Sheep three-medullary peptide capable of enhancing immunity as well as preparation method and application of sheep three-medullary peptide

Abstract

The invention discloses sheep three-medullary peptide for enhancing immunity and a preparation method and application thereof, and relates to the technical field of sheep three-medullary peptide preparation. The preparation method of the sheep three-medullary peptide for enhancing immunity comprises the following steps: performing homogenization treatment on sheep three medullary, and removing a grease layer to obtain an extraction stock solution; mixing the extraction stock solution with a compound enzyme, and carrying out primary enzymolysis to obtain a first enzymatic hydrolysate, the compound enzyme comprising lipase, hyaluronidase and chondroitinase ABC; carrying out enzyme deactivation treatment on the first enzymatic hydrolysate, then mixing the first enzymatic hydrolysate with alkaline protease, and carrying out secondary enzymolysis to obtain a second enzymatic hydrolysate; mixing the second enzymatic hydrolysate with flavourzyme, and performing enzymolysis for three times to obtain a third enzymatic hydrolysate; centrifuging the third enzymatic hydrolysate, collecting supernate, and then carrying out ultrafiltration and concentration treatment to obtain a sheep three-medullary peptide concentrated solution; and carrying out spray drying on the sheep three-medullary peptide concentrated solution to obtain the sheep three-medullary peptide for enhancing the immunity. The sheep three-medullary peptide prepared by the invention has the effect of enhancing immunity.

Description

Technical Field

[0001] The invention relates to the technical field of sheep trimyelin preparation, and in particular to a sheep trimyelin capable of enhancing immunity, and a preparation method and application thereof. Background Art

[0002] Peptides are substances in which amino acids are connected by peptide bonds. They are a class of compounds between amino acids and proteins. At present, more and more peptide products are developed in the market of ordinary and functional foods. Sheep trimyelin peptide is a bioactive peptide extracted from the brain, spinal cord and bone marrow of sheep. It is rich in various amino acids and has potential nutritional value and pharmacological activity, especially in enhancing immune function. Sheep trimyelin peptide is believed to be able to regulate the human immune system and promote health due to its unique composition and structure.

[0003] Enzymatic hydrolysis is one of the common technical means for the food industry to produce sheep trimyelin peptides. It has the advantages of mild process conditions, little damage to amino acids, and high nutritional value of the protein peptide products produced. The functional characteristics, purity and physicochemical properties of sheep trimyelin peptides obtained by different enzymatic hydrolysis methods will be different. Although some sheep trimyelin peptide products have been developed on the market, the further application of sheep trimyelin peptides in real life is limited because these products have unsatisfactory effects on immune regulation. Summary of the invention

[0004] The main purpose of the present invention is to provide a sheep trimyelin peptide for enhancing immunity and a preparation method and application thereof, aiming to solve the problem of poor immunoregulatory effect of existing sheep trimyelin peptide products.

[0005] To achieve the above object, the present invention provides a method for preparing sheep trimyelin peptide for enhancing immunity, comprising the following steps: S1, mincing the sheep trismus, mixing the minced sheep trismus with pre-cooled deionized water, and homogenizing the mixture to obtain a sheep trismus homogenate; S2, centrifuging the sheep medullary homogenate to remove the fat layer to obtain an extraction stock solution; S3, mixing the extraction solution and the complex enzyme, performing an enzymatic hydrolysis, and obtaining a first enzymatic hydrolysis solution; the complex enzyme comprises lipase, hyaluronidase and chondroitinase ABC; S4, performing enzyme inactivation treatment on the first enzymatic solution, and then mixing the first enzymatic solution with alkaline protease for secondary enzymatic hydrolysis to obtain a second enzymatic solution; S5, mixing the second enzymatic hydrolysate and flavor protease, and performing enzymatic hydrolysis three times to obtain a third enzymatic hydrolysate; S6, centrifuging the third enzymatic hydrolyzate, collecting the supernatant, and sequentially performing ultrafiltration and concentration treatments on the supernatant to obtain a sheep trimyelin concentrated solution; S7, spray-drying the sheep trimyelin peptide concentrate to obtain the immunity-enhancing sheep trimyelin peptide.

[0006] In one embodiment, the step S1 comprises: The sheep trismus is chopped, and 2 to 3 volumes of pre-cooled deionized water are added to the chopped sheep trismus for homogenization to obtain sheep trismus homogenate, wherein the temperature of the pre-cooled deionized water is 0 to 4°C.

[0007] In one embodiment, step S3 includes: The extraction stock solution is subjected to ultrasonic treatment, and then the extraction stock solution is mixed with a composite enzyme and subjected to an enzymatic hydrolysis to obtain a first enzymatic hydrolysis solution.

[0008] In one embodiment, in step S3, the amount of the complex enzyme added is 1% to 2% of the total mass of the extraction solution, the mass ratio of the lipase, hyaluronidase and chondroitinase ABC is 1: (0.2 to 1): (0.2 to 1), the pH of the primary enzymatic hydrolysis is 6.0 to 7.5, the temperature of the primary enzymatic hydrolysis is 35 to 40° C., and the time of the primary enzymatic hydrolysis is 1 to 2 h.

[0009] In one embodiment, in step S3, the complex enzyme further comprises collagenase, and the mass ratio of the collagenase to the lipase is 2:(0.5-1).

[0010] In one embodiment, step S4 includes: The first enzymatic hydrolysate is subjected to enzyme inactivation treatment, and then the first enzymatic hydrolysate, polyethylene glycol and alkaline protease are mixed to perform secondary enzymatic hydrolysis to obtain a second enzymatic hydrolysate.

[0011] In one embodiment, in step S4, the amount of alkaline protease added is 1% to 2% of the total mass of the first enzymatic hydrolysis solution, the pH of the secondary enzymatic hydrolysis is 7.0 to 8.5, the temperature of the secondary enzymatic hydrolysis is 50 to 60° C., and the time of the secondary enzymatic hydrolysis is 1 to 2 h.

[0012] In one embodiment, in step S5, the amount of flavor protease added is 0.5%-1% of the total mass of the second enzymatic hydrolysis solution, the pH of the tertiary enzymatic hydrolysis is 6.5-7.5, the temperature of the tertiary enzymatic hydrolysis is 44-55° C., and the time of the tertiary enzymatic hydrolysis is 2-3 h.

[0013] The invention provides a sheep trimyelin peptide for enhancing immunity, and the sheep trimyelin peptide for enhancing immunity is prepared according to the preparation method.

[0014] The invention provides application of sheep trimyelin peptide for enhancing immunity in preparing food for enhancing immunity.

[0015] The preparation method provided by the technical solution of the present invention first chops the sheep three marrows for homogenization to obtain sheep three marrow homogenate; then performs preliminary fat removal by centrifugation, and then performs pre-enzymatic hydrolysis by lipase, hyaluronidase and chondroitinase ABC to release more proteins tightly bound to lipids and carbohydrates, wherein lipids are hydrolyzed by lipase, fat is further removed, and proteins bound to lipids are released, and proteins bound to carbohydrates are released by hyaluronidase and chondroitinase ABC; then alkaline protease and flavor protease are used to perform step-by-step enzymatic hydrolysis on the enzymatic hydrolyzate after pre-enzymatic hydrolysis, so that the protein in the sheep three marrows is released as much as possible and fully enzymatically hydrolyzed, and more sheep three marrow peptides with small molecular weight and uniform distribution are generated. In addition, the preparation method of the present invention reduces the high-temperature cooking treatment steps in the conventional preparation process, and by reducing the long-term high-temperature treatment process, the possibility of irreversible denaturation of proteins at high temperatures can be reduced, which helps to improve the extraction rate of the effective ingredients of sheep three marrow peptides. The sheep trimyelin peptide prepared by the preparation method of the present invention has a small molecular weight and good distribution uniformity, has good immune activity, can be used to solve the problem of poor immune regulation effect of the existing sheep trimyelin peptide, and promote the further application of the sheep trimyelin peptide. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0018] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0019] At present, more and more peptide products are being developed in the market of general and functional foods. Sheep Trimyelin is a bioactive peptide extracted from the brain, spinal cord and bone marrow of sheep. It is rich in various amino acids and has potential nutritional value and pharmacological activity, especially in enhancing immune function. Sheep Trimyelin is believed to be able to regulate the human immune system and promote health due to its unique composition and structure.

[0020] Enzymatic hydrolysis is one of the common technical means for the food industry to produce sheep trimyelin peptides. It has the advantages of mild process conditions, little damage to amino acids, and high nutritional value of the protein peptide products produced. The functional characteristics, purity and physicochemical properties of sheep trimyelin peptides obtained by different enzymatic hydrolysis methods will be different. Although some sheep trimyelin peptide products have been developed on the market, the further application of sheep trimyelin peptides in real life is limited because these products have unsatisfactory effects on immune regulation.

[0021] In view of this, the present invention proposes a method for preparing sheep trimyelin peptide for enhancing immunity, comprising the following steps: S1, mincing the sheep trismus, mixing the minced sheep trismus with pre-cooled deionized water, and homogenizing the mixture to obtain a sheep trismus homogenate; S2, centrifuging the sheep medullary homogenate to remove the fat layer to obtain an extraction stock solution; S3, mixing the extraction solution and the complex enzyme, performing an enzymatic hydrolysis, and obtaining a first enzymatic hydrolysis solution; the complex enzyme comprises lipase, hyaluronidase and chondroitinase ABC; S4, performing enzyme inactivation treatment on the first enzymatic solution, and then mixing the first enzymatic solution with alkaline protease for secondary enzymatic hydrolysis to obtain a second enzymatic solution; S5, mixing the second enzymatic hydrolysate and flavor protease, and performing enzymatic hydrolysis three times to obtain a third enzymatic hydrolysate; S6, centrifuging the third enzymatic hydrolyzate, collecting the supernatant, and sequentially performing ultrafiltration and concentration treatments on the supernatant to obtain a sheep trimyelin concentrated solution; S7, spray-drying the sheep trimyelin peptide concentrate to obtain the immunity-enhancing sheep trimyelin peptide.

[0022] The protein components in the brain, spinal cord and bone marrow of sheep are relatively complex, containing a large number of neurons and supporting cells, rich in lipids and complex glycoproteins, and the proteins are difficult to be released, which will reduce the efficiency of protease hydrolysis and the yield of peptides. The preparation method provided by the technical solution of the present invention first performs a preliminary fat removal treatment on the chopped sheep three marrow; then pre-enzymatic hydrolysis is performed by lipase, hyaluronidase and chondroitinase ABC to release more protein components, wherein lipids are hydrolyzed by lipase to further remove fat and release proteins bound to lipids, and some polysaccharides in the brain, spinal cord or bone marrow are hydrolyzed by hyaluronidase and chondroitinase ABC to loosen the tissue structure, which is conducive to releasing more proteins bound to carbohydrates; then alkaline protease and flavor protease are used to gradually enzymolyze the enzymatic solution after pre-enzymatic hydrolysis, so that the protein in the sheep three marrow is fully enzymatically hydrolyzed to generate sheep three marrow peptides with small molecular weight and uniform distribution, thereby effectively improving the extraction rate of the effective components of sheep three marrow peptides. In addition, the preparation method of the present invention reduces the high-temperature cooking treatment step in the conventional preparation process. By reducing the long-term high-temperature treatment process, the possibility of irreversible denaturation of proteins at high temperatures can be reduced, which helps to improve the extraction rate of the effective ingredients of sheep trimyelin peptide. The sheep trimyelin peptide prepared by the preparation method of the present invention has a small molecular weight and good distribution uniformity, and has good immune activity. It can be used to solve the problem of poor immune regulation effect of existing sheep trimyelin peptides, which is conducive to promoting the further application of sheep trimyelin peptides.

[0023] In an embodiment of the present invention, step S1 comprises: Chop the sheep trismus into small pieces, add 2 to 3 times the volume of pre-cooled deionized water to the chopped sheep trismus, and perform homogenization to obtain a sheep trismus homogenate; Wherein, the temperature of the pre-cooled deionized water is 0-4°C.

[0024] Crushing and homogenizing the sheep trismus can effectively release the proteins in the cells. The technical solution of the present invention does not limit the method of homogenization treatment, and the homogenization treatment can crush the sheep trismus into a homogenate in a tissue homogenizer. Different from the conventional degreasing method of high-temperature boiling of cattle or sheep trismus and then using a degreasing agent, the technical solution of the present invention utilizes the density difference between fat and protein to separate the fat layer by centrifugal force at low temperature. Preliminary degreasing with precooled deionized water can quickly solidify and remove the fat. The low-temperature centrifugal degreasing method is simple to operate and highly safe, reduces long-term high-temperature treatment, can reduce the use of organic solvents, and reduces the problem of solvent residues.

[0025] In the embodiment of the present invention, in step S2, the centrifugal force of the sheep medullary homogenate during centrifugation is 8000-10000 × g, and the centrifugation time is 20-30 min. After centrifugation, the upper fat layer is carefully removed with a pipette or a scraper, and the middle aqueous phase and the bottom residue are collected. If there is a lot of residual fat, the homogenization (adding water) and centrifugation steps can be repeated 1-2 times.

[0026] In order to loosen the protein structure and improve the efficiency of enzymatic hydrolysis before enzymatic hydrolysis, in an embodiment of the present invention, before step S3, the step further includes: subjecting the extracted stock solution to ultrasonic treatment.

[0027] The cavitation effect of ultrasound can loosen the protein structure and improve the enzymatic efficiency of protease. The frequency of the ultrasound treatment is 20-40 kHz, the power is 300-400 W, and the time is 10-15 min.

[0028] In an embodiment of the present invention, in step S3, the amount of the complex enzyme added is 1% to 2% of the total mass of the extraction solution, the mass ratio of the lipase, hyaluronidase and chondroitinase ABC is 1: (0.2 to 1): (0.2 to 1), the pH of the primary enzymatic hydrolysis is 6.0 to 7.5, the temperature of the primary enzymatic hydrolysis is 35 to 40° C., and the time of the primary enzymatic hydrolysis is 1 to 2 h.

[0029] Lipase can decompose lipids in sheep's three marrows, reduce the physical barrier of fat to protein, and improve the contact efficiency of subsequent proteases. The use of hyaluronidase and chondroitinase ABC is conducive to releasing more proteins tightly bound to carbohydrates. The present application compounds lipase, hyaluronidase and chondroitinase ABC for pre-enzymatic hydrolysis at the same time, loosens the tissue structure, and promotes protein release. The enzyme addition amount, pH value, temperature and time of the primary enzymatic hydrolysis are set within the above range, the process links are less, and the enzymatic hydrolysis effect of the composite enzyme is better. In one implementation of the present invention, the enzymatic hydrolysis conditions of the primary enzymatic hydrolysis are: enzymatic hydrolysis at 37 ° C and pH 7.0 for 2 h.

[0030] To further improve the efficiency of enzymolysis, in an embodiment of the present invention, in step S3, the composite enzyme also includes collagenase, and the mass ratio of the collagenase to the lipase is 2: (0.5~1). The sheep three marrow raw material contains abundant collagen and has a highly ordered triple helix structure. The use of protease alone may not be able to effectively degrade collagen. Collagenase can destroy the triple helix structure of collagen fibers in the sheep three marrow, release the encapsulated protein, expose the enzyme cleavage site, and generate intermediates that are more easily degraded by other proteases. Moreover, collagenase can specifically hydrolyze the Gly-Pro-X / Y sequence of collagen, release proline-rich small molecule peptides, and activate the phagocytic function of macrophages.

[0031] In an embodiment of the present invention, in step S4, the amount of alkaline protease added is 1% to 2% of the total mass of the first enzymatic hydrolysis solution, the pH of the secondary enzymatic hydrolysis is 7.0 to 8.5, the temperature of the secondary enzymatic hydrolysis is 50 to 60° C., and the time of the secondary enzymatic hydrolysis is 1 to 2 h.

[0032] The technical solution of the present invention uses alkaline protease (Alcalase) for preliminary hydrolysis to release soluble peptides. Considering that the alkaline protease produced by fermentation of Bacillus licheniformis usually maintains activity at 60~70°C, the present invention uses alkaline protease produced by fermentation of Bacillus licheniformis, which can maintain high activity at 50~60°C. After treatment with alkaline protease, macromolecular proteins can be quickly cut off to generate medium peptide segments (2000~5000Da). Although high-temperature enzymatic hydrolysis (>60°C) has higher hydrolysis efficiency, it may destroy some active sequences. The enzymatic hydrolysis temperature of the technical solution of the present invention is low, which helps to retain thermosensitive immune peptides. The technical solution of the present invention uses alkaline protease for preliminary hydrolysis, and enzymatic hydrolysis at 50~60°C helps to retain thermosensitive immune peptides. When the enzyme addition amount, pH value, temperature and time of the secondary enzymatic hydrolysis are within the above range, the activity of the alkaline protease is good and the enzymatic hydrolysis efficiency is high. In one embodiment of the present invention, the enzymatic hydrolysis conditions of the secondary enzymatic hydrolysis are: enzymatic hydrolysis at 55° C. and pH 7.5 for 2 h.

[0033] In an embodiment of the present invention, step S4 includes: The first enzymatic hydrolysate is subjected to enzyme inactivation treatment, and then the first enzymatic hydrolysate, polyethylene glycol and alkaline protease are mixed to perform secondary enzymatic hydrolysis to obtain a second enzymatic hydrolysate.

[0034] The technical solution of the present invention reduces the aggregation of small molecule peptides by adding polyethylene glycol, promotes more efficient enzymolysis, and reduces the possibility of structural changes caused by the aggregation of small molecule peptides, which affects the function and activity. In one embodiment of the present invention, polyethylene glycol with a molecular weight of 4000 is used, and the amount of polyethylene glycol added is 0.1% of the total mass of the first enzymolysis solution. The first enzymolysis solution is heated to 85-90°C and kept warm for 8-12 min for enzyme inactivation.

[0035] In an embodiment of the present invention, in step S5, the amount of flavor protease added is 0.5%-1% of the total mass of the second enzymatic hydrolysis solution, the pH of the tertiary enzymatic hydrolysis is 6.5-7.5, the temperature of the tertiary enzymatic hydrolysis is 45-55° C., and the time of the tertiary enzymatic hydrolysis is 2-3 h.

[0036] Flavourzyme can effectively hydrolyze protein under mild conditions. The flavour protease in the present invention is produced by fermentation of Aspergillus oryzae. Since flavour protease is a mixture of endoprotease and exopeptidase, it can decompose protein structure more comprehensively, further decompose medium peptide segments into small molecule peptides, generate more functional peptides, and the molecular weight of the decomposed small molecule peptides is small (<1000Da). After being treated with flavour protease, bitter peptides can also be reduced and the flavour can be enhanced. When the enzyme addition amount, pH value, temperature and time of the three enzymatic hydrolysis are set within the above range, the activity of flavour protease is better and the enzymatic hydrolysis efficiency is high. In one embodiment of the present invention, the enzymatic hydrolysis conditions of the three enzymatic hydrolysis are set as: enzymatic hydrolysis at 50°C and pH 7.0 for 2 h.

[0037] In order to reduce the influence of alkaline protease, in an embodiment of the present invention, after the step of mixing the second enzymatic hydrolysate with the flavor protease and performing enzymatic hydrolysis three times to obtain the third enzymatic hydrolysate, the method further comprises: The third enzymatic hydrolysate is heated to 85-90° C. and kept warm for 8-12 min to perform high temperature enzyme inactivation treatment.

[0038] In an embodiment of the present invention, in step S6, the third enzymatic hydrolysate is centrifuged at a speed of 10000-15000 r / min by a centrifuge. Ultrafiltration is performed in sequence through ultrafiltration membranes with a molecular weight of less than 10kDa, a molecular weight of less than 3kDa, and a molecular weight of less than 1kDa at a vacuum degree of 5-25°C and 0.1-0.5 Mpa. Unreacted macromolecules, non-target peptides, enzymes, and other impurities are removed by ultrafiltration membrane treatment to obtain small molecule peptides with a molecular weight of less than 1kDa and uniform distribution. The filtrate after ultrafiltration is concentrated by a vacuum concentrator, and the filtrate after ultrafiltration is concentrated to one third of the original volume at a vacuum degree of 0.1-0.5 Mpa and a temperature of 20-40°C.

[0039] In an embodiment of the present invention, in step S7, the inlet air temperature of the spray drying is set to 180-190°C, and the outlet air temperature is set to 80-90°C.

[0040] The invention provides a sheep trimyelin peptide for enhancing immunity, and the sheep trimyelin peptide for enhancing immunity is prepared according to the preparation method.

[0041] The invention also provides an application of sheep trimyelin peptide for enhancing immunity in preparing food for enhancing immunity.

[0042] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0043] In the following examples, lipase, hyaluronidase and flavor protease were purchased from Shanghai Huzhen Industrial Co., Ltd.; chondroitinase ABC was purchased from Jinan Glacon Biotechnology Co., Ltd.; and alkaline protease was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Sigma-Aldrich).

[0044] Example 1 A method for preparing sheep trimyelin peptide for enhancing immunity comprises the following steps: (1) Select freshly washed sheep trismus, chop it into small pieces, add 2.5 times the volume of pre-cooled deionized water to the chopped sheep trismus, and perform homogenization to obtain sheep trismus homogenate; (2) centrifuging the sheep medullary homogenate to remove the fat layer to obtain an extraction stock solution; (3) adding 1% of a complex enzyme (the mass ratio of lipase, hyaluronidase and chondroitinase ABC is 1:0.5:0.5) to the extraction solution, performing enzymolysis at 37° C. and pH 7.0 for 2 h to obtain a first enzymolysis solution; (4) The first enzymatic hydrolysate was heated to 85°C and kept warm for 10 min to inactivate the enzyme at high temperature, cooled, and then 2% alkaline protease was added to the first enzymatic hydrolysate after enzyme inactivation, and enzymatic hydrolysis was performed at 55°C and pH 7.5 for 2 h to obtain a second enzymatic hydrolysate; (5) adding 1% flavor protease to the second enzymatic hydrolyzate, performing enzymatic hydrolysis at 50°C and pH 7.0 for 2 h to obtain a third enzymatic hydrolyzate; heating the third enzymatic hydrolyzate to 85°C and keeping the temperature for 10 min to inactivate the enzyme at high temperature, and cooling the third enzymatic hydrolyzate to room temperature; (6) Centrifuging the cooled third enzymatic hydrolysate at a speed of 10,000 r / min for 20 min, collecting the supernatant, and ultrafiltering the supernatant using ultrafiltration membranes with a molecular weight of less than 10 kDa, a molecular weight of less than 3 kDa, and a molecular weight of less than 1 kDa in sequence, collecting the filtrate; concentrating the filtrate using a vacuum concentrator to one third of the original volume, thereby obtaining a sheep trimyelin peptide concentrate; (7) The sheep trimyelin peptide concentrate is spray-dried (inlet air temperature 185° C., outlet air temperature 85° C.) to obtain the sheep trimyelin peptide for enhancing immunity.

[0045] Example 2 Compared with Example 1, the difference is that the amount of the complex enzyme added in step (3) is 1.5%, the amount of alkaline protease added in step (4) is 1.5%, and the amount of flavor protease added in step (5) is 1.5%.

[0046] Example 3 Compared with Example 1, the difference is that the composite enzyme in step (3) also adds collagenase, and the mass ratio of collagenase to lipase is 2:1.

[0047] Example 4 Compared with Example 3, the difference is that step (3) further adds ultrasonic treatment. The specific steps of step (3) are: The extraction solution was ultrasonically treated for 15 min (frequency of 20 kHz, power of 400 W), and then 1% of the complex enzyme (the mass ratio of collagenase, lipase, hyaluronidase and chondroitinase ABC was 2:1:0.5:0.5) was added to the extraction solution, and enzymolysis was performed at 37°C and pH 7.0 for 2 h to obtain the first enzymolysis solution.

[0048] Example 5 Compared with Example 4, the difference is that polyethylene glycol is also added in step (4), and the specific steps are: (1) Select freshly washed sheep trismus, chop it into small pieces, add 2.5 times the volume of pre-cooled deionized water to the chopped sheep trismus, and perform homogenization to obtain sheep trismus homogenate; (2) centrifuging the sheep medullary homogenate to remove the fat layer to obtain an extraction stock solution; (3) The extraction solution was ultrasonically treated for 15 min (frequency: 20 kHz, power: 400 W), and then 1% of a complex enzyme (the mass ratio of collagenase, lipase, hyaluronidase and chondroitinase ABC was 2:1:0.5:0.5) was added to the extraction solution, and enzymolysis was performed at 37°C and pH 7.0 for 2 h to obtain a first enzymolysis solution; (4) The first enzymatic hydrolysate was heated to 85°C and kept warm for 10 min to inactivate the enzyme at high temperature, then cooled, and then 2% alkaline protease and 0.1% polyethylene glycol were added to the first enzymatic hydrolysate after enzyme inactivation, and enzymatic hydrolysis was performed at 55°C and pH 7.5 for 2 h to obtain a second enzymatic hydrolysate; (5) adding 1% flavor protease to the second enzymatic hydrolyzate, performing enzymatic hydrolysis at 50°C and pH 7.0 for 2 h to obtain a third enzymatic hydrolyzate; heating the third enzymatic hydrolyzate to 85°C and keeping the temperature for 10 min to inactivate the enzyme at high temperature, and cooling the third enzymatic hydrolyzate to room temperature; (6) Centrifuging the cooled third enzymatic hydrolysate at a speed of 10,000 r / min for 20 min, collecting the supernatant, and ultrafiltering the supernatant using ultrafiltration membranes with a molecular weight of less than 10 kDa, a molecular weight of less than 3 kDa, and a molecular weight of less than 1 kDa in sequence, collecting the filtrate; concentrating the filtrate using a vacuum concentrator to one third of the original volume, thereby obtaining a sheep trimyelin peptide concentrate; (7) The sheep trimyelin peptide concentrate is spray-dried (inlet air temperature 185° C., outlet air temperature 85° C.) to obtain the sheep trimyelin peptide for enhancing immunity.

[0049] Example 6 Compared with Example 5, the difference is that step (3) does not involve ultrasonic treatment.

[0050] Comparative Example 1 Compared with Example 1, the difference is that there is no step of performing enzymatic hydrolysis with a composite enzyme, and the specific steps are as follows: (1) Select freshly washed sheep trismus, chop it into small pieces, add 2.5 times the volume of pre-cooled deionized water to the chopped sheep trismus, and perform homogenization to obtain sheep trismus homogenate; (2) centrifuging the sheep medullary homogenate to remove the fat layer to obtain an extraction stock solution; (3) Add 2% alkaline protease to the original extraction solution, perform enzymolysis at 55°C and pH 7.5 for 2 h, and obtain an enzymolysis solution; (4) adding 1% flavor protease to the enzymatic hydrolysate of step (3), performing enzymatic hydrolysis at 50°C and pH 7.0 for 2 h to obtain an enzymatic hydrolysate; heating the enzymatic hydrolysate to 85°C and keeping the temperature for 10 min to inactivate the enzyme at high temperature, and cooling the enzymatic hydrolysate to room temperature; (5) Centrifuging the cooled third enzymatic hydrolysate at a speed of 10,000 r / min for 20 min, collecting the supernatant, and ultrafiltering the supernatant using ultrafiltration membranes with a molecular weight of less than 10 kDa, a molecular weight of less than 3 kDa, and a molecular weight of less than 1 kDa in sequence, and collecting the filtrate; concentrating the filtrate using a vacuum concentrator to one third of the original volume, thereby obtaining a sheep trimyelin peptide concentrate; (6) The sheep trimyelin peptide concentrate is spray-dried (inlet air temperature 185° C., outlet air temperature 85° C.) to obtain the sheep trimyelin peptide for enhancing immunity.

[0051] Comparative Example 2 Compared with Example 1, the difference is that the complex enzyme in step (3) does not contain lipase.

[0052] Comparative Example 3 Compared with Example 1, the difference is that the complex enzyme in step (3) does not contain hyaluronidase.

[0053] Comparative Example 4 Compared with Example 1, the difference is that the complex enzyme in step (3) does not contain chondroitinase ABC.

[0054] Performance Testing 1. Molecular weight test: The molecular weight distribution of the sheep trimyelin peptides prepared in Examples 1-6 and Comparative Examples 1-4 of the present invention was tested using GB / T 22729-2008. The test results are shown in Table 1.

[0055] Table 1 Molecular weight test results

[0056] As shown in Table 1, the sheep trimyelin peptides prepared in Examples 1-6 consist of more than 90% peptide segments <1000Da, of which 30%~50% <500Da. The human body has a high absorption and utilization rate for small molecule peptides with a molecular weight range of less than 1000Da, and the effect of taking is good. It can be seen that the sheep trimyelin peptide prepared by the present invention has a small molecular weight and good distribution uniformity, and has a high absorption and utilization rate after taking, which is conducive to exerting efficacy. In addition, it can be seen from the test results of Examples 1-6 that ultrasonic treatment is conducive to promoting enzymatic hydrolysis efficiency and improving the generation of small molecular weight peptides (Examples 1 and 4); collagenase is conducive to protein decomposition and the generation of sheep trimyelin peptides with lower molecular weight (Examples 1 and 3); the addition of polyethylene glycol is conducive to the generation of molecular weight peptides (Examples 3 and 6). It can be seen from the results of Example 1 and Comparative Examples 1-4 that the result of Example 1 is better than that of Comparative Examples 1-4, wherein, since Comparative Example 1 does not use a composite enzyme to pre-hydrolyze the sheep triple marrow, Comparative Example 2 does not contain lipase, Comparative Example 3 does not contain hyaluronidase, and Comparative Example 4 does not add chondroitinase ABC, the effective protein in the system is reduced, resulting in a decrease in the generation rate of small molecule peptides with lower molecular weight.

[0057] 2. Immunity Experiment: Experimental animals: nude mice, half male and half female, weighing 18-25 g.

[0058] Experimental grouping: The mice were divided into 11 groups, each group had 10 mice (half male and half female), and were fed with physiological saline (control group), sheep trimyelin of Examples 1-6 and Comparative Examples 1-4, respectively.

[0059] Feeding method: Mice were gavaged with normal saline or sheep trimyelin solution 0.5 mL / 20 g body weight once a day for 2 consecutive weeks; sheep trimyelin solution was prepared by mixing sheep trimyelin with water, with a concentration of 0.6 g / mL.

[0060] Test items and detection indicators: The phagocytic index of mouse immune organs was detected by the mouse peritoneal macrophage phagocytosis of chicken red blood cells experiment; the ear swelling test of dinitrofluorobenzene-induced mouse DTH (Delayed-Type Hypersensitivity) was used to detect the thickening of the ear. The test results are shown in Table 2.

[0061] Table 2 Immunity test results

[0062] In Table 2, * indicates comparison with the control group. p <0.05.

[0063] From the data in Table 2, it can be seen that in the experiment of phagocytosis of chicken red blood cells by mouse peritoneal macrophages, the phagocytic percentage and phagocytic index of the sheep trimyelin peptide prepared in Examples 1-6 were significantly different from those of the control group, indicating that the sheep trimyelin peptide prepared in the present invention can improve the phagocytic index of immune organs and improve the function of mononuclear macrophages. In the DTH test induced by dinitrofluorobenzene, the weight gain of the ear shell of the sheep trimyelin peptide prepared in Examples 1-6 was higher than that of the control group, and the difference was statistically significant ( p <0.05), indicating that the sheep three myelin peptide prepared by Example 1-6 can significantly improve the proliferation capacity of lymphocytes and improve cellular immune function. It is thus proved that the sheep three myelin peptide prepared by the present invention has the effect of enhancing immunity. In contrast, the test results of Comparative Examples 1-4 deteriorate, which may be because Comparative Example 1 does not use a composite enzyme to pre-enzyme the sheep three myelin, the composite enzyme of Comparative Example 2 does not use lipase, the composite enzyme of Comparative Example 3 does not use hyaluronidase, and the composite enzyme of Comparative Example 4 does not use chondroitinase ABC, resulting in a deterioration of the pre-enzyme effect, a reduction in the released protein, thereby reducing the generation rate of small molecule peptides, and the sheep three myelin peptide The strengthening effect on immunity is weakened, and the above results show that the lipase, hyaluronidase and chondroitinase ABC used in the present invention can produce a synergistic effect, and improve the efficacy of sheep three myelin peptide.

[0064] The above description is only an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the protection scope of the present invention.

Claims

1. A method for preparing sheep trimyelin peptide for enhancing immunity, characterized in that: The following steps are involved: S1, mincing the sheep trismus, mixing the minced sheep trismus with pre-cooled deionized water, and homogenizing to obtain a sheep trismus homogenate; S2, centrifuging the sheep medullary homogenate to remove the fat layer to obtain an extraction stock solution; S3, mixing the extraction solution and the complex enzyme, performing an enzymatic hydrolysis, and obtaining a first enzymatic hydrolysis solution; the complex enzyme comprises lipase, hyaluronidase and chondroitinase ABC; S4, performing enzyme inactivation treatment on the first enzymatic solution, and then mixing the first enzymatic solution with alkaline protease for secondary enzymatic hydrolysis to obtain a second enzymatic solution; S5, mixing the second enzymolysis solution and flavor protease, and performing enzymolysis three times to obtain a third enzymolysis solution; S6, centrifuging the third enzymatic hydrolyzate, collecting the supernatant, and sequentially performing ultrafiltration and concentration treatments on the supernatant to obtain a sheep trimyelin concentrated solution; S7, spray-drying the sheep trimyelin peptide concentrate to obtain the immunity-enhancing sheep trimyelin peptide.

2. The method for preparing the immunity-enhancing sheep trimyelin peptide according to claim 1, characterized in that: The step S1 comprises: The sheep trismus is chopped, and 2 to 3 volumes of pre-cooled deionized water are added to the chopped sheep trismus for homogenization to obtain sheep trismus homogenate, wherein the temperature of the pre-cooled deionized water is 0 to 4°C.

3. The method for preparing the immunity-enhancing sheep trimyelin peptide according to claim 1, characterized in that: The step S3 comprises: The extraction stock solution is subjected to ultrasonic treatment, and then the extraction stock solution is mixed with a composite enzyme and subjected to a first enzymolysis to obtain a first enzymolysis solution.

4. The method for preparing the immunity-enhancing sheep trimyelin peptide according to claim 1, characterized in that: In step S3, the amount of the complex enzyme added is 1% to 2% of the total mass of the extraction solution, the mass ratio of the lipase, hyaluronidase and chondroitinase ABC is 1: (0.2 to 1): (0.2 to 1), the pH of the primary enzymatic hydrolysis is 6.0 to 7.5, the temperature of the primary enzymatic hydrolysis is 35 to 40° C., and the time of the primary enzymatic hydrolysis is 1 to 2 h.

5. The method for preparing the immunity-enhancing sheep trimyelin peptide according to claim 1 or 3, characterized in that: In step S3, the complex enzyme also includes collagenase, and the mass ratio of the collagenase to lipase is 2:(0.5~1).

6. The method for preparing the immunity-enhancing sheep trimyelin peptide according to claim 5, characterized in that: The step S4 comprises: The first enzymatic hydrolysate is subjected to enzyme inactivation treatment, and then the first enzymatic hydrolysate, polyethylene glycol and alkaline protease are mixed to perform secondary enzymatic hydrolysis to obtain a second enzymatic hydrolysate.

7. The method for preparing the immunity-enhancing sheep trimyelin peptide according to claim 1, characterized in that: In step S4, the amount of alkaline protease added is 1% to 2% of the total mass of the first enzymatic hydrolysis solution, the pH of the secondary enzymatic hydrolysis is 7.0 to 8.5, the temperature of the secondary enzymatic hydrolysis is 50 to 60° C., and the time of the secondary enzymatic hydrolysis is 1 to 3 h.

8. The method for preparing the immunity-enhancing sheep trimyelin peptide according to claim 1, characterized in that: In step S5, the amount of flavor protease added is 0.5%-1% of the total mass of the second enzymatic hydrolysis solution, the pH of the tertiary enzymatic hydrolysis is 6.5-7.5, the temperature of the tertiary enzymatic hydrolysis is 45-55° C., and the time of the tertiary enzymatic hydrolysis is 2-3 h.

9. A sheep tripylin peptide for enhancing immunity, characterized in that: The immunity-enhancing sheep trimyelin peptide is prepared according to the preparation method of the immunity-enhancing sheep trimyelin peptide according to any one of claims 1 to 8.

10. Use of the immunity-enhancing sheep trimyelin peptide as claimed in claim 9 in the preparation of immunity-enhancing food.

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