Yak small molecule peptide as well as preparation method and application thereof
By using complex enzymes for enzymatic decomposition, the problems of low content of high-active peptides and long enzymatic decomposition time in yak small molecule peptides were solved, and the efficient extraction of high-active peptides was achieved and the production cycle was shortened.
Patent Information
- Application Number
- CN202510193970.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-21
- Publication Date
- 2025-05-27
AI Technical Summary
In the prior art, when extracting small molecule peptides from yak bone marrow, the content of high-active peptides is low and the enzymatic time is long, resulting in a longer production cycle.
Complex enzymes (including elastase, ginger protease, trypsin and flavor protease) are used for enzymatic decomposition. By adjusting the mass ratio of the enzyme and the enzymatic decomposition conditions, the enzymatic decomposition time is shortened and the proportion of highly active peptides is increased.
The proportion of ultra-low molecular weight peptides in yak small molecule peptides has been increased to exceed 50%, shortening the enzymatic decomposition time, and improving the biological activity and production efficiency of the product.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of small molecule peptide preparation, and particularly relates to a yak small molecule peptide, a preparation method thereof, and an application thereof. Background Art
[0002] Qinghai is the largest yak production base in China, accounting for more than one-third of the total yak population in the country, and is widely known as the "World Yak Capital". Yak is a unique cattle species in alpine regions and is the mammal that lives at the highest altitude in the world. It is mainly produced in areas above 3000 meters above sea level on the Qinghai-Tibet Plateau in China. Yaks can adapt to alpine ecological conditions, are resistant to roughage and fatigue, are good at walking on steep slopes, dangerous roads, snow-capped mountains and swamps, and can swim across rivers and rapids, and are known as the "ship of the plateau". The unique plateau environment enables yaks to have extremely strong immunity, and the strong ultraviolet radiation makes yaks a treasure house of sterile resources. The natural grazing method makes yaks have unique nutritional value and reliable safety. Therefore, yak is an excellent raw material for extracting collagen peptides.
[0003] Small molecule active peptide is a biochemical substance between amino acids and proteins. It has a smaller molecular weight than proteins and a larger molecular weight than amino acids, and is a fragment of a protein. When two or more amino acids are connected by peptide bonds, the formed "amino acid chain" or "amino acid string" is called a peptide. Among them, peptides composed of 10 to 15 or more amino acids are called polypeptides, those composed of 2 to 9 amino acids are called oligopeptides, and those composed of 2 to 15 amino acids are called small molecule peptides or small peptides.
[0004] In recent years, small molecule peptides have attracted extensive attention in the fields of biomedicine and nutritional health products. Their characteristics of small molecular weight, high biological activity, and easy absorption have shown great potential in promoting human health. Yak, as a unique species living in alpine regions, its bone marrow is rich in various bioactive components, especially small molecule peptides. Therefore, the research and development of the technology for extracting small molecule peptides from yak bone marrow has important practical significance.
[0005] The extraction process of small molecule peptides from yak bone marrow usually includes key steps such as crushing, enzymatic hydrolysis, separation and purification, etc. Among them, enzymatic hydrolysis is the key link. By selecting appropriate enzymes and optimizing the enzymatic hydrolysis conditions, the proteins in the bone marrow can be degraded into small molecule peptides. However, despite the continuous maturity of extraction technology, the prepared yak bone marrow peptides by existing methods have problems such as a relatively low proportion of small molecular weight peptides and a relatively long production cycle due to multiple enzymatic hydrolyses. Summary of the Invention
[0006] The purpose of the present invention is to provide a preparation method and an application of yak small molecule peptides, so as to solve the problems of relatively low content of highly active peptides and long enzymatic hydrolysis time in yak small molecule peptides.
[0007] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:
[0008] The present invention provides a preparation method of yak small molecule peptides, comprising the following steps:
[0009] S1. Using yak bones as raw materials, after crushing, boiling, and filtering, collect the filtrate;
[0010] S2. Perform degreasing treatment on the filtrate to obtain yak bone protein solution;
[0011] S3. Mix the yak bone protein solution with a composite enzyme and perform enzymatic hydrolysis to obtain an enzymatic hydrolysate;
[0012] S4. Perform enzyme inactivation treatment on the enzymatic hydrolysate, centrifuge, and ultrafilter to remove residues to obtain a small molecule peptide solution;
[0013] S5. Concentrate the small molecule peptide solution and then perform spray drying to obtain the product;
[0014] The composite enzyme includes elastase, ginger protease, trypsin, and flavor protease, and the mass ratio of elastase, ginger protease, trypsin, and flavor protease is 1:1.4 - 1.8:1.4 - 1.8:2.5 - 3.5.
[0015] Preferably, the mass - to - volume ratio of the yak bones to water is 1g:4 - 5mL, the boiling temperature is 100 - 120°C, and the boiling time is 3.5 - 4.5h.
[0016] Preferably, the degreasing treatment is centrifugation, the rotation speed of the centrifugation is 4000 - 8000rpm, and the centrifugation time is 20 - 30min.
[0017] Preferably, first adjust the pH of the yak bone protein solution to 5.8 - 6.5, and then add the composite enzyme for enzymatic hydrolysis. The addition amount of the composite enzyme is 0.5 - 1% of the yak bone protein solution.
[0018] Preferably, the temperature of the enzymatic hydrolysis is 48 - 52°C, and the enzymatic hydrolysis time is 140 - 180min.
[0019] Preferably, the temperature of the enzyme inactivation is 95 - 105°C, and the enzyme inactivation time is 18 - 22min.
[0020] Preferably, in S4, the rotation speed of the centrifugation is 12000 - 16000rpm, and the centrifugation time is 8 - 12min; after centrifugation, ultrafiltration is performed using a 2000Da ultrafiltration membrane.
[0021] The present invention also provides the yak small molecule peptides prepared by the above - mentioned preparation method.
[0022] The present invention also provides the application of the yak small molecule peptide in the preparation of a functional food for assisting in reducing blood sugar.
[0023] By adopting the above technical solutions, the present invention has the following beneficial effects: The preparation of the yak small molecule peptide of the present invention includes steps of boiling, degreasing treatment, complex enzyme hydrolysis, centrifugation, ultrafiltration, concentration and spray drying. The complex enzyme is composed of elastase, ginger protease, trypsin and flavor protease in a specific mass ratio. By specific complex enzymes and hydrolysis conditions, the hydrolysis time is shortened, and the proportion of ultra-low molecular weight peptides in the yak small molecule peptide exceeds 50%, which is beneficial to human absorption. The yak small molecule peptide of the present invention has the efficacy of assisting in reducing the postprandial blood sugar level of hyperglycemic patients, can effectively prevent the rapid rise of postprandial blood sugar value, and reduce the harm caused by hyperglycemia to the body. Detailed implementation manners
[0024] The present invention provides a method for preparing a yak small molecule peptide, including the following steps:
[0025] S1. Using yak bones as raw materials, after crushing, boiling and filtering, collecting the filtrate;
[0026] S2. Performing degreasing treatment on the filtrate to obtain yak bone protein solution;
[0027] S3. Mixing the yak bone protein solution with a complex enzyme for hydrolysis to obtain a hydrolysate;
[0028] S4. Performing enzyme inactivation treatment on the hydrolysate, centrifuging and ultrafiltering to remove residues to obtain a small molecule peptide solution;
[0029] S5. Concentrating the small molecule peptide solution and then performing spray drying to obtain the product;
[0030] The complex enzyme includes elastase, ginger protease, trypsin and flavor protease, and the mass ratio of elastase, ginger protease, trypsin and flavor protease is 1:1.4 - 1.8:1.4 - 1.8:2.5 - 3.5.
[0031] The present invention first cleans yak bones and then crushes them. The length of the crushed yak bones is 2-3 cm. The crushed yak bones are boiled, and the boiling liquid is filtered to collect the filtrate. The mass-volume ratio of yak bones to water is 1 g:4-5 mL, further preferably 1 g:4.3-4.7 mL, and more preferably 1 g:4.5 mL. The boiling temperature is 100-120 °C, further preferably 105-115 °C, and more preferably 110 °C. The boiling time is 3.5-4.5 h, more preferably 3 h. The boiling liquid is filtered to collect the filtrate. The filtrate is degreased to obtain yak bone protein solution. The degreasing treatment is centrifugation. The rotation speed of the centrifugation is 4000-8000 rpm, further preferably 5000-7000 rpm, and more preferably 6000 rpm. The centrifugation time is 20-30 min, further preferably 22-28 min, and more preferably 25 min. After centrifugation, the fat layer is discarded to obtain yak bone protein solution.
[0032] Adjust the pH of the yak bone protein solution to 5.8-6.5, further preferably 6.0-6.3, and more preferably 6.2. Then add a complex enzyme for enzymatic hydrolysis. The addition amount of the complex enzyme is 0.5-1% of the yak bone protein solution, further preferably 0.6-0.9%, and more preferably 0.8%. The complex enzyme includes elastase, ginger protease, trypsin, and flavor protease. The mass ratio of elastase, ginger protease, trypsin, and flavor protease is 1:1.4-1.8:1.4-1.8:2.5-3.5, further preferably 1:1.5-1.7:1.5-1.7:2.8-3.3, and more preferably 1:1.6:1.6:3. The enzymatic hydrolysis temperature is 48-52 °C, further preferably 49-51 °C, and more preferably 50 °C. The enzymatic hydrolysis time is 140-180 min, further preferably 150-170 min, and more preferably 160 min. After enzymatic hydrolysis is completed, an enzymatic hydrolysate is obtained.
[0033] The enzymatic hydrolysate is inactivated. The inactivation temperature is 95-105 °C, further preferably 97-102 °C, and more preferably 100 °C. The inactivation time is 18-22 min, further preferably 19-21 min, and more preferably 20 min. The inactivated enzymatic hydrolysate is centrifuged at high speed. The rotation speed of the high-speed centrifugation is 12000-16000 rpm, further preferably 13000-15000 rpm, and more preferably 14000 rpm. The high-speed centrifugation time is 8-12 min, further preferably 9-11 min, and more preferably 10 min. After high-speed centrifugation, the residue is discarded, and ultrafiltration is carried out with a 2000 Da ultrafiltration membrane to obtain a small molecule peptide solution.
[0034] After concentrating the small molecule peptide solution and spray drying, yak small molecule peptide is obtained. The concentration is preferably falling film concentration.
[0035] The present invention also provides yak small molecule peptide prepared by the above preparation method.
[0036] The present invention also provides the application of the yak small molecule peptide in the preparation of functional foods for assisting in reducing blood sugar.
[0037] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0038] The elastase in the embodiments of the present invention is purchased from Shanghai Kanglang Biotechnology Co., Ltd., and the enzyme activity is 30 U / mg; the flavor protease and ginger protease are purchased from Nanjing Tongying Biotechnology Co., Ltd., and the enzyme activities are 50 U / mg and 100 U / mg respectively; the trypsin is purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the enzyme activity is 30 U / mg.
[0039] Example 1
[0040] Wash 10 kg of yak bones, then crush them into pieces with a length of 2 - 3 cm, add 40 L of water, boil at 100 °C for 4.5 h, filter the boiling solution, and collect the filtrate. Centrifuge the filtrate at 4000 rpm for 30 min for defatting, discard the fat layer after centrifugation to obtain yak bone protein solution. Adjust the pH of the yak bone protein solution to 5.8, add a composite enzyme accounting for 0.5% of the total mass of the yak bone protein solution for enzymatic hydrolysis. The composite enzyme includes elastase, ginger protease, trypsin, and flavor protease, and the mass ratio of elastase, ginger protease, trypsin, and flavor protease is 1:1.4:1.4:2.5. After adding the enzymatic hydrolysis solution, carry out enzymatic hydrolysis at 48 °C for 180 min to obtain an enzymatic hydrolysis solution. Heat the enzymatic hydrolysis solution at 100 °C for 20 min for enzyme inactivation treatment, and then decolorize it with activated carbon. Centrifuge the enzyme-inactivated and decolorized enzymatic hydrolysis solution at 12000 rpm for 12 min, discard the residue, and then carry out ultrafiltration with a 2000 Da ultrafiltration membrane to obtain a small molecule peptide solution. After falling film concentration of the small molecule peptide solution and spray drying, yak small molecule peptide is obtained.
[0041] Example 2
[0042] Wash 10 kg of yak bones thoroughly, then crush them into pieces with a length of 2 - 3 cm, add 45 L of water, boil at 110 °C for 4 h, then filter the boiling solution and collect the filtrate. Centrifuge the filtrate at 6000 rpm for 25 min for defatting, discard the fat layer after centrifugation to obtain yak bone protein solution. Adjust the pH of the yak bone protein solution to 6.2, add a composite enzyme accounting for 0.8% of the total mass of the yak bone protein solution for enzymatic hydrolysis. The composite enzyme includes elastase, ginger protease, trypsin, and flavor protease, and the mass ratio of elastase, ginger protease, trypsin, and flavor protease is 1:1.6:1.6:3. After adding the enzymatic hydrolysis solution, carry out enzymatic hydrolysis at 50 °C for 160 min to obtain an enzymatic hydrolysis solution. Heat the enzymatic hydrolysis solution at 100 °C for 20 min for enzyme inactivation treatment, and then decolorize it with activated carbon. Centrifuge the enzyme-inactivated and decolorized enzymatic hydrolysis solution at 14000 rpm at high speed for 10 min, discard the residue, and then carry out ultrafiltration with a 2000 Da ultrafiltration membrane to obtain a small molecule peptide solution. After the small molecule peptide solution is concentrated by falling film and then spray-dried, yak small molecule peptides are obtained.
[0043] Example 3
[0044] Wash 10 kg of yak bones thoroughly, then crush them into pieces with a length of 2 - 3 cm, add 50 L of water, boil at 120 °C for 3.5 h, then filter the boiling solution and collect the filtrate. Centrifuge the filtrate at 8000 rpm for 20 min for defatting, discard the fat layer after centrifugation to obtain yak bone protein solution. Adjust the pH of the yak bone protein solution to 6.5, add a composite enzyme accounting for 1% of the total mass of the yak bone protein solution for enzymatic hydrolysis. The composite enzyme includes elastase, ginger protease, trypsin, and flavor protease, and the mass ratio of elastase, ginger protease, trypsin, and flavor protease is 1:1.8:1.8:3.5. After adding the enzymatic hydrolysis solution, carry out enzymatic hydrolysis at 52 °C for 140 min to obtain an enzymatic hydrolysis solution. Heat the enzymatic hydrolysis solution at 100 °C for 20 min for enzyme inactivation treatment, and then decolorize it with activated carbon. Centrifuge the enzyme-inactivated and decolorized enzymatic hydrolysis solution at 16000 rpm at high speed for 8 min, discard the residue, and then carry out ultrafiltration with a 2000 Da ultrafiltration membrane to obtain a small molecule peptide solution. After the small molecule peptide solution is concentrated by falling film and then spray-dried, yak small molecule peptides are obtained.
[0045] Comparative Example 1
[0046] Differing from Example 1, the composite enzyme does not contain elastase, and an equal amount of trypsin is supplemented.
[0047] Comparative Example 2
[0048] Differing from Example 1, an equal amount of neutral protease is used to replace flavor protease.
[0049] Comparative Example 3
[0050] Different from Example 1, the mass ratio of elastase, ginger protease, trypsin and flavor protease is 1:1.6:1.6:2.
[0051] Comparative Example 4
[0052] Different from Example 1, the temperature of enzymatic hydrolysis is 58 °C.
[0053] Experimental Example 1
[0054] The molecular weight distributions of the bovine bone marrow small molecule peptides prepared in Examples 1-3 and Comparative Examples 1-4 were measured. The measurement method was carried out according to the method in "Zhang Xiaonan et al. Determination of the molecular weight of small molecule polypeptides by Glycine-SDS-PAGE. 2001". The measurement results are shown in Table 1.
[0055] Table 1 Molecular weight distribution of bovine bone marrow small molecule peptides in different groups
[0056] Treatment group <180Da (%) 180 - 480Da (%) 480 - 1000Da (%) Example 1 13.92 56.31 12.57 Example 2 11.29 50.24 18.82 Example 3 12.46 55.18 15.26 Comparative Example 1 8.35 28.19 28.82 Comparative Example 2 10.83 36.25 20.37 Comparative Example 3 9.37 40.28 23.39 Comparative Example 4 5.62 30.27 24.81
[0057] The small molecule peptides with a molecular weight of 180-480 daltons are ultra-low molecular weight peptides composed of 2-4 amino acids, which are highly active peptides. As can be seen from Table 1, in the present invention, by selecting elastase, ginger protease, trypsin and flavor protease and adjusting the mass ratio, the yak bone protein solution is enzymatically hydrolyzed, shortening the enzymatic hydrolysis time and increasing the proportion of highly active peptides, exceeding 50%. However, by changing the type and ratio of enzymes, or changing the conditions of enzymatic hydrolysis, the content of highly active peptides decreases, indicating that under the enzymatic hydrolysis conditions of the present invention, highly active peptides with high content can be obtained.
[0058] Experimental Example 2. Hypoglycemic experiment of yak small molecule peptides
[0059] Experimental animals: SD rats, purchased from the Experimental Animal Center of Beijing University of Chinese Medicine, with half males and half females, weighing 180-220 g. The above SD rats were randomly divided into 9 groups, with 10 rats in each group, 5 males and 5 females in each group. The SD rats in each group could drink water and eat freely. After 1 week of adaptive feeding, the SD rats in each group were fasted for 4 h, and the fasting blood glucose was measured. Then, they were fasted for 24 h (drinking water freely during this period) and injected with alloxan at a dose of 125 mg / kg for modeling. After 6 days, the blood glucose was measured after the animals were fasted for 4 h. A blood glucose value > 11 mmol / L indicated successful establishment of the hyperglycemic model.
[0060] 80 hyperglycemic model rats were selected and randomly divided into 8 groups, including a blank control group and 7 treatment groups. They were housed individually, fed and watered normally, and adaptively fed for 4 days. From the 5th day, their blood glucose was measured regularly at 8:00 am every day and then they were given gastric gavage. The rats in treatment groups 1 - 3 were given gastric gavage with the yak small molecule peptides prepared in Examples 1 - 3 respectively, and the rats in treatment groups 4 - 7 were given gastric gavage with the yak small molecule peptides prepared in Comparative Examples 1 - 4 respectively. The gavage dose was 2 g / (kg·d), and the blank control group was given gastric gavage with 2 mL / (kg·d) of normal saline for 30 consecutive days. The hypoglycemic effects are shown in Table 2.
[0061] Table 2 Comparison of the hypoglycemic effects of diabetic rats in each treatment group
[0062]
[0063] As can be seen from Table 2, the yak small molecule peptides prepared in Examples 1 - 3 have significant hypoglycemic effects, and the effects are better than those in Comparative Examples 1 - 4. The maximum percentage reduction in the blood glucose concentration of the rats reaches 39.31%, indicating that the yak small molecule peptides prepared in this application can be used for the preparation of functional foods for assisting in hypoglycemia.
[0064] As can be seen from the above examples, the present invention provides a yak small molecule peptide, its preparation method and application. The yak small molecule peptide prepared by the present invention is rich in highly active peptides, which is beneficial to human absorption and can be used for the preparation of functional foods for assisting in hypoglycemia.
[0065] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. A method for preparing yak small molecule peptide, characterized in that: The following steps are involved: S1. Using yak bones as raw materials, crushing, boiling, filtering, and collecting the filtrate; S2. The filtrate is defatted to obtain yak bone protein solution; S3. The yak bone protein solution is mixed with a compound enzyme and enzymatically hydrolyzed to obtain an enzymatic solution; S4. The enzymatic hydrolysate is subjected to enzyme inactivation treatment, centrifugation, ultrafiltration, and removal of residues to obtain a small molecule peptide solution; S5. The small molecule peptide solution is concentrated and spray-dried to obtain; The complex enzyme comprises elastase, ginger protease, trypsin and flavor protease, and the mass ratio of elastase, ginger protease, trypsin and flavor protease is 1:1.4-1.8:1.4-1.8:2.5-3.
5.
2. The preparation method according to claim 1, characterized in that: The mass volume ratio of the yak bone to water is 1 g:4-5 mL, the boiling temperature is 100-120° C., and the boiling time is 3.5-4.5 h.
3. The preparation method according to claim 1, characterized in that: The degreasing treatment is centrifugation, the centrifugal speed is 4000-8000 rpm, and the centrifugal time is 20-30 minutes.
4. The preparation method according to claim 1, characterized in that: Firstly, the pH of the yak bone protein liquid is adjusted to 5.8-6.5, and then a compound enzyme is added for enzymolysis, wherein the addition amount of the compound enzyme is 0.5-1% of the mass of the yak bone protein liquid.
5. The preparation method according to claim 4, characterized in that: The temperature of the enzymolysis is 48-52° C., and the time of the enzymolysis is 140-180 min.
6. The preparation method according to claim 1, characterized in that: The enzyme inactivation temperature is 95-105° C., and the enzyme inactivation time is 18-22 minutes.
7. The preparation method according to claim 1, characterized in that: S4: The centrifugal speed is 12000-16000 rpm, and the centrifugal time is 8-12 min; after centrifugation, ultrafiltration is performed using a 2000Da ultrafiltration membrane.
8. Yak small molecule peptide prepared according to any one of the preparation methods of claims 1 to 7.
9. Use of the yak small molecule peptide according to claim 8 in the preparation of functional foods that assist in lowering blood sugar.