Sheep placenta polypeptide and its preparation method and application

Abstract

The invention discloses a sheep placenta polypeptide and a preparation method and application thereof, belonging to the technical field of sheep placenta polypeptide preparation, specifically involving mixing chitosan and glycidyl furfural ether to react to prepare functionalized chitosan, then preparing magnetic particles in a solution from ferric chloride hexahydrate and ferrous chloride tetrahydrate, then mixing the functionalized chitosan and the magnetic particles in a solution containing a cross-linking agent to react, so that the magnetic particles are distributed in the cross-linked functionalized chitosan to obtain a magnetic glycosylated substance, finally using the magnetic glycosylated substance as a filler of a chromatography column, separating the sheep placenta enzymolyzate after enzymolysis treatment, and obtaining the sheep placenta polypeptide. The sheep placenta polypeptide yield of the method of the invention is high, and the sheep placenta polypeptide has high acid resistance and stability.

Description

Technical Field

[0001] The invention belongs to the technical field of sheep placenta polypeptide preparation, and specifically relates to a sheep placenta polypeptide and a preparation method and application thereof. Background Art

[0002] Humans have a long history of using placenta. About 400 years ago, Li Shizhen, in his Compendium of Materia Medica, which is a collection of ancient and modern Chinese medicine, believed that the placenta of mammals such as humans, sheep, pigs, and cattle could be used as medicine, and named the placenta "Placenta". Modern science has proven that the placenta contains more than 50 kinds of biologically active substances and nutrients such as amino acids and minerals. It is an ideal immunomodulator and nutritional beauty and health care agent.

[0003] Sheep placenta is the tissue responsible for the exchange of blood and nutrients between the mother and the fetus when the ewe is pregnant. It can produce more active substances, and its nutritional composition is close to that of human placenta. Sheep placenta is rich in protein, vitamins, lipopolysaccharide, active peptides, growth factors, multiple amino acids and trace elements, as well as antibodies related to immune function and maintaining the normal operation of the body, which can be well absorbed. According to the Compendium of Materia Medica, the placenta, also known as the placenta or placenta, tastes sweet, salty and warm. It enters the lungs, liver and kidneys. It has many functions such as warming the middle and replenishing qi, nourishing yin and blood, beautifying and nourishing the skin, preventing cancer, anti-aging, regulating endocrine, and improving the body's immunity. It is widely used in medical care, food, cosmetics and other fields. In Switzerland, sheep placenta extract has been added to the production of cosmetics. At the same time, some domestic researchers have also applied sheep placenta antioxidant peptides to some anti-aging cosmetics. Antioxidant peptides have become a research hotspot because of their good antioxidant activity, high stability, easy absorption, safe nutrition and other advantages. Summary of the invention

[0004] The purpose of the present invention is to provide a sheep placenta polypeptide which can be used to prepare an acid-resistant immunomodulator and a nutritional beauty and health care agent, and a preparation method and application of a sheep placenta polypeptide with high yield and high acid-resistant stability.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A method for preparing a sheep placenta polypeptide comprises: enzymolysis treatment of a sheep placenta to obtain a sheep placenta hydrolysate, and then separation and purification using a magnetic glycoside to obtain a sheep placenta polypeptide; the magnetic glycoside has a cross-linked chitosan-based material and magnetic particles distributed in the chitosan-based material, and the amount of the magnetic particles is 20-60wt% of the chitosan-based material. In the present invention, functionalized chitosan is firstly obtained by functionalizing chitosan using a functionalizing agent including glycidyl furfuryl ether, and then the magnetic glycoside is prepared with magnetic particles under the action of a cross-linking agent including glutaraldehyde. The present invention finds that after a specific functionalizing agent is connected to chitosan, the obtained sheep placenta polypeptide has high acid resistance stability, still has a high free radical scavenging rate under acidic conditions, and has a good yield. In the enzymolysis treatment process, a variety of sheep placenta polypeptides may be produced, and the glycidyl furfuryl ether, magnetic particles and cross-linked structure with a specific structure in the present invention have excellent selectivity for specific sheep placenta polypeptides.

[0007] Preferably, in the preparation of the magnetic saccharide, chitosan is reacted with glycidyl furfural ether to prepare functionalized chitosan, and then the functionalized chitosan is mixed with magnetic particles in a solution containing a crosslinking agent to react to prepare the magnetic saccharide.

[0008] More preferably, the amount of glycidyl furfural ether used is 30-100wt% of chitosan; or, the magnetic particles are ferrosoferric oxide; or, the cross-linking agent is glutaraldehyde; or, the amount of the cross-linking agent used is 20-100wt% of the functionalized chitosan; or, the solution further contains liquid paraffin and Tween 60.

[0009] More preferably, the preparation temperature of the functionalized chitosan is 70-90°C; or, the preparation temperature of the magnetic saccharide is 50-70°C.

[0010] Preferably, the magnetic particles are made of ferric chloride hexahydrate and ferrous chloride tetrahydrate under the action of ammonia water.

[0011] More preferably, the molar amount of ferrous chloride tetrahydrate used is 50% of the molar amount of ferric chloride hexahydrate used; or, the concentration of ammonia water is 5-25wt%.

[0012] Preferably, papain and trypsin are used in the enzymatic hydrolysis treatment; or, in the enzymatic hydrolysis treatment, the amount of protease used is 1-5wt% of the sheep placenta.

[0013] More preferably, papain and trypsin are used in a mass ratio of 1:0.5-2 in the enzymatic hydrolysis treatment.

[0014] Preferably, the magnetic saccharide is used as a filler in the chromatography column.

[0015] Preferably, the sheep placenta is washed with physiological saline, made into a homogenate, and subjected to freeze-thaw treatment to obtain sheep placenta powder for enzymatic hydrolysis treatment.

[0016] Preferably, in the preparation of functionalized chitosan, chitosan is added to deionized water, and then glycidyl furfural ether is added, stirred at a temperature of 70-90° C. for 6-24 hours, dried, and washed with ethanol to obtain functionalized chitosan.

[0017] More preferably, in the preparation of functionalized chitosan, the amount of chitosan used is 0.25-5 wt % of deionized water.

[0018] More preferably, in the preparation of functionalized chitosan, the amount of glycidyl furfural ether used is 30-100 wt % of chitosan.

[0019] Preferably, 3-glycidyloxypropyltrimethoxysilane can be added in the preparation of functionalized chitosan, and the amount of 3-glycidyloxypropyltrimethoxysilane used is 10-30wt% of chitosan. After glycidyl furfuryl ether is used as a functional agent, 3-glycidyloxypropyltrimethoxysilane can be added to prepare functionalized chitosan. Under the cross-linking structure formed by the functionalized chitosan having glycidyl furfuryl ether groups and 3-glycidyloxypropyltrimethoxysilane groups and the action of magnetic particles, sheep placenta polypeptides with high acid resistance stability can be further selectively separated, and the yield of sheep placenta polypeptides can be improved.

[0020] Preferably, in the preparation of magnetic particles, ferric chloride hexahydrate and ferrous chloride tetrahydrate are added to deionized water, the deionized water is deoxygenated with nitrogen, and stirred and mixed under nitrogen protection at a temperature of 20-40°C, and after stirring until transparent, ammonia water is added until a black precipitate is produced, and the stirring reaction is continued for 20-60 minutes, and then aged at a temperature of 70-90°C for 20-60 minutes, and finally washed with anhydrous ethanol and deionized water, separated by a magnet, and dried to obtain magnetic particles.

[0021] More preferably, in the preparation of magnetic particles, the amount of ferric chloride hexahydrate used is 1-5 wt % of deionized water.

[0022] More preferably, in the preparation of magnetic particles, the molar amount of ferrous chloride tetrahydrate used is 50% of the molar amount of ferric chloride hexahydrate used. Ammonia water is used in an appropriate amount.

[0023] Preferably, in the preparation of the magnetic saccharide, functionalized chitosan is added to deionized water, and then magnetic particles are added, ultrasonicated for 1-5 minutes, and then liquid paraffin and Tween 60 are added, stirred for 20-60 minutes, and then a cross-linking agent is added, stirred for 0.5-5 hours, and finally treated at a temperature of 50-70°C for 1-5 hours, washed with petroleum ether, acetone and deionized water, and dried to obtain a magnetic saccharide.

[0024] More preferably, in the preparation of the magnetic sugar-based material, the amount of functionalized chitosan used is 0.25-5 wt % of deionized water.

[0025] More preferably, in the preparation of the magnetic sugar-based material, the amount of magnetic particles used is 20-60 wt % of the functionalized chitosan.

[0026] More preferably, in the preparation of the magnetic sugar-based material, the amount of liquid paraffin used is 60-200 times the mass amount of the functionalized chitosan used.

[0027] More preferably, in the preparation of the magnetic sugar-based material, the amount of Tween 60 used is 600-1000wt% of the functionalized chitosan,

[0028] More preferably, in the preparation of the magnetic sugar-based material, the cross-linking agent is glutaraldehyde, and the amount of the cross-linking agent used is 20-100 wt % of the functionalized chitosan.

[0029] Preferably, in the preparation of sheep placenta polypeptide, the sheep placenta is pretreated to prepare sheep placenta powder, then subjected to enzymatic treatment to prepare sheep placenta hydrolysate, and finally separated and purified to obtain the sheep placenta polypeptide.

[0030] More preferably, in the preparation of sheep placenta polypeptide, in the pretreatment, the sheep placenta is cleaned with physiological saline, homogenized to prepare a 5-20wt% homogenate, and then subjected to freeze-thaw treatment to obtain sheep placenta powder.

[0031] More preferably, in the preparation of sheep placenta polypeptide, during the enzymatic hydrolysis treatment, the sheep placenta powder is made into a 5-20wt% sheep placenta powder liquid with sterile physiological saline, the pH is adjusted to 6-8, and proteases are added at 35-60°C. The proteases are papain and trypsin. The mass ratio of papain and trypsin in the protease is 1:0.5-2. The amount of protease used is 1-5wt% of the sheep placenta powder. The enzymatic hydrolysis time is 2-8h. After the enzymatic hydrolysis is completed, it is inactivated, the supernatant is taken by centrifugation, and freeze-dried to obtain a sheep placenta hydrolysate.

[0032] More preferably, in the preparation and separation and purification of the sheep placenta polypeptide, a chromatography column is used for separation, and a magnetic saccharide is used as a filler in the chromatography column to obtain the sheep placenta polypeptide.

[0033] The present invention adopts the method of mixing chitosan and glycidyl furfural ether to make functionalized chitosan, then making magnetic particles from ferric chloride hexahydrate and ferrous chloride tetrahydrate in a solution, then mixing the functionalized chitosan and the magnetic particles in a solution containing a crosslinking agent to react, so that the magnetic particles are distributed in the crosslinked functionalized chitosan to obtain a magnetic glycosylated substance, and finally using the magnetic glycosylated substance as a filler of a chromatography column to separate the enzymatic hydrolyzate of the sheep placenta after enzymatic hydrolysis to obtain a sheep placenta polypeptide, thus having the following beneficial effects: the sheep placenta polypeptide of the method of the present invention has a high yield, and the sheep placenta polypeptide has high acid resistance and stability. Therefore, the present invention is a sheep placenta polypeptide that can be used to prepare an acid-resistant immunomodulator and a nutritional beauty and health care agent, and a sheep placenta polypeptide preparation method and application with a high sheep placenta polypeptide yield and high acid resistance and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the infrared spectrum;

[0035] Figure 2 is the yield graph;

[0036] Figure 3 This is the acid resistance stability diagram. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with specific implementations and drawings:

[0038] Example 1: A sheep placenta polypeptide and its preparation method and application

[0039] Preparation of functionalized chitosan: chitosan was added to deionized water, and then glycidyl furfural ether was added, stirred at 80°C for 12h, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100g, the amount of chitosan used was 3g, and the amount of glycidyl furfural ether used was 1.5g.

[0040] Preparation of magnetic particles: Add ferric chloride hexahydrate and ferrous chloride tetrahydrate to deionized water, deionized water is deoxygenated with nitrogen, stirred and mixed under nitrogen protection at 30°C, stirred until transparent, add ammonia water until black precipitate is produced, continue stirring and reacting for 30 minutes, then age at 80°C for 30 minutes, finally wash with anhydrous ethanol and deionized water, separate with a magnet, and dry to obtain magnetic particles. The amount of deionized water used is 100g, the amount of ferric chloride hexahydrate used is 3g, and the molar amount of ferrous chloride tetrahydrate used is 50% of the molar amount of ferric chloride hexahydrate used. Ammonia water is used in appropriate amounts.

[0041] Preparation of magnetic glycosides: functionalized chitosan is added to deionized water, and then magnetic particles are added, ultrasonicated for 3 minutes, and then liquid paraffin and Tween 60 are added, stirred for 30 minutes, and then a cross-linking agent is added, stirred for 3 hours, and finally treated at 60°C for 3 hours, washed with petroleum ether, acetone and deionized water, and dried to obtain magnetic glycosides. The amount of deionized water used is 100g, the amount of functionalized chitosan used is 3g, the amount of magnetic particles used is 1.2g, the amount of liquid paraffin used is 300g, the amount of Tween 60 used is 24g, the cross-linking agent is glutaraldehyde, and the amount of the cross-linking agent used is 1.5g.

[0042] Preparation of sheep placenta polypeptide: sheep placenta is pretreated to prepare sheep placenta powder, then enzymatically treated to prepare sheep placenta hydrolysate, and finally separated and purified to obtain sheep placenta polypeptide. In the pretreatment, the sheep placenta is cleaned with physiological saline, homogenized to prepare a 10wt% homogenate, and then subjected to freeze-thaw treatment to obtain sheep placenta powder; in the enzymatic hydrolysis treatment, the sheep placenta powder is prepared into a 10wt% sheep placenta powder liquid with sterile physiological saline, the pH is adjusted to 6.5, and protease is added at 50°C, the protease is papain and trypsin, papain and trypsin in the protease are used in a mass ratio of 1:1, the amount of protease used is 3wt% of the sheep placenta powder, the enzymatic hydrolysis time is 5h, the enzymatic hydrolysis is inactivated after completion, the supernatant is centrifuged, and freeze-dried to obtain the sheep placenta hydrolysate; in the separation and purification, a chromatographic column is used for separation, and a magnetic glycosyl is used as a filler in the chromatographic column to obtain sheep placenta polypeptide.

[0043] Example 2: A sheep placenta polypeptide and its preparation method and application

[0044] Compared with Example 1, the difference between this example and Example 1 lies in the preparation of functionalized chitosan.

[0045] Preparation of functionalized chitosan: chitosan was added to deionized water, and then glycidyl furfural ether was added, stirred at 80°C for 12 hours, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100 g, the amount of chitosan used was 3 g, and the amount of glycidyl furfural ether used was 1 g.

[0046] Example 3: A sheep placenta polypeptide and its preparation method and application

[0047] Compared with Example 1, the difference between this example and Example 1 lies in the preparation of functionalized chitosan.

[0048] Preparation of functionalized chitosan: chitosan was added to deionized water, and then glycidyl furfural ether was added, stirred at 80°C for 12h, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100g, the amount of chitosan used was 3g, and the amount of glycidyl furfural ether used was 2g.

[0049] Example 4: A sheep placenta polypeptide and its preparation method and application

[0050] Preparation of functionalized chitosan: chitosan was added to deionized water, then glycidyl furfural ether and 3-glycidyloxypropyl trimethoxysilane were added, stirred at 80°C for 12h, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100g, the amount of chitosan used was 3g, the amount of glycidyl furfural ether used was 1.5g, and the amount of 3-glycidyloxypropyl trimethoxysilane used was 0.6g.

[0051] Preparation of magnetic particles: Add ferric chloride hexahydrate and ferrous chloride tetrahydrate to deionized water, deionized water is deoxygenated with nitrogen, stirred and mixed under nitrogen protection at 30°C, stirred until transparent, add ammonia water until black precipitate is produced, continue stirring and reacting for 30 minutes, then age at 80°C for 30 minutes, finally wash with anhydrous ethanol and deionized water, separate with a magnet, and dry to obtain magnetic particles. The amount of deionized water used is 100g, the amount of ferric chloride hexahydrate used is 3g, and the molar amount of ferrous chloride tetrahydrate used is 50% of the molar amount of ferric chloride hexahydrate used. Ammonia water is used in appropriate amounts.

[0052] Preparation of magnetic glycosides: functionalized chitosan is added to deionized water, and then magnetic particles are added, ultrasonicated for 3 minutes, and then liquid paraffin and Tween 60 are added, stirred for 30 minutes, and then a cross-linking agent is added, stirred for 3 hours, and finally treated at 60°C for 3 hours, washed with petroleum ether, acetone and deionized water, and dried to obtain magnetic glycosides. The amount of deionized water used is 100g, the amount of functionalized chitosan used is 3g, the amount of magnetic particles used is 1.2g, the amount of liquid paraffin used is 300g, the amount of Tween 60 used is 24g, the cross-linking agent is glutaraldehyde, and the amount of the cross-linking agent used is 1.5g.

[0053] Preparation of sheep placenta polypeptide: sheep placenta is pretreated to prepare sheep placenta powder, then enzymatically treated to prepare sheep placenta hydrolysate, and finally separated and purified to obtain sheep placenta polypeptide. In the pretreatment, the sheep placenta is cleaned with physiological saline, homogenized to prepare a 10wt% homogenate, and then subjected to freeze-thaw treatment to obtain sheep placenta powder; in the enzymatic hydrolysis treatment, the sheep placenta powder is prepared into a 10wt% sheep placenta powder liquid with sterile physiological saline, the pH is adjusted to 6.5, and protease is added at 50°C, the protease is papain and trypsin, papain and trypsin in the protease are used in a mass ratio of 1:1, the amount of protease used is 3wt% of the sheep placenta powder, the enzymatic hydrolysis time is 5h, the enzymatic hydrolysis is inactivated after completion, the supernatant is centrifuged, and freeze-dried to obtain the sheep placenta hydrolysate; in the separation and purification, a chromatographic column is used for separation, and a magnetic glycosyl is used as a filler in the chromatographic column to obtain sheep placenta polypeptide.

[0054] Example 5: A sheep placenta polypeptide and its preparation method and application

[0055] The difference between this embodiment and embodiment 4 lies in the preparation of functionalized chitosan.

[0056] Preparation of functionalized chitosan: chitosan was added to deionized water, then glycidyl furfural ether and 3-glycidyloxypropyl trimethoxysilane were added, stirred at 80°C for 12h, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100g, the amount of chitosan used was 3g, the amount of glycidyl furfural ether used was 1g, and the amount of 3-glycidyloxypropyl trimethoxysilane used was 0.6g.

[0057] Example 6: A sheep placenta polypeptide and its preparation method and application

[0058] The difference between this embodiment and embodiment 4 lies in the preparation of functionalized chitosan.

[0059] Preparation of functionalized chitosan: chitosan was added to deionized water, then glycidyl furfural ether and 3-glycidyloxypropyl trimethoxysilane were added, stirred at 80°C for 12h, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100g, the amount of chitosan used was 3g, the amount of glycidyl furfural ether used was 2g, and the amount of 3-glycidyloxypropyl trimethoxysilane used was 0.6g.

[0060] Example 7: A sheep placenta polypeptide and its preparation method and application

[0061] The difference between this embodiment and embodiment 4 lies in the preparation of functionalized chitosan.

[0062] Preparation of functionalized chitosan: chitosan was added to deionized water, then glycidyl furfural ether and 3-glycidyloxypropyl trimethoxysilane were added, stirred at 80°C for 12h, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100g, the amount of chitosan used was 3g, the amount of glycidyl furfural ether used was 1.5g, and the amount of 3-glycidyloxypropyl trimethoxysilane used was 0.4g.

[0063] Example 8: A sheep placenta polypeptide and its preparation method and application

[0064] The difference between this embodiment and embodiment 4 lies in the preparation of functionalized chitosan.

[0065] Preparation of functionalized chitosan: chitosan was added to deionized water, then glycidyl furfural ether and 3-glycidyloxypropyl trimethoxysilane were added, stirred at 80°C for 12h, dried, and washed with ethanol to obtain functionalized chitosan. The amount of deionized water used was 100g, the amount of chitosan used was 3g, the amount of glycidyl furfural ether used was 1.5g, and the amount of 3-glycidyloxypropyl trimethoxysilane used was 0.8g.

[0066] Comparative Example 1: A sheep placenta polypeptide and its preparation method and application

[0067] Compared with Example 1, this comparative example differs in the preparation of sheep placenta polypeptide.

[0068] Preparation of sheep placenta polypeptide: The sheep placenta is pretreated to prepare sheep placenta powder, and then the sheep placenta hydrolysate is prepared by enzymolysis, and finally the sheep placenta polypeptide is separated and purified. In the pretreatment, the sheep placenta is cleaned with physiological saline, homogenized to prepare a 10wt% homogenate, and then subjected to freeze-thaw treatment to obtain sheep placenta powder; in the enzymolysis treatment, the sheep placenta powder is made into a 10wt% sheep placenta powder liquid with sterile physiological saline, the pH is adjusted to 6.5, and protease is added at 50°C. The protease is papain and trypsin. The papain and trypsin in the protease are used in a mass ratio of 1:1. The amount of protease used is 3wt% of the sheep placenta powder, the enzymolysis time is 5h, and the enzymolysis is inactivated after completion. The supernatant is centrifuged and freeze-dried to obtain the sheep placenta hydrolysate; in the separation and purification, a chromatographic column is used for separation, and functionalized chitosan is used as a filler in the chromatographic column to obtain the sheep placenta polypeptide.

[0069] Comparative Example 2: A sheep placenta polypeptide and its preparation method and application

[0070] The difference between this comparative example and Example 1 lies in the preparation of the magnetic glycoside.

[0071] Preparation of magnetic glycosides: functionalized chitosan is added to deionized water, and then magnetic particles are added, ultrasonicated for 3 minutes, and then liquid paraffin and Tween 60 are added, stirred for 30 minutes, and then a cross-linking agent is added, stirred for 3 hours, and finally treated at 60°C for 3 hours, washed with petroleum ether, acetone and deionized water, and dried to obtain magnetic glycosides. The amount of deionized water used is 100g, the amount of functionalized chitosan used is 3g, the amount of magnetic particles used is 1.2g, the amount of liquid paraffin used is 300g, the amount of Tween 60 used is 24g, the cross-linking agent is glutaraldehyde, and the amount of the cross-linking agent used is 0.5g.

[0072] Comparative Example 3: A sheep placenta polypeptide and its preparation method and application

[0073] The difference between this comparative example and Example 1 lies in the preparation of the magnetic glycoside.

[0074] Preparation of magnetic glycosides: functionalized chitosan is added to deionized water, and then magnetic particles are added, ultrasonicated for 3 minutes, and then liquid paraffin and Tween 60 are added, stirred for 30 minutes, and then a cross-linking agent is added, stirred for 3 hours, and finally treated at 60°C for 3 hours, washed with petroleum ether, acetone and deionized water, and dried to obtain magnetic glycosides. The amount of deionized water used is 100g, the amount of functionalized chitosan used is 3g, the amount of magnetic particles used is 1.2g, the amount of liquid paraffin used is 300g, the amount of Tween 60 used is 24g, the cross-linking agent is glutaraldehyde, and the amount of the cross-linking agent used is 3.5g.

[0075] Test example:

[0076] The present invention performs infrared characterization on the magnetic sugar base prepared in Example 1, and the results are as follows: Figure 1 As shown, at 588cm -1 is the infrared absorption peak of the iron-oxygen bond at 1080cm -1 The infrared absorption peak of carbon-oxygen-carbon bond is at 1724 cm -1 The infrared absorption peak of carbon-oxygen double bond is at 2800-2900cm -1 The infrared absorption peaks of methyl and methylene are at 3393 cm -1 The infrared absorption peak of hydroxyl group is shown in Fig. 3, indicating that a magnetic glycoside with a cross-linked structure and dispersed magnetic particles is obtained.

[0077] The present invention calculated the yield of the sheep placenta polypeptide prepared in each embodiment and comparative example, and the results are as follows: Figure 2As shown, wherein S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. In the present invention, chitosan and glycidyl furfural ether are mixed and reacted to prepare functionalized chitosan, and then ferric chloride hexahydrate and ferrous chloride tetrahydrate are used to prepare magnetic particles in a solution, and then the functionalized chitosan and the magnetic particles are mixed in a solution containing a crosslinking agent to react, so that the magnetic particles are distributed in the crosslinked functionalized chitosan to obtain a magnetic saccharide. Finally, the magnetic glycoside is used as the filler of the chromatography column to separate the enzymatic hydrolyzate of the sheep placenta after enzymatic hydrolysis to obtain the sheep placenta polypeptide. The yield of the sheep placenta polypeptide prepared by the above method is good. The present invention has been tested and found that after glycidyl furfural ether is introduced into chitosan to prepare functionalized chitosan, and under the cross-linking effect of magnetic particles and cross-linking agents, the higher the amount of glycidyl furfural ether used, the higher the yield of the obtained sheep placenta polypeptide, indicating that the higher the functional degree of the functionalized chitosan, under the action of functionalized chitosan, magnetic particles and cross-linking structure, the sheep placenta polypeptide can be better allowed to pass through, thereby removing the sheep placenta polypeptide from The present invention is separated from the enzymatic hydrolyzate of sheep placenta; when separating sheep placental polypeptide, after replacing the magnetic saccharide with functionalized chitosan, it is found that the separation performance of functionalized chitosan on sheep placental polypeptide is poor; if the amount of cross-linking agent used is changed, it is found that the separation effect of the magnetic saccharide on sheep placental polypeptide becomes worse, indicating that the cross-linking degree in the magnetic saccharide also affects the separation of sheep placental polypeptide; based on the discovery that the higher the functional degree of functionalized chitosan is, the higher the separation effect of sheep placental polypeptide is, the present invention further uses glycidyl furfural ether and 3-glycidyl etheroxypropyltrimethoxysilane to functionalize chitosan, and finds that the separation effect of the functionalized chitosan on sheep placental polypeptide is better. After the prepared magnetic glycoside was applied to the separation of sheep placental polypeptides, it was found that the yield of sheep placental polypeptides was higher, indicating that under the joint action of glycidyl furfural ether and 3-glycidyloxypropyltrimethoxysilane, the magnetic glycoside was more effective in separating sheep placental polypeptides. When the amount of glycidyl furfural ether used remained unchanged, increasing or decreasing the amount of 3-glycidyloxypropyltrimethoxysilane had no significant effect on increasing or decreasing the yield of separated sheep placental polypeptides, indicating that 3-glycidyloxypropyltrimethoxysilane has an excellent separation effect on sheep placental polypeptides when used together with glycidyl furfural ether.

[0078] The present invention conducts an acid stability test on the sheep placenta polypeptides prepared in each embodiment and comparative example. Ordinary polypeptides will be hydrolyzed or reduced under acidic conditions. Sheep placenta polypeptide is added to distilled water to prepare a sheep placenta polypeptide solution with a concentration of 5 mg / mL, and the pH is adjusted to 2. The solution is treated at 25°C for 12 hours, and then the pH is adjusted to neutral by sampling. The antioxidant activity is tested and characterized by DPPH clearance rate. A 1 mg / mL sheep placenta polypeptide solution and a 0.1 mmol DPPH solution are prepared, and then a DPPH clearance test is performed. The difference between the DPPH clearance rate of the sheep placenta polypeptide without acid treatment and the DPPH clearance rate of the sheep placenta polypeptide after acid treatment is used for characterization. The smaller the difference, the higher the acid stability. The results are as follows: Figure 3As shown, wherein S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. In the present invention, chitosan and glycidyl furfural ether are mixed and reacted to prepare functionalized chitosan, and then ferric chloride hexahydrate and ferrous chloride tetrahydrate are used to prepare magnetic particles in a solution, and then the functionalized chitosan and the magnetic particles are mixed in a solution containing a crosslinking agent to react, so that the magnetic particles are distributed in the crosslinked functionalized chitosan to obtain a magnetic saccharide, and finally the magnetic saccharide is used as a layer The sheep placenta polypeptide prepared by the above method has good acid resistance stability. The present invention has been tested and found that after glycidyl furfural ether is introduced into chitosan to prepare functionalized chitosan, and under the cross-linking effect of magnetic particles and cross-linking agent, the higher the amount of glycidyl furfural ether used, the higher the acid resistance stability of the obtained sheep placenta polypeptide, indicating that the content of peptides with good acid resistance effect is higher, indicating that the functional degree of functionalized chitosan is higher, and under the action of functionalized chitosan, magnetic particles and cross-linking structure, more peptides with good acid resistance effect can be obtained; when separating the sheep placenta polypeptide, the present invention adopts After replacing the magnetic saccharide with functionalized chitosan, it is found that the functionalized chitosan has poor separation performance for the acid-resistant peptides in sheep placenta polypeptides; if the amount of cross-linking agent used is changed, it is found that the separation effect of the magnetic saccharide on the sheep placenta polypeptide becomes worse, indicating that the degree of cross-linking in the magnetic saccharide also affects the separation of the acid-resistant peptides in sheep placenta polypeptides; the present invention further uses glycidyl furfural ether and 3-glycidyl etheroxypropyltrimethoxysilane to functionalize the chitosan, and finds that the magnetic saccharide prepared by the functionalized chitosan is applied to sheep placenta polypeptides. After the separation of sheep placental polypeptides, it was found that the acid-resistant stability of sheep placental polypeptides was higher, indicating that under the joint action of glycidyl furfural ether and 3-glycidyloxypropyltrimethoxysilane, the magnetic glycoside was better in separating acid-resistant peptides in sheep placental polypeptides. When the amount of glycidyl furfural ether used remained unchanged, increasing or decreasing the amount of 3-glycidyloxypropyltrimethoxysilane had no significant effect on increasing or decreasing the yield of acid-resistant peptides in sheep placental polypeptides, indicating that 3-glycidyloxypropyltrimethoxysilane and glycidyl furfural ether used together had an excellent separation effect on acid-resistant peptides in sheep placental polypeptides.

[0079] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0080] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing sheep placenta polypeptide, comprising: The sheep placenta is subjected to enzymatic hydrolysis to obtain a sheep placenta hydrolysate, and then a magnetic sugar-based material is used as a chromatography column filler for separation and purification to obtain a sheep placenta polypeptide; the magnetic sugar-based material comprises a cross-linked chitosan-based material and magnetic particles distributed in the chitosan-based material, and the amount of the magnetic particles is 20-60wt% of the chitosan-based material; In the preparation of the magnetic sugar-based material, chitosan and glycidyl furfural ether are reacted to prepare functionalized chitosan, and then the functionalized chitosan and magnetic particles are mixed in a solution containing a cross-linking agent to react to prepare the magnetic sugar-based material, wherein the glycidyl furfural ether is used in an amount of 30-100wt% of the chitosan, and the magnetic particles are ferrosoferric oxide; the cross-linking agent is glutaraldehyde, and the amount of the cross-linking agent is used in an amount of 20-100wt% of the functionalized chitosan; The preparation temperature of the functionalized chitosan is 70-90°C; the preparation temperature of the magnetic saccharide is 50-70°C; The magnetic particles are made of ferric chloride hexahydrate and ferrous chloride tetrahydrate under the action of ammonia water; Papain and trypsin are used in the enzymatic hydrolysis treatment, and the mass ratio of papain and trypsin is 1:0.5-2. The pH of the enzymatic hydrolysis treatment is 6-8, and the temperature of the enzymatic hydrolysis treatment is 35-60°C. 3-glycidyloxypropyltrimethoxysilane is also added in the preparation of functionalized chitosan, and the usage amount of 3-glycidyloxypropyltrimethoxysilane is 10-30wt% of chitosan.

2. The method for preparing a sheep placenta polypeptide according to claim 1, characterized in that: The reaction solution of the magnetic sugar-based substance also contains liquid paraffin and Tween 60.

3. The method for preparing a sheep placenta polypeptide according to claim 1, characterized in that: The molar amount of ferrous chloride tetrahydrate used is 50% of the molar amount of ferric chloride hexahydrate used; or, the concentration of ammonia water is 5-25wt%.

4. The method for preparing a sheep placenta polypeptide according to claim 1, characterized in that: In the enzymatic hydrolysis treatment, the amount of protease used is 1-5wt% of the sheep placenta.

5. The method for preparing a sheep placenta polypeptide according to claim 1, characterized in that: The sheep placenta is washed with physiological saline, made into a homogenate, and subjected to freeze-thaw treatment to obtain sheep placenta powder for enzymatic hydrolysis treatment.

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