CN113956341B - Preparation method of Tianshan red deer abomasum glycoprotein with hyaluronidase inhibition activity - Google Patents
Preparation method of Tianshan red deer abomasum glycoprotein with hyaluronidase inhibition activity Download PDFInfo
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- CN113956341B CN113956341B CN202111360384.4A CN202111360384A CN113956341B CN 113956341 B CN113956341 B CN 113956341B CN 202111360384 A CN202111360384 A CN 202111360384A CN 113956341 B CN113956341 B CN 113956341B
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- 210000003165 abomasum Anatomy 0.000 title claims abstract description 128
- 241000282985 Cervus Species 0.000 title claims abstract description 104
- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 85
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 85
- 108010003272 Hyaluronate lyase Proteins 0.000 title claims abstract description 45
- 102000001974 Hyaluronidases Human genes 0.000 title claims abstract description 45
- 229960002773 hyaluronidase Drugs 0.000 title claims abstract description 45
- 230000000694 effects Effects 0.000 title claims abstract description 18
- 230000005764 inhibitory process Effects 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 35
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 24
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- 239000012153 distilled water Substances 0.000 claims description 21
- 238000004108 freeze drying Methods 0.000 claims description 21
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
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- 238000011033 desalting Methods 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 10
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- 238000000746 purification Methods 0.000 claims description 7
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 6
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 5
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 5
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- 239000002808 molecular sieve Substances 0.000 abstract 1
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- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 abstract 1
- 239000008399 tap water Substances 0.000 abstract 1
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
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- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to a preparation method of a Tianshan red deer abomasum glycoprotein with hyaluronidase inhibition activity, which comprises the steps of taking Tianshan red deer abomasum, washing with tap water, shearing, freezing, vacuum freeze-drying at low temperature, and crushing into powder; adding petroleum ether, placing in a Soxhlet extractor, heating, refluxing, degreasing, extracting the degreased filter residue with stirring at room temperature, filtering with an oscillating screen, and suction filtering; precipitating to obtain crude glycoprotein; then, carrying out primary separation by anion exchange resin column chromatography, and screening hyaluronidase inhibition activity; the gel column HW-55F molecular sieve column chromatography system is adopted to separate and purify the active components to obtain the Tianshan red deer abomasum glycoprotein, and the hyaluronidase inhibition function is evaluated by an in vitro activity screening method. The Tianshan red deer abomasum glycoprotein obtained by the method has high purity, the protein content is 91.63%, the sugar content is 3.09%, and the method has the advantages of simple process, strong operability, easy realization, full utilization of raw materials, and wide industrial application prospect.
Description
Technical Field
The invention relates to the technical field of animal food processing and biological activity, in particular to a preparation method of Tianshan red deer abomasum glycoprotein with hyaluronidase inhibition activity.
Background
Glycoproteins are formed by covalent linkage of oligosaccharides to polypeptide chains. Glycoproteins are widely present in eukaryotes, most of which have glycosylation modifications, and they play an important role in organisms and are closely related to the occurrence of some diseases. The sugar chains in the glycoprotein have the characteristics of micro-heterogeneity and non-template synthesis, and determine the complexity and composition diversity of the glycoprotein structure, so that the analysis of the sugar chains cannot make comprehensive and complete structural identification until now. The extraction of glycoprotein is generally carried out by dilute salt solution extraction, acid-base solution extraction, enzyme extraction, etc., and the extraction method can be determined according to the characteristics of the extracted object. With the rapid development of glycobiology research, glycoproteins have become a hotspot for biochemical research, attracting many disciplines, including chemical, biological, biochemical, immunological, cell biological, pharmaceutical, and food science workers to address this area of research. The glycoprotein has the characteristics of enhancing immunity, inhibiting tumors, reducing blood sugar and blood fat, resisting oxidation, resisting aging and the like and low toxicity, so that the glycoprotein has wider and wider research application in the fields of medicine and food science and has wide application prospect in the development of novel characteristic medicines and functional foods.
The Chinese is the main feeding country of the world red deer, 110 or more than ten thousands of artificial breeding stock accounts for 97 percent of the total world feeding amount. Xinjiang has rich red deer resources. At present, xinjiang has 31 breeding enterprises in the Xinjiang Qinghai general farm, and the large and small red deer farms such as the Altai, the Changji, the Yili and the Bazhou, and the artificially domesticated red deer has more than 10 ten thousand heads, so that the wild animal with the largest domesticating and breeding rule in Xinjiang is a wild animal. Abomasum is the fourth stomach of deer, also called deer fourth stomach, a stomach structure characteristic of ruminants. Deer abomasum has gastric glands, similar to common monogastric animals, which can secrete digestive juice, so it is also called true stomach. The deer abomasum has rich protein and protease. The deer stomach mainly has the effects of warming cold and nourishing stomach, and has good effects on gastritis, stomachache, gastric ulcer, gastrectasia, gastroptosis, gastric mucosa prolapse and gastric hyperacidity. The deer stomach also has the effects of tonifying middle-jiao and Qi, strengthening stomach and strengthening body constitution, promoting digestion, promoting absorption, treating dyspepsia, tonifying deficiency and enriching blood, has high medicinal value and is also a good health food.
Hyaluronidase inhibitors are substances that have an inhibitory effect on the activation of hyaluronidase. Hyaluronidase is a specific cleavage enzyme of hyaluronic acid, and hyaluronic acid plays an important role in many development and regulation processes of human body, and inhibiting the activity of hyaluronidase can prevent the decomposition of hyaluronic acid and maintain normal physiological functions.
Disclosure of Invention
The invention aims at providing a preparation method of a Tianshan red deer abomasum glycoprotein with hyaluronidase inhibition activity, which comprises the steps of firstly, carrying out reflux degreasing on a pretreated fresh Tianshan red deer abomasum by using petroleum ether, extracting, centrifuging to obtain a supernatant, concentrating under reduced pressure, precipitating with ethanol, and freeze-drying to obtain a crude glycoprotein; and separating and purifying the crude glycoprotein by anion exchange chromatography and gel chromatography to obtain high-purity Tianshan red deer abomasum glycoprotein. The obtained Tianshan red deer abomasum glycoprotein has the yield of 4.7%, the protein content of 91.63%, the sugar content of 3.09%, the molecular weight of 15.11Da and the hyaluronidase inhibition rate of 79.23%. The method has the advantages of simple process, convenient operation, high extraction rate, high purity, stable property, no need of using a large amount of toxic solvents, low cost, easy popularization and application and the like.
The preparation method of the Tianshan red deer abomasum glycoprotein with hyaluronidase inhibition activity comprises the following steps:
a. taking the abomasum of the Tianshan red deer, flushing the abomasum by flowing water to remove the content in the abomasum, and storing the abomasum in a refrigerator at the temperature of minus 20 ℃ for later use;
b. c, removing fat and connective tissues from the red deer abomasum cleaned by precooling distilled water flowing water in the step a, shearing the red deer abomasum to 5-8mm in width, freezing the red deer abomasum at the temperature of-80 ℃ for 12 hours, and then freeze-drying to obtain red deer abomasum dry powder;
c. c, crushing the red deer abomasum dry powder obtained in the step b at a low temperature by utilizing liquid nitrogen, crushing the red deer abomasum dry powder by using a crusher at a rotation speed of 5000r/min, suspending for 0.5min every 1min, performing cumulative crushing for 3-5min, sieving by using a 80-mesh sieve to obtain red deer abomasum powder, and storing in a refrigerator at a temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c according to a feed liquid ratio of 1:10-15 at the temperature of 4 ℃, mixing, placing in a Soxhlet extractor, heating for 3-5h at the temperature of 60-75 ℃, carrying out reflux degreasing treatment, removing petroleum ether by suction filtration, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the defatted abomasum powder obtained in the step d with 2-5% NaCl solution according to the mass ratio of 1:10-15, stirring and extracting at room temperature for 3-5h, filtering the extracting solution by using a vibrating screen, centrifuging the filtrate at 6000-8000r/min and the temperature of 4 ℃ for 10-20min to obtain supernatant, concentrating the supernatant under reduced pressure by using a rotary evaporator at 35-60mbar and the temperature of 50 ℃ to 1/10-1/15 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, and centrifuging the desalted solution at 6000-8000r/min and the temperature of 4 ℃ for 10-20min to obtain the total extracting solution;
f. adding absolute ethyl alcohol into the extract obtained in the step e at the temperature of 0-4 ℃ to ensure that the volume concentration of the ethyl alcohol is 70-80%, standing for 24 hours to completely precipitate, dissolving the precipitate with distilled water, centrifuging for 15 minutes at the temperature of 8000r/min and the temperature of 4 ℃, taking supernatant, and obtaining the Tianshan red deer abomasum glycoprotein after freeze drying for 10 Pa-80 ℃ and 24 hours;
g. purifying the crude glycoprotein obtained in the step f for the first time, completely dissolving the crude glycoprotein to a concentration of 15mg/mL by using a phosphate buffer solution with the pH of 7.8 and 0.02M, centrifuging, taking a supernatant, carrying out column chromatography by using a DEAE-Sephadex A-25 anion exchange resin, eluting by using NaCl solutions with the concentration of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL respectively, detecting the absorption peak of sugar at 492nm by using a phenol-sulfuric acid method, detecting the absorption peak of protein at 280nm by using an ultraviolet spectrophotometer, collecting eluent of mutually overlapped parts of the absorption peaks of the sugar and the protein, concentrating the eluent of the mutually overlapped parts under reduced pressure at 35-60mbar, concentrating at the temperature of 50 ℃, desalting the concentrate by using a 3500Da dialysis bag for 48h, and then carrying out freeze drying at the temperature of-80 ℃ and the pressure of 10Pa for 8h to obtain the first purified glycoprotein;
h. detecting the hyaluronidase inhibitory activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibitory activity;
i. and (3) performing secondary purification on the component with the hyaluronidase inhibitory activity collected in the step (h), dissolving the component with the hyaluronidase inhibitory activity in distilled water, separating and purifying the component with the distilled water by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, concentrating the pure red deer abomasum glycoprotein solution at the pressure of 35-60mbar and the temperature of 50 ℃, and performing freeze drying at the temperature of-80 ℃ and the pressure of 10Pa to obtain the red deer abomasum glycoprotein.
The Tianshan red deer abomasum glycoprotein obtained by the method is used for preparing anti-inflammatory medicaments.
The invention relates to a preparation method of a Tianshan red deer abomasum hyaluronidase inhibition activity glycoprotein, in the method, detection of the hyaluronidase inhibition activity is carried out by adopting 500unit/mL of a sample solution of 0.5mL and 0.5mL of hyaluronidase, and culturing for 20min at 37 ℃; adding 2.5mol/L CaCl 2 0.1mL, and preserving heat for 20min at 37 ℃; adding 0.5mg/mL of potassium hyaluronate, preserving heat at 37 ℃ for 40min, standing at normal temperature for 5min, adding 2 drops of 5mol/L NaOH, adding 0.5mL of acetylacetone solution, accurately boiling water for 15min, immediately cooling with ice water for 5min, standing at normal temperature for 10min, adding 0.5mL of deionized water and 0.5mL of Ehrlich reagent, adding absolute ethyl alcohol to 10mL, shaking, mixing uniformly, standing for 30min, developing, and measuring the ABS value at 530 nm.
Drawings
FIG. 1 is a schematic illustration of the process flow of the present invention;
FIG. 2 is a chromatogram of the present invention for separating and purifying a Tianshan red deer abomasum glycoprotein using an anion exchange resin;
FIG. 3 is a chromatogram of the purification of a Tianshan red deer abomasum glycoprotein using gel chromatography in accordance with the present invention;
FIG. 4 is a graph showing HPLC molecular weight measurement and purity identification of the present invention for the sika deer abomasum glycoprotein;
FIG. 5 is an infrared spectrum of the present invention of a Tianshan red deer abomasum glycoprotein;
FIG. 6 is a GC diagram for analysis of a glucose glycoprotein monosaccharide of a Tianshan red deer of the present invention, wherein 1 is rhamnose, 2 is arabinose, 3 is xylose, 4 is mannose, 5 is glucose, and 6 is galactose;
FIG. 7 is a full ultraviolet wavelength view of the sika deer abomasum glycoprotein of the present invention;
FIG. 8 is a 15% SDS-polyacrylamide gel electrophoresis of the present invention of a Tianshan red deer abomasum glycoprotein;
FIG. 9 shows the hyaluronidase inhibitory activity of the present invention Tianshan red deer abomasum glycoprotein.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited to any form.
Example 1
a. Taking the abomasum of the Tianshan red deer, flushing the abomasum by flowing water to remove the content in the abomasum, and storing the abomasum in a refrigerator at the temperature of minus 20 ℃ for later use;
b. c, removing fat and connective tissues from the red deer abomasum cleaned by precooling distilled water flowing water in the step a, shearing the red deer abomasum to be 5mm in width, freezing the red deer abomasum at the temperature of-80 ℃ for 12 hours, and then freeze-drying to obtain red deer abomasum dry powder;
c. c, crushing the red deer abomasum dry powder obtained in the step b at a low temperature by utilizing liquid nitrogen, crushing the red deer abomasum dry powder by using a crusher at a rotation speed of 5000r/min, suspending for 0.5min every 1min, performing cumulative crushing for 3-5min, sieving by using a 80-mesh sieve to obtain red deer abomasum powder, and storing in a refrigerator at a temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c according to a feed liquid ratio of 1:10 at the temperature of 4 ℃, mixing, placing in a Soxhlet extractor, heating at the temperature of 60 ℃ for 5 hours, carrying out reflux degreasing treatment, filtering to remove petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the defatted abomasum powder obtained in the step d with 2% NaCl solution according to a mass ratio of 1:10, stirring and extracting for 4 hours at room temperature, filtering the extracting solution by using an oscillating screen, filtering the filtering solution at 6000r/min and a temperature of 4 ℃, centrifuging for 10 minutes to obtain supernatant, concentrating the supernatant under reduced pressure by using a rotary evaporator at a pressure of 35mbar and a temperature of 50 ℃ to 1/10 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, centrifuging for 10 minutes at the temperature of 6000r/min, and obtaining the total extracting solution;
f. adding absolute ethyl alcohol into the extracting solution obtained in the step e according to the volume ratio of 1:4, mixing at the temperature of 4 ℃ to ensure that the volume concentration of the ethyl alcohol is 70%, standing for 24 hours to completely precipitate, dissolving the precipitate with distilled water, centrifuging for 15 minutes at the temperature of 8000r/min, taking supernatant, and obtaining the Tianshan red deer abomasum crude glycoprotein after freeze drying for 24 hours at the temperature of-80 ℃ under the pressure of 10Pa, wherein the extraction rate is 10.29%, the protein content is 41.73%, and the sugar content is 8.06%;
g. purifying the crude glycoprotein obtained in the step f for the first time, completely dissolving the crude glycoprotein with 0.02M phosphate buffer solution with pH of 7.8 to a concentration of 15mg/mL, centrifuging, collecting supernatant, subjecting to DEAE-Sephadex A-25 anion exchange resin column chromatography, eluting with NaCl solutions with concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL respectively, detecting the absorption peak of sugar at 492nm by phenol-sulfuric acid method, detecting the absorption peak of protein at 280nm by ultraviolet spectrophotometer, collecting eluent of overlapping parts of the absorption peaks of sugar and protein, concentrating the eluent of overlapping parts under reduced pressure at 35mbar, concentrating at 50 ℃, desalting the concentrate with 3500Da dialysis bag for 48h, and freeze-drying at-80 ℃ and 10Pa for 8h to obtain the first purified glycoprotein (figure 2);
h. detecting the hyaluronidase inhibitory activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibitory activity;
i. performing secondary purification on the component with the hyaluronidase inhibitory activity collected in the step h, dissolving the component with the hyaluronidase inhibitory activity in distilled water, separating and purifying the component with the hyaluronidase inhibitory activity by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, concentrating the solution at the pressure of 35mbar and the temperature of 50 ℃, and performing freeze drying at the temperature of-80 ℃ and the pressure of 10Pa to obtain the red deer abomasum glycoprotein; designated RDA4-1 (fig. 3).
Example 2
a. Taking the abomasum of the Tianshan red deer, flushing the abomasum by flowing water to remove the content in the abomasum, and storing the abomasum in a refrigerator at the temperature of minus 20 ℃ for later use;
b. c, removing fat and connective tissues from the red deer abomasum cleaned by precooling distilled water flowing water in the step a, cutting the red deer abomasum to be 6mm in width, freezing the red deer abomasum at the temperature of-80 ℃ for 12 hours, and then freeze-drying to obtain red deer abomasum dry powder;
c. c, crushing the red deer abomasum dry powder obtained in the step b at a low temperature by utilizing liquid nitrogen, crushing the red deer abomasum dry powder by using a crusher at a rotation speed of 5000r/min, suspending for 0.5min every 1min, performing cumulative crushing for 4min, sieving by using a 80-mesh sieve to obtain red deer abomasum powder, and storing in a refrigerator at a temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c according to a feed-liquid ratio of 1:13 at the temperature of 4 ℃, mixing, placing in a Soxhlet extractor, heating for 3 hours at the temperature of 70 ℃, carrying out reflux degreasing treatment, filtering to remove petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the defatted abomasum powder obtained in the step d with a NaCl solution with the concentration of 3% according to the mass ratio of 1:13, stirring and extracting for 3 hours at room temperature, filtering the extracting solution by using an oscillating screen, filtering the filtering solution at 7000r/min and the temperature of 4 ℃, centrifuging for 15 minutes to obtain a supernatant, concentrating the supernatant under reduced pressure by using a rotary evaporator at the pressure of 50mbar and the temperature of 50 ℃ to 1/13 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, centrifuging for 15 minutes at 7000r/min and the temperature of 4 ℃ to obtain the total extracting solution;
f. adding absolute ethyl alcohol into the extracting solution obtained in the step e according to the volume ratio of 1:4 at the temperature of 0 ℃ to ensure that the volume concentration of the ethyl alcohol is 75%, standing for 24 hours to completely precipitate, dissolving the precipitate by using distilled water, centrifuging for 15 minutes at the temperature of 8000r/min and the temperature of 4 ℃, taking supernatant, and obtaining the Tianshan red deer abomasum crude glycoprotein after freeze drying for 24 hours at the temperature of-80 ℃ under the pressure of 10Pa, wherein the extraction rate is 8.54%, the protein content is 35.63%, and the sugar content is 5.91%;
g. purifying the crude glycoprotein obtained in the step f for the first time, completely dissolving the crude glycoprotein with 0.02M phosphate buffer solution with pH of 7.8 to a concentration of 15mg/mL, centrifuging, collecting supernatant, subjecting to DEAE-Sephadex A-25 anion exchange resin column chromatography, eluting with NaCl solutions with concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL, detecting the absorption peak of sugar at 492nm by phenol-sulfuric acid method, detecting the absorption peak of protein at 280nm by ultraviolet spectrophotometer, collecting eluent of overlapping parts of the absorption peaks of sugar and protein, concentrating the eluent of overlapping parts under reduced pressure at 50mbar, concentrating at 50 ℃ and desalting with 3500Da dialysis bag for 48h, and freeze-drying at-80 ℃ and 10Pa for 8h to obtain the first purified glycoprotein (figure 2);
h. detecting the hyaluronidase inhibitory activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibitory activity;
i. and (3) performing secondary purification on the component with the hyaluronidase inhibitory activity collected in the step h, dissolving the component with the hyaluronidase inhibitory activity in distilled water, separating and purifying the component with the hyaluronidase inhibitory activity by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, concentrating the solution at the temperature of 50mbar and 50 ℃ under the pressure of-80 ℃ and performing freeze drying under the pressure of 10Pa to obtain the red deer abomasum glycoprotein named RDA4-1 (figure 3).
Example 3
a. Taking the abomasum of the Tianshan red deer, flushing the abomasum by flowing water to remove the content in the abomasum, and storing the abomasum in a refrigerator at the temperature of minus 20 ℃ for later use;
b. c, removing fat and connective tissues from the red deer abomasum cleaned by precooling distilled water flowing water in the step a, cutting the red deer abomasum to 8mm in width, freezing the red deer abomasum at the temperature of-80 ℃ for 12 hours, and then freeze-drying to obtain red deer abomasum dry powder;
c. crushing the dry powder of the red deer abomasum obtained in the step b at a rotation speed of 5000r/min, suspending for 0.5min every 1min, adding up and crushing for 5min, sieving with a 80-mesh sieve to obtain the powder of the red deer abomasum, and storing in a refrigerator at the temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c according to a feed-liquid ratio of 1:15 at the temperature of 4 ℃, mixing, placing in a Soxhlet extractor, heating for 5 hours at the temperature of 75 ℃, carrying out reflux degreasing treatment, filtering to remove petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the defatted abomasum powder obtained in the step d with 5% NaCl solution according to a mass ratio of 1:15, stirring and extracting at room temperature for 3-5h, filtering the extracting solution by using a vibrating screen, filtering the filtrate at 8000r/min and a temperature of 4 ℃, centrifuging for 20min to obtain supernatant, concentrating the supernatant under reduced pressure by using a rotary evaporator at 60mbar and a temperature of 50 ℃ to 1/15 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, centrifuging at 8000r/min and a temperature of 4 ℃ for 20min to obtain the total extracting solution;
f. adding absolute ethyl alcohol into the extract obtained in the step e at the temperature of 2 ℃ to ensure that the volume concentration of the ethyl alcohol is 80%, standing for 24 hours to completely precipitate, dissolving the precipitate with distilled water, and freeze-drying at the temperature of-80 ℃ under the pressure of 10Pa for 48 hours to obtain the Tianshan red deer abomasum glycoprotein;
g. purifying the crude glycoprotein obtained in the step f for the first time, completely dissolving the crude glycoprotein with 0.02M phosphate buffer solution with pH of 7.8 to a concentration of 15mg/mL, centrifuging, collecting supernatant, subjecting to DEAE-Sephadex A-25 anion exchange resin column chromatography, eluting with NaCl solutions with concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL respectively, detecting the absorption peak of sugar at 492nm by phenol-sulfuric acid method, detecting the absorption peak of protein at 280nm by ultraviolet spectrophotometer, collecting eluent of overlapping parts of the absorption peaks of sugar and protein, concentrating the eluent of overlapping parts under reduced pressure at 60mbar, concentrating at 50 ℃, desalting the concentrate with 3500Da dialysis bag for 48h, and freeze-drying at-80 ℃ and 10Pa for 8h to obtain the first purified glycoprotein (figure 2);
h. detecting the hyaluronidase inhibitory activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibitory activity;
i. and (3) performing secondary purification on the component with the hyaluronidase inhibitory activity collected in the step h, dissolving the component with the hyaluronidase inhibitory activity in distilled water, separating and purifying the component with the hyaluronidase inhibitory activity by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, concentrating the solution at the temperature of 50 ℃ under the pressure of 60mbar and the temperature of-80 ℃ under the pressure of 10Pa, and performing freeze drying on the concentrated solution to obtain the red deer abomasum glycoprotein named RDA4-1 (shown in figure 3).
Example 4
The implementation effect is as follows:
HPLC identification of purity and determination of molecular weight:
the chromatographic pure dextran and blue dextran purchased by Sigma and having molecular weights of 5kDa, 25kDa, 50kDa, 80kDa, 150kDa and 410kDa are prepared into polysaccharide molecular weight standard sample with concentration of 2mg/mL by using ultrapure water solution, and the sample is introduced after being filtered by a 0.45 μm filter membrane, and the retention time V of the blue dextran is adopted 0 Pore volume was calibrated as log of molecular weight (1 g Mw ) Drawing a standard curve with the retention time as an abscissa and the retention time as an ordinate, and obtaining a standard curve regression equation of y= -4.2574x+10.494, wherein R is as follows 2 =0.9904; meanwhile, sampling the Tianshan red deer abomasum glycoprotein obtained in any one of examples 1-3, and calculating the relative molecular mass according to the retention time; chromatographic conditions: the chromatographic column is TSK-G3000 PWXL (7.8X100 mm), the mobile phase is ultrapure water, the flow rate is 0.6mL/min, the temperature of a column temperature box is 25 ℃, and the detector is a differential detector; from HPLC analysis (FIG. 4), the molecular weight and purity of the Tianshan red deer abomasum glycoprotein prepared by the invention are 15.11kDa and 95.41% respectively;
infrared (FT-IR) analysis:
the method comprises mixing the above-mentioned glycoprotein obtained in examples 1-3 with 4000-500CM -1 Characteristic absorption peaks of saccharide and protein compounds appear therebetween, as shown in FIG. 5, which is in 3304CM -1 There is a distinct broad peak, which is the superposition of stretching vibration of O-H bond and N-H bond, indicating the existence of intermolecular or intramolecular hydrogen bond, which is the characteristic absorption peak of polysaccharide and protein molecules. 2927CM -1 Weaker bending is C-H vibration; at 1654CM -1 The absorption at this location is the deformation vibration of N-H, indicating the presence of protein in the sample; 1541CM -1 The absorption band at the position is C-N telescopic vibration; 1238CM -1 And 1077CM -1 The absorption peaks at are the stretching vibration of the c=o group and the c—n stretching vibration; at 536CM -1 Nearby absorption of N-H bending vibrations attributed to amide groups;
protein content determination:
the BCA method was used: accurately measuring 2mg/mL of standard bovine serum albumin solution, and preparing 25-2000 mug/mL of standard solution; preparing working solution according to the instruction of the BCA protein measurement kit, wherein BCA reagent is divided into reagent A and reagent B, and 1 time of reagent B is added to 50 times of reagent A in volume, and the mixture is fully shaken for later use; measuring 25uL standard solutions with different concentrations in a 96-well plate, respectively adding 175uL BCA working solution, slightly oscillating, fully and uniformly mixing, keeping the temperature in a constant temperature box at 37 ℃ for 30min, and measuring the absorbance of the solution at 562nm by using an enzyme-labeled instrument; drawing a standard curve by taking the absorbance of the standard substance as an abscissa and the mass concentration of bovine serum albumin as an ordinate; accurately weighing 2.0mg of purified Tianshan red deer abomasum glycoprotein sample, adding 2mL of distilled water for dissolution, taking 25 mu L of sample solution into a 96-well plate, measuring the absorbance value of the sample solution by the standard curve operation method, and calculating to know that the protein content of glycoprotein RDA4-1 is 91.63%;
determination of total sugar content:
the total sugar content was determined using the phenol-sulfuric acid method: accurately weighing 10mg of glucose standard substance, adding distilled water for dissolution, fixing volume to 50mL, and accurately sucking 0, 0.4, 0.8, 1.2, 1.6 and 2.0mL into different test tubes respectively, wherein the final concentration of the standard substance is 0.2 mg/mL; distilled water is added to make the volume of each tube reach 2mL; accurately sucking 50uL into a 96-well plate, rapidly adding 150uL of concentrated sulfuric acid into each well to be tested, adding 30uL of 5% phenol tube by tube, shaking uniformly, standing at room temperature for 20min, standing at room temperature for 10min, and measuring absorbance at 490nm by using an enzyme-labeled instrument; absorbance is taken as an abscissa, the mass concentration of the standard solution is taken as an ordinate, distilled water is taken as a blank control, and a standard curve is prepared; sample measurement: preparing a Tianshan red deer abomasum glycoprotein sample solution with the concentration of 200 mug/mL, measuring absorbance by the standard curve operation method, calculating the total sugar content, and calculating the total sugar content of Tianshan red deer abomasum glycoprotein RDA4-1 to be 3.09%;
monosaccharide composition analysis:
hydrolysis: taking 5mg of purified Tianshan red deer abomasum glycoprotein sample, adding 4mL of 2mol/L trifluoroacetic acid into a headspace bottle, sealing, hydrolyzing at constant temperature of 110 ℃ for 6h, adding methanol, evaporating under reduced pressure, and repeating for 3 times; acetylated derivatives: to the hydrolysate was added 8mg of glycolic acid, 1mL of pyridine and 1mL of acetic anhydride, heated at 90℃for 1 hour, cooled and dried with nitrogen, and the acetylated monosaccharide alcohol was diluted with chloroform and subjected to gas chromatography-mass spectrometry GS analysis (FIG. 6), and the analysis by GC revealed that the Tianshan red deer abomasum glycoprotein RDA4-1 consisted of rhamnose, arabinose, mannose, xylose, glucose and galactose (Table 1).
TABLE 1 composition of the red deer abomasum glycoprotein monosaccharides (mole ratio%)
Amino acid analysis:
the measuring method comprises the following steps: weighing 5mg of purified red deer abomasum glycoprotein, putting the purified red deer abomasum glycoprotein into a hydrolysis tube, adding 3mL of hydrochloric acid solution with the concentration of 6mol/L into the hydrolysis tube, filling nitrogen for 2min, vacuumizing and sealing the tube, putting the hydrolysis tube into an oven at the temperature of 110 ℃ for hydrolysis for 24h, decompressing and evaporating the hydrolyzed solution after hydrolysis by a rotary evaporator to fully evaporate hydrochloric acid, thereby removing acid in the hydrolyzed solution, washing the hydrolysis solution for 3 times by methanol, continuously evaporating residual hydrochloric acid, then fixing the volume to 1mL by distilled water, filtering the solution by a microporous filter membrane with the concentration of 0.45 mu m, and carrying out amino acid content determination analysis on the filtrate by an amino acid analyzer, wherein the determination result is shown in Table 2:
TABLE 2 results of analysis of amino acids of the glycoprotein of the abomasum of Anshan deer
As can be seen from Table 2, the protein chain in the Tianshan red deer abomasum glycoprotein obtained by the method of the present invention contains 17 amino acids, of which the main amino acids are glycine 25.257%, alanine 20.048%, tyrosine 12.903%, valine 8.115%, and lysine 6.710%;
full wavelength ultraviolet scanning:
scanning with ultraviolet spectrophotometer full wavelength: and switching on a power supply, opening an instrument switch, lifting a dark box cover of the sample chamber, and preheating for 10min. And pouring the blank liquid and the measuring liquid into a colorimetric cuvette respectively, wiping the outer wall by using a piece of mirror wiping paper, and placing the colorimetric cuvette into a sample chamber to align the blank pipe with the light path. After the ultraviolet scanner is corrected by the blank solution, the maximum absorption peak of the sample at the vicinity of 280nm is measured, and the maximum absorption characteristic of the ultraviolet scanner is also in accordance with the ultraviolet maximum absorption characteristic of protein substances, and the ultraviolet scanning spectrum is shown in figure 7.
SDS-PAGE electrophoresis:
to examine the molecular weight range and the separation and purification effect of the Tianshan red deer abomasum glycoprotein, 12% SDS-PAGE vertical electrophoresis was performed by referring to the Laemmli method (FIG. 8); sample buffer: 0.5mL of 0.5mol/L Tris-HCl solution at pH 6.8, 2mL of 10% SDS,1mL of glycerol, 0.5mL of beta-mercaptoethanol, 1mL of ultrapure water, and a trace amount of bromophenol blue; electrophoresis conditions: the concentration of the sample is 2.5mg/mL, the sample loading amount is 10 mu L, the sample is heated for 5min before the sample loading, the voltage is taken as a standard, the voltage for starting sample injection is 75V, and the voltage is increased to 150V after the sample is concentrated into a line in the concentrated gel part; electrophoresis buffer solution: 25mM Tris,192mM glycine,0.1%SDS,pH 8.3; fixing solution: 20% (v/v) ethanol, 10% (v/v) acetic acid; coomassie brilliant blue staining solution: 0.6g of Coomassie brilliant blue R250 was dissolved in 200mL of decolorized solution; decolorization liquid: 25% (v/v) ethanol, 8% (v/v) acetic acid;
hyaluronidase inhibitory Activity:
separating and purifying the obtained Tianshan red deer abomasum glycoprotein, and culturing for 20min at 37 ℃ by adopting 500 units/mL of Elson-Morgan, 0.5mL of sample solution and 0.5mL of hyaluronidase; adding 2.5mol/L CaCl 2 0.1mL, and preserving heat for 20min at 37 ℃; adding 0.5mg/mL of potassium hyaluronate, maintaining the temperature at 37deg.C for 40min, standing at normal temperature for 5min, adding 2 drops of 5m0.5mL of ol/L NaOH is added into the solution, the solution is accurately boiled for 15min, the solution is immediately cooled by ice water for 5min, the solution is placed for 10min at normal temperature, 0.5mL of deionized water and 0.5mL of Ehrlich reagent are added, absolute ethyl alcohol is added to 10mL of the solution, the solution is uniformly mixed by shaking, the solution is placed for 30min for color development, and the ABS value is measured at 530nm, as can be seen from FIG. 9, the Tianshan red deer abomasum glycoprotein prepared by the invention has good hyaluronidase inhibition activity, namely 79.23%.
The above examples are embodiments of the present invention, but are not limited thereto.
Claims (1)
1. A method for preparing a Tianshan red deer abomasum glycoprotein with hyaluronidase inhibition activity, which is characterized by comprising the following steps:
a. taking the abomasum of the Tianshan red deer, flushing the abomasum by flowing water to remove the content in the abomasum, and storing the abomasum in a refrigerator at the temperature of minus 20 ℃ for later use;
b. c, removing fat and connective tissues from the red deer abomasum washed by precooling distilled water flowing water in the step a, shearing the red deer abomasum to 5-8mm, freezing the red deer abomasum at the temperature of-80 ℃ for 12-h, and then freeze-drying to obtain red deer abomasum dry powder;
c. c, crushing the red deer abomasum dry powder obtained in the step b at a low temperature by utilizing liquid nitrogen, crushing the red deer abomasum dry powder by using a crusher at a rotation speed of 5000r/min, suspending for 0.5min every 1min of crushing, performing cumulative crushing for 3-5min, sieving by using a 80-mesh sieve to obtain red deer abomasum powder, and storing in a refrigerator at a temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c according to the feed liquid ratio of 1:10-15 at the temperature of 4 ℃, mixing, placing in a Soxhlet extractor, heating at the temperature of 60-75 ℃ for 3-5h, carrying out reflux degreasing treatment, removing petroleum ether by suction filtration, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the defatted abomasum powder obtained in the step d with 2-5% NaCl solution according to the mass ratio of 1:10-15, stirring at room temperature to extract 3-5h, filtering the extract by using a vibrating screen, centrifuging the filtrate at 6000-8000r/min and the temperature of 4 ℃ for 10-20min to obtain supernatant, concentrating the supernatant under reduced pressure by using a rotary evaporator at 35-60mbar and the temperature of 50 ℃ to 1/10-1/15 times of the original volume, desalting the concentrate by using a 3500Da dialysis bag for 48 hours, centrifuging the desalted concentrate at 6000-8000r/min and the temperature of 4 ℃ for 10-20min to obtain the total extract;
f. adding absolute ethanol into the extract obtained in the step e at 0-4 ℃ to ensure that the volume concentration of the ethanol is 70-80%, standing for 24-h to completely precipitate, dissolving the precipitate with distilled water, centrifuging for 15min at 8000r/min and 4 ℃, taking supernatant, and freeze-drying for 10 Pa-80 ℃ and 24-h to obtain the Tianshan red deer abomasum crude glycoprotein;
g. purifying the crude glycoprotein obtained in the step f for the first time, completely dissolving the crude glycoprotein with 0.02M phosphate buffer solution with pH of 7.8 until the concentration is 15mg/mL, centrifuging, collecting supernatant, subjecting the supernatant to DEAE-Sephadex A-25 anion exchange resin column chromatography, eluting with NaCl solution with concentration of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL respectively, detecting the absorption peak of sugar at 492nm by phenol-sulfuric acid method, detecting the absorption peak of protein at 280nm by ultraviolet spectrophotometer, collecting eluate of the overlapping part of the absorption peaks of sugar and protein, concentrating the eluate under reduced pressure, concentrating at temperature of 35-60mbar, desalting the concentrate with 3500Da dialysis bag 48h, and freeze-drying at temperature of-80 ℃ and pressure of 10Pa to obtain the first purified glycoprotein;
h. detecting the hyaluronidase inhibitory activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibitory activity;
i. and (3) performing secondary purification on the component with the hyaluronidase inhibitory activity collected in the step (h), dissolving the component with the hyaluronidase inhibitory activity in distilled water, separating and purifying the component with the hyaluronidase inhibitory activity by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, concentrating the solution at the temperature of 50 ℃ under the pressure of 35-60mbar, and performing freeze drying at the temperature of-80 ℃ under the pressure of 10Pa to obtain the red deer abomasum glycoprotein.
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| CN115353545A (en) * | 2022-09-22 | 2022-11-18 | 中国科学院新疆理化技术研究所 | Method for preparing lamb abomasum glycoprotein part by response surface method and application |
| CN116178482A (en) * | 2022-12-07 | 2023-05-30 | 中国科学院新疆理化技术研究所 | Method for optimizing ultrasonic-assisted extraction of Tianshan red deer abomasum glycoprotein based on response surface method and application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB575457A (en) * | 1944-02-10 | 1946-02-19 | Francisco Spitzer | Process for treating abomasum for manufacture of rennet |
| CN1724530A (en) * | 2005-07-13 | 2006-01-25 | 东北林业大学 | Method of chromatography preparing high purity EGCG by continous medium-pressure column |
| WO2021109714A1 (en) * | 2019-12-06 | 2021-06-10 | 华南理工大学 | Millettia speciosa polysaccharides, preparation method therefor, and application thereof in antibacterial aspects |
| CN113278047A (en) * | 2021-06-28 | 2021-08-20 | 深圳中正生物技术有限责任公司 | Processing method for extracting composite active polypeptide from fresh pilose antler |
| CN113444141A (en) * | 2021-08-12 | 2021-09-28 | 中国科学院新疆理化技术研究所 | Method for extracting, separating and purifying lamb abomasum glycoprotein and application thereof |
| CN113527411A (en) * | 2021-07-24 | 2021-10-22 | 中国科学院新疆理化技术研究所 | Separation and purification method of dioscorea opposita glycopeptide |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003215232A1 (en) * | 2002-02-12 | 2003-09-04 | Alltech, Inc. | Amylase feed supplements for improved ruminant nutrition |
-
2021
- 2021-11-17 CN CN202111360384.4A patent/CN113956341B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB575457A (en) * | 1944-02-10 | 1946-02-19 | Francisco Spitzer | Process for treating abomasum for manufacture of rennet |
| CN1724530A (en) * | 2005-07-13 | 2006-01-25 | 东北林业大学 | Method of chromatography preparing high purity EGCG by continous medium-pressure column |
| WO2021109714A1 (en) * | 2019-12-06 | 2021-06-10 | 华南理工大学 | Millettia speciosa polysaccharides, preparation method therefor, and application thereof in antibacterial aspects |
| CN113278047A (en) * | 2021-06-28 | 2021-08-20 | 深圳中正生物技术有限责任公司 | Processing method for extracting composite active polypeptide from fresh pilose antler |
| CN113527411A (en) * | 2021-07-24 | 2021-10-22 | 中国科学院新疆理化技术研究所 | Separation and purification method of dioscorea opposita glycopeptide |
| CN113444141A (en) * | 2021-08-12 | 2021-09-28 | 中国科学院新疆理化技术研究所 | Method for extracting, separating and purifying lamb abomasum glycoprotein and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 梅花鹿鹿茸蛋白聚糖的提取与分离;熊和丽等;西北农业学报;第16卷(第04期);第80页右栏第一段 * |
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