Method for extracting biologically active substances from sheep placenta

Abstract

The invention discloses a method for extracting bioactive substances from sheep placenta, and relates to the technical field of animal placenta extraction. The method uses immobilized lipase to enzymolyze the crushed fresh sheep placenta, then adds protease to continue enzymolysis, and after the enzyme is inactivated, the purified bioactive substances have a high yield; and the immobilized lipase prepared by the chitosan derivative modified by the thiazole group has good application stability; in addition, the present invention also uses S-benzyl-L-cysteine ​​methyl ester hydrochloride to undergo a substitution reaction with a polyamide nanofiltration membrane, and the obtained nanofiltration membrane is used in the purification process of the bioactive substance, which has a good decolorization effect.

Description

Technical Field

[0001] The invention belongs to the technical field of animal placenta extraction, and in particular relates to a method for extracting bioactive substances from sheep placenta. Background Art

[0002] With the continuous development of human society's material civilization and the continuous improvement of scientific and technological levels, people have been inspired to enjoy life and explore the miracles of life. As a result, functional foods that enhance physical fitness and beautify the skin have emerged and have considerable economic value.

[0003] A large number of sheep placentas are produced every year, but most of them are not fully developed and utilized, resulting in a huge waste of resources. The value of the bioactive substances extracted from a sheep placenta is far greater than its edible value. If the sheep placenta is comprehensively developed and utilized, it will produce huge economic and social benefits.

[0004] Studies have found that the cell tissue structure of sheep placenta is most similar to that of human placenta, and it has the functions of regulating the body's immunity, improving microcirculation, lowering cholesterol in the blood, regulating human hormones and endocrine secretion, and effectively inhibiting the decomposition of melanin formation. The trace elements contained in sheep placenta, such as Al, Ba, P, Si, etc., are closely related to the life activities of animals and are trace elements necessary for animal physiological metabolism. Sheep placenta is rich in superoxide dismutase (SOD). SOD, as a natural scavenger of excess free radicals in the body, protects the body's cells and plays an important role in defending against the toxicity of free radicals, anti-aging, preventing tumors and anti-inflammation. The functional characteristics of sheep placenta are increasingly recognized by people, and objectively put forward the requirements for deep processing of sheep placenta.

[0005] At present, the development of sheep placenta food at home and abroad focuses on the extraction and application of active substances, such as sheep placenta whole powder, active sheep placenta, etc. In addition to its own biological activity, the sheep placenta has a protein content of more than 80%. Therefore, the sheep placenta resources should be fully utilized, and modern biological enzymatic hydrolysis technology should be used to convert a large amount of protein in the sheep placenta into sheep placenta bioactive peptides. Bioactive peptides have unique physical and chemical properties and biological activity, and their molecular weight is relatively low. They can avoid allergic reactions to the human body caused by proteins or high molecular peptides, and are easier to be digested and absorbed by the human body. Summary of the invention

[0006] The object of the present invention is to provide a method for extracting bioactive substances from sheep placenta, wherein the bioactive substances purified by the method have a higher yield.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0008] An immobilized lipase:

[0009] The above-mentioned immobilized lipase is a lipase fixed by a carrier;

[0010] The carrier comprises chitosan microspheres;

[0011] The chitosan microspheres are prepared from chitosan derivatives modified with thiazole groups.

[0012] A method for preparing immobilized lipase, comprising:

[0013] Add phosphate buffer to the lipase powder (the mass volume ratio of the two is: 1g:0.1-0.2mL), stir and grind, filter under reduced pressure, and continue to add phosphate buffer to the filtrate to obtain an enzyme solution with a concentration of 0.25-0.5g / L; then add chitosan microspheres to the enzyme solution, oscillate at 28-32°C for 1-2h, wash with ultrapure water, filter, and dry to obtain immobilized lipase.

[0014] For the present invention, the pH of the phosphate buffer is neutral.

[0015] In the present invention, the concentration of the chitosan microspheres in the enzyme solution is 0.2-0.3 wt %.

[0016] A method for preparing chitosan microspheres comprises: adding acetic acid solution to a chitosan derivative modified with a thiazole group, then adding liquid paraffin and sorbitan oleate, stirring and mixing, then adding glutaraldehyde solution, stirring and reacting, cooling, washing, and drying to obtain chitosan microspheres.

[0017] Specifically, the method for preparing the chitosan microspheres comprises the following steps:

[0018] An acetic acid solution is added to the chitosan derivative modified with a thiazole group, and the mixture is stirred at room temperature for 10-15 hours. Liquid paraffin and sorbitan oleate are then added, and the mixture is stirred and mixed at room temperature for 1.5-3 hours. Then, a glutaraldehyde solution having a concentration of 0.2-0.3wt% is added, and the mixture is stirred and reacted at 40-50°C for 1-2.5 hours. The mixture is cooled to room temperature, washed with acetone for 3-5 times, filtered, and dried to obtain chitosan microspheres.

[0019] In the present invention, the mass volume ratio of the chitosan derivative modified with the thiazole group to the acetic acid solution is: 1 g: 3.5-5 mL.

[0020] For the present invention, the concentration of the acetic acid solution is 1.8-2.5wt%.

[0021] In the present invention, the mass volume ratio of the chitosan derivative modified with the thiazole group to the liquid paraffin is: 1g:12-18mL.

[0022] In the present invention, the volume ratio of the liquid paraffin to sorbitan oleate is 1:0.027-0.035.

[0023] In the present invention, the mass volume ratio of the chitosan derivative modified with thiazole groups to the glutaraldehyde solution is: 1 g: 1.5-2 mL.

[0024] The present invention also discloses a method for preparing a chitosan derivative modified with a thiazole group, comprising: carrying out a condensation reaction between 2,4-dimethyl-1,3-thiazole-5-carboxylic acid and chitosan to prepare the chitosan derivative modified with a thiazole group.

[0025] The invention provides a method for preparing a chitosan derivative modified by a thiazole group. 2,4-dimethyl-1,3-thiazole-5-carboxylic acid is used as a modifier, and the modified chitosan derivative modified by the thiazole group is used for preparing an immobilized lipase. The immobilized lipase is then used in the enzymatic hydrolysis process of extracting biologically active substances, so that the active peptide has a higher yield. The immobilized lipase prepared by using the chitosan derivative modified by the thiazole group has good application stability.

[0026] Specifically, the preparation method of the above-mentioned thiazole group-modified chitosan derivative comprises the following steps:

[0027] A hydrochloric acid solution with a concentration of 0.08-0.1 g / mL is added to chitosan, and the mixture is stirred until completely dissolved to obtain a chitosan solution. Then, 2,4-dimethyl-1,3-thiazole-5-carboxylic acid is added to DMF (the mass volume ratio of the two is: 1 g:42-50 mL), and then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added. After stirring and mixing evenly, the mixture is added to the chitosan solution, and the pH is adjusted to 5.3-5.8. The mixture is stirred and reacted for 20-30 hours at room temperature, dialyzed with ultrapure water, and freeze-dried to obtain a chitosan derivative modified with a thiazole group.

[0028] In the present invention, the mass volume ratio of the chitosan to the hydrochloric acid solution is: 1g:42-50mL.

[0029] In the present invention, the mass ratio of the chitosan to 2,4-dimethyl-1,3-thiazole-5-carboxylic acid is 1:1.5-2.3.

[0030] In the present invention, the molar ratio of the above-mentioned N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is: 1:0.8-1.3.

[0031] In the present invention, the mass ratio of the chitosan to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1-1.2.

[0032] The invention also discloses the use of the chitosan derivative modified with thiazole groups in extracting biologically active substances.

[0033] The present invention also discloses a method for extracting bioactive substances from sheep placenta, comprising: crushing fresh sheep placenta and adding ultrapure water; then adding the above-mentioned immobilized lipase for enzymolysis treatment, then adding protease to continue enzymolysis, inactivating the enzyme, filtering, centrifuging, nanofiltering, purifying, drying, and obtaining the bioactive substances.

[0034] Specifically, the method for extracting bioactive substances from sheep placenta comprises the following steps:

[0035] Fresh sheep placenta is crushed and ultrapure water is added; then immobilized lipase is added, and enzymolysis is carried out at 45-60°C for 30-50 minutes, and then protease is added, and enzymolysis is continued at a constant temperature for 3-4.5 hours, and then the enzyme is inactivated at 100-110°C for 10-20 minutes, filtered, centrifuged, and the enzymolysis solution is nanofiltered using a nanofiltration membrane, purified using an ion filter, concentrated, pre-cooled at -40°C to -48°C for 1-1.5 hours, and then dried at a vacuum degree of 0.13-0.17 mbar and a temperature of -30°C to -18°C for 8-9 hours, and dried at a vacuum degree of 0.18-0.23 mbar and a temperature of -15°C to -25°C for 2-3 hours to obtain biologically active substances.

[0036] For the present invention, the mass volume ratio of the fresh sheep placenta to ultrapure water is: 1g:4-8mL;

[0037] In the present invention, the mass ratio of the fresh sheep placenta to the immobilized lipase is 1:0.01-0.02.

[0038] For the present invention, the above-mentioned protease comprises papain.

[0039] For the present invention, the mass ratio of the fresh sheep placenta to the protease is 1:0.015-0.025.

[0040] In the present invention, the above-mentioned biologically active substances include active peptides.

[0041] The invention also discloses the use of the bioactive substance prepared by the method in preparing cosmetics.

[0042] The invention also discloses a method for preparing a nanofiltration membrane, comprising: using S-benzyl-L-cysteine ​​methyl ester hydrochloride and a polyamide nanofiltration membrane to undergo a substitution reaction to obtain the nanofiltration membrane.

[0043] The invention provides a method for preparing a nanofiltration membrane. S-benzyl-L-cysteine ​​methyl ester hydrochloride is used as a modifier. The prepared nanofiltration membrane is used in the purification process of extracting biologically active substances from sheep placenta, and the enzymatic hydrolysate is subjected to nanofiltration treatment to have a good decolorization effect.

[0044] Specifically, the method for preparing the nanofiltration membrane comprises the following steps:

[0045] The polyamide nanofiltration membrane is dried, then washed with n-hexane, dried again, and then an acetone solution of S-benzyl-L-cysteine ​​methyl ester hydrochloride is poured on the surface of the polyamide nanofiltration membrane. After 4-8 minutes, the membrane is dried at 55-65° C. for 4-8 minutes, and then the membrane is immersed in RO water for standby use to obtain a nanofiltration membrane.

[0046] For the present invention, the concentration of the acetone solution of S-benzyl-L-cysteine ​​methyl ester hydrochloride is 1.5-3wt%.

[0047] In the present invention, 0.1-0.3 mL of acetone solution of S-benzyl-L-cysteine ​​methyl ester hydrochloride is poured per square centimeter on the surface of the polyamide nanofiltration membrane.

[0048] The invention also discloses the use of the nanofiltration membrane in extracting biologically active substances.

[0049] The beneficial effects of the present invention include:

[0050] The invention provides a method for extracting bioactive substances from sheep placenta. The method uses immobilized lipase to perform enzymolysis treatment on crushed fresh sheep placenta, then adds protease to continue enzymolysis, and after the enzyme is inactivated, the purified bioactive substances have a high yield; and the immobilized lipase prepared by using a chitosan derivative modified with a thiazole group has good application stability; in addition, the invention also uses S-benzyl-L-cysteine ​​methyl ester hydrochloride to undergo a substitution reaction with a polyamide nanofiltration membrane, and the prepared nanofiltration membrane is used in the purification process of the bioactive substances, and has a good decolorization effect.

[0051] Therefore, the present invention provides a method for extracting bioactive substances from sheep placenta, and the bioactive substances purified by the method have a higher yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The infrared spectrum test results of the chitosan derivative modified with thiazole group and chitosan prepared in Example 1;

[0053] Figure 2 These are the infrared spectra test results of the nanofiltration membrane and polyamide nanofiltration membrane prepared in Example 1. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the present invention more clear, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods:

[0055] Embodiment 1:

[0056] The method for extracting bioactive substances from sheep placenta comprises the following steps:

[0057] Fresh sheep placenta was crushed and added with ultrapure water; then immobilized lipase was added and enzymolysis was carried out at 50℃ for 35min, papain was added and enzymolysis was continued at constant temperature for 3h, then the enzyme was inactivated at 100℃ for 10min, filtered and centrifuged, the enzymolysis solution was nanofiltered with a nanofiltration membrane, purified with an ion filter, concentrated, precooled at -40℃ for 1.5h, then dried at a vacuum degree of 0.13mbar and a temperature of -18℃ for 9h, and dried at a vacuum degree of 0.18mbar and a temperature of -25℃ for 2h to obtain active peptides of biologically active substances. Among them, the mass volume ratio of fresh sheep placenta to ultrapure water is: 1g:4mL; the mass ratio of fresh sheep placenta to immobilized lipase is: 1:0.01; the mass ratio of fresh sheep placenta to protease is: 1:0.015.

[0058] A method for preparing immobilized lipase, comprising:

[0059] Neutral phosphate buffer was added to the lipase powder (the mass volume ratio of the two was: 1g:0.1mL), stirred and ground, filtered under reduced pressure, and phosphate buffer was continued to be added to the filtrate to obtain an enzyme solution with a concentration of 0.25g / L; then chitosan microspheres were added to the enzyme solution, and the concentration of chitosan microspheres in the enzyme solution was 0.2wt%, and the solution was oscillated at 28°C for 1h, washed with ultrapure water, filtered, and dried to obtain immobilized lipase.

[0060] The preparation method of chitosan microspheres comprises the following steps:

[0061] An acetic acid solution was added to a chitosan derivative modified with a thiazole group, and after stirring at room temperature for 10 hours, liquid paraffin and sorbitan oleate were added, and the mixture was stirred at room temperature for 1.5 hours. Then, a glutaraldehyde solution with a concentration of 0.2wt% was added, and the mixture was stirred and reacted at 40°C for 1 hour. The mixture was cooled to room temperature, washed with acetone for 3 times, filtered, and dried to obtain chitosan microspheres. The mass volume ratio of the chitosan derivative modified with the thiazole group to the acetic acid solution was 1 g:3.5 mL. The concentration of the acetic acid solution was 1.8wt%. The mass volume ratio of the chitosan derivative modified with the thiazole group to the liquid paraffin was 1 g:12 mL. The volume ratio of the liquid paraffin to the sorbitan oleate was 1:0.027. The mass volume ratio of the chitosan derivative modified with the thiazole group to the glutaraldehyde solution was 1 g:1.5 mL.

[0062] A method for preparing a thiazole group-modified chitosan derivative, comprising:

[0063] A hydrochloric acid solution with a concentration of 0.08 g / mL was added to chitosan, and the mixture was stirred until completely dissolved to obtain a chitosan solution. Then, 2,4-dimethyl-1,3-thiazole-5-carboxylic acid was added to DMF (the mass volume ratio of the two was 1 g:42 mL), and then N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added. After stirring and mixing evenly, the mixture was added to the chitosan solution, and the pH was adjusted to 5.3. The mixture was stirred and reacted for 20 hours at room temperature. The chitosan derivative modified with thiazole group was obtained by dialyzing with ultrapure water and freeze drying; the mass volume ratio of chitosan to hydrochloric acid solution was 1 g:42 mL; the mass ratio of chitosan to 2,4-dimethyl-1,3-thiazole-5-carboxylic acid was 1:1.5; the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 1:0.8; the mass ratio of chitosan to 1-ethyl-(3-dimethylaminopropyl)carbodiimide was 1:1.

[0064] The method for preparing a nanofiltration membrane comprises the following steps:

[0065] The polyamide nanofiltration membrane is dried, then washed with n-hexane, and dried again, and then an acetone solution of S-benzyl-L-cysteine ​​methyl ester hydrochloride is poured on the surface of the polyamide nanofiltration membrane. After 4 minutes, the membrane is dried at 55°C for 8 minutes, and then the membrane is immersed in RO water for standby use to obtain a nanofiltration membrane; wherein the concentration of the acetone solution of S-benzyl-L-cysteine ​​methyl ester hydrochloride is 1.5wt%; 0.1mL of the acetone solution of S-benzyl-L-cysteine ​​methyl ester hydrochloride is poured per square centimeter on the surface of the polyamide nanofiltration membrane.

[0066] Embodiment 2:

[0067] The difference between the method for extracting biologically active substances from sheep placenta and Example 1 is that the preparation method of immobilized lipase is different.

[0068] The difference between the preparation method of immobilized lipase and Example 1: the preparation method of chitosan microspheres is different.

[0069] The difference between the preparation method of chitosan microspheres and Example 1 is that chitosan is used to replace the chitosan derivative modified with thiazole groups.

[0070] Embodiment 3:

[0071] The difference between the method for extracting bioactive substances from sheep placenta and Example 1 is that a polyamide nanofiltration membrane is used instead of a nanofiltration membrane.

[0072] Embodiment 4:

[0073] The difference between the method for extracting bioactive substances from sheep placenta and Example 2 is that a polyamide nanofiltration membrane is used instead of a nanofiltration membrane.

[0074] Embodiment 5:

[0075] The difference between the method for extracting bioactive substances from sheep placenta and Example 1 is that lipase is used instead of immobilized lipase.

[0076] Test example:

[0077] 1. Infrared spectrum test

[0078] The test was conducted using a TENSOR37 Fourier transform infrared spectrometer from the German BRUKER company and a potassium bromide tablet method.

[0079] The above test was performed on the chitosan derivative modified with thiazole group and chitosan prepared in Example 1. The results are as follows: Figure 1 As shown by Figure 1 It can be seen that compared with the infrared spectrum of chitosan, the chitosan derivative modified with thiazole group has an infrared spectrum of 1678 cm -1 There is an infrared characteristic absorption peak of C=O bond at the position, indicating that 2,4-dimethyl-1,3-thiazole-5-carboxylic acid participates in the formation reaction of thiazole group-modified chitosan derivatives.

[0080] The above test was performed on the nanofiltration membrane and polyamide nanofiltration membrane prepared in Example 1. The results are as follows: Figure 2 As shown by Figure 2 It can be seen that compared with the infrared spectrum of the polyamide nanofiltration membrane, the nanofiltration membrane prepared in Example 1 has an infrared spectrum of 1052 cm -1 The presence of the infrared characteristic absorption peak of the CS bond at the position indicates that S-benzyl-L-cysteine ​​methyl ester hydrochloride participates in the formation reaction of the nanofiltration membrane.

[0081] 2. Active peptide yield test

[0082] The protein content in fresh sheep placenta was determined by Kjeldahl nitrogen determination method before the experiment; the biologically active substances extracted in each embodiment were freeze-dried, and then added with deionized water to prepare a concentration of 5 mg / mL; then the gel chromatography method was used for determination, and the yield of active peptides with a molecular weight between 180-500 Da was calculated, and the calculation formula was as follows:

[0083] F / %=(R / K)×100%

[0084] Among them, F is the yield of active peptides; R is the amount of active peptides between 180-500Da; K is the protein content.

[0085] Table 1 Test results of active peptide yield

[0086]

[0087] The above test was performed on the active peptides obtained in Examples 1-2 and 5, and the results are shown in Table 1. As can be seen from Table 1, compared with Example 2, Example 1, the yield of active peptides was significantly improved, indicating that the chitosan derivative modified with thiazole group prepared by 2,4-dimethyl-1,3-thiazole-5-carboxylic acid was used for the preparation of immobilized lipase, and then the preparation of immobilized lipase was used in the enzymatic hydrolysis process of extracting biologically active substances, so that the active peptides had a higher yield; compared with Example 5, the yield of active peptides in Example 2 was also improved, indicating that the use of immobilized lipase in the enzymatic hydrolysis process of extracting biologically active substances also resulted in a higher yield of active peptides.

[0088] 3. Decolorization effect test

[0089] The enzymatic hydrolysate before and after nanofiltration treatment was tested using a spectrophotometer.

[0090] S / %=[(T0-T1) / T0]×100%

[0091] Wherein, S is the decolorization rate; T0 is the absorbance of the enzymatic hydrolyzate before nanofiltration treatment; T1 is the absorbance of the enzymatic hydrolyzate after nanofiltration treatment.

[0092] Table 2 Decolorization effect test results

[0093]

[0094] The above test was performed on the enzymatic hydrolysate before and after the nanofiltration treatment of Example 1 and Example 3, and the results are shown in Table 2. As can be seen from Table 2, compared with Example 3, the decolorization rate of Example 1 is significantly improved, indicating that the nanofiltration membrane prepared using S-benzyl-L-cysteine ​​methyl ester hydrochloride has a good decolorization effect.

[0095] 4. Application stability of immobilized lipase

[0096] Add 40 mL of methanol to 10 mL of soybean oil, then add 12 g of immobilized lipase, react at 40 ° C for 60 hours, filter to separate the immobilized lipase, wash with tert-butanol, wash with neutral PBS buffer, and dry. Then use the dried immobilized lipase to perform the ester exchange reaction between soybean oil and methanol again; repeat the above operation 5 times, and record the conversion rate of the 5th ester exchange reaction.

[0097] Table 3 Application stability test results of immobilized lipase

[0098]

[0099] The above test was performed on the immobilized lipase prepared in Example 3 and Example 4, and the results are shown in Table 3. As can be seen from Table 3, compared with Example 4, the conversion rate of Example 3 is significantly improved, indicating that the chitosan derivative modified with thiazole group prepared by 2,4-dimethyl-1,3-thiazole-5-carboxylic acid is used for the preparation of immobilized lipase, which can make the immobilized lipase have good application stability.

[0100] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0101] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for extracting bioactive substances from sheep placenta, comprising: Crush the fresh sheep placenta and add ultrapure water; Then, immobilized lipase is added for enzymolysis treatment, and papain is added for further enzymolysis, the enzyme is inactivated, filtered, centrifuged, nanofiltered, purified, and dried to obtain a bioactive substance; the mass ratio of fresh sheep placenta to immobilized lipase is 1:0.01, and the conditions for enzymolysis treatment of the immobilized lipase are: temperature 45-60°C, time 30-50min; the mass ratio of fresh sheep placenta to papain is 1:0.015; The immobilized lipase is a lipase fixed by a carrier, and the carrier comprises chitosan microspheres; The preparation method of chitosan microspheres comprises: adding acetic acid solution to a chitosan derivative modified with a thiazole group, then adding liquid paraffin and sorbitan oleate, stirring and mixing, then adding glutaraldehyde solution, stirring and reacting, cooling, washing, and drying to obtain chitosan microspheres; The method for preparing the chitosan derivative modified by the thiazole group comprises: using 2,4-dimethyl-1,3-thiazole-5-carboxylic acid to react with chitosan to obtain the chitosan derivative modified by the thiazole group; The preparation method of immobilized lipase comprises: adding phosphate buffer to lipase powder, stirring and grinding, decompressing and filtering, and continuously adding phosphate buffer to the filtrate to obtain an enzyme solution with a concentration of 0.25 g / L; then adding chitosan microspheres to the enzyme solution, shaking at 28-32° C. for 1-2 hours, washing with ultrapure water, filtering, and drying to obtain immobilized lipase; the concentration of chitosan microspheres in the enzyme solution is 0.2wt%; A nanofiltration membrane is used in nanofiltration, and a preparation method of the nanofiltration membrane comprises: using S-benzyl-L-cysteine ​​methyl ester hydrochloride to undergo a substitution reaction with a polyamide nanofiltration membrane to obtain the nanofiltration membrane.

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