Lactoprotein zymolyte with peptide chain absolute net charge density ridge-shaped region distribution and application of lactoprotein zymolyte in anti-muscle attenuation activity

Abstract

The invention discloses lactoprotein hydrolysate with peptide chain absolute net charge density ridge-shaped region distribution and application in muscle attenuation resistance activity, the lactoprotein hydrolysate comprises polypeptide with peptide chain absolute net charge density ridge-shaped region distribution formed by head, middle and tail peptide fragments, the head peptide fragment is j1, the middle peptide fragment is j2, the tail peptide fragment is j3, the middle peptide fragment is j1, and the tail peptide fragment is j2. The charge density of the j1 ranges from-0.0241 e <-1 > / 3 to-0.1224 e <-1 > / 3, the charge density of the j2 ranges from-0.0083 e <-1 > / 3 to-0.1238 e <-1 > / 3, the charge density of the j3 ranges from-0.0472 e <-1 > / 3 to-0.1752 e <-1 > / 3, the charge density of the j1 and the charge density of the j3 are both larger than the charge density of the j2, or the charge density of the j1 and the charge density of the j3 are both smaller than the charge density of the j2. The lactoprotein zymolyte provided by the invention is suitable for preparing products with muscle quality and functional decline characteristics such as prevention or delay of muscle attenuation and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active peptides, in particular to a peptide chain absolute net charge density ridge region distribution of milk protein enzymatic hydrolysate and application in anti-muscle attenuation activity. BACKGROUND

[0002] Sarcopenia, simply referred to as muscle deficiency, is a metabolic disease that is accompanied by the characteristics of aging, which is prone to cause body dysfunction and increase the risk of falling, disability and death. The intracellular mass control mechanism determined by protein homeostasis balance is a target of skeletal muscle dysfunction in sarcopenia and has attracted much attention. Protein homeostasis balance is crucial for the integrity of muscle fiber mass and function, and the number, type and function of skeletal muscle cells are the result of competition between the rates of muscle protein synthesis metabolism (MPS) and catabolism (MPB).

[0003] As a disease related to the gradual decline of muscle mass and function with age, its occurrence is closely related to lipid accumulation and inflammatory infiltration. With age, adipose tissue gradually deposits in muscle (referred to as intramuscular fat), causing muscle fibers to be replaced by adipocytes, weakening the contraction function of muscle, and excessive lipid accumulation induces lipotoxicity, damaging the mitochondrial function of muscle cells and reducing energy metabolism efficiency. In addition, chronic low-grade inflammation plays an important role in muscle attenuation-related disorders, and the levels of pro-inflammatory factors (such as TNF-α, IL-6) are elevated, which directly inhibits muscle protein synthesis and promotes catabolism, and the infiltration of immune cells in muscle tissue further aggravates the inflammatory response. Lipid accumulation and inflammatory infiltration promote each other, forming a vicious cycle, and together accelerate the decline of muscle mass and function. CD36, as a key target for lipid metabolism and inflammation regulation, activates NF-κB through assisting TLR4 in a MyD88-dependent pathway, causing the body to produce an inflammatory response; when cells are stimulated by external stimuli (such as various modified lipoproteins), CD36 induces a signal cascade, causing CD36 / p38 MAPK, CD36 / ERK MAPK, and other signal pathways to be activated, ultimately leading to lipid metabolism disorder, inflammatory response and induction of muscle attenuation-related symptoms, suggesting that regulating the CD36 key target protein can become a key link in regulating muscle attenuation.

[0004] Currently, polypeptides with anti-muscle attenuation activity have been disclosed, such as the peptides with specific sequences disclosed in Chinese patent documents CN202280057794.8 and CN202080070699.2, which can be used to treat muscle atrophy, but the peptide sequences do not exist in food-derived protein sequences, resulting in the problem that the application of these peptide segments is still limited by the source. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a safe milk protein hydrolysate with improved muscle mass and muscle functional decline function of peptide chain absolute net charge density crenel region distribution.

[0006] A milk protein hydrolysate with peptide chain absolute net charge density crenel region distribution, comprising a polypeptide composed of a first segment peptide segment, a middle segment peptide segment and a tail segment peptide segment, wherein the first segment peptide segment is j1, the middle segment peptide segment is j2, and the tail segment peptide segment is j3, the charge density of j1 ranges from -0.0241 e - / Š3 to -0.1224 e - / Š3 , the charge density of j2 ranges from -0.0083 e - / ų to -0.1238 e - / ų, and the charge density of j3 ranges from -0.0472 e - / ų to -0.1752 e - / ų, the charge densities of j1 and j3 are both greater than the charge density of j2, or the charge densities of j1 and j3 are both less than the charge density of j2, and the absolute net charge density of the polypeptide peptide chain is in crenel region distribution through the difference in charge density between j1, j2 and j3.

[0007] According to the overall structure of the peptide chain, the three-segment (j=1, j=2, j=3) division is carried out in a way of equal division according to the molecular length, so as to ensure that each segment can effectively reflect the charge characteristics of different regions. The above-mentioned j=1 segment refers to the first equal-division-length amino acid residue segment in the polypeptide chain, i.e. the first segment peptide segment j1; the above-mentioned j=2 segment refers to the second equal-division-length amino acid residue segment in the polypeptide chain, i.e. the middle segment peptide segment j2; and the j=3 segment refers to the third equal-division-length amino acid residue segment in the polypeptide chain, i.e. the tail segment peptide segment j3.

[0008] The branched-chain amino acid (BCAA) is an essential amino acid composed of leucine (Leu), isoleucine (Ile) and valine (Val).

[0009] The net charge density (also referred to as charge density) refers to the algebraic sum of positive and negative charges per unit volume; weak negative net charge density refers to the presence of lower density negative charge in the structure; and strong negative net charge density refers to the presence of higher density negative charge in the structure.

[0010] Preferably, the difference in charge density between j2 / j1 or j3 / j2 ranges from 0.08 to 0.50 or 2 to 15.

[0011] Further, the peptide segments of j1 and j3 are arranged with branched amino acid residues in between or in succession, so that the branched amino acid residues form a natural conformation of chain-like ridge; that is, the structure of the branched amino acid residues is BCAA-nX, nX-BCAA-nX or BCAA-nX-BCAA-nX; wherein X is any of the 20 amino acids, and n is 0-4.

[0012] Further, the branched amino acid residues form a natural conformation of chain-like ridge, and specifically bind to the B-chain key hydrophobic center (ILE598-PRO744) of the key target protein CD36 of muscle atrophy syndrome through hydrophobic interaction; the average hydrophobic interaction frequency of a single peptide ligand with the ligand amino acid is 6.4 times per peptide-12.5 times per peptide, the hydrophobic interaction contact area is 43.26 Å2-617.83 Å2, and the ligand hydrophobic surface coverage is 48.93%-69.02%. Among the known specific ligand-protein ligand binders, the specific ligand hydrophobic surface coverage is usually 30%-70% (too low may be weakly bound, and too high may have poor solubility). The known hydrophobic interaction frequency reaches more than 5 times, and the known protein-ligand hydrophobic interaction area is in the range of 50 Å2-500 Å2, which is more likely to reflect specific binding. The characteristics of the enzyme hydrolysate in the present application include the known specific interaction frequency range.

[0013] Further, based on the identifier P02754 of the beta-lactoglobulin in the UniProt database, the characteristic peptide segments of the polypeptide (highly abundant characteristic peptide segments detected, accounting for 37.32% of the characteristic peptide segments) are from the 17th-21st, 92nd-98th, 111th-118th, 153rd-161st and 163rd-170th amino acids of the beta-lactoglobulin. Therefore, the polypeptide is mapped to the source protein, and the beta-lactoglobulin accounts for >50% of the source protein.

[0014] Still further, the beta-lactoglobulin is derived from ruminant milk, preferably one or more of cow milk, sheep milk, camel milk, donkey milk, and horse milk.

[0015] The preparation method of the above-mentioned milk protein enzyme hydrolysate comprises: Mixing the pretreated raw material containing milk protein with distilled water to adjust the pH value; Raising the temperature to the enzyme hydrolysis temperature, and adding the first protease and the second protease in two steps respectively to perform enzyme hydrolysis, and then inactivating the enzyme to obtain an enzyme hydrolysate; Centrifuging and membrane-filtering the enzyme hydrolysate to obtain a preliminary enzyme hydrolysate containing the target functional active peptide.

[0016] The adjusted pH value only needs to be suitable for the pH value required for the enzyme hydrolysis of the first protease and the second protease, for example, the pH value is adjusted to about 6-8.5.

[0017] Furthermore, the first protease includes, but is not limited to, serine proteases, plant proteases, animal proteolytic enzymes, Bacillus licheniformis proteases, and Bacillus thermophilus proteases; the second protease includes, but is not limited to, Bacillus subtilis proteases, neutral complex proteases, serine complex proteases, trypsin, alkaline proteases, streptomycin proteases, papain, and Aspergillus niger fermentation proteases. Among them, plant proteases include, but are not limited to, bromelain and papain.

[0018] Furthermore, the sources of the raw materials containing milk protein include all edible dairy products such as cow's milk, goat's milk, camel's milk, human milk, donkey milk, and horse milk.

[0019] Furthermore, the distilled water is mixed with the milk protein in the raw material containing milk protein at a ratio of (10-20):1; And / or, the pH is adjusted to 6.0-8.5; And / or, the enzymatic hydrolysis temperature is 52-58℃; And / or, the total enzyme-to-bottom ratio in the enzymatic hydrolysis process is 1:(20-100), and the total enzymatic hydrolysis lasts for 4-6 hours.

[0020] The enzyme-to-substrate ratio refers to the mass ratio (w / w) between the enzyme and the substrate. In this invention, the substrate is measured by weight of milk protein (skimmed) powder.

[0021] The use of the above-mentioned lactase hydrolysate or the lactase hydrolysate prepared by the above-mentioned preparation method in the preparation of products with anti-muscle mass loss and functional decline effects.

[0022] The dosage forms of the products in this invention are powder, tablets, capsules, oral liquids, and injections.

[0023] The lactase hydrolysate of this invention can also be compounded with other food bases to prepare dietary supplements with synergistic effects.

[0024] The lactase hydrolysate synergistically treats muscle mass loss and muscle functional decline by regulating the reprogramming of muscle mitochondrial lipid metabolism and improving the balance of muscle protein metabolism.

[0025] The muscle mass loss and functional impairment mentioned include, but are not limited to, sarcopenia syndrome, Duchenne muscular dystrophy, and other muscle loss characteristics characterized by decreased muscle mass and muscle functional decline. The functional active peptides can effectively inhibit muscle mass and functional decline, thereby slowing down its progression.

[0026] The technical solution of this invention has the following advantages: 1. This invention provides a lactase hydrolysate with a ridged region distribution of absolute net charge density of the peptide chain, a novel functional bioactive peptide derived from ruminant milk and its hydrolysate. The bioactive peptide has a specific chain-like ridged charge density conformation and can interact with CD36, a core target protein for lipid metabolism and inflammation regulation related to sarcopenia, through hydrophobic interactions. It is suitable for preparing products that prevent or delay muscle loss and other characteristics of muscle mass and functional decline.

[0027] 2. The lactase hydrolysate provided by this invention, whose peptide sequence is derived from whey protein, is prepared through a specific enzymatic hydrolysis process. Peptides obtained from whey protein exhibit unique advantages in the treatment of sarcopenia. First, whey protein is widely available, easily accessible, and has relatively low production costs, which facilitates large-scale production and widespread application. Second, whey protein peptides have good safety and digestibility, maintaining stability and bioactivity in the human body, thereby reducing potential safety risks. Furthermore, studies have shown that anti-sarcopenia peptides derived from whey protein mainly include whey protein hydrolysate (WPH), soluble whey protein hydrolysate (WPS), whey protein isolate (WPI) digestive subsets (ALPM, GDLE, VGIN, and AVEGPK), and angiopoietin RNase5 from bovine milk protein. These peptides primarily promote muscle protein synthesis and inhibit its degradation by activating the PI3K / Akt / mTOR and MAPK pathways, and inhibiting the ubiquitin-proteasome system, AMPK pathway, and autophagy pathway, thus playing a key role in improving sarcopenia. A study on the effects of 35g of whey protein hydrolysate (WPH) supplementation over 26 weeks on women over 60 years of age showed that, compared with the control group, WPH supplementation resulted in a reduction in the 10-minute walk speed test and increases in knee extension, chest press, and total strength. Compared with pre-intervention levels, the WPH intervention group showed a significant reduction in the time required to rise from a seated position and a significant increase in the degree of posterior arm curl. Furthermore, compared with maltodextrin placebo, the WPH intervention group showed a significant increase in the percentage of skeletal muscle mass. Therefore, milk-derived bioactive peptides, especially characteristic milk protein peptides, hold promise as a more effective novel nutritional supplement for treating sarcopenia. In summary, compared with existing peptide intervention methods, the milk protein peptides and their enzymatic hydrolysates provided by this invention have higher bioactivity, lower toxicity, and better bioavailability, and are expected to represent a new breakthrough in the treatment of sarcopenia syndrome.

[0028] 3. This invention extracts milk-derived peptides with a directional chain crested charge density distribution designed with a hydrophobic core of the target protein from milk protein. This not only holds promise for providing new ideas and methods for the treatment of sarcopenia, but may also make them more suitable as potential drugs or nutritional supplements for improving the regulation of muscle loss by optimizing the production process and reducing costs. Attached Figure Description

[0029] 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.

[0030] Figure 1 The effects of the enzymatic hydrolysates of Examples 1-3 and Comparative Examples 1-5 on the body undulation rate of nematodes.

[0031] Figure 2 The effects of the enzymatic hydrolysates of Examples 1-3 and Comparative Examples 1-5 on the amplitude of body fluctuations in nematodes.

[0032] Figure 3 The effects of the enzymatic hydrolysates of Examples 1-3 and Comparative Examples 1-5 on the activity level of nematode body movement.

[0033] Figure 4 The enrichment sites and distribution characteristics of the milk precursor proteins in the peptide mapping results of the lactase hydrolysate in Example 1 are shown.

[0034] Figure 5 These are the sites of peptide enrichment in the precursor protein β-lactoglobulin of the lactase hydrolysate identified in Example 1.

[0035] Figure 6 The N-terminal amino acid distribution characteristics of peptides 2-8 in the peptide profile identification results of the lactase hydrolysate in Example 1.

[0036] Figure 7 The amino acid distribution characteristics of peptides 2-8 in the peptide profile identification results of the lactase hydrolysate in Example 1 are shown.

[0037] Figure 8 The diagram shows the chain crest charge density distribution of peptides in the lactase hydrolysate in the examples. In the diagram, a corresponds to the chain crest charge density distribution of Example 1, b corresponds to the chain crest charge density distribution of Example 2, and c corresponds to the chain crest charge density distribution of Example 3.

[0038] Figure 9 The diagram shows the charge density distribution of peptides in comparative lactase hydrolysates, where a corresponds to the charge density distribution of Comparative Example 1 and b corresponds to the charge density distribution of Comparative Example 5.

[0039] Figure 10 This is a schematic diagram of the first, middle, and last segments (segment 1, segment 2, and segment 3) of the characteristic peptide segment IRLSNPT in the lactase hydrolysate of Example 1.

[0040] Figure 11This is the charge density distribution of single atoms of characteristic peptide segments in the lactase hydrolysate of Example 1.

[0041] Figure 12 This is a hydrophobic surface diagram of the lactase hydrolysate and the core hydrophobic amino acids of the CD36 protein B chain in Example 1.

[0042] Figure 13 The distribution of the total hydrophobic interaction frequency between the top 10 peptide sequences with the highest abundance of lactase hydrolysate in Example 1 and the core hydrophobic group of the CD36 protein B chain. Detailed Implementation

[0043] 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.

[0044] The specific types of the first and second proteases that can be used in this invention are as follows: Serine proteases (e.g., food-grade serine proteases with enzyme activity ≥10U / mg produced by Shanghai Zhongfeng Biotechnology, and food-grade serine proteases with enzyme activity ≥20U / mg produced by Xingtai Wanda). Animal protein hydrolytic enzymes (e.g., food-grade animal protein hydrolytic enzymes with an enzyme activity of 100,000 U / g produced by Nanning Pangbo Biotechnology, food-grade animal protein hydrolytic enzymes with an enzyme activity of 100,000 U / g produced by Nantong Yingruida, and food-grade animal protein hydrolytic enzymes with an enzyme activity of 50,000 U / g produced by Shandong Fengtai Biotechnology). Plant proteases (e.g., food-grade papain with an enzyme activity of 100,000 U / g produced by Nanning Pangbo Biotechnology, food-grade fig protease with an enzyme activity of 100,000 U / g produced by Henan Dechen Biotechnology, and food-grade bromelain with an enzyme activity of 50,000 U / g produced by Hebei Jijie Biotechnology). Bacillus subtilis protease (e.g., food-grade Bacillus subtilis protease with an enzyme activity of 20 U / mg produced by Jiangsu Jiujia Biotechnology, and food-grade Bacillus subtilis protease with an enzyme activity of 20 U / mg produced by Shandong Pingju Biotechnology).

[0045] Bacillus licheniformis protease (e.g., food-grade Bacillus licheniformis protease with an enzyme activity of 200 U / mg produced by Hebei Jijie Biotechnology, and food-grade Bacillus licheniformis protease with an enzyme activity of 200 U / mg produced by Anhui Zhonghong Bioengineering).

[0046] 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.

[0047] I. Preparation process of enzymatic hydrolysate Example 1 A lactase hydrolysate with a peptide chain absolute net charge density cristae distribution is prepared as follows: Weigh out defatted milk protein powder and dissolve it in distilled water at a ratio of 1g:15mL. Adjust the pH to 7.0 using sodium carbonate solution (0.1 mol / L). Prepare a 50 mg / mL solution of the first and second proteases and activate it at 37℃ for 15 min before use. A two-step enzymatic hydrolysis scheme was used, with a total enzyme-to-protein ratio (w / w) of 1:25. First, preheat the dissolved milk protein solution to 55℃. In the first step, add the first protease at a w / w ratio of 1:50 and hydrolyze for 4 h. In the second step, add the second protease and activate it at 37℃ before use at a w / w ratio of 1:50 for 2 h. After hydrolysis, inactivate the protease at 85℃ for 30 min, cool, and centrifuge at 25℃, 4000 rpm, for 30 min. Store the hydrolysate at -80℃ for later analysis. Specifically, the first protease is a food-grade serine protease with an enzyme activity of ≥10U / mg produced by Shanghai Zhongfeng Biotechnology, and the second protease is a food-grade Bacillus subtilis protease with an enzyme activity of 20U / mg produced by Jiangsu Jiujia Biotechnology.

[0048] Example 2 Weigh out defatted milk protein powder and dissolve it in distilled water at a ratio of 1 g:10 mL. Adjust the pH to 6.0 using sodium carbonate solution (0.1 mol / L). Prepare a 50 mg / mL solution of the first and second proteases and activate it at 37°C for 15 min before use. A two-step enzymatic hydrolysis scheme was used, with a total enzyme-to-protein ratio (w / w) of 1:20. First, preheat the dissolved milk protein solution to 52°C. In the first step, add the first protease at a w / w ratio of 1:100 and hydrolyze for 6 h. In the second step, add the second protease and activate it at 37°C before use at a w / w ratio of 1:25 and hydrolyze for 3 h. After hydrolysis, inactivate the protease at 85°C for 30 min, cool, and centrifuge at 25°C, 4000 rpm for 30 min. Store the hydrolysate at -80°C for later analysis. Specifically, the first protease is food-grade bromelain with an enzyme activity of 50,000 U / g produced by Hebei Jijie Biotechnology, and the second protease is food-grade Bacillus subtilis protease with an enzyme activity of 20 U / mg produced by Jiangsu Jiujia Biotechnology.

[0049] Example 3 Weigh out defatted milk protein powder and dissolve it in distilled water at a ratio of 1 g:20 mL. Adjust the pH to 8.5 using sodium carbonate solution (0.1 mol / L). Prepare a 50 mg / mL solution of the first and second proteases and activate it at 37°C for 15 min before use. A two-step enzymatic hydrolysis scheme was used, with a total enzyme-to-protein ratio (w / w) of 1:50. First, preheat the dissolved milk protein solution to 58°C. In the first step, add the first protease at a w / w ratio of 1:67 and hydrolyze for 5 h. In the second step, add the second protease and activate it at 37°C before use at a w / w ratio of 1:200 and hydrolyze for 3 h. After hydrolysis, inactivate the protease at 85°C for 30 min, cool, and centrifuge at 25°C, 4000 rpm for 30 min. Store the hydrolysate at -80°C for later analysis. Specifically, the first protease is a food-grade animal protein hydrolase with an enzyme activity of 100,000 U / g produced by Nanning Pangbo Biotechnology, and the second protease is a food-grade Bacillus licheniformis protease with an enzyme activity of 200 U / mg produced by Anhui Zhonghong Bioengineering.

[0050] Comparative Example 1 Weigh out defatted milk protein powder and dissolve it in distilled water at a feed-to-liquid ratio of 1 g:15 mL. Adjust the pH to 7.0 using sodium carbonate solution (0.1 mol / L). Prepare a 50 mg / mL solution of Bacillus subtilis protease from Example 1 and activate it at 37°C for 15 min before use. First, preheat the dissolved milk protein solution to 55°C. Add Bacillus subtilis protease during enzymatic hydrolysis at a feed-to-protein ratio of 1:25 and hydrolysis time of 6 h. After hydrolysis, inactivate the protease at 85°C for 30 min, cool, and centrifuge at 25°C, 4000 rpm, for 30 min. Store the hydrolysate at -80°C for later analysis.

[0051] Comparative Examples 2-5 The only difference from Comparative Example 1 is the enzyme used; the differences between Comparative Examples 1-5 are shown in Table 1 below: Table 1

[0052] All other parameters and conditions are exactly the same as those in Comparative Example 1.

[0053] II. Evaluation of the kinematic capabilities of nematodes (1) Evaluation process 1) Nematode resuscitation and routine culture: Remove the cryopreserved nematodes from the -80 ℃ ultra-low temperature freezer and thaw naturally at room temperature. After complete thawing, transfer them to centrifuge tubes and centrifuge at 1000 r / min for 30 s. Discard the supernatant, mix the remaining liquid, and pipette it onto NGM plates coated with E. coli OP50. Rotate the plate to distribute the nematodes evenly and incubate at 20 ℃ with humidity controlled at 40-60%. Subculture according to the food on the plates and experimental needs.

[0054] 2) Nematode Synchronization Procedure: To ensure experimental accuracy, nematodes need to be synchronized before each batch of experiments to ensure consistent growth stages. Prepare 2-3 culture dishes for culturing nematodes. Once some nematodes enter the oviposition stage, wash all nematodes with M9 buffer and transfer them to centrifuge tubes. Centrifuge at 1000 r / min for 30 s, discard the supernatant, and add the lysate to the centrifuge tubes at a ratio of 5M sodium hydroxide: 5% sodium hypochlorite: M9 buffer of 1:1:2. Invert the tubes to mix thoroughly, ensuring full contact between the nematodes and the lysis buffer. Observe carefully during the process; the lysis endpoint is reached when the liquid changes from turbid to clear. Immediately centrifuge at 1000 r / min for 30 s, discard the supernatant, add M9 buffer, mix well, and centrifuge to wash the eggs. Repeat this process 3 times. Aspirate the precipitate and place it on an NGM plate containing OP50. After approximately 14 hours, the eggs will hatch, yielding synchronized nematodes.

[0055] 3) Nematode intervention method: After the nematode eggs hatch, culture them for about 8 days to the L4 stage, pick the nematodes onto NGM plates containing 5 μg / mL of 5'-fluorodeoxyuridine, and coat the surface of the plates with 100 μL of inactivated OP50 solution. The inactivated OP50 solution contains 4 mg / mL of lactase hydrolysate. Change the nematode plates daily.

[0056] 4) Evaluation of nematode motility: When nematodes were cultured at 20 °C for 5 and 6.5 days, they were picked into M9 buffer and allowed to adapt for 30 seconds. Then, a biological microscope with a camera was used to record a complete tracking video of more than 30 seconds. The motility of the nematodes was quantified using Celset software.

[0057] (2) Evaluation results: like Figure 1 , Figure 2 and Figure 3 As shown, when the intervention concentration in Example 1 was 4 mg / mL, compared with the untreated nematodes, the frequency of body undulations in the nematodes treated with the lactase hydrolysate obtained in Example 1 was accelerated (p<0.01). Figure 1 Increased amplitude of body fluctuations (p<0.01) Figure 2 ), level of physical activity (p<0.01) Figure 3The locomotor abilities of the nematodes in Examples 2-3 were significantly improved, including slower body undulation frequency (p<0.05), reduced body undulation amplitude (p<0.05), and increased locomotor activity (p<0.05).

[0058] like Figure 1 and, Figure 2 and Figure 3 When the intervention concentration of Comparative Example 1 was 4 mg / mL, compared with the untreated nematodes, the frequency of somatic fluctuations in nematodes treated with the lactase hydrolysate obtained in Comparative Example 1 was significantly higher (p>0.05). Figure 1 ), body fluctuation amplitude (p>0.05) Figure 2 ), level of physical activity (p>0.05) Figure 3 There were no significant differences in the frequency (p>0.05), amplitude (p>0.05), and activity level (p>0.05) of the body undulations of nematodes treated with the lactase hydrolysate obtained in Comparative Examples 2-5 compared with the untreated nematodes, and their locomotion ability was not improved.

[0059] III. Peptide Spectroscopy Identification (1) Identification process: Mass spectrometry identification procedures for Examples 1, 2, 3, Comparative Examples 1 and 5: 1) Peptide extraction: Use a 10kD ultrafiltration tube to remove protein and extract peptides; 2) Reductive alkylation: Take 100 μg of sample, dilute to 100 μL with water, accurately pipette 1 μL of 1M DTT (dithiothreitol) solution into the sample to make the final DTT concentration 10 mmol / L, and reduce in a 56℃ water bath for 1 h; then, accurately pipette 2 μL of 1M IAM solution into the sample to make the final IAM (iodoacetamide) concentration 20 mmol / L, and react in the dark at room temperature (25℃) for 40 min; finally, accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L to neutralize unreacted IAA.

[0060] 3) C18 desalting: Desalting with C18 stage-tip and vacuum drying at 45℃.

[0061] 4) Liquid Chromatography Conditions: The pre-packed column had an inner diameter of 150 μm and a length of 170 mm. The packing material was Reprosil-Pur120 C18-AQ 1.9 μm. Mobile phase A was 0.1% (v / v) formic acid aqueous solution, and mobile phase B was 0.1% (v / v) formic acid acetonitrile aqueous solution (acetonitrile was 80%, % refers to v / v). The column temperature was 25ºC, the injection volume was 5 μL, the elution flow rate was 600 nL / min, and the elution time was 66 min. The elution sequence was as follows: 0–2 min, mobile phase B increased from 4% (v / v) to 8% (v / v); 2–45 min, mobile phase B increased from 8% (v / v) to 28% (v / v); 45–55 min, mobile phase B increased from 28% (v / v) to 40% (v / v); 55–56 min, mobile phase B increased from 28% (v / v) to 40% (v / v). At 50 min, the mobile phase B was increased from 40% (v / v) to 95% (v / v); from 56 to 66 min, the mobile phase B was maintained at 95%.

[0062] 5) Mass spectrometry conditions: The first-stage mass spectrometry uses detection mode with a scanning range of 100~1500 m / z, a mass spectrometry resolution of 70,000 (m / z 200), AGC target of 3e6, and Maximum IT of 100 ms; the second-stage mass spectrometry uses DIA data acquisition mode with a mass spectrometry resolution of 15,000 (m / z 200), AGC target of 1e5, Maximum IT of 50 ms, TopN of 20, and NCE / steppedNCE of 28.

[0063] 6) Search for peptides and compare them: The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: ① Database: Uniprot_Bos taurus.

[0064] ②Peptide Mass Tolerance (PMT): 20 ppm; ③ Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.02 Da.

[0065] (2) Evaluation results: like Figure 4 As shown, the protein mapping results of characteristic peptides in Example 1 are as follows: 343 peptides (approximately 1 / 3) are derived from common nutrient-supplying secretory milk proteins, and their response intensity accounts for 60% of the total peptides (e.g., Figure 4The peptides are mainly concentrated in nutrient-supplying milk-derived polypeptides such as β-lactoglobulin (50.20%), albumin (2%), lactoferrin (0.75%), β-casein (0.96%), α-lactalbumin (4.3%), β-2-microglobulin (0.5%), α-S1-casein (0.5%), α-S2-casein (0.07%), latex (0.25%), and κ-casein (0.38%). A portion of the composition comes from functional immunoglobulins (2.39%), albumin (2.39%), lactoferrin (0.7%), GlyCAM-1 glycosylation-dependent cell adhesion molecule 1 (1.75%), complement C3 (0.85%), lactoglucosin (0.84%), and α-S2-fetoglobulin (0.28%).

[0066] Protein source analysis showed that most of the peptides detected in Example 1 were derived from beta-lactoglobulin, with a total peptide count of 120 (13.09%), but their total response value accounted for 50.20% of the total sample response value. Figure 5 As shown.

[0067] In addition, through weblogo visualization analysis, the high-frequency amino acids at the N-terminus N1 to N7 of the short peptides (2-8 peptides) in the lactase hydrolysate of Example 1 are mainly hydrophobic amino acids of leucine, proline, isoleucine, alanine, valine, and phenylalanine. Figure 6 Furthermore, the amino acid positioning frequency distribution of short peptides (2-8 peptides) is mainly dominated by valine, phenylalanine, leucine, and proline. Figure 7 Furthermore, peptides containing high hydrophobic and high-density charge (BCAA) at the j=1 and j=3 segments accounted for 97.23% of the total peptide spectrum detection intensity, indicating that BCAA-containing peptides dominate the enzymatic hydrolysate and are mostly distributed at the j=1 and j=3 segments. Therefore, the enzymatic hydrolysate may contain a large number of peptides with chain-like "ridge"-shaped charge density distribution structures.

[0068] IV. Characterization and Evaluation of Peptide Structural Features (1) Evaluation process: 1) Molecular docking of BCAA-containing peptides with CD36 protein: First, the receptor protein CD36 (ID: 5lgd) was downloaded from the PDB database. Water molecules were removed using PyMOL software, and hydrogen atoms and Gasteiger charges were added using AutoDock Tools, converting the result to a PDBQT file. Then, ligand molecules were generated in batches using a script, and energy minimization was performed using an MM94F force field, converting the result to a PDBQT file. Next, the core docking sites were obtained in PyMOL, and the docking box size was set. Finally, molecular docking was performed using AutoDock Vina.

[0069] 2) Extraction of peptide chain charge value: Input the peptide sequence information from a peptide profile file, and use Python to extract the charge values ​​of all atoms in the peptide chain. This step relies on the construction of a molecular model to ensure accurate acquisition of charge data for each atom. The charge values ​​are calculated using Python's `chem` library, specifically by calculating the Gasteiger charge for each atom. Simultaneously, the three-dimensional coordinates of each atom are extracted. The coordinates will be used for subsequent charge analysis and visualization.

[0070] 3) Classification by peptide chain charge characteristics: Based on the overall structure of the peptide chain, it is divided into equal molecular segments to ensure that each segment effectively reflects the charge characteristics of different regions. The divisions are roughly as follows: Figure 10 As shown, different colors represent different segments. Assuming each segment has n1, n2, and n3 atoms, this ensures that subsequent mean calculations are representative and cover the charge characteristics of the entire peptide chain. The mean charge of each segment is calculated; for each segment j (j=1, 2, 3), its mean charge is calculated. : , Where n j q is the number of atoms in the j-th segment. k This is the charge value of each atom within the segment; this step will provide information on the charge distribution of each segment, which helps to understand its relative characteristics in space.

[0071] 4) Calculate the overall average charge of the peptide chain: The charge value of each atom and the average charge value of each segment are displayed using plotting libraries such as Matplotlib (the average charge value in Example 1 is shown below). Figure 11 Visualization helps to intuitively identify and analyze the heterogeneity of charge distribution in peptide sequences. , Where N is the total number of atoms in the peptide chain. This calculation will help to comprehensively assess the charge characteristics of the peptide chain.

[0072] (2) Evaluation results: The charge density distribution of the lactase hydrolysate peptide chains in Examples 1, 2, and 3 is as follows: Figure 8 As shown, segments j1 and j3 of the peptide contain 1 to 5 branched-chain amino acid residues (any one of BCAA, Ile, Leu, and Val). The Ile, Leu, and Val residues in the peptide are arranged in a spaced or continuous pattern (e.g., BCAA-nX, nX-BCAA-nX, or BCAA-nX-BCAA-nX). The Ile, Leu, and Val residues in the polypeptide can spontaneously form a chain-like "ridge"-shaped charge density distribution conformation with a strong charge density measure. In this invention, the specific positioning of BCAA residues in the enzymatic hydrolysate peptide causes a significant characteristic chain-like "ridge"-shaped charge density distribution, while the charge density distribution of the lactase hydrolysate peptides in Comparative Example 1 and Comparative Example 5 does not exhibit the "ridge"-shaped charge density distribution characteristics of the examples. Figure 9 As shown.

[0073] like Figure 8 As shown, the charge densities of j1 and j3 are both greater than the charge density of j2, or the charge densities of j1 and j3 are both less than the charge density of j2. Specifically, in Example 1, the first, middle, and last segments (j1...) j2 j3) The charge density range is [-0.0241 e - / Š3 ~-0.1168 e - / Š3 ] -0.0203 e - / ų~-0.1238 e - / ų] -0.0507 e - / ų~-0.1284 e - / ų], charge density change (j2 / j1) The difference fold between j3 and j2 ranges from 0.3039 to 4.8091. [0.4729~4.3167]; the range of j2 / j1 is 1.01~4.81, or the range of j2 / j1 is 0.30~0.99; the range of j3 / j2 is 1.07~4.32, or the range of j2 / j1 is 0.47~0.99. Example 2: First, middle, and last segments (j1) j2 j3) The charge density range is [-0.0291e - / Š3 ~-0.1224 e - / Š3 ] -0.0083 e- / ų~-0.1233 e - / ų] -0.0563 e - / ų~-0.1752e - / ų], charge density change (j2 / j1) The difference fold between j3 and j2 ranges from 0.0803 to 4.2371. [0.4615~14.3607]; j2 / j1 ranges from 1.01 to 4.24, or j2 / j1 ranges from 0.08 to 0.99; j3 / j2 ranges from 1.05 to 14.37, or j2 / j1 ranges from 0.46 to 0.97. Example 3: First, middle, and last segments (j1) The charge density range of j2 U j3 is [-0.0401 e - / Š3 ~-0.0991 e - / Š3 ] -0.0330 e - / ų~-0.1018 e - / ų] -0.0472 e - / ų~-0.1076 e - / ų], charge density change (j2 / j1) The difference fold between j3 and j2 ranges from 0.4682 to 2.1397. [0.5403~2.2576]; j2 / j1 ranges from 1.01 to 2.14, or j2 / j1 ranges from 0.46 to 0.98; j3 / j2 ranges from 1.04 to 2.26, or j3 / j2 ranges from 0.53 to 0.94. The above symbols " "" indicates parallel. Based on comprehensive data analysis, it is found that the charge density variation factor of most (over 90%) j2 / j1 or j3 / j2 ranges from 0.08 to 0.50 or from 2 to 15.

[0074] Furthermore, in Example 1, the ligand peptide chain with a chain-like "ridge" charge density distribution binds hydrophobically to the target protein CD36, as shown in the following structure. Figure 12 and Figure 13 As shown, the key hydrophobic center (ILE598-PRO744) of the B chain of the critical target protein CD36 interacts with each other via hydrophobic interactions. Figure 13As shown, the horizontal axis represents the key amino acid residues of the key hydrophobic center (ILE598-PRO744) of the B chain of the key target protein CD36, and the vertical axis represents the frequency (in times) of hydrophobic interactions between the top 10 ligand peptides with the ligand target protein in the lactase hydrolysate of Example 1. In Examples 1, 2, and 3, the average frequency of hydrophobic interactions between a single peptide ligand and the ligand amino acid was 12.4, 8.7, and 6.4 times / peptide, respectively, with ligand hydrophobic surface coverage of 69.02%, 56.21%, and 48.93%, respectively. The hydrophobic contact areas between their key residues were [78.92 Ų - 617.83 Ų], respectively. [43.26 Ų - 523.76 Ų] [67.24 Ų - 453.74 Ų]. Currently, it is known that the hydrophobic surface coverage of ligands is typically 30%-70%, and the known frequency of hydrophobic interactions reaches more than 5 times, particularly concentrated in the core region of the binding pocket, accompanied by other types of specific interactions, which are more likely to reflect specific binding. The known protein-ligand hydrophobic interaction area ranges from 50 Ų to 500 Ų. The enzymatic hydrolysate characteristics of this invention include the known interaction area range, indicating that the specific positioning of BCAA residues in the peptide fragments of this invention causes a chain-like "ridge" charge density distribution, which can undergo specific hydrophobic interactions with the target protein, thereby exerting the corresponding biological activity.

[0075] like Figure 9 As shown, the first, middle, and last segments of Comparative Example 1 (j1) The charge density range of j2 U j3 is [-0.0509 e - / Š3 ~-0.0921 e - / Š3 ] -0.0455 e - / ų~-0.0977 e - / ų] -0.0563 e - / ų~-0.1012 e - / ų], charge density change (j2 / j1) The difference fold between j3 and j2 ranges from 0.7492 to 1.3131. [0.7841~1.3494]; Comparative Example 5: First, Middle and Last Segments (j1) j2 j3) The charge density range is [-0.0529 e - / Š3 ~-0.0886 e - / Š3] -0.0490e - / ų~-0.0922 e - / ų] -0.0563 e - / ų~-0.0966 e - / ų], charge density change (j2 / j1) The difference fold between j3 and j2 ranges from 0.7894 to 1.3212. [0.7573~1.3413]; Furthermore, the lactase hydrolysate peptide chains in Comparative Examples 1 and 5 had hydrophobic contact areas of 17.63 Ų to 274.15 Ų, and ligand hydrophobic surface coverage of 35.75%. This indicates that the enzymatic hydrolysate peptides in the comparative examples of this invention do not possess the characteristic chain-like "ridge" charge density distribution, resulting in poor characteristic hydrophobic interaction with the target protein, thereby affecting its corresponding biological activity.

[0076] In summary, the lactase hydrolysate prepared by the method of the present invention can not only improve muscle function, such as increasing hind limb pulling strength, but also directly act on muscle tissue to promote the recovery of muscle quality and function, thereby achieving an anti-muscle loss effect.

[0077] 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 lactoprotein enzymatic hydrolysate having a distribution of regions of absolute net charge density ridges of the peptide chains, characterized in that, The polypeptide comprises a head section peptide segment, a middle section peptide segment and a tail section peptide segment, and the absolute net charge density crenel region distribution of the peptide chain is composed of the polypeptide, wherein the head section peptide segment is j1, the middle section peptide segment is j2, and the tail section peptide segment is j3, the charge density range of j1 is -0.0241 e - / Š3 -0.1224 e - / Š3 , the charge density range of j2 is -0.0083 e - / ų-0.1238 e - / ų, the charge density range of j3 is -0.0472 e - / ų-0.1752 e - / ų, and the charge density of j1 and j3 is greater than the charge density of j2, or the charge density of j1 and j3 is less than the charge density of j2.

2. The milk protein hydrolysate according to claim 1, characterized in that, The peptide segments of j1 and j3 are arranged in a spacer or in series with branched amino acid residues, and the structure of the branched amino acid residues is BCAA-nX, nX-BCAA-nX or BCAA-nX-BCAA-nX; wherein X is any of the 20 amino acids, and n is 0-4.

3. The milk protein hydrolysate according to claim 2, characterized in that, The polypeptide forms a chain-like ridge natural conformation through branched amino acid residues, and is hydrophobically interacted with the B-chain key hydrophobic center of the specific binding muscle attenuation syndrome key target protein CD36; The average hydrophobic interaction frequency of the peptide ligand of the B-chain key hydrophobic center and the ligand amino acid residue is 6-13 times per peptide; Hydrophobic interaction contact area is 40 A 2 - 650 A 2 Average hydrophobic interaction contact area is > 250 A 2 ; The specific binding muscle attenuation syndrome key target protein CD36 is a ligand, and the ligand hydrophobic surface coverage is 40%-80%.

4. The milk protein hydrolysate according to any one of claims 1 to 3, characterized in that, The polypeptide is mapped to the source protein, and the proportion of beta-lactoglobulin in the source protein is >50%; The source of the beta-lactoglobulin is ruminant milk.

5. A process for the preparation of a milk protein hydrolysate according to any one of claims 1 to 4, characterised in that, It comprises: Mixing the pretreated milk protein-containing raw material with distilled water to adjust the pH; Raising the temperature to the enzyme hydrolysis temperature, adding the first protease and the second protease in two steps respectively for enzyme hydrolysis, and then inactivating the enzyme to obtain an enzyme hydrolysis liquid; The enzyme hydrolysis liquid is centrifuged and filtered to obtain a preliminary enzyme hydrolysis product containing the target functional active peptide.

6. The preparation method according to claim 5, characterized in that, The first protease comprises one or more of serine protease, plant protease, animal proteolytic enzyme, bacillus licheniformis protease, and thermophilic bacillus protease; and / or, the second protease comprises one or more of bacillus subtilis protease, bacillus licheniformis protease, plant protease, animal proteolytic enzyme, alkaline protease, streptomyces protease, and black koji fermentation protease.

7. The preparation method according to claim 5, characterized in that, The source of the milk protein-containing raw material comprises one or more of cow milk, goat milk, camel milk, donkey milk, and horse milk.

8. The preparation method according to claim 5, characterized in that, The distilled water is mixed with the milk protein in the milk protein-containing raw material at a ratio of (10-20):1; And / or, the pH is adjusted to 6.0-8.5; And / or, the enzyme hydrolysis temperature is 52-58℃; And / or, during the enzyme hydrolysis, the total enzyme substrate ratio is 1:(20-100), and the enzyme hydrolysis is performed for 4-6 hours.

9. Use of the milk protein hydrolysate of any one of claims 1-4 or prepared by the preparation method of any one of claims 5-8 in the preparation of a product for resisting muscle mass loss and / or functional attenuation efficacy.

10. Use according to claim 9, characterized in that, The dosage form of the product is powder, tablet, capsule, oral liquid, injection; And / or, the milk protein hydrolysate is used in combination to synergistically treat muscle mass loss and muscle functional attenuation dysfunction by regulating muscle mitochondrial lipid metabolism reprogramming and improving muscle protein metabolism homeostasis balance.

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