Deer bone protein for resisting exercise-induced fatigue as well as preparation method and application of deer bone protein

Abstract

The invention discloses a deer bone protein for resisting exercise-induced fatigue as well as a preparation method and application thereof, relates to the technical field of biology, and aims to provide a deer bone protein with an exercise-induced fatigue resisting effect and a preparation method thereof so as to lay a foundation and a theoretical basis for development and clinical application of the deer bone protein. The method comprises the following steps: 1, obtaining a deer bone alkali extract from deer bone powder by adopting an alkali extraction method; 2, adjusting the isoelectric point of the deer bone alkali extract by using an HCl solution to obtain an alkali extract solution; 3, salting out the alkali extract solution by adopting ammonium sulfate to obtain deer bone crude protein CNBP-C; and 4, purifying the deer bone crude protein CNBP-C by adopting a cellulose ion exchange chromatographic column to obtain the deer bone protein CNBP-C-I. The method is easy and safe to operate, high in extraction rate, low in cost and not prone to protein denaturation. The deer bone protein extracted by the method disclosed by the invention has relatively high antioxidant activity, also has the effect of resisting exercise-induced fatigue, and can effectively solve the problem of utilization of a sika deer by-product deer bone.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a deer bone protein for resisting sports fatigue and a preparation method and application thereof. Background Art

[0002] Deer bones are bones of red deer (Cervus elaphus Linnaeus) or sika deer (Cervus NipponTemminck), animals of the Cervidae family. They are sweet and slightly hot, and non-toxic. The Compendium of Materia Medica records that deer bones have the effects of replenishing internal deficiency, healing broken bones, and strengthening bones and tendons. The Tang Materia Medica records that deer bones can cure asthenia, can be made into wine, cure wind deficiency, and replenish bone marrow.

[0003] In recent years, with the increase in the number of people who exercise regularly, the proportion of people who suffer from sports fatigue has reached 85%. It is estimated that by 2025, the number of people who suffer from sports fatigue will theoretically reach 458 million, because it can cause diseases such as hypertension and diabetes, which are harmful to people's physical and mental health. Chinese medicine relieves sports fatigue from the perspective of warming the kidney and replenishing yang, regulating qi and nourishing blood, which is consistent with the clinical efficacy of deer bones in treating long-term illness, weakness, lack of essence, and anemia.

[0004] In the past few decades, my country has mainly focused on the sales and deep processing of deer by-products such as velvet antlers, deer penises and deer fetuses, and has rarely conducted research and development on deer bones, deer skins and other by-products. Bones are a by-product of deer, with a huge annual output, but most of them are not fully utilized and are only used in the primary processing of bone products such as bone ultrafine powder and fertilizer. In recent years, some scholars have isolated biologically active proteins or polypeptide components from deer bones, and verified their different effects through pharmacological experiments, but there are no reports on the treatment of sports fatigue. Summary of the invention

[0005] The purpose of the present invention is to provide a deer bone protein with anti-exercise fatigue effect and a preparation method thereof, so as to lay a foundation and theoretical basis for the development and clinical application of deer bone protein.

[0006] The deer bone protein for resisting sports fatigue of the present invention comprises four secondary structures, namely, 20.45% of α-helix, 39.89% of β-fold, 15.42% of β-turn and 24.22% of random coil structure. The molecular weight of the deer bone protein is 10.96 kDa.

[0007] The deer bone protein of the present invention includes a high-confidence protein Celaphus_00011443 (Protein ID: A0A212DFL6) having a specific peptide sequence KYEDEINKR.

[0008] The method for preparing the deer bone protein for resisting sports fatigue of the present invention comprises the following steps:

[0009] Step 1: obtaining deer bone alkaline extract from deer bone powder by alkaline extraction method;

[0010] Step 2: adjusting the isoelectric point of the deer bone alkaline extract with HCl solution to obtain an alkaline extract solution;

[0011] Step 3: salting out the alkaline extract solution with ammonium sulfate to obtain deer bone crude protein CNBP-C;

[0012] Step 4: Purify the deer bone crude protein CNBP-C using a cellulose ion exchange chromatography column to obtain deer bone protein CNBP-C-Ⅰ.

[0013] Furthermore, the solid-liquid ratio of the alkaline extraction method in step 1 is 1:20-1:30, the extraction temperature is 35-55° C., and the extraction time is 2-4 hours.

[0014] Preferably, the solid-liquid ratio is 1:25, the extraction temperature is 47° C., and the extraction time is 3.6 h.

[0015] Furthermore, the specific step of adjusting the isoelectric point of the protein with HCl solution in step 2 is: adjusting the concentrated deer bone alkali extract to pH=3.1 with 0.05-0.5 mol / L HCl solution.

[0016] Furthermore, the specific steps of the ammonium sulfate salting out in step three are: adding ammonium sulfate to a solution with a pH value of 3.1 to make its saturation 50%, and then placing it in a 4°C environment for 10-12 hours to obtain a protein suspension; the protein suspension is centrifuged at 3500-5000rpm for 15-30min, the supernatant is retained, and the saturation of the supernatant is adjusted to 80% with ammonium sulfate, and then placed in a 4°C environment for 10-12h, and centrifuged again at 3500-5000rpm for 15-30min, and the precipitate is the deer bone protein precipitate; distilled water is added until the precipitate is completely dissolved, and a 500-1500Da dialysis bag is used for 48-72h dialysis, and deer bone crude protein CNBP-C is obtained after freeze-drying.

[0017] Preferably, the freeze-drying temperature is ≤-50°C and the freeze-drying time is 24-48h.

[0018] Furthermore, the specific steps of purifying deer bone protein with a cellulose ion exchange chromatography column in step 4 are as follows: adding the deer bone crude protein CNBP-C solution to a cellulose ion exchange chromatography column, and eluting in stages with 0.01 mol / L Tris-HCl containing 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl, with a flow rate of 0.5-1.5 mL / min, 4-6 mL per tube, and a total of 100 tubes. The DEAE-52 cellulose chromatographic separation curve is drawn with the number of tubes detected as the abscissa and the absorbance value at 595 nm as the ordinate, and the first peak is recovered and separated. The collected elution component is the deer bone protein CNBP-C-Ⅰ.

[0019] The deer bone crude protein CNBP-C solution is obtained by dissolving deer bone crude protein CNBP-C in Tris-HCl, and the concentration is 15-25 mg / mL.

[0020] The deer bone protein of the present invention is used in preparing medicines or health products for resisting sports fatigue.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a method for preparing deer bone protein for resisting sports fatigue, obtains the chemical structure of deer bone protein and identifies it in combination with proteomics. The present invention utilizes the different solubility of protein at different pH values ​​and salt concentrations, adopts alkaline extraction method through isoelectric point treatment combined with ammonium sulfate precipitation of protein, which is not only simple and safe to operate, but also has high extraction rate, low cost, and is not easy to denature protein.

[0023] The deer bone protein extracted by the present invention has high antioxidant activity and anti-sports fatigue effect, which can effectively solve the utilization of deer bones, a byproduct of sika deer, and provide an experimental basis for anti-sports fatigue. It lays an experimental foundation and theoretical basis for the development and clinical application of deer bone protein, and will also promote the development of the deer bone industry and open up its broader market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the response curve diagram and contour diagram of the extraction process optimized by the response surface method of the present invention;

[0025] Figure 2 This is a line graph of the isoelectric point of deer bone protein determined by the Zeta potential method of the present invention;

[0026] Figure 3 This is the isoelectric point potential diagram of deer bone protein determined by the Zeta potential method of the present invention;

[0027] Figure 4 This is an elution curve diagram of CNBP-C of the present invention separated and purified into protein CNBP-C-Ⅰ by DEAE cellulose ion exchange chromatography column;

[0028] Figure 5 This is a graph showing the analysis results of the amino acid components derived from the ninhydrin column at 570 nm of the present invention;

[0029] Figure 6 This is a graph showing the analysis results of amino acid components derived from ninhydrin column at 440 nm of the present invention;

[0030] Figure 7 Fourier transform infrared spectroscopy analysis of CNBP-C-Ⅰ of the present invention;

[0031] Figure 8 It is the infrared fitting diagram of the secondary structure of CNBP-C-Ⅰ of the present invention;

[0032] Fig. 9 The DPPH free radical scavenging rate test result of CNBP-C-Ⅰ of the present invention;

[0033] Fig.10 The ABTS free radical scavenging rate test result of CNBP-C-Ⅰ of the present invention;

[0034] Fig.11 The hydroxyl radical scavenging rate test result of CNBP-C-Ⅰ of the present invention;

[0035] Fig.12 The molecular weight spectrum of the protein standard determined by HPGFC of the present invention;

[0036] Fig.13 This is a standard curve diagram of the molecular weight of protein standards determined by HPGFC of the present invention;

[0037] Fig.14 The molecular weight spectrum of CNBP-C-Ⅰ determined by HPGFC of the present invention;

[0038] Fig.15 It is the LC-MS total ion current spectrum of the present invention;

[0039] Fig.16 LC-MS / MS spectrum of the specific peptide KYEDEINKR;

[0040] Fig.17 LC-MS / MS spectrum of the specific peptide DYQELMNVK;

[0041] Fig.18 LC-MS / MS spectrum of the specific peptide TAAENEFVTLK;

[0042] Fig.19 LC-MS / MS spectrum of the specific peptide LLRDYQELMNVK;

[0043] Fig. 20 HE staining results of the blank group (Con group) in the anti-exercise fatigue experiment (100×);

[0044] Fig.21 HE staining results of the model group (Mod group) in the anti-exercise fatigue experiment (100×);

[0045] Fig. 22 HE staining results of whey protein group (Pos group) in the anti-exercise fatigue experiment (100×);

[0046] Fig.23 HE staining results of CNBP-C-Ⅰ group in the anti-exercise fatigue experiment (100×);

[0047] Fig.24 The effect of CNBP-C-Ⅰ on the content of GSH-Px in exercise-induced fatigue model mice;

[0048] Fig.25 The effect of CNBP-C-Ⅰ on the MDA content in exercise-induced fatigue model mice;

[0049] Fig.26 The effect of CNBP-C-Ⅰ on the SOD content in exercise-induced fatigue model mice;

[0050] Fig. 27 The effect of CNBP-C-Ⅰ on LG content in exercise-induced fatigue model mice;

[0051] Fig.28 The effect of CNBP-C-Ⅰ on the G content in exercise-induced fatigue model mice. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0053] Embodiment 1:

[0054] The preparation method of deer bone protein in this embodiment comprises the following steps:

[0055] (1) Extraction of deer bone protein

[0056] Weigh 500 g of deer bone powder, add 12.5 L of NaOH solution, the concentration of the NaOH solution is 0.01 mol / L, and the pH value is 8.1; heat to 47° C., stir magnetically for 3.6 hours, centrifuge at room temperature at 3500 rpm for 15 minutes, and retain the supernatant; add 12.5 L of NaOH solution to the precipitate again, heat to 47° C., stir magnetically for 3.6 hours, centrifuge at room temperature at 3500 rpm for 15 minutes, and retain the supernatant; concentrate the supernatant twice using a rotary evaporator to obtain a deer bone alkali extract.

[0057] The response surface method was used to optimize the extraction process of the alkaline extraction method in this embodiment, and the response curve and contour map are shown in Figure 1 The optimal extraction conditions are extraction temperature 47℃, solid-liquid ratio 1:25, and extraction time 3.6h.

[0058] The deer bone alkali extract is adjusted to the protein isoelectric point, i.e., pH=3.1, with a 0.01 mol / L HCl solution, and then ammonium sulfate is added to make its saturation 50%, and then the mixed solution is placed in a 4°C environment for 12 hours to obtain a protein suspension; the protein suspension is centrifuged at 4000 rpm for 20 minutes, the supernatant is retained, and the saturation of the supernatant is adjusted to 80% with ammonium sulfate, and then placed in a 4°C environment for 12 hours, and centrifuged again at 4000 rpm for 20 minutes, and the precipitate is the deer bone protein precipitate; distilled water is added until the precipitate is completely dissolved, and a 1000Da dialysis bag is used for 56 hours for desalination, and then freeze-dried for 24 hours, and the freezing temperature is ≤-50°C to obtain deer bone crude protein CNBP-C.

[0059] Among them, the isoelectric point of deer bone protein was determined by Zeta potential. The method was as follows: weigh an appropriate amount of deer bone crude protein sample CNBP-C and prepare it into 1 mg·mL -1 The samples were prepared into solutions with pH values ​​of 4.5, 4.8, 5.1, 5.4, 5.7, 6, and 6.3 using 0.1 mol / L HCl. The samples were placed in a ZS-90 dynamic light scattering instrument to detect the isoelectric point and potential of the protein. The isoelectric point and particle size of the protein were detected using a nanoparticle size analyzer at room temperature. The isoelectric point of deer bone protein determined by the Zeta potential method is shown in the following figure. Figure 2 As shown, the potential diagram is Figure 3 As shown, according to the data in the figure, the isoelectric point of deer bone crude protein can be determined to be 3.1.

[0060] (2) Separation and purification of CNBP-C column chromatography and preparation of protein CNBP-C-Ⅰ:

[0061] DEAE-52 cellulose ion exchange chromatography column separation and purification of CNBP-C-Ⅰ:

[0062] 1) DEAE-52 cellulose ion exchange resin treatment and column balance

[0063] Weigh 500g of DEAE-52 cellulose ion exchange resin, add sufficient deionized water, stir evenly to allow it to fully swell until the volume no longer changes, remove fine particles and impurities, and vacuum filter. Use 0.5mol·L -1 After stirring and soaking for 1 h, filter and wash with water until neutral. -1 HCl was stirred and soaked for 1 h, then filtered and washed with water until neutral. -1 After stirring and soaking for 1 hour, filter and wash with water until neutral. -1 Soak in Tris-HCl (pH = 5.1) and stir evenly. -1 The column was washed with Tris-HCl (pH = 5.1), and then 0.01 mol·L -1 When Tris-HCl (pH = 5.1) drops to 5 cm from the bottom, pour in DEAE cellulose filler. The filled column should be dense and uniform, without bubbles or stratification. The buffer should be left on the top of the column to cover it. Use 0.01 mol·L -1 The cell was washed with Tris-HCl (pH=5.1) until the pH of the effluent was 5.1, and then equilibrated with the buffer for 12 h.

[0064] 2) Loading and elution

[0065] Use 0.01 mol·L -1 Dissolve 5 g of CNBP-C in Tris-HCl (pH = 5.1) to a protein concentration of 20 mg mL -1 After dissolution, centrifuge at 3500rpm for 15min, take the supernatant and filter it through a 0.45μm filter membrane before loading. Before loading, drain the liquid on the upper layer of the column bed to slightly above the column bed, close the lower end of the chromatography column, slowly and evenly add the sample with a pipette, open the outlet at the lower end, and let the protein solution enter the column bed by gravity. When the protein solution is about to be added, rinse the column wall with 1-2mL of buffer solution. The protein solution is adsorbed for 3h, and then washed with 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mol·L -1 0.01 mol·L of NaCl -1 Tris-HCl (pH = 5.1) was used for step elution, the elution volume was 1.5 times the column volume, and the flow rate was 1 mL min -1 , one tube 5mL, a total of 100 tubes, Coomassie brilliant blue was used to detect the absorbance at 595nm, and the DEAE-52 cellulose chromatographic separation curve was drawn with the number of tubes detected as the horizontal axis and the absorbance value at 595nm as the vertical axis. The results are shown in Figure 4The elution curve of CNBP-C separated and purified by DEAE-52 cellulose ion exchange chromatography column was obtained. According to the curve, the first separated peak was recovered, and the collected elution components were concentrated, dialyzed and desalted for 72 hours, and freeze-dried to obtain deer bone protein CNBP-C-Ⅰ.

[0066] The deer bone protein CNBP-C-Ⅰ obtained in this example was subjected to the following analyses and determinations:

[0067] 1. Analysis of amino acid composition of deer bone protein CNBP-C-Ⅰ

[0068] Weigh 20mg of deer bone protein CNBP-C-Ⅰ sample accurately and add it to the hydrolysis tube, then add 10mL of 6mol / L HCl solution and mix well, and add 3 drops of phenol. Put the hydrolysis tube into the freezer and freeze for 5min, connect it to the vacuum pump's exhaust pipe, evacuate, and then fill it with nitrogen. Repeat 3 times, and seal it while filling it with nitrogen. Put the sealed hydrolysis tube in a 110℃ electric blast thermostat, hydrolyze for 22h, take it out, and cool it to room temperature. Filter the hydrolyzate into a 50mL volumetric flask, accurately draw 1mL of filtrate, dry it under reduced pressure at 50℃, dissolve the dried residue with 1mL of distilled water solution, dry it under reduced pressure again, and finally add 1mL of sodium citrate buffer solution with pH=2.2. After shaking evenly, filter it with a 0.22μm filter membrane and enter the fully automatic amino acid analyzer.

[0069] A Biochrom Na type cation exchange resin column (4.6 mm×20 cm) was used; running time: 57 min; reaction unit temperature: 135° C.; detection wavelengths: 570 nm, 440 nm; the separation procedure is shown in Table 1.

[0070] Table 1 Separation procedure

[0071]

[0072] Figure 5 This is the analysis result of amino acid components derived from ninhydrin column at 570nm. Figure 6 The following is the analysis result of amino acid components derived from ninhydrin column at 570nm. A is 570nm, B is 440nm, and a total of 17 amino acids (Gly, Pro, Glu, Ala, Arg, Asp, Lys, Ser, Leu, Val, Thr, Phe, Ile, Tyr, His, Met, and Cys) were detected. After acid hydrolysis, Asn was converted to Asp, Gln was converted to Glu, Trp was completely destroyed, and Met and Cys were severely oxidized, so they were not shown in the results. The relevant contents are shown in Table 2.

[0073] Table 2 Analysis of amino acid composition of CNBP-C-Ⅰ

[0074]

[0075]

[0076] 2. Functional group analysis and secondary structure prediction of CNBP-C-Ⅰ by Fourier transform infrared spectroscopy

[0077] Weigh an appropriate amount of CNBP-C-Ⅰ sample and mix it with dry potassium bromide in a ratio of 1:200, grind it into powder, take an appropriate amount of powder and press it into thin sheets in a tablet press, and use a Fourier exchange infrared spectrometer for full-band scanning.

[0078] The measurement conditions were set as follows: ambient temperature 25°C, wave number 4000-500cm -1 , resolution 4cm -1 The secondary structure was analyzed using Peakfit V4.12 software. Figure 7 As can be seen from the figure, the protein secondary structure prediction results are shown in Table 3.

[0079] Table 3 Prediction of the secondary structure of CNBP-C-Ⅰ

[0080] α-helix β-sheet β-turn Irregular curl CNBP-C-Ⅰ 20.45％ 39.89％ 15.42％ 24.22％

[0081] 3. Antioxidant Determination

[0082] (1) DPPH free radical scavenging rate

[0083] Dissolve 3.9 mg DPPH in anhydrous ethanol to obtain a solution containing 0.2 mmol·L -1 DPPH ethanol solution and stored in a refrigerator at 4°C. 1 mL of CNBP-C-Ⅰ solution of different concentrations was added to the test tube, and then 0.2 mmol·L -1 1 mL of DPPH anhydrous ethanol solution was mixed with a vortex machine, protected from light for 30 minutes, and the absorbance was measured at 517 nm. After replacing the protein with an equal volume of distilled water, the above experiment was repeated. During this period, vitamin C was used as a positive control to calculate the scavenging rate of CNBP-C-Ⅰ on DPPH free radicals. The calculation formula is as follows, and the results are shown in Fig. 9 At a concentration of 5 mg / mL, the scavenging rate of CNBP-C-Ⅰ on DPPH free radicals reached a maximum of 82%, proving that CNBP-C-Ⅰ has a good scavenging effect on DPPH free radicals and has antioxidant properties.

[0084] DPPH free radical scavenging rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100%

[0085] In the formula, A 样 Indicates the absorbance of the sample to be tested; A 对 A represents the absorbance of Vc in the control group; 空 Represents the absorbance of water in the blank group.

[0086] (2) ABTS free radical scavenging rate

[0087] Accurately weigh 83.2 mg ABTS, dissolve it in distilled water, and then transfer it to a 10 mL volumetric flask to make up the volume. Weigh 1.5 g K2S2O8 and dissolve it in distilled water to make up the volume in a 10 mL volumetric flask. Then mix the ABTS-K2S2O8 mixed solution with a vortex machine and place it away from light for 10 hours. Before the experiment, dilute the ABTS-K2S2O8 mixed solution with 95% anhydrous ethanol, and the absorbance of the solution is required to be 0.7±0.02 at 734 nm to prepare the ABTS working solution.

[0088] Take 0.6mL of CNBP-C-Ⅰ solution of different concentrations from the test tube, then add 2.4mL of ABTS working solution and mix well. After standing at room temperature for 6 minutes, the absorbance at 734nm is measured. Use an equal amount of anhydrous ethanol to replace the sample for absorbance measurement. C As a positive control, the scavenging rate of CNBP-C-Ⅰ on ABTS free radicals was calculated. The calculation formula is shown below and the results are shown in Fig.10 At a concentration of 5 mg / mL, the scavenging rate of CNBP-C-Ⅰ on ABTS free radicals reached a maximum of 71.6%, proving that CNBP-C-Ⅰ has a good scavenging effect on ABTS free radicals and has antioxidant properties.

[0089] ABTS free radical scavenging rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100%

[0090] In the formula, A 样 Indicates the absorbance of the sample to be tested, A 对 represents the absorbance of Vc in the control group, A 空 It represents the absorbance of anhydrous ethanol in the blank group.

[0091] (3) Hydroxyl radical scavenging rate

[0092] Add 1 mL of CNBP-C-Ⅰ solution and 1 mL of 9 mmol·L -1 of salicylic acid solution and 1mL of 9mmol·L -11 mL of 0.03% ferrous sulfate solution was added to the solution, and 1 mL of 0.03% hydrogen peroxide solution was added to the solution. The solution was mixed with a vortex machine, and the solution was placed at room temperature for 30 min. The absorbance was then measured at 510 nm. In the same way, an equal amount of distilled water was used to replace the protein sample, and then hydrogen peroxide was used to replace the distilled water. The absorbance was measured at V C As a positive control, the scavenging rate of CNBP-C-Ⅰ on hydroxyl free radicals was calculated. The calculation formula is as follows. The results are shown in Fig.11 At a concentration of 5 mg / mL, the scavenging rate of CNBP-C-Ⅰ on hydroxyl radicals reached a maximum of 65.4%, proving that CNBP-C-Ⅰ has a good scavenging effect on hydroxyl radicals and has antioxidant properties.

[0093] Hydroxyl radical scavenging rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100%

[0094] In the formula, A 样 Indicates the absorbance of the sample to be tested, A 对 represents the absorbance of Vc in the control group, A 空 Represents the absorbance of water in the blank group.

[0095] 3. Determination of molecular weight of CNBP-C-Ⅰ by HPGFC

[0096] Chromatographic conditions and system suitability test: Chromatographic column: Ultrahydrogel TM (Linear) water-soluble gel column (7.8×300mm); mobile phase: 0.02mol / L Phosphate Buffer (pH=7.25)+0.1mol / L Na2SO4; detection wavelength 220nm; flow rate 1.0mL / min; column temperature: 27℃; injection volume: 20μL.

[0097] Five standard proteins, bovine serum albumin (66.4 kDa), egg white albumin (44.5 kDa), cytochrome C (12.4 kDa), bovine insulin (5.7 kDa), and bacitracin (1.4 kDa), were selected to establish a molecular weight standard curve. The molecular weight spectrum of the protein standard is shown in Fig.12 As shown, black indicates the peak of bovine serum albumin, red indicates the peak of egg white albumin, blue indicates the peak of cytochrome C, green indicates the peak of bovine insulin, and purple indicates the peak of bacitracin. The molecular weight standard curve of protein standard is shown in Fig.13 As shown, the molecular weight spectrum of CNBP-C-Ⅰ is as follows Fig.14 According to the sample peak time, the molecular weight of CNBP-C-Ⅰ was calculated to be 10.96 kDa.

[0098] IV. CNBP-C-Ⅰ protein identification

[0099] Identification by LC-MS / MS mass spectrometry:

[0100] 1) Decolorization of stained gel strips: Cut the protein strips into 1 mm pieces 3 The colloid particles were placed in 2mL EP tubes. 1mL 50% ACN-50% 50mM NH4HCO3 solution was added to decolorize until the blue color of the colloid particles disappeared. 1mL 100% ACN was added until the colloid particles turned white and clumping, and the ACN was discarded and placed to dry at room temperature;

[0101] 2) Reductive alkylation: Use a pipette to accurately transfer 200 μL of 10 mmol / L DTT solution to the sample, reduce in a 56°C water bath for 1 hour, aspirate and discard, use a pipette to accurately transfer 200 μL of 55 mmol / L IAM solution to the sample, react in the dark at room temperature for 1 hour, aspirate and discard, use a pipette to accurately transfer 200 μL of decolorizing solution to the sample, aspirate and discard after vortexing, use a pipette to accurately transfer 1000 μL of 100% ACN to the sample, wait until the colloid particles turn white and shrink to a clumping shape, discard the ACN, and place at room temperature to dry;

[0102] 3) In-gel digestion: Add 50 μL of Trypsin (concentration: 0.025 μg / μL) and 100 μL of Trypsin buffer (50 mM NH4HCO3) to the dried particles, allow them to fully absorb, and then incubate in a 37°C constant temperature incubator for 16 h;

[0103] 4) Peptide extraction: add 200 μL / tube of extraction solution (5% TFA-50% ACN-45% water), and place in a 37°C water bath for 1 hour. Ultrasonicate for 5 minutes, centrifuge for 5 minutes, transfer the extraction solution to another new EP tube, and repeat the extraction once. Combine the extraction solutions, and dry them by vacuum centrifugation at 57°C;

[0104] 5) C18 desalting (Stage-Tip): Activation: Activate the desalting column twice with 300 μL 100% ACN, Equilibrate the desalting column twice with 200 μL 0.1% TFA, Load: Load twice, Desalting: Desalt three times with 200 μL 0.1% TFA, Elution: Elute once with 300 μL 80% ACN-20% 0.1% TFA, and dry under vacuum at 45°C;

[0105] 6) On the machine: analytical column: 100 μm id×180 mm, packing: Reprosil-Pur 120C18-AQ 3 μm; mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid-80% ACN; flow rate: 600 nL / min; analysis time for each component: 66 min; specific chromatographic conditions are shown in Table 4;

[0106] Table 4 Chromatographic conditions

[0107] Time (min) Phase B 0 4％ 2 8％ 35 28％ 55 40％ 56 95％ 66 95％

[0108] 7) Mass spectrometry conditions: The full scan range of the mass spectrometry was 300-1800 m / z, the primary mass spectrometry resolution was set to 70000, AGC was 3e6, and MaximumIT was 100ms; the parent ion with an ion intensity of 20 in the full scan was selected and fragmented using the high energy collision fragmentation (HCD) method for secondary mass spectrometry detection, the secondary mass spectrometry resolution was set to Resolution: 17500, AGC was 1e5, MaximumIT was 50ms, and the peptide fragmentation collision energy was set to 28% to generate mass spectrometry detection raw data (.raw);

[0109] 8) Search conditions: The original mass spectrometry files were searched against the target protein database using MaxQuant (2.4.9.0) with the following search parameters: a) Fixed modifications: Carbamidomethyl (C); b) Variable modifications: Oxidation (M); Acetyl (Protein N-term); c) Enzyme: Trypsin / P; d) Database: Corylus (genus); e) Maximum Missed Cleavages: 2; f) Peptide Mass Tolerance: 20 ppm; g) Fragment Mass Tolerance: 20 ppm; h) Peptide / fragment ion mass values: Monoisotopic; i) Significance threshold: 0.01. The high-confidence protein Celaphus_00011443 (Protein ID: A0A212DFL6) was matched, with a Scroe of 323.1 and an Intensity of 7039600000, and 17 peptides were detected, including 4 specific peptides, as shown in Table 5. The LC-MS total ion current spectrum is shown in Fig.15, Celaphus_00011443 (Protein ID: A0A212DFL6) measured 4 specific peptides LC-MS / MS spectra as shown Figure 16-Figure 19 As shown, Fig.16 This is the LC-MS / MS spectrum of the specific peptide KYEDEINKR. Fig.17 is the LC-MS / MS spectrum of the specific peptide DYQELMNVK. Fig.18 is the LC-MS / MS spectrum of the specific peptide TAAENEFVTLK. Fig.19 This is the LC-MS / MS spectrum of the specific peptide LLRDYQELMNVK.

[0110] Table 5 Information on 17 peptides in A0A212DFL6

[0111]

[0112] 5. Pharmacodynamic evaluation of CNBP-C-Ⅰ against exercise-induced fatigue

[0113] The present invention establishes a sports fatigue mouse model, and evaluates the pharmacodynamics of CNBP-C-Ⅰ against sports fatigue in mice by observing the pathology of mouse gastrocnemius muscle and measuring the levels of GSH-Px, SOD and MDA in mouse serum, the level of LG in liver tissue and the level of MG in gastrocnemius muscle tissue by enzyme-linked immunosorbent assay. By measuring the levels of GSH-Px, SOD and MDA in mouse serum, the level of LG in liver tissue and the level of MG in gastrocnemius muscle tissue, not only the success of establishing the ICR mouse sports fatigue model can be evaluated, but also the therapeutic effect of deer bone protein on sports fatigue can be evaluated.

[0114] Forty SPF male ICR mice weighing (30±2g) were randomly divided into four groups according to body weight after 7 days of adaptive feeding, namely, blank group (Con group), model group (Mod group), whey protein group (Pos group), and CNBP-C-Ⅰ group (n=10); the mice in the same group were distinguished by ear tag method, the mice in the blank group did not exercise, and the mice in the other three groups were modeled by exhaustive swimming test every day for 14 days. The Con group and Mod group were gavaged with normal saline, the Pos group was gavaged with whey protein (0.50g / kg·bw), and the CNBP-C-Ⅰ group was gavaged with CNBP-C-Ⅰ (0.50g / kg·bw) for 14 days. Except for the Con group, the other three groups were modeled while gavage. After the experiment was terminated, the mice were anesthetized with sodium pentobarbital. The injection volume of sodium pentobarbital was determined according to the weight of the mice. After anesthesia, the eyeballs were removed to collect blood, which was placed in an EP tube without heparin. After standing for 2 hours, the blood was rotated at 3000 r / min. -1Centrifuge for 15 minutes and collect the upper serum. After taking the blood from the eyeball, the mice were killed by dislocation of the neck. The right gastrocnemius muscle and liver tissue of the mice were washed with saline to remove the blood stains on the surface. The excess liquid was absorbed by filter paper and placed in dry ice. The serum was used for GSH-Px, MDA, and SOD ELISA, the liver tissue was used for LG ELISA, and the right gastrocnemius muscle was used for MG ELISA. The left gastrocnemius muscle of the mouse was washed with PBS and placed in an EP tube, fixed in 10% paraformaldehyde solution, used for HE staining, and tissue sections were made.

[0115] (1) Pathological observation results of gastrocnemius muscle tissue by HE staining

[0116] The results of CNBP-C-Ⅰ anti-mouse exercise fatigue pathological observation are as follows Figure 20-23 As shown in the figure, it can be seen that compared with the Con and Mod groups, the muscle cell morphology of the Pos group and the CNBP-C-Ⅰ group was more complete and had fewer gaps. This indicates that CNBP-C-Ⅰ can repair the morphology of gastrocnemius muscle cells and reduce cell damage caused by exercise fatigue, proving that CNBP-C-Ⅰ has an anti-exercise fatigue effect.

[0117] (2) The kit was used to measure the levels of GSH-Px, MDA, SOD, LG and MG in the serum, liver tissue and gastrocnemius muscle of ICR mice. The results were as follows: Figure 24-28 As shown. Fig.24 , 25 , 26 is the antioxidant index in the body, Fig. 27 , 28 As the glycogen index, compared with Con, GSH-Px and SOD in Mod group were significantly decreased (P<0.01), and MDA was increased (P<0.05), indicating that the model was successfully established; compared with Con group, GSH-Px and SOD in Pos and CNBP-C-Ⅰ groups were significantly increased (P<0.01), and MDA in CNBP-C-Ⅰ group was significantly decreased (P<0.01); compared with Con group, LG level in Pos group was increased (P<0.05), and MG level was significantly increased (P<0.01), and LG level in CNBP-C-Ⅰ group was significantly increased (P<0.01), and MG level was increased (P<0.05), and the difference was statistically significant. The results showed that at the same dose, CNBP-C-Ⅰ had a better anti-exercise fatigue effect than Pos group.

[0118] Branched-chain amino acids (Ile, Leu, Val) can promote the expression of antioxidant enzymes, increase cellular energy supply, enhance peroxisome and mitochondrial function, and improve antioxidant properties. Leu protects cells from hydrogen peroxide-induced oxidative stress by regulating propionate metabolism, branched-chain amino acid degradation and heat production pathways; Ile improves the antioxidant properties of cells by regulating peroxisomal pathways and propionic acid metabolic pathways; Val reduces oxidative stress and lipid peroxidation by improving the function and number of mitochondria. Moreover, branched-chain amino acids can also reduce the consumption and utilization of muscle glycogen by oxidative energy supply in skeletal muscle. The experimental results show that CNBP-C-Ⅰ has a high proportion of branched-chain amino acids of 18.49%, which can significantly reduce the level of MDA and increase the levels of GSH-Px, SOD, LG and MG, improve the state of exercise fatigue in mice, and have a significant therapeutic effect on model mice. It can be seen that the anti-exercise fatigue of CNBP-C-Ⅰ may be a biological effect on gastrocnemius muscle through antioxidant and increased glycogen levels in vivo.

[0119] In summary, the present invention firstly adopts response surface method to optimize the alkaline extraction process of deer bone protein, and the optimal extraction conditions are extraction temperature of 47°C, solid-liquid ratio of 1:25, and extraction time of 3.6h, which greatly reduces the waste of pre-experimental time and raw materials.

[0120] The principle of precipitating deer bone protein by alkali extraction through isoelectric point treatment combined with ammonium sulfate salting out is to utilize the property that protein is easily soluble in alkaline solution and easily precipitates when the pH is isoelectric point. At the same time, the protein can be precipitated by salting out in neutral salt to precipitate the protein. This is not only simple and safe to operate, without the need to add other interfering chemical reagents, but also can stabilize the active substances in the protein and is not easy to denature the protein.

[0121] In the analysis of amino acid composition, the ninhydrin post-column derivatization method was used to analyze the amino acid composition of deer bone protein hydrolysis. This method is mature and reliable, with high stability, reproducibility and linearity, and is an international and national standard method; infrared spectroscopy is one of the main methods for determining protein functional groups and secondary structures. It has the advantages of rapid detection and small sample volume, and there is a direct correlation between the frequency of the amide I band and the secondary structure of the protein. Due to the C=O stretching vibration of the peptide bond, the amide I band is at 1600-1700cm -1 It contains the secondary structure information of protein, including α-helix, β-fold, β-turn and random coil structure.

[0122] The invention prepares a crude deer bone protein extract, separates and purifies the deer bone protein extract through a DEAE-52 cellulose ion exchange chromatography column to obtain CNBP-C-Ⅰ, wherein the protein content of the CNBP-C-Ⅰ is 89.6%, RP-HPLC determines that the CNBP-C-Ⅰ contains 17 kinds of amino acids, infrared spectrum analyzes the functional groups and secondary structure of the CNBP-C-Ⅰ, and predicts that the proportions of the CNBP-C-Ⅰ are 20.45% of alpha-helix, 39.89% of beta-fold, 15.42% of beta-turn and 24.22% of random coil structure, and HPGFC determines that the molecular weight of the CNBP-C-Ⅰ is 10.96 kDa. CNBP-C-Ⅰ was then identified by LC-MS / MS, and the protein was reduced, alkylated, and digested with enzymes, and analyzed on a nano LC-MS / MS system. Under fixed parameter settings, MaxQuant (2.4.9.0) software analyzed the data and compared and matched with the Uniprot protein database to obtain a protein Celaphus_00011443 (Protein ID: A0A212DFL6) with a high-confidence amino acid sequence of KYEDEINKR.

[0123] The present invention establishes a sports fatigue mouse model, and determines the levels of GSH-Px, SOD and MDA in mouse serum, the level of LG in liver tissue and the level of MG in gastrocnemius muscle tissue by enzyme-linked immunosorbent assay through pathological observation of mouse gastrocnemius muscle, and proves that CNBP-C-Ⅰ can significantly reduce the level of MDA and increase the levels of GSH-Px, SOD, LG and MG, improve the state of sports fatigue in mice, and has a more obvious therapeutic effect on model mice.

Claims

1. A method for preparing deer bone protein for resisting sports fatigue, characterized in that: The method comprises the following steps: Step 1: obtaining deer bone alkaline extract from deer bone powder by alkaline extraction method; Step 2: adjusting the isoelectric point of the deer bone alkaline extract with HCl solution to obtain an alkaline extract solution; Step 3: salting out the alkaline extract solution with ammonium sulfate to obtain deer bone crude protein CNBP-C; Step 4: Purify the deer bone crude protein CNBP-C using a cellulose ion exchange chromatography column to obtain deer bone protein CNBP-C-Ⅰ.

2. The method for preparing a deer bone protein for resisting sports fatigue according to claim 1, characterized in that: The solid-liquid ratio of the alkaline extraction method in step 1 is 1:20-1:30, the extraction temperature is 35-55° C., and the extraction time is 2-4 hours.

3. The method for preparing the deer bone protein for resisting sports fatigue according to claim 1, characterized in that: The solid-liquid ratio of the alkaline extraction method in step 1 is 1:25, the extraction temperature is 47° C., and the extraction time is 3.6 h.

4. The method for preparing a deer bone protein for resisting sports fatigue according to claim 1, characterized in that: The specific steps of adjusting the isoelectric point of the protein with HCl solution in step 2 are: adjusting the concentrated deer bone alkali extract to pH=3.1 with 0.05-0.5 mol / L HCl solution.

5. The method for preparing the deer bone protein for resisting sports fatigue according to claim 1, characterized in that: The specific steps of the ammonium sulfate salting out in step 3 are: adding ammonium sulfate to a solution with a pH value of 3.1 to make its saturation 50%, and then placing it in a 4°C environment for 10-12 hours to obtain a protein suspension; the protein suspension is centrifuged at 3500-5000rpm for 15-30 minutes, the supernatant is retained, and the saturation of the supernatant is adjusted to 80% with ammonium sulfate, and then placed in a 4°C environment for 10-12 hours, and centrifuged again at 3500-5000rpm for 15-30 minutes, and the precipitate is the deer bone protein precipitate; distilled water is added until the precipitate is completely dissolved, and a 500-1500Da dialysis bag is used for 48-72 hours of dialysis, and deer bone crude protein CNBP-C is obtained after freeze-drying.

6. The method for preparing the deer bone protein for resisting sports fatigue according to claim 5, characterized in that: The freeze drying temperature is ≤-50°C, and the freeze drying time is 24-48h.

7. The method for preparing the anti-sports fatigue deer bone protein according to claim 1, characterized in that: The specific steps of purifying deer bone protein with a cellulose ion exchange chromatography column in step 4 are as follows: adding the deer bone crude protein CNBP-C solution to the cellulose ion exchange chromatography column, and eluting in stages with 0.01 mol / L Tris-HCl containing 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl, with a flow rate of 0.5-1.5 mL / min, 4-6 mL per tube, and a total of 100 tubes. The DEAE-52 cellulose chromatographic separation curve is drawn with the number of tubes detected as the abscissa and the absorbance value at 595 nm as the ordinate, and the first peak is recovered and separated. The collected elution component is the deer bone protein CNBP-C-Ⅰ.

8. The method for preparing the deer bone protein for resisting sports fatigue according to claim 7, characterized in that: The deer bone crude protein CNBP-C solution is obtained by dissolving deer bone crude protein CNBP-C in Tris-HCl, and the concentration is 15-25 mg / mL.

9. Deer bone protein prepared by the method for preparing deer bone protein for resisting sports fatigue according to any one of claims 1 to 8.

10. Use of the deer bone protein prepared by the method according to any one of claims 1 to 8 in the preparation of drugs or health products for resisting sports fatigue.

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