A method for preparing sheep ruminant effective component extract
Modified agarose resin was prepared as chromatographic filler by reacting agarose resin with divinyl sulfone and isopropyl 3-mercaptopropionate, which solved the problem of insufficient extraction rate and reduction ability of goat ruminant peptin, and achieved high efficiency and high quality protein separation effect.
Patent Information
- Application Number
- CN202311741884.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-18
- Publication Date
- 2025-01-03
- Estimated Expiration
- 2043-12-18
AI Technical Summary
The prior art is difficult to efficiently extract proteins from sheep rumination, especially sheep rumination pepin with high extraction rate and strong reduction ability, and it is difficult to effectively isolate a variety of proteins during the extraction process.
The modified agarose resin was prepared as chromatographic filler by reacting agarose resin with divinyl sulfone and isopropyl 3-mercaptopropionate. The chromatographic separation technology was used to extract goat ruminin, which improved the extraction rate and reduction ability.
The efficient extraction and strong reduction ability of sheep ruminant pepin was achieved, which significantly improved the extraction rate and separation effect, and ensured high-quality separation of proteins.
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Figure CN117736258B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of extraction of effective components from sheep ruminant, and particularly relates to a method for preparing an extract of effective components from sheep ruminant. Background Art
[0002] The scientific name of the fourth stomach of sheep is the ruminant, which is a stomach structure unique to ruminants. Ruminants have four stomachs: the rumen, reticulum, omasum and abomasum. The first three stomachs do not have gastric glands, while the fourth stomach has gastric glands, similar to ordinary monogastric animals, and can secrete digestive juices, so the fourth stomach is also called the true stomach. At present, the main application of sheep stomach is the extraction of rennet, which is used in cheese production, and most people's concept of sheep stomach only stays in the aspect of eating. "Compendium of Materia Medica" records: "Cow and sheep stomach strengthen the spleen and stomach, and avoid food damage caused by drinking and stagnation", which shows that sheep stomach contains active ingredients that are beneficial to the human body, and the active ingredients in the fourth stomach of lambs are more active and deserve to be better utilized. Existing data reports show that the biologically active ingredients in the fourth stomach of lambs mainly contain pepsin, rennet, mucin, protein and bifidobacterial factors. Lamb stomach extract has been used as a drug raw material for treating gastric diseases. Among them, rennet combined with pepsin can be used to break down protein to promote protein absorption, improve the coagulation effect, and improve gastric dysfunction related to protein dissolution; mucin can cover the surface of the gastric mucosa, protect the damaged part of the stomach, increase the proliferation of gastric wall cells, and promote mucosal repair; bifidobacterium factor can promote the growth of Bifidobacterium and fight against harmful bacteria such as Helicobacter pylori. Summary of the invention
[0003] The object of the present invention is to provide a method for preparing a sheep ruminant effective component extract which can be used for separating sheep ruminant protein, has high sheep ruminant protein extraction rate and strong total reducing ability of sheep ruminant protein.
[0004] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:
[0005] A method for preparing an effective component extract of sheep ruminant, comprising: performing a crude extraction treatment on the sheep ruminant to obtain a crude extract of the effective component extract of the sheep ruminant, and purifying the crude extract of the effective component extract of the sheep ruminant to obtain the effective component extract of the sheep ruminant; in the purification treatment, an agarose-based material is used as a chromatographic filler, and the agarose-based material has a sulfone group and a 3-mercaptopropionic acid isopropyl ester group. The present invention first reacts divinyl sulfone with agarose, and then reacts with 3-mercaptopropionic acid isopropyl ester to graft the 3-mercaptopropionic acid isopropyl ester group to obtain a modified agarose resin, and the modified agarose resin is used as an agarose-based material in chromatographic separation, so that sheep ruminant protein with high reducing ability can be obtained, and the extraction rate of the obtained sheep ruminant protein is high. When the sheep ruminant is extracted, a variety of proteins can be obtained, and the modified agarose resin prepared by the method of the present invention has a higher selective separation effect on sheep ruminant protein with reducing ability.
[0006] Preferably, the active ingredient extract is sheep ruminant protein; or, the agarose-based material is formed by bonding divinyl sulfone to agarose resin and 3-mercaptopropionate.
[0007] Preferably, in the preparation of the agarose-based material, firstly, agarose resin is reacted with divinyl sulfone to prepare activated agarose resin, and then the activated agarose resin is reacted with a thiol reagent to prepare the agarose-based material.
[0008] More preferably, in the preparation of the activated agarose resin, the agarose resin and the divinyl sulfone solution are added to a carbonate buffer solution, and the activated agarose resin is prepared by reaction; or, the agarose-based material is a modified agarose resin, and in the preparation of the modified agarose resin, the activated agarose resin and 3-mercaptopropionate are added to a HEPES buffer solution, and the modified agarose resin is prepared by reaction.
[0009] More preferably, the concentration of the carbonate buffer is 0.2-1M; or, the pH of the carbonate buffer is 10-11; or, the amount of agarose resin used is 10-30wt% of the carbonate buffer; or, the divinyl sulfone solution is a mixture of divinyl sulfone and DMSO, and the content of divinyl sulfone in the divinyl sulfone solution is 40-60wt%; or, the amount of divinyl sulfone used is 80-120wt% of the agarose resin.
[0010] More preferably, the concentration of HEPES buffer is 10-30 mM; or, the pH of HEPES buffer is 7.5-8.5; or, the amount of activated agarose resin used is 10-30 wt % of the HEPES buffer; or, the amount of 3-mercaptopropionic acid isopropyl ester used is 2-10 wt % of the activated agarose resin.
[0011] Preferably, the crude extraction process includes pretreatment and extraction process, and the pretreatment includes powdering process and defatting and decolorizing process.
[0012] More preferably, the fat and connective tissue in the sheep ruminant are removed and powdered during the powdering process; or, petroleum ether and anhydrous ethanol are used for defatting and decolorizing; or, sodium chloride solution is used to extract the active ingredients during the extraction process, and chloroform solution is used for extraction and separation.
[0013] More preferably, sodium chloride solution is used for elution in the purification process.
[0014] Preferably, in the preparation of activated agarose resin, the agarose resin is added to a carbonate buffer solution, and then a divinyl sulfone solution is added, and the reaction is carried out at a temperature of 20-40° C. for 6-24 hours. After the reaction is completed, acetonitrile is used to wash to remove unreacted divinyl sulfone, and then deionized water is used to wash to remove acetonitrile, and then dried to obtain the activated agarose resin.
[0015] More preferably, in the preparation of the activated agarose resin, the concentration of the carbonate buffer is 0.2-1 M, and the pH of the carbonate buffer is 10-11.
[0016] More preferably, in the preparation of the activated agarose resin, the amount of the agarose resin used is 10-30 wt % of the carbonate buffer.
[0017] More preferably, in the preparation of the activated agarose resin, the divinyl sulfone solution is a mixture of divinyl sulfone and DMSO, and the content of divinyl sulfone in the divinyl sulfone solution is 40-60wt%.
[0018] More preferably, in the preparation of the activated agarose resin, the amount of divinyl sulfone solution used is based on the amount of divinyl sulfone therein, and the amount of divinyl sulfone used is 80-120 wt % of the agarose resin.
[0019] Preferably, in the preparation of the modified agarose resin, the activated agarose resin is added to a HEPES buffer, and then 3-mercaptopropionic acid isopropyl is added, and the reaction is carried out at a temperature of 20-40° C. for 3-12 hours. After the reaction is completed, the resin is washed with deionized water and dried to obtain the modified agarose resin.
[0020] More preferably, in the preparation of the modified agarose resin, the concentration of the HEPES buffer is 10-30 mM, and the pH of the HEPES buffer is 7.5-8.5.
[0021] More preferably, in the preparation of the modified agarose resin, the amount of the activated agarose resin used is 10-30 wt % of the HEPES buffer.
[0022] More preferably, in the preparation of the modified agarose resin, the amount of isopropyl 3-mercaptopropionate used is 2-10 wt % of the activated agarose resin.
[0023] Preferably, 2-mercaptoimidazole can also be added in the preparation of the modified agarose resin, and the amount of 2-mercaptoimidazole used is 0.4-2wt% of the activated agarose resin. When the present invention uses 3-mercaptopropionic acid isopropyl to modify the activated agarose, 2-mercaptoimidazole is further used for co-modification, thereby improving the extraction rate of sheep ruminant protein with high reducing ability.
[0024] Preferably, in the extraction of sheep ruminant protein, the sheep rumen is pretreated to obtain sheep ruminant powder, the sheep ruminant powder is extracted to obtain a sheep ruminant protein crude extract, and the sheep ruminant protein crude extract is purified to obtain sheep ruminant protein.
[0025] More preferably, in the extraction of sheep ruminant protein, the pretreatment includes powdering treatment and defatting and decolorization treatment. In the powdering treatment, the fresh sheep rumen is washed with water, and then the fat and connective tissue are removed, and it is frozen at minus 100 to minus 60°C for 1-5h, crushed, and freeze-dried to obtain sheep ruminant frozen powder; in the defatting and decolorization treatment, the sheep ruminant frozen powder is defatted with petroleum ether, and then anhydrous ethanol is used to remove alcohol-soluble small molecular impurities, filtered under reduced pressure, and naturally air-dried to obtain sheep ruminant powder.
[0026] More preferably, in the extraction of sheep ruminant protein, in the extraction process, sheep ruminant powder is added to a 0.5-2wt% sodium chloride solution to prepare a first solution with a sheep ruminant powder content of 2-5wt%, ultrasonically treated at a temperature of 50-65°C for 15-60min, then centrifuged to obtain a supernatant, the supernatant is concentrated to a volume of 20-40% at a temperature of 40-60°C, 3-5 times the volume of anhydrous ethanol is added, and the solution is allowed to stand at a temperature of 0-10°C for 6-24h, centrifuged to obtain a precipitate, the precipitate is dissolved in a 0.5-2wt% sodium chloride solution to prepare a second solution with a sheep ruminant powder content of 2-5wt%, and then a 20-40% volume of chloroform solution is added and fully stirred and mixed, the upper aqueous phase is extracted and separated, dialyzed for desalination, and freeze-dried to obtain a crude extract of sheep ruminant protein, and the chloroform solution is prepared by mixing chloroform and n-butanol, and the chloroform and n-butanol in the chloroform solution are mixed in a volume ratio of 1:0.2-0.5.
[0027] More preferably, in the extraction of sheep ruminant protein, in the purification process, the sheep ruminant protein crude extract is separated by a chromatography column, the sheep ruminant protein crude extract is prepared into a sheep ruminant protein crude extract solution with a concentration of 5-20 mg / mL using a 0.5-2wt% sodium chloride solution, and the 0.5-2wt% sodium chloride solution is used as an eluent for elution separation, the eluent detects the characteristic absorption of sugar at 490nm and the absorption of protein at 280nm, and the elution peak of the sheep ruminant protein is selected for collection, dialysis, desalting, freezing, and concentration to obtain the protein. Modified agarose resin is used as a filler in the chromatography column.
[0028] The invention discloses use of an agarose-based material in extracting sheep ruminant protein. The agarose-based material has a sulfone group and a 3-mercaptopropionic acid isopropyl ester group.
[0029] The present invention adopts the method of mixing agarose resin and divinyl sulfone in carbonate buffer, reacting to prepare activated agarose resin containing vinyl sulfone structure, and then reacting the activated agarose resin with 3-mercaptopropionic acid isopropyl to prepare modified agarose resin, and then using the modified agarose resin as the filler of the chromatography column to separate the sheep ruminant protein, and the sheep ruminant protein is prepared from fresh sheep rumen through a series of steps, so it has the following beneficial effects: the sheep ruminant protein has a high extraction rate and the sheep ruminant protein has a strong total reducing ability. Therefore, the present invention is a preparation method of a sheep ruminant effective component extract that can be used to separate sheep ruminant protein, has a high sheep ruminant protein extraction rate, and has a strong total reducing ability of sheep ruminant protein. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 It is an electron microscope image;
[0031] Figure 2 is the extraction rate diagram;
[0032] Figure 3 The total reduction capacity diagram. DETAILED DESCRIPTION
[0033] The technical solution of the present invention is further described in detail below in conjunction with specific implementations and drawings:
[0034] Example 1: A method for preparing sheep ruminant protein
[0035] Preparation of activated agarose resin: Add agarose resin to carbonate buffer, then add divinyl sulfone solution, react at 30°C for 12h, wash with acetonitrile to remove unreacted divinyl sulfone after the reaction is completed, then wash with deionized water to remove acetonitrile, and dry to obtain activated agarose resin. The concentration of carbonate buffer is 0.5M, the pH of carbonate buffer is 11, the amount of carbonate buffer used is 100g, the amount of agarose resin used is 20g, the divinyl sulfone solution is a mixture of divinyl sulfone and DMSO, the content of divinyl sulfone in the divinyl sulfone solution is 50wt%, the amount of divinyl sulfone solution used is based on the amount of divinyl sulfone therein, and the amount of divinyl sulfone used is 20g.
[0036] Preparation of modified agarose resin: Add activated agarose resin to HEPES buffer, then add isopropyl 3-mercaptopropionate, react at 30°C for 6h, wash with deionized water after the reaction is completed, and dry to obtain modified agarose resin. The concentration of HEPES buffer is 20mM, the pH of HEPES buffer is 8, the amount of HEPES buffer used is 100g, the amount of activated agarose resin used is 20g, and the amount of isopropyl 3-mercaptopropionate used is 1g.
[0037] Extraction of sheep ruminant protein: pre-treat the sheep ruminant to obtain sheep ruminant powder, extract the sheep ruminant powder to obtain a sheep ruminant protein crude extract, purify the sheep ruminant protein crude extract to obtain sheep ruminant protein. The pretreatment includes powdering treatment and defatting and decolorizing treatment. In the powdering treatment, the fresh sheep rumen is washed with water, and then the fat and connective tissue are removed, frozen at minus 80°C for 3 hours, crushed, and freeze-dried to obtain sheep rumen frozen powder; in the defatting and decolorizing treatment, the sheep rumen frozen powder is defatted with petroleum ether, and then anhydrous ethanol is used to remove alcohol-soluble small molecular impurities, filtered under reduced pressure, and naturally air-dried to obtain sheep rumen powder; in the extraction treatment, the sheep rumen powder is added to a 0.5wt% sodium chloride solution to prepare a first solution with a sheep rumen powder content of 3wt%, ultrasonically treated at a temperature of 60°C for 30 minutes, and then centrifuged to obtain the supernatant, the supernatant is concentrated to 30% volume at a temperature of 50°C, 4 times the volume of anhydrous ethanol is added, and the solution is allowed to stand at a temperature of 5°C for 12 hours, and centrifuged to obtain a precipitate, and the precipitate is 1wt% Sodium chloride solution is dissolved to prepare a second solution with a content of 3wt% of sheep ruminant powder, and then 30% volume of chloroform solution is added and stirred and mixed, and the upper aqueous phase is extracted and separated, dialyzed for desalination, and freeze-dried to obtain a sheep ruminant protein crude extract, wherein the chloroform solution is a mixture of chloroform and n-butanol, and the chloroform and n-butanol in the chloroform solution are mixed at a volume ratio of 1:0.25; in the purification process, the sheep ruminant protein crude extract is separated by a chromatography column, and the sheep ruminant protein crude extract is prepared by using 1wt% sodium chloride solution to prepare a sheep ruminant protein crude extract solution with a concentration of 10mg / mL, and 1wt% sodium chloride solution is used as an eluent for elution separation, and the eluent detects the characteristic absorption of sugar at 490nm and the absorption of protein at 280nm, and the elution peak of the sheep ruminant protein is selected for collection, dialyzed, desalted, frozen, and concentrated to obtain the protein. Modified agarose resin is used as a filler in the chromatography column.
[0038] Example 2: A method for preparing sheep ruminant protein
[0039] Compared with Example 1, the difference between this example and Example 1 lies in the preparation of modified agarose resin.
[0040] Preparation of modified agarose resin: Add activated agarose resin to HEPES buffer, then add isopropyl 3-mercaptopropionate, react at 30°C for 6h, wash with deionized water after the reaction is completed, and dry to obtain modified agarose resin. The concentration of HEPES buffer is 20mM, the pH of HEPES buffer is 8, the amount of HEPES buffer used is 100g, the amount of activated agarose resin used is 20g, and the amount of isopropyl 3-mercaptopropionate used is 0.5g.
[0041] Example 3: A method for preparing sheep ruminant protein
[0042] Compared with Example 1, the difference between this example and Example 1 lies in the preparation of modified agarose resin.
[0043] Preparation of modified agarose resin: Add activated agarose resin to HEPES buffer, then add isopropyl 3-mercaptopropionate, react at 30°C for 6h, wash with deionized water after the reaction is completed, and dry to obtain modified agarose resin. The concentration of HEPES buffer is 20mM, the pH of HEPES buffer is 8, the amount of HEPES buffer used is 100g, the amount of activated agarose resin used is 20g, and the amount of isopropyl 3-mercaptopropionate used is 1.5g.
[0044] Example 4: A method for preparing sheep ruminant protein
[0045] Preparation of activated agarose resin: Add agarose resin to carbonate buffer, then add divinyl sulfone solution, react at 30°C for 12h, wash with acetonitrile to remove unreacted divinyl sulfone after the reaction is completed, then wash with deionized water to remove acetonitrile, and dry to obtain activated agarose resin. The concentration of carbonate buffer is 0.5M, the pH of carbonate buffer is 11, the amount of carbonate buffer used is 100g, the amount of agarose resin used is 20g, the divinyl sulfone solution is a mixture of divinyl sulfone and DMSO, the content of divinyl sulfone in the divinyl sulfone solution is 50wt%, the amount of divinyl sulfone solution used is based on the amount of divinyl sulfone therein, and the amount of divinyl sulfone used is 20g.
[0046] Preparation of modified agarose resin: Add activated agarose resin to HEPES buffer, then add isopropyl 3-mercaptopropionate and 2-mercaptoimidazole, react at 30°C for 6h, wash with deionized water after the reaction is completed, and dry to obtain modified agarose resin. The concentration of HEPES buffer is 20mM, the pH of HEPES buffer is 8, the amount of HEPES buffer used is 100g, the amount of activated agarose resin used is 20g, the amount of isopropyl 3-mercaptopropionate used is 1g, and the amount of 2-mercaptoimidazole used is 0.2g.
[0047] Extraction of sheep ruminant protein: pre-treat the sheep ruminant to obtain sheep ruminant powder, extract the sheep ruminant powder to obtain a sheep ruminant protein crude extract, purify the sheep ruminant protein crude extract to obtain sheep ruminant protein. The pretreatment includes powdering treatment and defatting and decolorizing treatment. In the powdering treatment, the fresh sheep rumen is washed with water, and then the fat and connective tissue are removed, frozen at minus 80°C for 3 hours, crushed, and freeze-dried to obtain sheep rumen frozen powder; in the defatting and decolorizing treatment, the sheep rumen frozen powder is defatted with petroleum ether, and then anhydrous ethanol is used to remove alcohol-soluble small molecular impurities, filtered under reduced pressure, and naturally air-dried to obtain sheep rumen powder; in the extraction treatment, the sheep rumen powder is added to a 0.5wt% sodium chloride solution to prepare a first solution with a sheep rumen powder content of 3wt%, ultrasonically treated at a temperature of 60°C for 30 minutes, and then centrifuged to obtain the supernatant, the supernatant is concentrated to 30% volume at a temperature of 50°C, 4 times the volume of anhydrous ethanol is added, and the solution is allowed to stand at a temperature of 5°C for 12 hours, and centrifuged to obtain a precipitate, and the precipitate is 1wt% Sodium chloride solution is dissolved to prepare a second solution with a content of 3wt% of sheep ruminant powder, and then 30% volume of chloroform solution is added and stirred and mixed, and the upper aqueous phase is extracted and separated, dialyzed for desalination, and freeze-dried to obtain a sheep ruminant protein crude extract, wherein the chloroform solution is a mixture of chloroform and n-butanol, and the chloroform and n-butanol in the chloroform solution are mixed at a volume ratio of 1:0.25; in the purification process, the sheep ruminant protein crude extract is separated by a chromatography column, and the sheep ruminant protein crude extract is prepared by using 1wt% sodium chloride solution to prepare a sheep ruminant protein crude extract solution with a concentration of 10mg / mL, and 1wt% sodium chloride solution is used as an eluent for elution separation, and the eluent detects the characteristic absorption of sugar at 490nm and the absorption of protein at 280nm, and the elution peak of the sheep ruminant protein is selected for collection, dialyzed, desalted, frozen, and concentrated to obtain the protein. Modified agarose resin is used as a filler in the chromatography column.
[0048] Example 5: A method for preparing sheep ruminant protein
[0049] Compared with Example 4, the difference between this example and Example 4 lies in the preparation of the modified agarose resin.
[0050] Preparation of modified agarose resin: Add activated agarose resin to HEPES buffer, then add isopropyl 3-mercaptopropionate and 2-mercaptoimidazole, react at 30°C for 6h, wash with deionized water after the reaction is completed, and dry to obtain modified agarose resin. The concentration of HEPES buffer is 20mM, the pH of HEPES buffer is 8, the amount of HEPES buffer used is 100g, the amount of activated agarose resin used is 20g, the amount of isopropyl 3-mercaptopropionate used is 0.5g, and the amount of 2-mercaptoimidazole used is 0.2g.
[0051] Example 6: A method for preparing sheep ruminant protein
[0052] Compared with Example 4, the difference between this example and Example 4 lies in the preparation of the modified agarose resin.
[0053] Preparation of modified agarose resin: Add activated agarose resin to HEPES buffer, then add isopropyl 3-mercaptopropionate and 2-mercaptoimidazole, react at 30°C for 6h, wash with deionized water after the reaction is completed, and dry to obtain modified agarose resin. The concentration of HEPES buffer is 20mM, the pH of HEPES buffer is 8, the amount of HEPES buffer used is 100g, the amount of activated agarose resin used is 20g, the amount of isopropyl 3-mercaptopropionate used is 1.5g, and the amount of 2-mercaptoimidazole used is 0.2g.
[0054] Comparative Example 1:
[0055] The difference between this comparative example and Example 1 lies in the extraction of sheep ruminant protein.
[0056] Extraction of sheep ruminant protein: pre-treat the sheep ruminant to obtain sheep ruminant powder, extract the sheep ruminant powder to obtain a sheep ruminant protein crude extract, purify the sheep ruminant protein crude extract to obtain sheep ruminant protein. The pretreatment includes powdering treatment and defatting and decolorizing treatment. In the powdering treatment, the fresh sheep rumen is washed with water, and then the fat and connective tissue are removed, frozen at minus 80°C for 3 hours, crushed, and freeze-dried to obtain sheep rumen frozen powder; in the defatting and decolorizing treatment, the sheep rumen frozen powder is defatted with petroleum ether, and then anhydrous ethanol is used to remove alcohol-soluble small molecular impurities, filtered under reduced pressure, and naturally air-dried to obtain sheep rumen powder; in the extraction treatment, the sheep rumen powder is added to a 0.5wt% sodium chloride solution to prepare a first solution with a sheep rumen powder content of 3wt%, ultrasonically treated at a temperature of 60°C for 30 minutes, and then centrifuged to obtain the supernatant, the supernatant is concentrated to 30% volume at a temperature of 50°C, 4 times the volume of anhydrous ethanol is added, and the solution is allowed to stand at a temperature of 5°C for 12 hours, and centrifuged to obtain a precipitate, and the precipitate is 1wt% Sodium chloride solution is dissolved to prepare a second solution with a content of 3wt% of sheep ruminant powder, and then 30% volume of chloroform solution is added and stirred and mixed, and the upper aqueous phase is extracted and separated, dialyzed for desalination, and freeze-dried to obtain a sheep ruminant protein crude extract, wherein the chloroform solution is a mixture of chloroform and n-butanol, and the chloroform and n-butanol in the chloroform solution are mixed at a volume ratio of 1:0.25; in the purification process, the sheep ruminant protein crude extract is separated by a chromatography column, and the sheep ruminant protein crude extract is prepared into a sheep ruminant protein crude extract solution with a concentration of 10mg / mL by using 1wt% sodium chloride solution, and eluted and separated by using 1wt% sodium chloride solution as eluent, and the eluent detects the characteristic absorption of sugar at 490nm and the absorption of protein at 280nm, and the elution peak of the sheep ruminant protein is selected for collection, dialyzed, desalted, frozen, and concentrated to obtain the protein. Activated agarose resin is used as filler in the chromatography column.
[0057] Comparative Example 2:
[0058] The difference between this comparative example and Example 1 lies in the extraction of sheep ruminant protein.
[0059] Extraction of sheep ruminant protein: pre-treat the sheep ruminant to obtain sheep ruminant powder, extract the sheep ruminant powder to obtain a sheep ruminant protein crude extract, purify the sheep ruminant protein crude extract to obtain sheep ruminant protein. The pretreatment includes powdering treatment and defatting and decolorizing treatment. In the powdering treatment, the fresh sheep rumen is washed with water, and then the fat and connective tissue are removed, frozen at minus 80°C for 3 hours, crushed, and freeze-dried to obtain sheep rumen frozen powder; in the defatting and decolorizing treatment, the sheep rumen frozen powder is defatted with petroleum ether, and then anhydrous ethanol is used to remove alcohol-soluble small molecular impurities, filtered under reduced pressure, and naturally air-dried to obtain sheep rumen powder; in the extraction treatment, the sheep rumen powder is added to a 0.5wt% sodium chloride solution to prepare a first solution with a sheep rumen powder content of 3wt%, ultrasonically treated at a temperature of 60°C for 30 minutes, and then centrifuged to obtain the supernatant, the supernatant is concentrated to 30% volume at a temperature of 50°C, 4 times the volume of anhydrous ethanol is added, and the solution is allowed to stand at a temperature of 5°C for 12 hours, and centrifuged to obtain a precipitate, and the precipitate is 1wt% Sodium chloride solution is dissolved to prepare a second solution with a content of 3wt% of sheep ruminant powder, and then 30% volume of chloroform solution is added and stirred and mixed, and the upper aqueous phase is extracted and separated, dialyzed for desalination, and freeze-dried to obtain a sheep ruminant protein crude extract, wherein the chloroform solution is a mixture of chloroform and n-butanol, and the chloroform and n-butanol in the chloroform solution are mixed at a volume ratio of 1:0.25; in the purification process, the sheep ruminant protein crude extract is separated by a chromatography column, and the sheep ruminant protein crude extract is prepared by using 1wt% sodium chloride solution to prepare a sheep ruminant protein crude extract solution with a concentration of 10mg / mL, and 1wt% sodium chloride solution is used as an eluent for elution separation, and the eluent detects the characteristic absorption of sugar at 490nm and the absorption of protein at 280nm, and the elution peak of the sheep ruminant protein is selected for collection, dialyzed, desalted, frozen, and concentrated to obtain the protein. Agarose resin is used as a filler in the chromatography column.
[0060] Test example:
[0061] The modified agarose resin prepared in Example 1 was characterized by SEM. Figure 1 As shown, the surface of the modified agarose resin is completely formed without obvious collapse and the pore structure is clear.
[0062] The present invention calculated the extraction rate of sheep ruminant protein extracted from each embodiment and comparative example, and the results are as follows: Figure 2As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, D1 is Comparative Example 1, and D2 is Comparative Example 2. The present invention mixes agarose resin and divinyl sulfone in a carbonate buffer, reacts to prepare an activated agarose resin containing an olefinic sulfone structure, and then reacts the activated agarose resin with 3-mercaptopropionic acid isopropyl to prepare a modified agarose resin. The modified agarose resin is then used as a filler for a chromatography column to separate sheep ruminant protein. The sheep ruminant protein is separated from the fresh sheep rumen through a step-by-step process. The present invention finds that divinyl sulfone combines agarose resin and 3-mercaptopropionic acid isopropyl through chemical bonds to obtain a modified agarose resin. Compared with the activated agarose resin obtained by modifying the agarose resin with divinyl sulfone alone, the modified agarose resin is more The agarose resin is used as a chromatography column filler, and the extraction effect of sheep ruminant protein is better, that is, the extraction rate of the obtained sheep ruminant protein is higher. If agarose that is not treated with divinyl is used as a chromatography column filler, the extraction effect of sheep ruminant protein is poor, indicating that after the agarose resin is modified by divinyl sulfone, 3-mercaptopropionate isopropyl is further reacted, and under the action of the agarose resin structure and the 3-mercaptopropionate isopropyl structure, the sheep ruminant protein has a better extraction effect; on this basis, after obtaining the activated agarose resin, the present invention further uses 3-mercaptopropionate isopropyl and 2-mercaptoimidazole to jointly treat the activated agarose to obtain a modified agarose resin obtained by the joint reaction of 3-mercaptopropionate isopropyl and 2-mercaptoimidazole, and then uses the modified agarose resin as a chromatography column filler, which has a better extraction effect on sheep ruminant protein.
[0063] The present invention tests the total reducing ability of the sheep ruminant protein extracted from each embodiment and the comparative example. The sheep ruminant protein obtained from each embodiment and the comparative example is added to a 0.025M phosphate buffer with a pH of 6.86, and the concentration of the sheep ruminant protein reaches 0.2 mg / mL. Potassium ferrocyanide is added, and the concentration of potassium ferrocyanide reaches 0.3wt%. The reaction is treated at 50° C. for 30 minutes, and then trichloroacetic acid is added to terminate the reaction, and the concentration of trichloroacetic acid reaches 2.5wt%. The supernatant is centrifuged, an equal volume of distilled water and a quarter volume of a 0.1wt% ferric chloride solution are added, and the mixture is evenly mixed and then treated for 10 minutes. The absorbance at 700 nm is detected, and the reducing ability value is characterized by the ratio of the absorbance difference before and after reduction to the absorbance difference before and after reduction of vitamin C at the same concentration. The total reducing ability test result is as follows: Figure 3As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, D1 is Comparative Example 1, and D2 is Comparative Example 2. The present invention mixes agarose resin and divinyl sulfone in a carbonate buffer, reacts to prepare an activated agarose resin containing an olefinic sulfone structure, and then reacts the activated agarose resin with 3-mercaptopropionic acid isopropyl to prepare a modified agarose resin. The modified agarose resin is then used as a filler for a chromatography column to separate sheep ruminant protein. The sheep ruminant protein is separated from fresh sheep rumen through a step-by-step process. The present invention finds that divinyl sulfone combines agarose resin and 3-mercaptopropionic acid isopropyl through chemical bonds to obtain a modified agarose resin. Compared with the modification of agarose resin using only divinyl sulfone, The obtained activated agarose resin is used as a chromatography column filler, and the obtained sheep ruminant protein has higher reduction performance. If agarose not treated with divinyl is used as a chromatography column filler, the obtained sheep ruminant protein has poor reduction performance, indicating that after the agarose resin is modified with divinyl sulfone, 3-mercaptopropionate is further reacted, and under the action of the agarose resin structure and the 3-mercaptopropionate isopropyl structure, the obtained sheep ruminant protein has better reduction performance; on this basis, after obtaining the activated agarose resin, the present invention further uses 3-mercaptopropionate isopropyl and 2-mercaptoimidazole to jointly treat the activated agarose to obtain a modified agarose resin obtained by the joint reaction of 3-mercaptopropionate isopropyl and 2-mercaptoimidazole, and then uses the modified agarose resin as a chromatography column filler, and the obtained sheep ruminant protein has stronger reduction performance.
[0064] 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.
[0065] 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 an extract of active ingredients from sheep ruminant stomach, comprising: The sheep rumen is subjected to a crude extraction treatment to obtain a crude extract of the sheep rumen effective component extract, and the crude extract of the sheep rumen effective component extract is subjected to a purification treatment to obtain the sheep rumen effective component extract; in the purification treatment, an agarose-based material is used as a chromatographic filler, the agarose-based material has a sulfone group and a 3-mercaptopropionic acid isopropyl ester group, and the agarose-based material is formed by bonding divinyl sulfone with agarose resin and 3-mercaptopropionic acid isopropyl ester respectively; in the preparation of the agarose-based material, firstly, agarose resin is reacted with divinyl sulfone to obtain an activated agarose resin, and then the activated agarose resin is reacted with a sulfhydryl reagent to obtain the agarose-based material; In the preparation of the activated agarose resin, agarose resin and divinyl sulfone solution are added to a carbonate buffer solution to react and prepare the activated agarose resin; the agarose-based material is a modified agarose resin, and in the preparation of the modified agarose resin, activated agarose resin and 3-mercaptopropionic acid isopropyl are added to a HEPES buffer solution to react and prepare the modified agarose resin; The concentration of the carbonate buffer is 0.2-1M; the pH of the carbonate buffer is 10-11; the amount of agarose resin used is 10-30wt% of the carbonate buffer; the divinyl sulfone solution is a mixture of divinyl sulfone and DMSO, and the content of divinyl sulfone in the divinyl sulfone solution is 40-60wt%; the amount of divinyl sulfone used is 80-120wt% of the agarose resin; The concentration of the HEPES buffer is 10-30 mM; the pH of the HEPES buffer is 7.5-8.5; the amount of activated agarose resin used is 10-30 wt % of the HEPES buffer; the amount of 3-mercaptopropionic acid isopropyl used is 2-10 wt % of the activated agarose resin; In the purification process, the sheep ruminant protein crude extract is separated by a chromatography column, the sheep ruminant protein crude extract is prepared into a sheep ruminant protein crude extract solution with a concentration of 5-20 mg / mL by using a 0.5-2wt% sodium chloride solution, the 0.5-2wt% sodium chloride solution is used as an eluent, elution separation is performed, the eluent detects the characteristic absorption of sugar at 490nm and the absorption of protein at 280nm, the elution peak of the sheep ruminant protein is selected for collection, dialyzed, desalted, frozen, concentrated, and the protein is obtained, and the modified agarose resin is used as a filler in the chromatography column.
2. The method for preparing a sheep ruminant effective ingredient extract according to claim 1, characterized in that: The active ingredient extract is sheep ruminant protein.
3. The method for preparing a sheep ruminant effective ingredient extract according to claim 1, characterized in that: The crude extraction process includes pretreatment and extraction process, and the pretreatment includes powdering process and defatting and decoloring process.
4. The method for preparing a sheep ruminant effective component extract according to claim 3, characterized in that: In the powdering process, fat and connective tissue in the sheep ruminant are removed and powdered; or, petroleum ether and anhydrous ethanol are used in the defatting and decolorizing process; or, sodium chloride solution is used to extract the effective ingredients in the extraction process, and chloroform solution is used for extraction and separation.
5. The method for preparing a sheep ruminant effective component extract according to claim 1, characterized in that: In the purification process, sodium chloride solution is used for elution.
6. Use of the agarose-based material according to claim 1 in extracting sheep ruminant protein.