A method for extracting small molecule peptides from yak spinal cord with high biological activity and stability

Abstract

The invention discloses a method for extracting high biological activity stability yak spinal cord small molecule peptides, belonging to the technical field of peptide extraction, and the method comprises the following steps: pre-treating yak bone marrow tissue; enzymatic hydrolysis of the pre-treated tissue; separation to obtain a small molecule peptide solution after enzyme inactivation; high-purity small molecule peptides after purification; wherein, the pre-treatment sequentially uses N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate to pre-treat yak tissue. The present invention pre-treats yak bone marrow tissue by using a specific reagent combination and a specific processing sequence, thereby increasing the stability of small molecule peptides and proteins in yak bone marrow tissue, being not easily denatured during subsequent processing, and being conducive to extracting and obtaining high-purity small molecule peptides.

Description

Technical Field

[0001] The invention belongs to the technical field of peptide extraction, and in particular relates to a method for extracting yak spinal cord small-molecule peptides with high biological activity and stability. Background Art

[0002] Small molecule peptides, a bioactive substance, have attracted widespread attention in the field of biomedicine and nutritional health products in recent years. Their small molecular weight, high bioactivity and easy absorption make small molecule peptides show great potential in promoting human health. Yaks, as a unique species living in high-altitude cold areas, have bone marrow rich in a variety of bioactive ingredients, especially small molecule peptides. Therefore, the technical research and development of extracting small molecule peptides from yak bone marrow has important practical significance.

[0003] The extraction process of small molecule peptides from yak bone marrow usually includes key steps such as crushing, enzymatic hydrolysis, separation and purification. Among them, enzymatic hydrolysis is the key link. By selecting appropriate enzymes and optimizing enzymatic hydrolysis conditions, the protein in the bone marrow can be efficiently degraded into small molecule peptides. However, despite the continuous maturity of extraction technology, there are still deficiencies in the biological activity stability of the product small molecule peptides.

[0004] From the relevant background, small molecule peptides have broad application prospects in nutrition supplementation, health promotion, disease prevention, etc. due to their unique biological activity. Especially in the fields of anti-aging, enhancing immunity, promoting bone health, etc., the role of small molecule peptides has been widely recognized. Therefore, the technology of extracting small molecule peptides from yak bone marrow not only helps to meet market demand, but also provides strong support for the development of related industries.

[0005] However, the current technology for extracting small molecule peptides from yak bone marrow still has some shortcomings and bottlenecks. The most prominent problem is the poor stability of the biological activity of the small molecule peptides. During the extraction and purification process, due to operating conditions, environmental factors and imperfect extraction technology, the structure of the small molecule peptides may change, thereby affecting their biological activity. In addition, small molecule peptides are also easily affected by temperature, humidity, light and other conditions during storage and transportation, resulting in reduced activity or even inactivation.

[0006] In response to these issues, research and development hotspots are mainly focused on optimizing extraction processes, improving purification efficiency, exploring small molecule peptide stabilization technology, etc. Through in-depth research on the relationship between the structure and function of small molecule peptides, as well as the laws of their stability changes under different environments, it is expected to provide theoretical support and practical guidance for improving the stability of product biological activity.

[0007] In short, the extraction of small molecule peptides from yak bone marrow is a technology with broad application prospects. Although there are still deficiencies and bottlenecks in the stability of product biological activity, with the continuous advancement of technology and the deepening of research and development, I believe these problems will be gradually solved and make greater contributions to human health. Summary of the invention

[0008] In view of the above problems, the present invention provides a method for extracting yak brain and bone marrow small molecule peptides from a pretreatment perspective to improve the stability of biological activity. The specific scheme is as follows:

[0009] A method for extracting yak spinal cord small molecule peptides with high biological activity and stability, comprising the following steps:

[0010] S1. Pre-treating yak bone marrow tissue;

[0011] S2, performing enzymatic hydrolysis on the pretreated tissue;

[0012] S3, after inactivating the enzyme, separate and obtain a small molecule peptide solution;

[0013] S4, after purification, high-purity small molecule peptides are obtained;

[0014] In the enzymatic hydrolysis in step S2, a composite enzyme of trypsin and papain is used, and the enzymatic activity ratio of the two enzymes is (1-2):1.

[0015] Preferably, in the enzymolysis in step S2, the amount of enzyme used is 1.5-2.0wt% of the yak tissue, the enzymolysis temperature is 35-45°C, and the enzymolysis time is 2-4h.

[0016] Preferably, in step S3, the enzyme is inactivated by heating the enzymatic hydrolyzate obtained after enzymatic hydrolysis to 80-90° C., maintaining the temperature for 10-20 min, and then cooling the solution to room temperature.

[0017] Preferably, in step S3, the separation is filtered and then centrifuged to obtain the supernatant as the small molecule peptide solution. The centrifugation is performed at 2000-5000 rps for 10-15 min.

[0018] Preferably, the purification in step S4 includes ultrafiltration, using an ultrafiltration membrane with a molecular weight cutoff between 3-5 kDa and an operating pressure of 0.1-0.2 MPa.

[0019] Preferably, the purification in step S4 further comprises ultrafiltration for dialysis desalination and freeze-drying; the dialysis bag with a molecular weight cutoff of 1 kDa is selected, the liquid is changed every 1 hour, and the dialysis is performed for 8-12 hours; the small molecule peptide solution obtained by dialysis is freeze-dried to obtain a high-purity small molecule peptide.

[0020] Preferably, in step S1, N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate are sequentially used to pretreat the yak tissue.

[0021] Preferably, the preprocessing in step S1 comprises the following steps:

[0022] (1) N-acetylglucosamine treatment: Place yak bone marrow tissue in PBS buffer, add N-acetylglucosamine to the yak bone marrow tissue, and stir;

[0023] (2) Trehalose treatment: adding trehalose to the tissue treated in step (1), stirring and mixing, and then allowing to stand for treatment;

[0024] (3) Sodium chloride treatment: adding sodium chloride to the tissue treated in step (2) and stirring to mix;

[0025] (4) Sodium dodecyl sulfate treatment: Add sodium dodecyl sulfate to the tissue treated in step (3), stir and mix, and then let stand.

[0026] Preferably, the above preprocessing further includes:

[0027] (5) Heat treatment: Heat the tissue treated in step (4) in a water bath at 55-65°C for 30-40 min.

[0028] Preferably, the yak bone marrow tissue used in the above step (1) is yak bone marrow tissue processed by the following steps: cutting the yak bone marrow tissue into small pieces, grinding the tissue into powder with a mortar and pestle under liquid nitrogen freezing conditions, and restoring it to room temperature for use.

[0029] Preferably, the amount of PBS buffer used in step (1) is 30-50 mL PBS buffer per 10 g yak bone marrow tissue.

[0030] Preferably, the amount of N-acetylglucosamine used in step (1) is 1-1.5 wt % of yak bone marrow tissue, the treatment temperature is room temperature, and the stirring time is 10-15 min.

[0031] Preferably, the amount of trehalose used in step (2) is 5-6 wt % of the yak bone marrow tissue, the treatment temperature is room temperature, and the standing time is 15-20 min, so that the trehalose fully penetrates the tissue.

[0032] Preferably, the amount of sodium chloride used in step (3) is 0.9-1.3 wt % of yak bone marrow tissue.

[0033] Preferably, the amount of sodium dodecyl sulfate in step (4) is 0.5-1.0 wt% of the yak bone marrow tissue. Stirring and mixing, the sodium dodecyl sulfate solution evenly covers the tissue surface. The standing temperature is room temperature, and the standing time is 20-30 minutes.

[0034] Beneficial Effects

[0035] The present invention pre-treats yak bone marrow tissue by adopting a specific reagent combination and a specific treatment sequence, thereby increasing the stability of small molecule peptides and proteins in the yak bone marrow tissue, making them less susceptible to denaturation during subsequent treatments, and at the same time improving the biological activity stability of the product through the influence of specific reagents in the pre-treatment on the product peptide chain. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0039] Example 1 Extraction of high biological activity and stability of yak spinal cord small molecule peptides:

[0040] S1. Pre-treating yak bone marrow tissue;

[0041] S2, performing enzymatic hydrolysis on the pretreated tissue;

[0042] S3, after inactivating the enzyme, separate and obtain a small molecule peptide solution;

[0043] S4, after purification, high-purity small molecule peptides are obtained;

[0044] Wherein, step S1 sequentially uses N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate to pretreat the yak tissue.

[0045] The enzymatic hydrolysis in step S2 uses a composite enzyme of trypsin and papain, and the enzyme activity ratio of the two enzymes is 1:1.

[0046] In the enzymolysis of step S2, the amount of enzyme used is 1.5wt% of the yak tissue, the enzymolysis temperature is 35°C, and the enzymolysis time is 4h.

[0047] In step S3, the enzyme is inactivated by heating the enzymatic hydrolyzate obtained after enzymatic hydrolysis to 80° C., maintaining the temperature for 20 min, and then cooling the solution to room temperature.

[0048] In step S3, the separation is performed, filtered and then centrifuged, and the supernatant is taken as the small molecule peptide solution. The centrifugation is performed at 2000 rps for 10 min.

[0049] The purification in step S4 includes ultrafiltration, using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa and an operating pressure of 0.1 MPa.

[0050] The purification in step S4 also includes ultrafiltration for dialysis desalination and freeze-drying; the dialysis bag with a molecular weight cutoff of 1 kDa is selected, the liquid is changed every 1 hour, and the dialysis is performed for 8 hours; the small molecule peptide solution obtained by dialysis is freeze-dried to obtain a high-purity small molecule peptide.

[0051] The pre-processing in step S1 comprises the following steps:

[0052] (1) N-acetylglucosamine treatment: Place yak bone marrow tissue in PBS buffer, add N-acetylglucosamine to the yak bone marrow tissue, and stir;

[0053] (2) Trehalose treatment: adding trehalose to the tissue treated in step (1), stirring and mixing, and then allowing to stand for treatment;

[0054] (3) Sodium chloride treatment: adding sodium chloride to the tissue treated in step (2) and stirring to mix;

[0055] (4) Sodium dodecyl sulfate treatment: Add sodium dodecyl sulfate to the tissue treated in step (3), stir and mix, and then let stand.

[0056] The yak bone marrow tissue used in the above step (1) is the yak bone marrow tissue processed by the following steps: the yak bone marrow tissue is cut into small pieces, and then ground into powder using a mortar and pestle under liquid nitrogen freezing conditions, and then restored to room temperature for use.

[0057] The amount of PBS buffer used in step (1) is 30 mL PBS buffer per 10 g yak bone marrow tissue.

[0058] The amount of N-acetylglucosamine used in step (1) is 1wt% of yak bone marrow tissue, the treatment temperature is room temperature, and the stirring time is 10 minutes.

[0059] The amount of trehalose used in step (2) is 5wt% of the yak bone marrow tissue, the treatment temperature is room temperature, and the standing time is 15 minutes, so that the trehalose can fully penetrate the tissue.

[0060] The amount of sodium chloride used in step (3) is 0.9wt% of yak bone marrow tissue.

[0061] In step (4), the amount of sodium dodecyl sulfate used is 0.5 wt % of the yak bone marrow tissue. The mixture is stirred and mixed so that the sodium dodecyl sulfate solution evenly covers the tissue surface. The standing temperature is room temperature and the standing time is 20 min.

[0062] Example 2 Extraction of high biological activity and stability of yak spinal cord small molecule peptides:

[0063] S1. Pre-treating yak bone marrow tissue;

[0064] S2, performing enzymatic hydrolysis on the pretreated tissue;

[0065] S3, after inactivating the enzyme, separate and obtain a small molecule peptide solution;

[0066] S4, after purification, high-purity small molecule peptides are obtained;

[0067] Wherein, step S1 sequentially uses N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate to pretreat the yak tissue.

[0068] The enzymatic hydrolysis in step S2 uses a composite enzyme of trypsin and papain, and the enzyme activity ratio of the two enzymes is (1-2):1.

[0069] In the enzymolysis of step S2, the amount of enzyme used is 2.0wt% of the yak tissue, the enzymolysis temperature is 45°C, and the enzymolysis time is 2h.

[0070] In step S3, the enzyme is inactivated by heating the enzymatic hydrolyzate obtained after enzymatic hydrolysis to 90° C., maintaining the temperature for 10 min, and then cooling the solution to room temperature.

[0071] The separation in step S3 is followed by filtration and centrifugation, and the supernatant is taken as the small molecule peptide solution. The centrifugation is performed at 5000 rps for 10 min.

[0072] The purification in step S4 includes ultrafiltration, using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa and an operating pressure of 0.2 MPa.

[0073] The purification in step S4 also includes ultrafiltration for dialysis desalination and freeze-drying; the dialysis bag with a molecular weight cutoff of 1 kDa is selected, the liquid is changed every 1 hour, and the dialysis is performed for 12 hours; the small molecule peptide solution obtained by dialysis is freeze-dried to obtain a high-purity small molecule peptide.

[0074] The pre-processing in step S1 comprises the following steps:

[0075] (1) N-acetylglucosamine treatment: Place yak bone marrow tissue in PBS buffer, add N-acetylglucosamine to the yak bone marrow tissue, and stir;

[0076] (2) Trehalose treatment: adding trehalose to the tissue treated in step (1), stirring and mixing, and then allowing to stand for treatment;

[0077] (3) Sodium chloride treatment: adding sodium chloride to the tissue treated in step (2) and stirring to mix;

[0078] (4) Sodium dodecyl sulfate treatment: Add sodium dodecyl sulfate to the tissue treated in step (3), stir and mix, and then let stand.

[0079] The yak bone marrow tissue used in the above step (1) is the yak bone marrow tissue processed by the following steps: the yak bone marrow tissue is cut into small pieces, and then ground into powder using a mortar and pestle under liquid nitrogen freezing conditions, and then restored to room temperature for use.

[0080] The amount of PBS buffer used in step (1) is 30 mL PBS buffer per 10 g yak bone marrow tissue.

[0081] The amount of N-acetylglucosamine used in step (1) is 1wt% of yak bone marrow tissue, the treatment temperature is room temperature, and the stirring time is 10 minutes.

[0082] The amount of trehalose used in step (2) is 5wt% of the yak bone marrow tissue, the treatment temperature is room temperature, and the standing time is 15 minutes, so that the trehalose can fully penetrate the tissue.

[0083] The amount of sodium chloride used in step (3) is 0.9wt% of yak bone marrow tissue.

[0084] In step (4), the amount of sodium dodecyl sulfate used is 0.5 wt % of the yak bone marrow tissue. The mixture is stirred and mixed so that the sodium dodecyl sulfate solution evenly covers the tissue surface. The standing temperature is room temperature and the standing time is 20 min.

[0085] Example 3 Extraction of high biological activity and stability of yak spinal cord small molecule peptides:

[0086] S1. Pre-treating yak bone marrow tissue;

[0087] S2, performing enzymatic hydrolysis on the pretreated tissue;

[0088] S3, after inactivating the enzyme, separate and obtain a small molecule peptide solution;

[0089] S4, after purification, high-purity small molecule peptides are obtained;

[0090] Wherein, step S1 sequentially uses N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate to pretreat the yak tissue.

[0091] The enzymatic hydrolysis in step S2 uses a composite enzyme of trypsin and papain, and the enzyme activity ratio of the two enzymes is (1-2):1.

[0092] In the enzymolysis of step S2, the amount of enzyme used is 2.0wt% of the yak tissue, the enzymolysis temperature is 45°C, and the enzymolysis time is 2h.

[0093] In step S3, the enzyme is inactivated by heating the enzymatic hydrolyzate obtained after enzymatic hydrolysis to 90° C., maintaining the temperature for 10 min, and then cooling the solution to room temperature.

[0094] The separation in step S3 is followed by filtration and centrifugation, and the supernatant is taken as the small molecule peptide solution. The centrifugation is performed at 5000 rps for 10 min.

[0095] The purification in step S4 includes ultrafiltration, using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa and an operating pressure of 0.2 MPa.

[0096] The purification in step S4 also includes ultrafiltration for dialysis desalination and freeze-drying; the dialysis bag with a molecular weight cutoff of 1 kDa is selected, the liquid is changed every 1 hour, and the dialysis is performed for 12 hours; the small molecule peptide solution obtained by dialysis is freeze-dried to obtain a high-purity small molecule peptide.

[0097] The pre-processing in step S1 comprises the following steps:

[0098] (1) N-acetylglucosamine treatment: Place yak bone marrow tissue in PBS buffer, add N-acetylglucosamine to the yak bone marrow tissue, and stir;

[0099] (2) Trehalose treatment: adding trehalose to the tissue treated in step (1), stirring and mixing, and then allowing to stand for treatment;

[0100] (3) Sodium chloride treatment: adding sodium chloride to the tissue treated in step (2) and stirring to mix;

[0101] (4) Sodium dodecyl sulfate treatment: Add sodium dodecyl sulfate to the tissue treated in step (3), stir and mix, and then let stand.

[0102] The yak bone marrow tissue used in the above step (1) is the yak bone marrow tissue processed by the following steps: the yak bone marrow tissue is cut into small pieces, and then ground into powder using a mortar and pestle under liquid nitrogen freezing conditions, and then restored to room temperature for use.

[0103] The amount of PBS buffer used in step (1) is 50 mL PBS buffer per 10 g yak bone marrow tissue.

[0104] The amount of N-acetylglucosamine used in step (1) is 1.5wt% of yak bone marrow tissue, the processing temperature is room temperature, and the stirring time is 15 minutes.

[0105] The amount of trehalose used in step (2) is 6 wt % of the yak bone marrow tissue, the treatment temperature is room temperature, and the standing time is 20 min, so that the trehalose can fully penetrate the tissue.

[0106] The amount of sodium chloride used in step (3) is 1.3wt% of yak bone marrow tissue.

[0107] In step (4), the amount of sodium dodecyl sulfate used is 1.0 wt% of the yak bone marrow tissue. Stirring and mixing, the sodium dodecyl sulfate solution evenly covers the tissue surface. The standing temperature is room temperature, and the standing time is 30 minutes.

[0108] Example 4 Extraction of high biological activity and stability of yak spinal cord small molecule peptides:

[0109] S1. Pre-treating yak bone marrow tissue;

[0110] S2, performing enzymatic hydrolysis on the pretreated tissue;

[0111] S3, after inactivating the enzyme, separate and obtain a small molecule peptide solution;

[0112] S4, after purification, high-purity small molecule peptides are obtained;

[0113] Wherein, step S1 sequentially uses N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate to pretreat the yak tissue.

[0114] The enzymatic hydrolysis in step S2 uses a composite enzyme of trypsin and papain, and the enzyme activity ratio of the two enzymes is (1-2):1.

[0115] In the enzymolysis of step S2, the amount of enzyme used is 2.0wt% of the yak tissue, the enzymolysis temperature is 45°C, and the enzymolysis time is 2h.

[0116] In step S3, the enzyme is inactivated by heating the enzymatic hydrolyzate obtained after enzymatic hydrolysis to 90° C., maintaining the temperature for 10 min, and then cooling the solution to room temperature.

[0117] The separation in step S3 is followed by filtration and centrifugation, and the supernatant is taken as the small molecule peptide solution. The centrifugation is performed at 5000 rps for 10 min.

[0118] The purification in step S4 includes ultrafiltration, using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa and an operating pressure of 0.2 MPa.

[0119] The purification in step S4 also includes ultrafiltration for dialysis desalination and freeze-drying; the dialysis bag with a molecular weight cutoff of 1 kDa is selected, the liquid is changed every 1 hour, and the dialysis is performed for 12 hours; the small molecule peptide solution obtained by dialysis is freeze-dried to obtain a high-purity small molecule peptide.

[0120] The pre-processing in step S1 comprises the following steps:

[0121] (1) N-acetylglucosamine treatment: Place yak bone marrow tissue in PBS buffer, add N-acetylglucosamine to the yak bone marrow tissue, and stir;

[0122] (2) Trehalose treatment: adding trehalose to the tissue treated in step (1), stirring and mixing, and then allowing to stand for treatment;

[0123] (3) Sodium chloride treatment: adding sodium chloride to the tissue treated in step (2) and stirring to mix;

[0124] (4) Sodium dodecyl sulfate treatment: Add sodium dodecyl sulfate to the tissue treated in step (3), stir and mix, and then let stand.

[0125] The above preprocessing also includes:

[0126] (5) Heat treatment: Heat the tissue treated in step (4) in a 55°C water bath for 30 min.

[0127] The yak bone marrow tissue used in the above step (1) is the yak bone marrow tissue processed by the following steps: the yak bone marrow tissue is cut into small pieces, and then ground into powder using a mortar and pestle under liquid nitrogen freezing conditions, and then restored to room temperature for use.

[0128] The amount of PBS buffer used in step (1) is 50 mL PBS buffer per 10 g yak bone marrow tissue.

[0129] The amount of N-acetylglucosamine used in step (1) is 1.5wt% of yak bone marrow tissue, the processing temperature is room temperature, and the stirring time is 15 minutes.

[0130] The amount of trehalose used in step (2) is 6 wt % of the yak bone marrow tissue, the treatment temperature is room temperature, and the standing time is 20 min, so that the trehalose can fully penetrate the tissue.

[0131] The amount of sodium chloride used in step (3) is 1.3wt% of yak bone marrow tissue.

[0132] In step (4), the amount of sodium dodecyl sulfate used is 1.0 wt% of the yak bone marrow tissue. Stirring and mixing, the sodium dodecyl sulfate solution evenly covers the tissue surface. The standing temperature is room temperature, and the standing time is 30 minutes.

[0133] Example 5 Extraction of high biological activity and stability of yak spinal cord small molecule peptides:

[0134] S1. Pre-treating yak bone marrow tissue;

[0135] S2, performing enzymatic hydrolysis on the pretreated tissue;

[0136] S3, after inactivating the enzyme, separate and obtain a small molecule peptide solution;

[0137] S4, after purification, high-purity small molecule peptides are obtained;

[0138] Wherein, step S1 sequentially uses N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate to pretreat the yak tissue.

[0139] The enzymatic hydrolysis in step S2 uses a composite enzyme of trypsin and papain, and the enzyme activity ratio of the two enzymes is (1-2):1.

[0140] In the enzymolysis of step S2, the amount of enzyme used is 2.0wt% of the yak tissue, the enzymolysis temperature is 45°C, and the enzymolysis time is 2h.

[0141] In step S3, the enzyme is inactivated by heating the enzymatic hydrolyzate obtained after enzymatic hydrolysis to 90° C., maintaining the temperature for 10 min, and then cooling the solution to room temperature.

[0142] The separation in step S3 is followed by filtration and centrifugation, and the supernatant is taken as the small molecule peptide solution. The centrifugation is performed at 5000 rps for 10 min.

[0143] The purification in step S4 includes ultrafiltration, using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa and an operating pressure of 0.2 MPa.

[0144] The purification in step S4 also includes ultrafiltration for dialysis desalination and freeze-drying; the dialysis bag with a molecular weight cutoff of 1 kDa is selected, the liquid is changed every 1 hour, and the dialysis is performed for 12 hours; the small molecule peptide solution obtained by dialysis is freeze-dried to obtain a high-purity small molecule peptide.

[0145] The pre-processing in step S1 comprises the following steps:

[0146] (1) N-acetylglucosamine treatment: Place yak bone marrow tissue in PBS buffer, add N-acetylglucosamine to the yak bone marrow tissue, and stir;

[0147] (2) Trehalose treatment: adding trehalose to the tissue treated in step (1), stirring and mixing, and then allowing to stand for treatment;

[0148] (3) Sodium chloride treatment: adding sodium chloride to the tissue treated in step (2) and stirring to mix;

[0149] (4) Sodium dodecyl sulfate treatment: Add sodium dodecyl sulfate to the tissue treated in step (3), stir and mix, and then let stand.

[0150] The above preprocessing also includes:

[0151] (5) Heat treatment: The tissue treated in step (4) was heat treated in a 65°C water bath for 40 min.

[0152] The yak bone marrow tissue used in the above step (1) is the yak bone marrow tissue processed by the following steps: the yak bone marrow tissue is cut into small pieces, and then ground into powder using a mortar and pestle under liquid nitrogen freezing conditions, and then restored to room temperature for use.

[0153] The amount of PBS buffer used in step (1) is 50 mL PBS buffer per 10 g yak bone marrow tissue.

[0154] The amount of N-acetylglucosamine used in step (1) is 1.5wt% of yak bone marrow tissue, the processing temperature is room temperature, and the stirring time is 15 minutes.

[0155] The amount of trehalose used in step (2) is 6 wt % of the yak bone marrow tissue, the treatment temperature is room temperature, and the standing time is 20 min, so that the trehalose can fully penetrate the tissue.

[0156] The amount of sodium chloride used in step (3) is 1.3wt% of yak bone marrow tissue.

[0157] In step (4), the amount of sodium dodecyl sulfate used is 1.0 wt% of the yak bone marrow tissue. Stirring and mixing, the sodium dodecyl sulfate solution evenly covers the tissue surface. The standing temperature is room temperature, and the standing time is 30 minutes.

[0158] (The difference between Example 1 and Example 2: the basic processing step parameters are different, and the pre-processing parameters are the same; the difference between Example 3 and Example 2: the basic processing step parameters are the same, and the pre-processing step parameters are different; the difference between Example 4 and Example 3: a heat treatment step is added; the difference between Example 5 and Example 4: the heat treatment parameters are different.)

[0159] The difference between Comparative Example 1 and Example 5 is that the pretreatment in step S1 does not include the "adding N-acetylglucosamine and stirring treatment" in step (1), and the other steps and parameters are the same as those in Example 5.

[0160] The difference between Comparative Example 2 and Example 5 is that the pretreatment in step S1 does not include step (2), and the other steps and parameters are the same as those in Example 5.

[0161] The difference between Comparative Example 3 and Example 5 is that the pretreatment in step S1 does not include step (3), and the other steps and parameters are the same as those in Example 5.

[0162] The difference between Comparative Example 4 and Example 5 is that the pretreatment in step S1 does not include step (4), and the other steps and parameters are the same as those in Example 5.

[0163] The difference between Comparative Example 5 and Example 5 is that the pretreatment in step S1 does not include "adding N-acetylglucosamine and stirring" in (1) and (2), and the other steps and parameters are the same as those in Example 5.

[0164] The difference between Comparative Example 6 and Example 5 is that the pretreatment in step S1 does not include "adding N-acetylglucosamine and stirring" in (1) and (3), and the other steps and parameters are the same as those in Example 5.

[0165] The difference between Comparative Example 7 and Example 5 is that the pretreatment in step S1 does not include "adding N-acetylglucosamine and stirring" in (1) and (4), and the other steps and parameters are the same as those in Example 5.

[0166] The difference between Comparative Example 8 and Example 5 is that the pretreatment in step S1 does not include (2) and (3), and the other steps and parameters are the same as those in Example 5.

[0167] The difference between Comparative Example 9 and Example 5 is that the pretreatment in step S1 does not include (2) and (4), and the other steps and parameters are the same as those in Example 5.

[0168] The difference between Comparative Example 10 and Example 5 is that the pretreatment in step S1 does not include (3) and (4), and the other steps and parameters are the same as those in Example 5.

[0169] The difference between Comparative Example 11 and Example 5 is that the pretreatment in step S1 does not include "adding N-acetylglucosamine and stirring" in (1), (2), and (3), and the other steps and parameters are the same as those in Example 5.

[0170] The difference between Comparative Example 12 and Example 5 is that the pretreatment in step S1 does not include "adding N-acetylglucosamine and stirring" in (1), (2), and (4), and the other steps and parameters are the same as those in Example 5.

[0171] The difference between Comparative Example 13 and Example 5 is that the pretreatment in step S1 does not include "adding N-acetylglucosamine and stirring" in (1), (3), and (4), and the other steps and parameters are the same as those in Example 5.

[0172] The difference between Comparative Example 14 and Example 5 is that the pretreatment described in step S1 does not include (2), (3) and (4), and the other steps and parameters are the same as those in Example 5.

[0173] The difference between Comparative Example 15 and Example 5 is that in the pretreatment described in step S1, the order of "adding N-acetylglucosamine and stirring" in (1) and (2) is swapped.

[0174] The difference between Comparative Example 16 and Example 5 is that in the pretreatment described in step S1, the order of "adding N-acetylglucosamine and stirring" in (1) is swapped with that in (3).

[0175] The difference between Comparative Example 17 and Example 5 is that in the pretreatment described in step S1, the order of "adding N-acetylglucosamine and stirring" in (1) is swapped with that in (4).

[0176] The difference between Comparative Example 18 and Example 5 is that in the pretreatment described in step S1, the order of (2) and (3) is swapped.

[0177] The difference between Comparative Example 19 and Example 5 is that in the pretreatment described in step S1, the order of (2) and (4) is swapped.

[0178] The difference between Comparative Example 20 and Example 5 is that in the pretreatment of step S1, the order of (3) and (4) is swapped.

[0179] The small molecule peptides obtained from all the above examples and comparative examples were tested for biological activity stability:

[0180] Experimental Materials:

[0181] Small molecule peptide samples (products obtained from all the above examples and comparative examples)

[0182] Trypsin (commercial source, such as Sigma-Aldrich)

[0183] Substrate: Tyrosine ethyl ester

[0184] Reaction buffer: Tris-HCl buffer (pH 8.0)

[0185] Instruments: spectrophotometer, micropipette, centrifuge tube, constant temperature water bath

[0186] Experimental and control group design:

[0187] Experimental group 1: freshly extracted small molecule peptides + trypsin + substrate

[0188] Experimental group 2: small peptide (buffer solution) + trypsin + substrate stored under specific conditions (4°C) for 24 hours

[0189] Experimental group 3: small peptide (buffer solution) + trypsin + substrate stored under specific conditions (4°C) for 72 hours

[0190] Experimental group 4: small molecule peptide + trypsin + substrate stored under specific conditions (-20℃) for 72 hours

[0191] Control group: buffer (instead of small molecule peptide) + trypsin + substrate

[0192] Experimental steps:

[0193] Prepare the reaction system:

[0194] Add appropriate amount of Tris-HCl buffer (200 μL for each reaction system) into the centrifuge tubes.

[0195] The same concentration of trypsin solution was added to the experimental group and the control group.

[0196] Add appropriate amount of small molecule peptide sample to the experimental group (equal amount of small molecule peptide in each group, the small molecule peptide is dissolved in buffer and added).

[0197] An equal volume of buffer was added to the control group instead of the small molecule peptide solution.

[0198] Preheat the reaction system: Place the centrifuge tube in a constant temperature water bath and preheat to the reaction temperature (37°C).

[0199] Start the reaction: Add substrate solution to each centrifuge tube and start the timer.

[0200] Reaction: Allow the reaction to proceed in a constant temperature water bath for a period of time.

[0201] Termination of reaction: Terminate the reaction by adding an appropriate amount of reaction termination solution.

[0202] Detection and analysis: The absorbance of the reaction product was measured using a spectrophotometer (tyrosine 275 nm).

[0203] The absorbance values ​​were converted to product concentrations according to the standard curve.

[0204] Calculate enzyme activity: enzyme activity (unit / time) = (change in product concentration) / (reaction time).

[0205] Comparison of enzyme activity: Compare the enzyme activity values ​​of the experimental group and the control group to analyze the effect of small molecule peptides on trypsin activity.

[0206] We found that the product concentrations of the experimental groups were all lower than those of the control group, indicating that the small molecule peptides obtained in the examples and comparative examples inhibited trypsin. Therefore, we used the following method to calculate the biological activity preservation rate of the small molecule peptides after storage for a certain period of time and under certain conditions:

[0207] Calculate the biological activity preservation rate: biological activity preservation rate (%) = (enzyme activity of the control group - enzyme activity of the experimental group X) / (enzyme activity of the control group - enzyme activity of the experimental group 1) × 100%

[0208] Each sample is calculated separately. Since this calculation method is performed between the same samples, the influence of sample purity on the calculation results can be ignored.

[0209] Table 1 Biological activity preservation rate of all examples and comparative examples in experimental group 2

[0210] Sample No. Saving rate % Sample No. Saving rate % Example 1 98.56 Comparative Example 9 92.68 Example 2 98.64 Comparative Example 10 92.56 Example 3 98.52 Comparative Example 11 92.77 Example 4 99.32 Comparative Example 12 92.60 Example 5 99.37 Comparative Example 13 93.84 Comparative Example 1 92.65 Comparative Example 14 93.59 Comparative Example 2 92.46 Comparative Example 15 93.46 Comparative Example 3 92.39 Comparative Example 16 93.35 Comparative Example 4 92.26 Comparative Example 17 93.77 Comparative Example 5 92.70 Comparative Example 18 93.60 Comparative Example 6 92.54 Comparative Example 19 93.64 Comparative Example 7 92.41 Comparative Example 20 93.45 Comparative Example 8 92.79

[0211] Table 2 Biological activity preservation rate of all examples and comparative examples in experimental group 3

[0212] Sample No. Saving rate % Sample No. Saving rate % Example 1 98.17 Comparative Example 9 83.13 Example 2 98.22 Comparative Example 10 83.20 Example 3 98.15 Comparative Example 11 82.91 Example 4 98.86 Comparative Example 12 82.66 Example 5 98.89 Comparative Example 13 85.12 Comparative Example 1 82.74 Comparative Example 14 85.17 Comparative Example 2 83.12 Comparative Example 15 85.26 Comparative Example 3 83.28 Comparative Example 16 84.88 Comparative Example 4 82.63 Comparative Example 17 85.37 Comparative Example 5 82.85 Comparative Example 18 84.75 Comparative Example 6 82.97 Comparative Example 19 85.22 Comparative Example 7 82.77 Comparative Example 20 85.18 Comparative Example 8 82.71

[0213] Since the calculated biological activity preservation rates of experimental group 4 were all greater than 99.61%, and no significant differences or regularities were observed between the samples, the calculated results of experimental group 4 are not displayed.

[0214] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0215] The present invention and its implementation methods are described above, and such description is not restrictive, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design methods and embodiments similar to the technical solution without creativity, which should all fall within the protection scope of the present invention.

Claims

1. A method for extracting yak spinal cord small molecule peptides with high biological activity and stability, characterized in that: The following steps are involved: S1. Pre-treating yak bone marrow tissue; S2, performing enzymatic hydrolysis on the pretreated tissue; S3, after inactivating the enzyme, separate and obtain a small molecule peptide solution; S4, after purification, high-purity small molecule peptides are obtained; Wherein, the enzymatic hydrolysis in step S2 uses a composite enzyme of trypsin and papain, and the enzyme activity ratio of the two enzymes is (1-2):1; In step S2, the enzyme dosage is 1.5-2.0wt% of the yak tissue, the enzymatic hydrolysis temperature is 35-45°C, and the enzymatic hydrolysis time is 2-4h; Step S1 sequentially uses N-acetylglucosamine, trehalose, sodium chloride and sodium dodecyl sulfate to pretreat the yak tissue; the pretreat in step S1 comprises the following steps: (1) N-acetylglucosamine treatment: Place yak bone marrow tissue in PBS buffer, add N-acetylglucosamine to the yak bone marrow tissue, and stir; (2) Trehalose treatment: adding trehalose to the tissue treated in step (1), stirring and mixing, and then allowing to stand for treatment; (3) Sodium chloride treatment: adding sodium chloride to the tissue treated in step (2) and stirring to mix; (4) Sodium dodecyl sulfate treatment: add sodium dodecyl sulfate to the tissue treated in step (3), stir and mix, and then let stand; The pretreatment further comprises: (5) heat treatment: heat treating the tissue treated in step (4) in a water bath at 55-65° C. for 30-40 min; The amount of N-acetylglucosamine used in step (1) is 1-1.5wt% of the yak bone marrow tissue, the treatment temperature is room temperature, and the stirring time is 10-15min; the amount of trehalose used in step (2) is 5-6wt% of the yak bone marrow tissue, the treatment temperature is room temperature, and the standing time is 15-20min; the trehalose is allowed to fully penetrate the tissue; the amount of sodium chloride used in step (3) is 0.9-1.3wt% of the yak bone marrow tissue; the amount of sodium dodecyl sulfate used in step (4) is 0.5-1.0wt% of the yak bone marrow tissue; the mixture is stirred and mixed so that the sodium dodecyl sulfate solution evenly covers the tissue surface; the standing temperature is room temperature, and the standing time is 20-30min.

2. The method for extracting yak spinal cord small molecule peptides with high biological activity and stability according to claim 1, characterized in that: In step S3, the enzyme is inactivated by heating the enzymatic solution obtained after enzymatic hydrolysis to 80-90° C., maintaining the temperature for 10-20 minutes, and then cooling the solution to room temperature.

3. The method for extracting yak spinal cord small molecule peptides with high biological activity and stability according to claim 1, characterized in that: In step S3, the separation is performed, and the filtration is followed by centrifugation, and the supernatant is taken as the small molecule peptide solution; the centrifugation is performed at 2000-5000 rps for 10-15 min.

4. The method for extracting yak spinal cord small molecule peptides with high biological activity and stability according to claim 1, characterized in that: The purification in step S4 includes ultrafiltration, using an ultrafiltration membrane with a molecular weight cutoff between 3 and 5 kDa and an operating pressure of 0.1 to 0.2 MPa.

5. The method for extracting yak spinal cord small molecule peptides with high biological activity and stability according to claim 1, characterized in that: The purification in step S4 also includes ultrafiltration for dialysis desalination and freeze-drying; the dialysis bag with a molecular weight cutoff of 1 kDa is selected, the liquid is changed every 1 hour, and the dialysis is performed for 8-12 hours; the small molecule peptide solution obtained by dialysis is freeze-dried to obtain a high-purity small molecule peptide.