Preparation method and application of corn peptide LQQQLL for promoting skeletal muscle protein synthesis

Abstract

The invention provides a preparation method and application of a corn peptide LQQQLL for promoting synthesis of skeletal muscle protein, and the preparation method comprises the following steps: pretreating corn protein to obtain a corn protein solution, firstly adding papain, then adding alkaline protease, and carrying out double-enzyme segmented hydrolysis to prepare the corn peptide LQQQLL. The corn peptide prepared in the invention has the efficacy of promoting synthesis of skeletal muscle protein, and can be used for preparing the anti-skeletal muscle attenuation medicine for treating skeletal muscle attenuation syndrome. The corn peptide LQQQLL disclosed by the invention is convenient to synthesize, can be industrially produced, and has a good application prospect in the fields of medicines, health-care products and the like.

Description

Technical Field

[0001] The invention belongs to the field of bioactive peptides, and specifically relates to a preparation method and application of a corn peptide LQQQLL for promoting skeletal muscle protein synthesis. Background Art

[0002] Sarcopenia is a disease that gradually worsens and affects skeletal muscles throughout the body. It not only leads to a significant decrease in skeletal muscle mass and strength, but is also closely related to a series of adverse health outcomes, including increased risk of falls, increased susceptibility to fractures, gradual loss of physical function, and may even lead to death. The drug treatment strategy for sarcopenia mainly focuses on two aspects: one is to supplement myosin synthesis hormones to stimulate muscle growth, and the other is to regulate the balance of protein metabolism in the body to maintain muscle health. Among them, supplementation of testosterone and growth hormone is the main means. However, these treatments have many adverse reactions, so further in-depth research and exploration are still needed to seek safer and more effective treatment strategies. Existing research evidence shows that BCAAs (especially leucine) and glutamine (Gln) have the ability to directly activate the signaling pathways related to skeletal muscle protein synthesis. This effect is to remind by strengthening the initiation signal of protein translation, thereby promoting the protein synthesis process. Although amino acid supplementation has a certain promoting effect on skeletal muscle protein synthesis, amino acid supplementation alone cannot meet all the substrates required for skeletal muscle protein synthesis, and therefore cannot effectively and significantly improve the quality of skeletal muscle. Therefore, in order to promote the synthesis of skeletal muscle protein and improve its quality, it is a wise move to choose food-derived protein rich in BCAAs and / or Gln. Corn gluten meal (corn yellow meal) is a byproduct of corn processing. It is rich in protein, but its water solubility is poor and the amino acid distribution is uneven, so its application range is limited. However, it contains a lot of BCAAs and Gln. Anti-nutritional factors that are difficult to remove can be passivated by exogenous protease hydrolysis, and small peptides have the advantage of being easily absorbed, thus making up for the shortcomings of plant protein; at the same time, it can effectively replace high-quality animal protein and realize the high-value utilization of plant protein. Summary of the invention

[0003] Technical problem to be solved: The purpose of the present invention is to provide a new use of a bioactive peptide in combating skeletal muscle attenuation syndrome.

[0004] Technical solution: A corn peptide LQQQLL that promotes skeletal muscle protein synthesis, wherein the amino acid sequence of the corn peptide LQQQLL is Leu-Glu-Glu-Glu-Leu-Leu. A method for preparing corn peptide LQQQLL for promoting skeletal muscle protein synthesis comprises the following steps: S1. Pretreatment: Add corn protein powder to an appropriate amount of water, stir thoroughly to dissolve it completely, heat it in boiling water for 15 minutes, and cool it to room temperature to obtain a corn protein solution; S2. First enzymatic hydrolysis: adjust the pH of the corn protein solution to 5-7, add papain, stir and hydrolyze in a constant temperature water bath, heat to 100°C and keep for 15 minutes to inactivate the enzyme, and obtain the first enzymatic hydrolyzate; S3. Secondary enzymolysis: the pH of the first enzymolysis solution was adjusted to 9-11, alkaline protease was added, and the enzymolysis was stirred in a constant temperature water bath, and the solution was heated to 100°C and kept for 15 minutes to inactivate the enzyme, thereby obtaining a secondary enzymolysis solution; S4. Preparation of corn peptide LQQQLL: Centrifuge the secondary enzymatic hydrolyzate and take the supernatant to obtain the corn peptide LQQQLL. Furthermore, the concentration of the corn protein solution in step S1 is 1.5-3.5%. Furthermore, in step S2, the amount of papain added is 2-10%, and the enzymolysis time is 0.5-2.5h. Furthermore, in step S3, the concentration of alkaline protease is 1-5%, and the enzymolysis time is 0.5-2.5h. Furthermore, a corn peptide LQQQLL for promoting skeletal muscle protein synthesis is used in the preparation of drugs and health products for promoting skeletal muscle protein synthesis. Furthermore, the drug also contains a pharmaceutically acceptable carrier. Furthermore, the application includes anti-skeletal muscle attenuation. Furthermore, the health care product includes a functional fermented dairy product. Furthermore, a corn peptide LQQQLL that promotes skeletal muscle protein synthesis is used in the preparation of materials that promote bone synthesis and calcium absorption. Beneficial effects: 1. The corn peptides in the present invention have a significant promoting effect on the proliferation of C2C12 cells, and can also significantly improve the aging oxidative damage of C2C12 cells induced by D-galactose, among which LQQQLL has the best proliferation promoting effect; 2. The corn peptide LQQQLL provided by the present invention can not only increase the relative content of type II muscle fibers suitable for fast movement, but also has significant positive effects. It can effectively alleviate the muscle attenuation phenomenon of mice during the aging process and help restore their muscle function to normal levels. It can also improve the muscle mass and muscle fiber morphology of mice with skeletal muscle attenuation and restore them to normal levels, which is beneficial to the improvement of the mice's athletic ability. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1The effects of different enzymatic hydrolysis sequences, substrate concentrations, papain addition and enzymatic hydrolysis time, as well as alkaline protease addition and enzymatic hydrolysis time on the scavenging rate of DPPH free radicals in the hydrolyzate, where A is the enzymatic hydrolysis sequence; B is the substrate concentration; C is the papain addition; D is the papain enzymatic hydrolysis time; E is the alkaline protease addition; and F is the alkaline protease enzymatic hydrolysis time. Figure 2 The effect of corn peptide on the proliferation activity of C2C12 cells induced by 0.32 mol / L D-galactose, wherein A is QQPIVGGA; B is QYQLPSY; C is LQQQLL; D is LQQQQL; E is QQRQQLLNPL; F is NPLAV; Figure 3 The effect of corn peptide on the proliferation activity of C2C12 cells induced by 0.16 mol / L D-galactose, wherein A is QQPIVGGA; B is QYQLPSY; C is LQQQLL; D is LQQQQL; E is QQRQQLLNPL; F is NPLAV; Figure 4 This is the distribution map of type I and type II muscle fibers in the gastrocnemius muscle tissue of mice; Figure 5 Distribution ratio of type I and type II muscle fibers in mouse gastrocnemius muscle tissue. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 Preparation of corn peptide LQQQLL (1) Computer virtual enzymatic hydrolysis First, the sequences of three kinds of corn proteins were searched in the UniProt database, including Zein-alpha (UniProt ID: Q5TJC1), Zein-beta (UniProt ID: A0A3L6E6C9), and Glutenin (UniProt ID: A0A0D4WUF9). Four common commercial enzymes, trypsin (Try), pepsin (Pep), papain (Pap), and alkaline protease (Alk), were selected, and then the "Profiles of potential biological activity" module and "enzyme action" in the BIOPEP database were used to simulate enzymatic hydrolysis of the selected three protein amino acid sequences. The results are shown in Table 1. The virtual enzymatic hydrolysis results of different combinations were recorded, and the release frequency AE values ​​generated by enzymatic hydrolysis were statistically analyzed. The two enzymes most suitable for hydrolyzing corn proteins were papain and alkaline protease. Table 1 BIOPEP virtual enzymolysis results BIOPEP virtual enzymolysis results (2) Screening of the order of adding two enzymes The double enzyme segmented hydrolysis process was used for enzymatic hydrolysis. The double enzyme addition method was set as follows: first add papain and then add alkaline protease, or first add alkaline protease and then add papain. The enzyme amount was 5%. At the respective optimal enzymatic hydrolysis temperature and pH value, the DPPH free radical scavenging rate of the enzymatic hydrolyzate was determined after 2 hours of enzymatic hydrolysis. Figure 1 A. The appropriate enzymatic hydrolysis order can be selected as adding papain first and then alkaline protease. Among them, the determination method of DPPH free radical scavenging rate is as follows: ① Use anhydrous ethanol as solvent to prepare 1×10 -4 mol / L DPPH solution, store the prepared solvent away from light; ② Mix 2 mL of enzymatic hydrolysate and 2 mL of DPPH solution in the same test tube and shake well. The mixed solution needs to react for 30 minutes under light-proof conditions to obtain a mixed solution; ③ Determine the absorbance of the mixed solution at a wavelength of 517nm and record it as A i For comparison, the absorbance A0 of 2 mL DPPH solution mixed with 2 mL anhydrous ethanol and the absorbance A of 2 mL sample solution mixed with 2 mL anhydrous ethanol were measured. j , the calculation formula is as follows: (3) Single factor test of double enzyme hydrolysis of corn protein 1. Substrate concentration The enzyme-substrate ratio (papain 8% and alkaline protease 2%) was fixed, the enzymatic hydrolysis time of papain and alkaline protease was 2h, and corn proteinase hydrolysis reaction was carried out at different substrate concentrations (1.5%, 2%, 2.5%, 3%, 3.5%) at the optimal enzymatic hydrolysis temperature and pH value of the two enzymes to investigate the effect of different substrate concentrations on the DPPH free radical scavenging rate of corn peptides. Figure 1 As shown in B, the optimal substrate concentration was finally determined to be 2.5%. 2. Papain Addition Amount The substrate concentration was fixed at 2.5%, the enzymatic hydrolysis time of papain and alkaline protease was 2h, the alkaline protease addition amount was 2%, and the corn proteinase hydrolysis reaction was carried out with different papain addition amounts (2%, 4%, 6%, 8%, 10%) at the optimal enzymatic hydrolysis temperature and pH value of the two enzymes to investigate the effect of different papain addition amounts on the DPPH free radical scavenging rate of corn peptides. Figure 1 As shown in C, the optimal addition amount of papain was finally determined to be 8%. 3. Papain enzymatic hydrolysis time The substrate concentration was fixed at 2.5%, the amount of papain added was 8%, the amount of alkaline protease added was 2%, and the alkaline protease enzymolysis time was kept constant at 2h. At the optimal enzymolysis temperature and pH value of the two enzymes, corn proteinase enzymolysis reactions were carried out with different papain enzymolysis times (0.5h, 1h, 1.5h, 2h, 2.5h) to investigate the effect of different papain enzymolysis times on the DPPH free radical scavenging rate of corn peptides. Figure 1 As shown in D, the optimal enzymatic hydrolysis time of papain was finally determined to be 2h. 4. Amount of alkaline protease added The substrate concentration was fixed at 2.5%, the amount of papain added was 8%, the enzymatic hydrolysis time of papain and alkaline protease was 2h, and the corn proteinase hydrolysis reaction was carried out at different alkaline protease addition amounts (1%, 2%, 3%, 4%, 5%) at the optimal enzymatic hydrolysis temperature and pH value of the two enzymes to investigate the effect of different alkaline protease addition amounts on the DPPH free radical scavenging rate of corn peptides. Figure 1 As shown in Figure E, the optimal addition amount of alkaline protease was finally determined to be 2%. 5. Alkaline protease enzymatic hydrolysis time The substrate concentration was fixed at 2.5%, the amount of papain added was 8%, the papain enzymatic hydrolysis time was 2h, and the amount of alkaline protease added was unchanged at 2%. At the optimal enzymatic hydrolysis temperature and pH value of the two enzymes, corn proteinase hydrolysis reaction was carried out with different alkaline protease enzymatic hydrolysis times (0.5h, 1h, 1.5h, 2h, 2.5h) to investigate the effect of different alkaline protease enzymatic hydrolysis times on the DPPH free radical scavenging rate of corn peptides. Figure 1 As shown in Figure F, the optimal enzymatic hydrolysis time of alkaline protease was determined to be 2h. Structural identification and mass spectrometry analysis of corn peptides (1) Preparation of test and reference solutions Use ultrapure water to dissolve the freeze-dried corn peptide powder and reference substance respectively, and filter through a filter membrane to remove possible impurities; (2) Chromatographic analysis conditions Chromatographic column: Accucore C-18 (2.6 μm, 100×2.1 mm), mobile phase: A: 0.1% formic acid-acetonitrile, B: 0.1% formic acid aqueous solution, injection concentration: 1 mg / mL, column temperature T=45°C, flow rate 0.2 mL / min, injection volume 5 μL, wavelength 214 / 280 nm, gradient elution conditions: 0-45 min: 1% A+99% B; 45-50 min: 30% A+70% B; 50-52 min: 90% A+10% B; 52-55 min: 90% A+10% B; 55-60 min: 1% A+99% B; (3) Mass spectrometry analysis conditions Ion Source:ESI+MS,Spray Voltage:3800V,Max Spray Current:100.00,SheathGas:40.00,Aux Gas:10.00,Aux gas Heater Temp:150.00,S-Lens RF Level:60.00,CapillaryTemperature:275℃; (4) Mass spectrometry analysis The reagents used for mass spectrometry were all Fisher reagents, and the data were collected and analyzed using the software (Thermo Fisher Proteome Discoverer 2.4) UniProt database. As shown in Table 2, 57 corn peptides were identified in the corn protein hydrolysate, indicating that Zein-alpha and Zein-beta were well hydrolyzed, while the hydrolysis effect of gluten was poor, which may be due to the fact that the content ratio of zein (68%) and gluten (22%) in corn protein powder is 3:1. The structure of the filtered corn peptide solution was identified using high performance liquid chromatography-tandem mass spectrometry (UPLC-Q-Extractive Orbitrap MS) to obtain its precise amino acid sequence information. Table 2 Sequences of corn peptides identified by UPLC-Q-Extractive Orbitrap MS Molecular docking and virtual screening (1) Preparation of receptor molecules The peptide structure was constructed in SYBYL-X 2.0 software, and after energy minimization, hydrogen was added, charges were calculated, and atom types were added, and the output files were saved in mol2 format; (2) Selection and preparation of ligand molecules mTORC1 protein: mTOR protein is a multi-domain protein, which contains two complexes, mTORC1 and mTORC2. The mTORC1 signaling pathway plays a key role in skeletal muscle protein synthesis. When cells receive growth signals, mTORC1 is activated, which in turn promotes the expression and translation of protein synthesis-related genes, and ultimately increases the content of skeletal muscle protein. Therefore, the mTORC1 signaling pathway is of great significance for maintaining and enhancing skeletal muscle strength and function; (3) Molecular docking The Surflex-dock module was used to screen out the best corn peptide ligands and the receptor structure of macromolecular proteins, and the automatic search mode was used to generate the binding pocket. The docking mode was then set to Surflex-Dock (SFXC), and the corn peptide ligands were docked with the two macromolecular proteins. The Total-Score and other parameter values ​​of the corn ligands and the two macromolecular proteins were analyzed to select the best corn peptide ligands, as shown in Table 3. The Total Scores of mTORC1 and FOXO of the four corn peptides QQPIVGGA, QYQLPSY, LQQQLL, and LQQQQL were 22.0787, 20.9057, 19.541, and 19.4094, respectively, with the highest total scores. NPLAV, QQRQQLLNPL have lower affinity with mTORC1 and FOXO. Table 3 Scoring of the docking model of corn peptides and mTORC1· / FOXO Example 2 Corn peptide activity verification (1) Cell recovery First, place the C2C12 cell cryopreservation tube in a 37°C water bath, accelerate the thawing process by shaking, and then quickly transfer it to a centrifuge tube, add complete culture medium, and centrifuge at 1200r / min for 3 minutes; after centrifugation, pour out the supernatant, re-pump the cells with complete culture medium until they are suspended, and finally transfer them to a cell culture bottle, which is then placed in a constant temperature incubator for culture; (2) Cell culture When C2C12 cells are subcultured, first, pour out the old culture medium, wash with PBS, add 1 mL of preheated 37°C trypsin to digest for 30 seconds, aspirate the trypsin with a sterile pipette, and then put the culture bottle into a constant temperature incubator for 30 seconds. Observe under an inverted optical microscope, wait until the cell edges shrink and become round, add 3 mL of complete culture medium to stop digestion, and pipette to a cell suspension. The subculture ratio of this treatment is 1:3; (3) Effect of corn peptides on C2C12 cell proliferation Select C2C12 cells in the logarithmic growth phase to prepare a cell suspension, and dilute the cell suspension to a specific concentration, i.e., 1.5×10 5 cells / mL, inoculated in 96-well plates, 100μL per well, after cell attachment, the supernatant was discarded, and two concentrations of D-galactose, 0.16mol / L and 0.32mol / L, were selected to construct the model for subsequent experiments. The blank group only added culture medium, the control group added cells without D-galactose, the model group added 0.16 or 0.32mol / L D-galactose, and the remaining experimental groups added D-galactose (0.16 or 0.32mol / L) and different concentrations of QQPIVGGA, QYQLPSY, LQQQLL, LQQQQL, QQRQQLLNPL, NPLAV solutions (0.02-0.8mM), and 6 replicates were set up in each group to select the corn peptide with the highest proliferation rate of C2C12 cells. After 24 hours of culture, the old culture medium was removed, and 100 μL of MTT solution (a dye commonly used in cell proliferation and cytotoxicity assays) was added to each well, and the cells were cultured in the incubator for 3-5 hours to allow MTT to react with dehydrogenases in living cells and be reduced to formazan crystals. MTT was aspirated, and 150 μL of dimethyl sulfoxide was added to each well. The cells were reacted in the dark for 5-8 minutes, and the absorbance A570 of each well was measured at 570 nm. The results were repeated 3 times, and the average value was taken and calculated according to the following formula: (4) Effect of corn peptides on the proliferation activity of senescent C2C12 cells induced by D-galactose The MTT method was used to detect the cell proliferation rate after 24 hours of culture to verify the effect of corn peptide on skeletal muscle attenuation. Figure 2 , 3 The results show that corn peptides have a promoting effect on the proliferation of C2C12 cells induced by 0.32mol / L and 0.16mol / LD-galactose, respectively. This is consistent with the results of virtual screening. At the same time, some concentrations of corn peptides can significantly improve the aging oxidative damage of C2C12 cells induced by 0.16 / 0.32mol / LD-galactose, among which LQQQLL has the best proliferation activity on C2C12 cells and C2C12 senescent cells, which verifies that the corn peptide LQQQLL can improve the proliferation activity of senescent cells and has the potential to resist skeletal muscle attenuation. (5) Effect of corn peptides on the distribution of skeletal muscle fiber types in mice After one week of adaptation, the experimental mice were randomly divided into 7 groups, namely normal group (Normal), D-galactose model group (Model), positive group (whey protein isolate / WPI), corn-derived pure peptide group (LQQQLL), corn peptide low-dose group (300 mg / kg / d.bw) and corn peptide high-dose group (600 mg / kg / d.bw). The experimental period was 8 weeks. The specific grouping and dosing are shown in Table 4. Muscle fibers are the microscopic units of muscle tissue, and muscle fibers are generally divided into type I and type II. Type I is rich in mitochondria and capillaries, resistant to fatigue, and suitable for slow exercise; type II is rich in myofibrils, has strong anaerobic respiration capacity, and is more suitable for fast and intense exercise. Figure 4 It can be seen that after Masson staining, the type I muscle fibers of the mouse gastrocnemius muscle tissue sections appear blue, while the type II muscle fibers appear red and blue. Figure 4 Type I and type II muscle fibers are interspersed with each other. Figure 4 and 5 As shown in the figure, compared with the normal group, the content of type I fibers in the model group increased significantly (P<0.05), and the content of type II fibers decreased significantly (P<0.05). This may be because D-galactose caused the aging of mice, and the motor ability of mice decreased, which led to the decrease in the relative content of type II fibers suitable for fast exercise in mice, and converted them into type I fibers suitable for slow exercise. Compared with the model group, after oral administration of LQQQLL, the content of type I fibers in mice decreased significantly (P<0.05), and the content of type II fibers increased significantly (P<0.05). This may be because leucine and glutamine can improve the motor ability of animals, so the type II fibers suitable for fast and strong exercise will increase accordingly. After oral administration of WPI and low-dose corn peptides, the content of type I fibers in mice decreased significantly (P<0.05) and the content of type II fibers increased significantly (P<0.05) compared with the model group; the content of type II fibers in mice with high-dose corn peptides was not significantly different from that in the model group (P>0.05). This result shows that WPI and low-dose corn peptides can significantly improve the skeletal muscle attenuation of mice and restore it to normal levels, which is beneficial to the improvement of mice's athletic ability. Compared with the WPI group, there was no significant difference in the content of type I and type II muscle fibers in the LQQQLL group and corn peptide group, which is consistent with the previous results. Table 4 Grouping and treatment of experimental animals The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A corn peptide LQQQLL that promotes skeletal muscle protein synthesis, characterized in that: The amino acid sequence of the corn peptide LQQQLL is Leu-Glu-Glu-Glu-Leu-Leu.

2. The method for preparing a corn peptide LQQQLL that promotes skeletal muscle protein synthesis according to claim 1, characterized in that: The following steps are involved: S1. Pretreatment: Add corn protein powder to an appropriate amount of water, stir thoroughly to dissolve it completely, heat it in boiling water for 15 minutes, and cool it to room temperature to obtain a corn protein solution; S2. First enzymatic hydrolysis: adjust the pH of the corn protein solution to 5-7, add papain, stir and hydrolyze in a constant temperature water bath, heat to 100°C and keep for 15 minutes to inactivate the enzyme, and obtain the first enzymatic hydrolyzate; S3. Secondary enzymolysis: the pH of the first enzymolysis solution was adjusted to 9-11, alkaline protease was added, and the enzymolysis was stirred in a constant temperature water bath, and the solution was heated to 100°C and kept for 15 minutes to inactivate the enzyme, thereby obtaining a secondary enzymolysis solution; S4. Preparation of corn peptide LQQQLL: Centrifuge the secondary enzymatic hydrolyzate and take the supernatant to obtain the corn peptide LQQQLL.

3. The method for preparing a corn peptide LQQQLL that promotes skeletal muscle protein synthesis according to claim 1, characterized in that: The concentration of the zein solution in step S1 is 1.5-3.5%.

4. The method for preparing a corn peptide LQQQLL that promotes skeletal muscle protein synthesis according to claim 1, characterized in that: In step S2, the amount of papain added is 2-10%, and the enzymolysis time is 0.5-2.5h.

5. The method for preparing a corn peptide LQQQLL that promotes skeletal muscle protein synthesis according to claim 1, characterized in that: In step S3, the concentration of alkaline protease is 1-5%, and the enzymolysis time is 0.5-2.5h.

6. Use of the corn peptide LQQQLL for promoting skeletal muscle protein synthesis according to claim 1 in the preparation of drugs and health products for promoting skeletal muscle protein synthesis.

7. The use according to claim 6, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

8. The use according to claim 6 or 7, characterized in that: The applications include combating skeletal muscle weakening.

9. The use according to claim 6, characterized in that: The health care products include functional fermented dairy products.

10. Use of the corn peptide LQQQLL for promoting skeletal muscle protein synthesis according to claim 1 in the preparation of materials for promoting bone synthesis and calcium absorption.

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