A method for extracting hypoglycemic active peptides from sheep bones and its application

Abstract

The present invention relates to the technical field of enzymatic extraction of functional peptides, and more particularly to a method and application thereof for extracting hypoglycemic active peptides from sheep bones, S1: bone residue is prepared; S2: bone powder is prepared; S3: first defatting treatment is carried out with a composite degreasing agent, and the composite degreasing agent is mixed by food-grade lipase and sodium glycocholate according to a mass ratio of 1: (0.1-0.15); S4: second defatting treatment is carried out with anhydrous ethanol; S5: enzymolysis treatment is carried out to the obtained defatted bone powder B in sequence by the first protease and the second protease, and the first protease is collagenase, and the second composite protease is mixed by aminopeptidase and serine protease according to a mass ratio of 1: (1-3); S6: after the obtained active peptide suspension is de-enzymed, centrifuged and freeze-dried, hypoglycemic active peptides are obtained. The present invention uses sheep bones as raw materials for the first time, and is efficiently extracted with small molecule hypoglycemic active peptides through secondary defatting and two protease segmented enzymolysis treatments.

Description

Technical Field

[0001] The invention relates to the technical field of enzymatic extraction of functional peptides, in particular to a method for extracting hypoglycemic active peptides from sheep bones and application thereof. Background Art

[0002] Diabetes is a common chronic disease worldwide, the main feature of which is abnormally high blood sugar levels. According to reports, as of 2021, a total of about 537 million adults in the world suffer from diabetes. It is estimated that by 2045, the number of adults with diabetes in the world will increase by about 46%, and the number of adult patients with diabetes will reach about 783 million by then. Therefore, various types of sugar control products have extremely high market prospects. For example, the sales of semaglutide alone by Danish pharmaceutical giant Novo Nordisk in the first half of the year reached about US$12.98 billion.

[0003] In recent years, as hyperglycemia, diabetes and other diseases have attracted widespread attention from all walks of life, hypoglycemic active peptides have been widely developed and applied as a potential bioactive substance for the treatment of diabetes. For example, the invention patent with application number CN202011086514.5 discloses a sheepskin collagen peptide with DPP-IV inhibitory activity and a preparation method thereof, and the sheepskin collagen peptide is prepared by combining chemical hydrolysis and enzymatic hydrolysis; the invention patent with application number CN202110489295.3 discloses a yak bone peptide with hypoglycemic and antioxidant functions and a preparation method thereof, and the yak bone peptide is prepared by a two-step enzymatic hydrolysis method; the invention patent with application number CN202311533246.0 discloses a buckwheat peptide with DPP-IV inhibitory activity and its application, and the buckwheat peptide with DPP-IV inhibitory activity is prepared by buckwheat protein preparation, enzymatic hydrolysis, extraction, separation and purification, etc. As a resource rich in collagen, osteopontin, elastin and other active ingredients, the development of hypoglycemic active peptides using sheep bone raw materials is still a research gap at home and abroad and has not yet been reported in papers or patents. Summary of the invention

[0004] In view of this, the purpose of the present invention is to propose a method for extracting hypoglycemic active peptides from sheep bones, taking sheep bones as raw materials, undergoing secondary defatting with a composite degreasing agent and anhydrous ethanol and two protease segmented enzymatic treatments, and finally extracting hypoglycemic active peptides.

[0005] Based on the above purpose, the present invention provides a method for extracting hypoglycemic active peptides from sheep bones, comprising the following steps:

[0006] S1: Steam the sheep bones under high pressure until the bone joints can be easily crushed, remove impurities, wash and crush into bone residue;

[0007] S2: drying the bone residue obtained in S1, placing it in a grinder, grinding it into bone powder, and then passing it through a 60-80 mesh sieve to obtain bone powder;

[0008] S3: placing the bone powder in pure water, adding a composite degreasing agent, and treating with magnetic stirring at 35-50°C for 2-4 hours to degrease, and then filtering and drying to obtain defatted bone powder A;

[0009] The composite degreasing agent is prepared by mixing food-grade lipase and sodium glycocholate in a mass ratio of 1:(0.1-0.15);

[0010] S4: adding defatted bone powder A to anhydrous ethanol, treating with magnetic stirring at 20-40° C. for 12-24 hours, performing secondary defatting, filtering, washing, filtering again, and drying to obtain defatted bone powder B;

[0011] The degreasing efficiency was greatly improved through secondary degreasing treatment with a composite degreasing agent and anhydrous ethanol, with a degreasing rate of up to 99.4%, without the need for subsequent purification. At the same time, the study found that under the condition of two protease segmented enzymatic hydrolysis treatments in the subsequent S5 step, with the increase of the degreasing rate, the extraction rate of hypoglycemic active peptides also increased, and the two showed a certain positive correlation.

[0012] The reason may be that the fat in bone meal is easy to form emulsion or precipitation during the protease hydrolysis process, affecting the contact between the protease and the target protein and reducing the enzymatic efficiency. At the same time, the presence of fat may also cause physical obstruction or chemical interference to the active site of the protease, further affecting the normal function of the protease. Therefore, by defatting the bone meal, the fat covering the surface of the bone meal is avoided, and the emulsification or precipitation effect of the fat is avoided, so that the protease can more directly contact the collagen, osteomucin, elastin and other proteins in the bone meal matrix. After defatting, the viscosity of the bone meal suspension is also reduced (fat will increase the viscosity of the bone meal suspension), which in turn improves the flow of enzyme molecules and improves the extraction rate of active peptides.

[0013] The inventors also found that compared with a single lipase, a composite degreasing agent that is rationally combined with lipase and sodium glycocholate can more efficiently decompose lipase, because sodium glycocholate can reduce the interfacial tension between water and oil, thereby promoting lipase to contact fat molecules more effectively; and in the presence of sodium glycocholate, fat is dispersed into smaller microemulsions and forms sodium glycocholate-fat molecule mixed micelles, allowing lipase to bind to fat molecules more efficiently, thereby enhancing the catalytic hydrolysis of fat in bone meal by lipase.

[0014] S5: Place defatted bone powder B in pure water, add the first protease, adjust the pH to 7.2-8.2, and treat with magnetic stirring at 35-39°C for 1-2h, then add the second composite protease, adjust the pH to 8.5-9.5, and treat with magnetic stirring at 40-50°C for 1-3h. After two segmented enzymatic hydrolysis treatments, a suspension containing active peptides is obtained;

[0015] The first protease is collagenase;

[0016] The second composite protease is prepared by mixing aminopeptidase and serine protease in a mass ratio of 1:(1-3);

[0017] The segmented enzymatic hydrolysis of bone powder by the first protease and the second protease can effectively improve the extraction rate of hypoglycemic active peptides. First, the first protease (collagenase) is used to hydrolyze bone powder at pH = 7.2-8.2 and 35-39°C, which can effectively extract the active peptide components in collagen protein. Then, the second composite protease (aminopeptidase + serine protease) is used to hydrolyze bone powder at pH = 8.5-9.5 and 40-50°C, which can effectively extract the active peptide components in non-collagen proteins. At the same time, the combined use of the first protease and the second protease makes the enzymatic hydrolysis system have more cleavage sites, making the hydrolysis of sheep bone powder more complete, further improving the extraction rate of active peptides.

[0018] S6: After the active peptide suspension is inactivated in a 100°C water bath for 15-30 minutes, it is cooled at -4°C and centrifuged at 4000-6000 r / min for 10-20 minutes. The supernatant is taken and freeze-dried to obtain a glucose-lowering active peptide with DPP-IV inhibitory effect.

[0019] Preferably, the impurities include but are not limited to residual meat and fascia.

[0020] Preferably, the mass ratio of the bone meal, pure water and composite degreasing agent is 1:10:(0.03-0.05).

[0021] Preferably, the enzyme activity of the food-grade lipase is ≥ 1×10 6 U / g.

[0022] Preferably, the mass ratio of the defatted bone powder A to anhydrous ethanol is 1:(8-12).

[0023] Preferably, the mass ratio of the defatted bone powder B, pure water, the first protease, and the second composite protease is 1:10:(0.05-0.09):(0.1-0.15).

[0024] Preferably, the enzyme activity of the collagenase is ≥10000 U / g.

[0025] Preferably, the enzyme activity of the aminopeptidase is ≥50000 U / g.

[0026] Preferably, the enzyme activity of the serine protease is ≥10000 U / g.

[0027] The present invention further provides the use of the hypoglycemic active peptides extracted according to the method for extracting hypoglycemic active peptides from sheep bones in the preparation of blood sugar-lowering health food.

[0028] Beneficial effects of the present invention:

[0029] The present invention uses sheep bones as raw materials for the first time, and extracts small molecule glucose-lowering active peptides through secondary degreasing with a composite degreasing agent and anhydrous ethanol and two protease segmented enzymatic hydrolysis treatments. The glucose-lowering active peptides have a high extraction rate and high DPP-IV inhibitory activity.

[0030] The synergistic degreasing treatment of composite degreasing agent and anhydrous ethanol greatly improves the degreasing efficiency of bone powder, eliminates the need for subsequent purification treatment, simplifies the procedure, and improves the extraction rate of protein peptides to a certain extent. The reason may be that the fat in bone powder is easy to form emulsion or precipitation during protease hydrolysis, affecting the contact between protease and target protein and reducing enzymatic efficiency. At the same time, the presence of fat may also cause physical hindrance or chemical interference to the active site of protease, further affecting the normal function of protease. Therefore, by degreasing the bone powder, fat covering the surface of bone powder is avoided, and the emulsification or precipitation effect of fat is avoided, so that the protease can more directly contact the collagen, osteomucin, elastic hard protein and other proteins in the bone powder matrix. After degreasing, the viscosity of the bone powder suspension is also reduced (fat will increase the viscosity of the bone powder suspension), which in turn improves the flow of enzyme molecules and improves the extraction rate of active peptides.

[0031] In addition, the extracted hypoglycemic active peptides after defatting also showed better stability. On the one hand, the fat may produce some by-products that affect the stability of the active peptides during the extraction process. On the other hand, the active peptide products that have not been defatted contain a large amount of fat. The chemical properties of fat are unstable and it is easy to oxidize and deteriorate during storage, which accelerates the deterioration of the active peptides. Therefore, the synergistic degreasing treatment of composite degreasing agents and anhydrous ethanol can also improve the stability of hypoglycemic active peptides and extend the shelf life.

[0032] The inventors also found that compared with a single lipase, a composite degreasing agent that is rationally combined with lipase and sodium glycocholate can more efficiently decompose lipase, because sodium glycocholate can reduce the interfacial tension between water and oil, thereby promoting lipase to contact fat molecules more effectively; and in the presence of sodium glycocholate, fat is dispersed into smaller microemulsions and forms sodium glycocholate-fat molecule mixed micelles, allowing lipase to bind to fat molecules more efficiently, thereby enhancing the catalytic hydrolysis of fat in bone meal by lipase.

[0033] Therefore, synergistic defatting treatment is an important step in the process of extracting hypoglycemic active peptides, which helps to improve the purity of active peptide extraction, improve the extraction effect, reduce the difficulty of subsequent processing, and improve the stability of hypoglycemic active peptides, thereby ensuring the quality and yield of hypoglycemic active peptides.

[0034] The present invention can effectively improve the extraction rate of hypoglycemic active peptides by segmented enzymolysis of bone powder by a first protease and a second protease. First, the first protease (collagenase) is used to hydrolyze the bone powder under the conditions of pH=7.2-8.2 and 35-39°C, which can effectively extract the active peptide components in the collagen protein. Then, the second composite protease (aminopeptidase + serine protease) is used to hydrolyze the bone powder under the conditions of pH=8.5-9.5 and 40-50°C, which can effectively extract the active peptide components in non-collagenous proteins. At the same time, the combined use of the first protease and the second protease increases the cleavage sites of the enzymatic hydrolysis system, so that the hydrolysis of the sheep bone powder is more sufficient, and the extraction rate of the active peptides is further improved.

[0035] The bovine bone active peptide of the present invention has high polypeptide content, small molecular weight, high activity, and excellent DPP-IV inhibitory activity. Only 1.0 mg / mL of active peptide can achieve a DPP-IV inhibition rate of 55.8-62.7%, and can be used to prepare a new blood sugar-lowering health food. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0037] The protease activities and suppliers used in the following examples and comparative examples of the present invention are as follows: the activity of serine protease is 10000U / g, purchased from Jiangsu Huace Biotechnology Co., Ltd.; the activity of aminopeptidase is 50000U / g, purchased from Jiangsu Caiwei Biotechnology Co., Ltd.; the activity of collagenase is 10000U / g, purchased from Henan Ruiren Bioengineering Co., Ltd.; the activity of food-grade lipase is 1.0×10 6 U / g, purchased from Jiangsu Caiwei Biotechnology Co., Ltd.

[0038] Example 1 Preparation of bone powder

[0039] Preparation of bone residue: Steam the sheep bones under high pressure until the bone joints can be easily crushed, remove the residual meat, fascia and other impurities, wash and crush into bone residue;

[0040] Preparation of bone powder: After drying the obtained bone residue, place it in a grinder, grind it into bone powder, and then pass it through an 80-mesh sieve to obtain bone powder, and divide the obtained bone powder into ten equal parts, wherein the mass of any one part is recorded as M0, and the protein content of one part of the bone powder raw material is determined according to the Kjeldahl nitrogen determination method of GB 5009.5-2016, recorded as Md1, and the fat content of another part of the bone powder raw material is determined according to the Soxhlet extraction method of GB 5009.6-2016, recorded as Md2, and the remaining eight equal parts of bone powder are used as the extraction raw materials of the hypoglycemic active peptides in the following Examples 2-4 and Comparative Examples 1-5, respectively.

[0041] Example 2 Extraction of hypoglycemic active peptides

[0042] A. First defatting treatment: an equal portion of the bone powder in Example 1 is placed in pure water, a composite defatting agent is added, and the composite defatting agent is prepared by mixing food-grade lipase and sodium glycocholate in a mass ratio of 1:0.1, and the mass ratio of bone powder, pure water, and composite defatting agent is controlled to be 1:10:0.03, and the mixture is subjected to magnetic stirring at 35° C. for 2 h for defatting, filtering, and drying to obtain defatted bone powder A;

[0043] B. Second defatting treatment: add defatted bone powder A to 8 times its mass of anhydrous ethanol, stir under magnetic stirring at 20℃ for 12h, perform secondary defatting treatment, filter, wash, filter again, and dry to obtain defatted bone powder B, which is weighed and recorded as M1;

[0044] C. Double enzymatic hydrolysis method to prepare active peptide suspension:

[0045] C1. Preparation:

[0046] First protease: collagenase;

[0047] The second composite protease is a mixture of aminopeptidase and serine protease in a mass ratio of 1:1;

[0048] C2. Double enzymatic hydrolysis: defatted bone powder B was placed in pure water, the first protease was added, the pH was adjusted to 7.2, and the mixture was treated with magnetic stirring at 35°C for 1 hour. The second composite protease was then added, and the mass ratio of defatted bone powder B, pure water, the first protease, and the second composite protease was 1:10:0.05:0.1. The pH was adjusted to 8.5, and the mixture was treated with magnetic stirring at 40°C for 1 hour. After two segmented enzymatic hydrolysis treatments, an active peptide suspension was obtained;

[0049] D. Preparation of hypoglycemic active peptides: The active peptide suspension was inactivated in a water bath at 100°C for 15 min, cooled at -4°C, centrifuged at 4000 r / min for 10 min, the supernatant was taken, and freeze-dried to obtain hypoglycemic active peptides with DPP-IV inhibitory effect. The protein content was determined according to the Kjeldahl method according to GB 5009.5-2016, recorded as Mt.

[0050] Example 3 Extraction of glucose-lowering active peptides

[0051] A. First defatting treatment: an equal portion of the bone powder in Example 1 is placed in pure water, a composite defatting agent is added, and the composite defatting agent is prepared by mixing food-grade lipase and sodium glycocholate in a mass ratio of 1:0.125, and the mass ratio of bone powder, pure water, and composite defatting agent is controlled to be 1:10:0.04, and the mixture is subjected to magnetic stirring at 40° C. for 3 h for defatting, filtering, and drying to obtain defatted bone powder A;

[0052] B. Second defatting treatment: add defatted bone powder A to 10 times its mass of anhydrous ethanol, stir under magnetic stirring at 30°C for 18 hours, perform secondary defatting treatment, filter, wash, filter again, and dry to obtain defatted bone powder B, which is weighed and recorded as M1;

[0053] C. Double enzymatic hydrolysis method to prepare active peptide suspension:

[0054] C1. Preparation:

[0055] First protease: collagenase;

[0056] The second composite protease is a mixture of aminopeptidase and serine protease in a mass ratio of 1:2;

[0057] C2. Double enzymatic hydrolysis: defatted bone powder B was placed in pure water, the first protease was added, the pH was adjusted to 7.7, and the mixture was treated with magnetic stirring at 37°C for 1.5 hours. The second composite protease was then added, and the mass ratio of defatted bone powder B, pure water, the first protease, and the second composite protease was 1:10:0.07:0.125, and the pH was adjusted to 9.0, and the mixture was treated with magnetic stirring at 45°C for 2 hours. After two segmented enzymatic hydrolysis treatments, an active peptide suspension was obtained;

[0058] D. Preparation of hypoglycemic active peptides: Inactivate the enzyme in a water bath containing active peptides for 20 min at 100°C, cool at -4°C, centrifuge at 5000 r / min for 15 min, take the supernatant, freeze-dry, and obtain hypoglycemic active peptides with DPP-IV inhibitory effect. Determine the protein content according to the Kjeldahl nitrogen determination method of GB 5009.5-2016, recorded as Mt.

[0059] Example 4 Extraction of glucose-lowering active peptides

[0060] A. First defatting treatment: Place an equal portion of the bone powder in Example 1 in pure water, add a composite defatting agent, and the composite defatting agent is prepared by mixing food-grade lipase and sodium glycocholate in a mass ratio of 1:0.15, and control the mass ratio of bone powder, pure water, and composite defatting agent to be 1:10:0.05, and treat with magnetic stirring at 50° C. for 4 h to perform defatting, and after filtering and drying, obtain defatted bone powder A;

[0061] B. Second defatting treatment: add defatted bone powder A to 12 times its mass of anhydrous ethanol, stir under magnetic stirring at 40°C for 24 hours, perform secondary defatting treatment, filter, wash, filter again, and dry to obtain defatted bone powder B, which is weighed and recorded as M1;

[0062] C. Double enzymatic hydrolysis method to prepare active peptide suspension:

[0063] C1. Preparation:

[0064] First protease: collagenase;

[0065] The second composite protease is a mixture of aminopeptidase and serine protease in a mass ratio of 1:3;

[0066] C2. Double enzymatic hydrolysis: defatted bone powder B was placed in pure water, the first protease was added, the pH was adjusted to 8.2, and the mixture was treated with magnetic stirring at 39°C for 2 hours. The second composite protease was then added, and the mass ratio of defatted bone powder B, pure water, the first protease, and the second composite protease was 1:10:0.09:0.15. The pH was adjusted to 9.5, and the mixture was treated with magnetic stirring at 50°C for 3 hours. After two segmented enzymatic hydrolysis treatments, an active peptide suspension was obtained;

[0067] D. Preparation of hypoglycemic active peptides: The active peptide suspension was inactivated in a water bath at 100°C for 30 min, cooled at -4°C, centrifuged at 6000 r / min for 20 min, the supernatant was taken, and freeze-dried to obtain hypoglycemic active peptides with DPP-IV inhibitory effect. The protein content was determined according to the Kjeldahl method according to GB 5009.5-2016, recorded as Mt.

[0068] Comparative Example 1 is the same as Example 2, except that there is no second degreasing process of S4, that is, degreasing is performed only with the composite degreasing agent, and the weight is recorded as M1.

[0069] Comparative Example 2 is the same as Example 2, except that there is no S3 first degreasing process, that is, degreasing is directly performed only with anhydrous ethanol.

[0070] Comparative Example 3 is the same as Example 2, except that food grade lipase is used instead of the composite degreasing agent.

[0071] Comparative Example 4 is the same as Example 2, except that sodium lauroyl glutamate is used instead of sodium glycocholate.

[0072] Comparative Example 5 is the same as Example 2, except that: during the S5 operation, collagenase was not used as the first protease for enzymatic hydrolysis; specifically, defatted bone powder B was placed in pure water, the second composite protease was added, and the mass ratio of defatted bone powder B, pure water, and the second composite protease was 1:10:0.1, and the pH was adjusted to 8.5, and the mixture was treated with magnetic stirring at 40°C for 1 hour. After two segmented enzymatic hydrolysis treatments, an active peptide suspension was obtained.

[0073] The degreasing rates of Examples 2-4 and Comparative Examples 1-5 were calculated according to the formula 100%×(M0-M1) / Md2;

[0074] The protein extraction rates of Examples 2-4 and Comparative Examples 1-5 were calculated according to the formula 100%×Mt / Md1.

[0075] Table 1 lists the degreasing rate and protein extraction rate of Examples 2-4 and Comparative Examples 1-5:

[0076] Table 1

[0077] As shown in Table 1, the degreasing rate and protein extraction rate of the method for extracting hypoglycemic active peptides in Example 2-4 are significantly better than those in Comparative Examples 1-5, wherein the degreasing rate is as high as 99.4% and the protein extraction rate is as high as 69.3%. At the same time, it can be seen from the data of Example 2-4 and Comparative Examples 1-4 that under the condition that the subsequent S5 step of the embodiment and the comparative example is subjected to two protease segmented enzymolysis treatments, as the degreasing rate increases, the protein extraction rate is also higher, and the two show a certain positive correlation, which also shows that the present invention also improves the protein peptide extraction rate to a certain extent by the synergistic degreasing treatment of the composite degreasing agent and anhydrous ethanol. In addition, compared to Example 2, the protein extraction rate of Comparative Example 5, which is not treated with collagenase, is the lowest, which shows that the present invention has an important influence on improving the extraction rate of active peptides by segmented enzymolysis of collagenase and "aminopeptidase + serine protease", and further combined with the data of Comparative Examples 1-4, it can be concluded that the present invention is treated by a specific "secondary degreasing + two segmented enzymolysis of protease", which helps to efficiently extract small molecule hypoglycemic active peptides.

[0078] From the data of Comparative Example 2 and Comparative Examples 1-2, it can be seen that the degreasing efficiency is greatly improved by the secondary degreasing treatment of the composite degreasing agent and anhydrous ethanol; from the data of Comparative Example 2 and Comparative Examples 3-4, it can be seen that sodium glycocholate and sodium lauroyl glutamate both have a certain promoting effect on the hydrolysis of fat by lipase, but the catalytic hydrolysis of sodium glycocholate on lipase is superior to that of sodium lauroyl glutamate. The reason may be that sodium glycocholate disperses fat into smaller microemulsions and forms sodium glycocholate-fat molecule mixed micelles, which enables lipase to bind to fat molecules more efficiently, thereby enhancing the catalytic hydrolysis of fat in bone meal by lipase.

[0079] The hypoglycemic effect of the active peptides prepared in Examples 1-3 was evaluated by measuring the DPP-IV inhibition rate: the active peptide lyophilized powder was prepared into a 1.0 mg / mL sample solution using a pH = 8.0 Tris-HCl buffer, and a pH = 8.0 Tris-HCl buffer was used as a control solution, and the samples were mixed with the substrate (Gly-Pro-pNA), incubated at 37°C, and then the DPP-IV enzyme solution was added, and the absorbance at 405 nm was measured. According to the formula 100% × (S 对照 -S 样品 ) / S 对照 Calculate the DPP-IV inhibition rate, where S 对照 and S 样品 Respectively represent the slope of absorbance change of Tris-HCl-substrate solution and active peptide-substrate solution over time.

[0080] Table 2 lists the glucose-lowering active peptide DPP-IV inhibition rates extracted from Examples 2-4:

[0081] Table 2

[0082] As shown in Table 2, the glucose-lowering active peptides extracted from Examples 2-4 have high DPP-IV inhibitory activity, and only 1.0 mg / mL of active peptides can achieve a DPP-IV inhibition rate of 55.8-62.7%, which can be used to prepare new glucose-lowering health foods.

[0083] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0084] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting hypoglycemic active peptides from sheep bones, characterized in that: The following steps are involved: S1: Steam the sheep bones under high pressure until the bone joints can be easily crushed, remove impurities, wash and crush into bone residue; S2: drying the bone residue obtained in S1, placing it in a grinder, grinding it into coarse bone powder, and then passing it through a 60-80 mesh sieve to obtain bone powder; S3: placing the bone powder in pure water, adding a composite degreasing agent, and treating with magnetic stirring at 35-50°C for 2-4 hours to degrease, and then filtering and drying to obtain defatted bone powder A; The composite degreasing agent is prepared by mixing food-grade lipase and sodium glycocholate in a mass ratio of 1:(0.1-0.15); S4: adding defatted bone powder A to anhydrous ethanol, treating with magnetic stirring at 20-40° C. for 12-24 hours, performing secondary defatting, filtering, washing, filtering again, and drying to obtain defatted bone powder B; S5: Place defatted bone powder B in pure water, add the first protease, adjust the pH to 7.2-8.2, and treat with magnetic stirring at 35-39°C for 1-2h, then add the second composite protease, adjust the pH to 8.5-9.5, and treat with magnetic stirring at 40-50°C for 1-3h. After two segmented enzymatic hydrolysis treatments, a suspension containing active peptides is obtained; The first protease is collagenase; The second composite protease is prepared by mixing aminopeptidase and serine protease in a mass ratio of 1:(1-3); S6: After the active peptide suspension is inactivated in a 100°C water bath for 15-30 minutes, it is cooled at -4°C and centrifuged at 4000-6000 r / min for 10-20 minutes. The supernatant is taken and freeze-dried to obtain a glucose-lowering active peptide with DPP-IV inhibitory effect.

2. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The impurities include but are not limited to residual meat and fascia.

3. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The mass ratio of the bone powder, pure water and composite degreasing agent is 1:10:(0.03-0.05).

4. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The enzyme activity of the food-grade lipase is ≥1×10 6 U / g.

5. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The mass ratio of the defatted bone powder A to anhydrous ethanol is 1:(8-12).

6. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The mass ratio of the defatted bone powder B, pure water, the first protease and the second composite protease is 1:10:(0.05-0.09):(0.1-0.15).

7. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The enzyme activity of the collagenase is ≥10000 U / g.

8. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The enzyme activity of the aminopeptidase is ≥50000 U / g.

9. The method for extracting hypoglycemic active peptides from sheep bones according to claim 1, characterized in that: The enzyme activity of the serine protease is ≥10000 U / g.

10. Use of the hypoglycemic active peptide extracted by the method for extracting hypoglycemic active peptide from sheep bones according to any one of claims 1 to 9 in the preparation of health food that assists in lowering blood sugar.

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