculate the theoretical molecular weight, isoelectric point (pI), hydrophilicity, and net charge of the peptides. The biological activity of the peptides was evaluated by PeptideRanker, and peptides (2 - 5 amino acids) with scores exceeding 0.5 were screened. The SwissADME tool was used to further evaluate the absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics of the peptides, with a focus on analyzing their water solubility and pharmacokinetic parameters.
[0049] The present invention screened out five small molecular peptides with relatively strong antioxidant potential (Table 1):
[0050] Table 1 Bioinformatics analysis of MFGM peptides
[0051]
[0052]
[0053] Note:
[0054] 1. Bioinformatics analysis of identified low molecular weight (<1000 Da) peptides;
[0055] 2. The data are from the peptide library and can be obtained at http: / / distilldeep.ucd.ie / PeptideRanker / ;
[0056] 3. The theoretical mass, pI, hydrophobicity and net charge of each peptide were calculated using PepDraw, which can be obtained at http: / / pepdraw.com / ;
[0057] 4. KCAAA refers to key antioxidant amino acids, including C (Cys), H (His), M (Met), W (Trp) and T (Tyr);
[0058] 5. HAA refers to hydrophobic amino acids, including L (Leu), V (Val), A (Ala), P (Pro), F (Phe), W (Trp), I (Ile), M (Met);
[0059] 6. - indicates that there is no amino residue in the polypeptide.
[0060] As can be seen from Table 1, TGIIT (503.29 Da), TSPLG (473.25 Da), IITQ (473.28 Da), YIC (397.17 Da) and DTP (331.14 Da). Evaluated by the ToxinPred database, these peptides are all non-toxic peptides, and DTP exhibits excellent water solubility and ADMET properties. Except for YIC, the remaining peptides are novel peptides and no corresponding records were found in the BIOPEP database. The MS / MS spectra of TGIIT and IITQ and their structure-activity relationships further verified the antioxidant potential of these peptides.
[0061] Example 3 Screening of milk-derived MFGM small molecular antioxidant peptides
[0062] Molecular docking was performed using AutoDockVina 1.1.2 to predict the binding affinity and binding mode between the protein and the ligand. Combining the potential energy model and the scoring function, the non-covalent interactions between the protein and the ligand were evaluated. The binding site coordinates of Keap1 are: The docking box size was set to to ensure the exploration of multiple binding orientations of the ligand to the protein and to consider the flexibility of the ligand and the protein. AutoDockVina uses a force field-based scoring function and combines widely used physical models to calculate the binding energy of the protein-ligand complex. The scoring function takes into account factors such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. By optimizing the spatial conformation of the ligand and the flexibility of the protein, Vina uses a gradient optimization algorithm to improve the calculation efficiency and obtain the conformation with the lowest binding energy. Analyses such as the binding energy, the interaction mode of key amino acid residues, and the binding stability further verified the accuracy and reliability of the docking results.
[0063] The interaction of MFGM high-abundance peptides with Keap1 and the evaluation of their binding free energy (Table 2 and Figure 2 )
[0064] Table 2 Potential binding sites of high-abundance MFGM peptides binding to Keap1
[0065]
[0066] The results showed that the TGIIT and IITQ peptides were rich in antioxidant and hydrophobic amino acids, and IITQ showed the lowest binding free energy and was thus considered the best peptide binding to Keap1. Molecular docking analysis showed that TGIIT interacted with the hydrophobic residues of Keap1 (such as ALA510, VAL463, GLY462) and formed hydrogen bonds with SER508, SER555, SER602, and the binding free energy was -7.1 kcal / mol; IITQ interacted hydrophobically with residues such as GLY509, GLY511, GLY364, and formed hydrogen bonds with SER555, ARG415, LEU557, and the binding free energy was -7.5 kcal / mol.
[0067] Therefore, when the milk-derived MFGM small molecule antioxidant peptides of the present invention include IITQ and TGIIT, they exhibit good antioxidant capacity in a simulated digestion environment, can significantly inhibit the generation of free radicals caused by oxidative stress, and activate the Nrf2 / ARE antioxidant signaling pathway by binding to the Keap1 protein. At the same time, Sangon Biotech Co., Ltd. (Shanghai, China) was commissioned to synthesize IITQ and TGIIT by the Fmoc solid-phase synthesis method, and the purity of the synthesized peptides was >98%.
[0068] In vitro verification of small molecule active peptides in Example 4
[0069] (1) MTT assay:
[0070] L6 cells were treated with different concentration gradients of MFGM small molecule peptides (IITQ and TGIIT) for 24 h, 48 h and 72 h, and the effects on cell viability were measured. Rat L6 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) at a concentration of 5×10 4 / mL were seeded in 96-well plates, and 200 μM Dex was used to induce oxidative stress. 100, 200, 500 μg / mL of MFGM peptides, TGIIT and IITQ were added respectively, and 0.01 mol / L PBS was used as the control group, and MFGM mixed peptides were used as the positive control group. After 24, 48 and 72 hours of treatment, 20 μL of MTT working solution (5 mg / mL) was added to each well and incubated for 4 hours. After removing the medium, 100 μL of DMSO was added, and the absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader to determine the MFGM small molecule peptides with the best dose-effect relationship.
[0071] Protective effects of MFGM peptides TGIIT and IITQ on dexamethasone (Dex)-induced oxidative damage in L6 cells( Figure 6 ). The cell viability of all treatment groups was significantly higher than that of the control group (Dex model group). Both TGIIT and IITQ could effectively increase cell viability. Among them, TGIIT increased by 2.57% to 8.27%, and IITQ increased by 7.21% to 15.32% (p<0.01). The cell viability of the IITQ group was 2.23 times higher than that of the MFGM mixed peptide group (p<0.01), and at a concentration of 200 μg / mL, the protective effect of IITQ was better than that of TGIIT (p<0.01). These results indicate that TGIIT and IITQ, especially IITQ, as antioxidant peptides in MFGM, can effectively promote cell survival. Given the large variety of MFGM proteins, directly screening small molecule antioxidant peptides can not only reduce costs, but also accelerate the discovery of active ingredients and mechanisms.
[0072] (2) Determination of DPPH free radical scavenging activity:
[0073] After mixing 100 μL of 0.1 mM DPPH ethanol solution with 100 μL of IITQ and TGIIT solutions at different concentrations (0.1, 0.2, 0.5, 1, 2 mg / mL), they were added to 96-well plates and left to stand in the dark for 30 minutes. Ethanol was used as the control group, and the absorbance at 517 nm was measured and the free radical scavenging rate was calculated. The experimental results( Figure 7)It was shown that the DPPH radical scavenging rates of TGIIT and IITQ increased with increasing concentration. The scavenging rate of IITQ was up to 46.7% at the highest in the range of 0.5–2 mg / mL, and was significantly higher than that of TGIIT. (3) Determination of ABTS+ radical scavenging activity:
[0074] ABTS stock solution reacted with potassium persulfate to generate ABTS+ radicals. After incubating in the dark for 16 hours, it was diluted with PBS (pH 7.4) to an absorbance at 734 nm. 1 mL of the diluted ABTS+ solution was mixed with 1 mL of samples at different concentrations (0.1, 0.2, 0.5, 1, 2 mg / mL). After incubating for 10 minutes, the absorbance at 734 nm was measured. The experimental results ( Figure 8 ) showed that the ABTS+ radical scavenging rates of TGIIT and IITQ increased with increasing concentration. The scavenging ability of IITQ was significantly better than that of TGIIT, being 59.1% - 1.67 times higher, which was consistent with the results of DPPH and cell protection experiments. In summary, as antioxidant peptides in MFGM, TGIIT and IITQ exhibited significant antioxidant activities.
[0075] Example 5 Regulation of Antioxidation of MFGM Small Peptides on Rat Skeletal Muscle
[0076] 1. Experimental animals and grouping:
[0077] Male SD rats (n = 40), purchased from Liaoning Changsheng Biotechnology Co., Ltd. (license number: SCXK 2020-0001), after a one-week adaptation period, were randomly divided into two groups for modeling. Model group: Rats were intraperitoneally injected with 1.0 mg / kg of Dex daily for four weeks; Control group: Rats were intraperitoneally injected with 0.01 mol / L phosphate buffer (PBS) daily. After successful modeling, the model group was randomly divided into three groups again (n = 6): Model group, IITQ group, and TGIIT group, and the experiment lasted for four weeks. Rats in the control group and the model group were orally administered 0.2 mL of normal saline daily, and rats in the IITQ group and the TGIIT group were orally administered IITQ and TGIIT (30 mg / kg), respectively.
[0078] 2. Experimental process and data collection:
[0079] The food intake and body weight of rats were measured weekly, and their physical signs, hair conditions, and mental states were observed. One day before the end of the experiment, after fasting for 12 hours, the rats were anesthetized by intraperitoneal injection with ketamine (100 mg / kg·bw) and blood was collected. After euthanasia, the body length of the rats was measured, and each tissue was collected and weighed, and stored in a -80 °C refrigerator.
[0080] 3. Histopathological analysis:
[0081] After the fresh muscle tissue was fixed in 10% formalin solution for 48 hours, hematoxylin-eosin (H&E) staining was performed, and the pathological changes such as the morphology, cross-sectional area, and inflammation of muscle fibers were observed through an optical microscope. The results are as Figure 3 shown.
[0082] As can be seen from the figure, the gastrocnemius muscle fibers of the control group rats were arranged regularly, the cytoplasm was red, the cell nuclei were blue-violet, the diameters of the muscle fibers were uniform, and the nuclear structure was clear. The gastrocnemius muscle fibers of the model group were arranged disorderly and loosely, the fiber diameters were uneven, the area was significantly reduced, and the cell gap was increased, indicating that oxidative stress led to mitochondrial dysfunction and lipid peroxidation, thus causing muscle damage. The muscle fibers of the IITQ group and the TGIIT group were arranged tightly, the cell gap was reduced, and the striated muscle edema disappeared, indicating that the two groups could effectively alleviate lipid deposition, mitochondrial dysfunction, and muscle structure damage caused by oxidative stress, and had good application prospects.
[0083] In summary, the present invention uses milk-derived MFGM as a raw material, and through an in vitro method simulating human digestion, membrane separation and purification, mass spectrometry identification combined with molecular docking technology, obtains MFGM small molecule antioxidant peptides that promote skeletal muscle development. In particular, both TGIIT and IITQ can effectively improve the cell survival rate. Among them, TGIIT increased by 2.57% to 8.27%, and IITQ increased by 7.21% to 15.32% (p<0.01). The DPPH free radical scavenging rate of TGIIT and IITQ increased with the increase of concentration. The scavenging rate of IITQ was up to 46.7% at most in the range of 0.5–2 mg / mL, and was significantly higher than that of TGIIT. At the same time, it was proved that the MFGM small molecule antioxidant peptide of the present invention has an application in the preparation of drugs for promoting skeletal muscle development, providing a new direction for solving sarcopenia.
Claims
1. A class of milk source MFGM small molecule antioxidant peptides, characterized in that, Comprising one or more combinations of TGIIT, TSPLG, IITQ, YIC or DTP.
2. The class of milk source MFGM small molecule antioxidant peptides according to claim 1, characterized in that, Comprising TGIIT and IITQ, wherein both TGIIT and IITQ have a high binding energy with keap1.
3. The preparation method of the milk-derived MFGM small molecule antioxidant peptide according to claim 1, characterized in that, Using milk-derived MFGM protein as a raw material, hydrolyzing the MFGM protein by a two-stage enzymatic hydrolysis method, and after membrane filtration, ultrafiltration concentration and vacuum freeze-drying, obtaining MFGM small molecule antioxidant peptides.
4. The preparation method according to claim 3, wherein Comprising the following steps: Step 1, Take milk-derived MFGM protein in a beaker, and under the condition of a constant temperature water bath at 42 - 45 °C, prepare a 5 mg / mL MFGM protein solution. Step 2, Add an equal volume of simulated gastric juice to the MFGM protein solution, adjust the pH of the solution to 3.0 with 1 mol / L HCl, then successively add CaCl2, pepsin and gastric lipase, and hydrolyze in a constant temperature magnetic stirrer at 37 °C for 2 hours, adjust the pH to 6.5 to inactivate pepsin, and obtain a mixed solution A. Step 3, Add an equal volume of simulated intestinal juice to the mixed solution A, adjust the pH to 7.0 with 1 mol / L NaOH solution, then successively add bile salts, CaCl2 solution and pancreatin, and hydrolyze in a constant temperature magnetic stirrer at 37 °C to obtain a mixed solution B. Step 4, After the enzymatic hydrolysis is completed, inactivate the enzymes in the mixed solution B at 95 °C to terminate the digestion process of the MFGM protein, and then perform centrifugation to remove pepsin, pancreatin, undigested protein, insoluble peptides and impurities. Step 5, Use membrane separation technology to separate the MFGM peptide fraction with a molecular weight lower than 3000 Da from the supernatant after centrifugation, concentrate by ultrafiltration, and vacuum freeze-dry to obtain milk-derived MFGM small molecule antioxidant peptides.
5. The preparation method according to claim 4, characterized in that The MFGM protein solution described in Step 1 is obtained by mixing pepsin and MFGM protein and dissolving them in 2.0 mg / mL NaCl, and the mass ratio of pepsin to MFGM protein is 13:
1.
6. The preparation method according to claim 4, wherein The rotation speed of centrifugation in Step 4 is 10000 rpm.
7. The preparation method according to claim 4, characterized in that, The temperature of vacuum freeze-drying in Step 5 is (-40 °C) - (-10 °C).
8. Application of the milk-derived MFGM small molecule antioxidant peptides prepared by the preparation method according to Claim 4 in the preparation of drugs for promoting skeletal muscle development.
9. Application of the milk-derived MFGM small molecule antioxidant peptides prepared by the preparation method according to Claim 4 in the preparation of nutrients.