CN118027135A - Method for extracting selenium polypeptide from selenium-enriched rice - Google Patents
Method for extracting selenium polypeptide from selenium-enriched rice Download PDFInfo
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- CN118027135A CN118027135A CN202410430861.7A CN202410430861A CN118027135A CN 118027135 A CN118027135 A CN 118027135A CN 202410430861 A CN202410430861 A CN 202410430861A CN 118027135 A CN118027135 A CN 118027135A
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- selenium
- polypeptide
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- enriched rice
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- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 127
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 127
- 239000011669 selenium Substances 0.000 title claims abstract description 127
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 63
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 63
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 63
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 55
- 235000009566 rice Nutrition 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 44
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 54
- 239000011347 resin Substances 0.000 claims abstract description 43
- 229920005989 resin Polymers 0.000 claims abstract description 43
- 238000001179 sorption measurement Methods 0.000 claims abstract description 36
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000002243 precursor Substances 0.000 claims abstract description 17
- 239000003960 organic solvent Substances 0.000 claims abstract description 15
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000002386 leaching Methods 0.000 claims abstract description 11
- 238000005238 degreasing Methods 0.000 claims abstract description 9
- 238000003795 desorption Methods 0.000 claims abstract description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 41
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 239000008367 deionised water Substances 0.000 claims description 16
- 229910021641 deionized water Inorganic materials 0.000 claims description 16
- 235000013312 flour Nutrition 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 239000004365 Protease Substances 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 12
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 10
- -1 benzophenone peroxide Chemical class 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 10
- RRSXICBKOPODSP-UHFFFAOYSA-N 1,4-bis(chloromethoxy)butane Chemical compound ClCOCCCCOCCl RRSXICBKOPODSP-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000012965 benzophenone Substances 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 239000004519 grease Substances 0.000 claims description 8
- 229920003063 hydroxymethyl cellulose Polymers 0.000 claims description 8
- 229940031574 hydroxymethyl cellulose Drugs 0.000 claims description 8
- 108010074686 Selenoproteins Proteins 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 102000008114 Selenoproteins Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 108090000145 Bacillolysin Proteins 0.000 claims description 5
- 108010004032 Bromelains Proteins 0.000 claims description 5
- 102000035092 Neutral proteases Human genes 0.000 claims description 5
- 108091005507 Neutral proteases Proteins 0.000 claims description 5
- 108090000526 Papain Proteins 0.000 claims description 5
- 235000019835 bromelain Nutrition 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 235000019834 papain Nutrition 0.000 claims description 5
- 229940055729 papain Drugs 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 108010007119 flavourzyme Proteins 0.000 claims description 4
- 238000007873 sieving Methods 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 108090000270 Ficain Proteins 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 235000019836 ficin Nutrition 0.000 claims description 3
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000010557 suspension polymerization reaction Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 abstract description 20
- 238000005516 engineering process Methods 0.000 abstract description 4
- 229910052739 hydrogen Chemical group 0.000 abstract description 4
- 125000004218 chloromethyl group Chemical group [H]C([H])(Cl)* 0.000 abstract description 3
- 125000003277 amino group Chemical group 0.000 abstract description 2
- 239000001257 hydrogen Chemical group 0.000 abstract description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 abstract description 2
- 230000036632 reaction speed Effects 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 26
- 239000012071 phase Substances 0.000 description 23
- 230000000052 comparative effect Effects 0.000 description 22
- 238000002835 absorbance Methods 0.000 description 6
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 6
- 230000002292 Radical scavenging effect Effects 0.000 description 5
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 238000003916 acid precipitation Methods 0.000 description 4
- 239000003463 adsorbent Substances 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 229960004889 salicylic acid Drugs 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 2
- 230000007760 free radical scavenging Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 102000011845 Iodide peroxidase Human genes 0.000 description 1
- 108010036012 Iodide peroxidase Proteins 0.000 description 1
- 101710186773 Selenide, water dikinase Proteins 0.000 description 1
- 102000004531 Selenoprotein P Human genes 0.000 description 1
- 108010042443 Selenoprotein P Proteins 0.000 description 1
- 102000013090 Thioredoxin-Disulfide Reductase Human genes 0.000 description 1
- 108010079911 Thioredoxin-disulfide reductase Proteins 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920000779 poly(divinylbenzene) Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting selenium polypeptide from selenium-enriched rice, which belongs to the technical field of selenium polypeptide extraction of selenium-enriched rice and comprises the following steps: degreasing; leaching; performing enzymolysis; adsorption and desorption. The invention has mild extraction process temperature, high specificity of enzymolysis extraction, controllable condition and high reaction speed, avoids the loss of organic selenium caused by the traditional technology, and has high selenium polypeptide extraction efficiency, high quality and small molecular weight; n-isopropyl acrylamide and divinylbenzene are used for synthesizing precursor resin, chloromethyl groups are introduced on benzene rings of the precursor resin, and react with octa-aminophenyl-POSS to obtain modified macroporous adsorption resin, the N-isopropyl acrylamide can promote the adsorption and desorption of selenium polypeptide through the temperature adjustment, octa-aminophenyl-POSS improves the specific surface area of the resin, the stability of the resin and the tolerance of an organic solvent are improved, unreacted amino groups and hydrogen bonds with selenium polypeptide molecules are formed, and the adsorption of the selenium polypeptide by the modified macroporous adsorption resin is promoted.
Description
Technical Field
The invention belongs to the technical field of selenium polypeptide extraction of selenium-enriched rice, and particularly relates to a method for extracting selenium polypeptide from selenium-enriched rice.
Background
Selenium, which is an indispensable trace element for human body, is deficient and induces various diseases. Since selenium is involved in 25 selenoprotein genes in the human genome, selenium is involved in a number of important physiological metabolic processes, such as synthesis of glutathione peroxidase, thioredoxin reductase, thyroid hormone deiodinase, selenophosphate synthase, selenoprotein P, etc. The selenoprotein has multiple functions of resisting oxidation, regulating cell growth and apoptosis, regulating hormone balance and the like, and is vital to maintaining human health. The rice is a main component of resident dietary structures in most areas, and becomes a main source for people to ingest selenium. Therefore, the selenium-enriched rice is widely planted in a plurality of provinces and areas, and becomes one of main selenium-enriched agricultural products. Selenium polypeptide in rice has many advantages over inorganic selenium, such as easy absorption, strong biological activity, high safety and good stability. However, the existing extraction method of selenium polypeptide in selenium-enriched rice mainly depends on the traditional alkali extraction and acid precipitation technology, and the method has low efficiency and causes the loss of a large amount of organic selenium. Therefore, the search for a more efficient and high-yield extraction method of selenium polypeptide in the selenium-enriched rice is particularly urgent, and has important significance for fully utilizing the nutritional ingredients in the selenium-enriched rice and meeting the requirements of people on selenium.
Disclosure of Invention
Aiming at the situation, in order to solve the problem that the existing extraction method of selenium polypeptide in the selenium-enriched rice mainly depends on the traditional alkali extraction and acid precipitation technology, the mode has low efficiency and causes loss of a large amount of organic selenium, the invention provides a method for extracting selenium polypeptide from the selenium-enriched rice, which comprises the following steps:
S1, degreasing: drying selenium-enriched rice for 40 hours at 50 ℃ by using a vacuum drying oven, crushing, sieving to obtain selenium-enriched rice flour, soaking and degreasing by using an organic solvent, timely removing upper-layer grease of the solution and replacing the solvent until no obvious grease exists on the upper layer of the solution, filtering to obtain solid, and drying and ventilating for 12 hours at room temperature to obtain defatted rice flour;
S2, leaching: homogenizing the defatted rice flour and deionized water, leaching for 2 hours at 2 ℃, repeatedly leaching for 2 times, and combining the filtrates to obtain selenium-rich protein extract;
S3, enzymolysis: adding the selenium-enriched protein extract into an immobilized fermentation tank, performing enzymolysis reaction, and performing centrifugal separation to obtain a supernatant;
s4, adsorption and desorption: and (3) adsorbing the supernatant by using modified macroporous adsorption resin, eluting after the adsorption is finished, collecting eluent, concentrating and drying to obtain a selenium polypeptide product.
Further, the modified macroporous adsorption resin is prepared by the following steps:
(1) Adding N-isopropyl acrylamide, divinylbenzene and benzophenone peroxide into methylene dichloride to obtain an oil phase, dissolving hydroxymethyl cellulose into deionized water to obtain a water phase, adding the oil phase into the water phase, heating to 60 ℃, carrying out suspension polymerization, reacting for 4 hours, filtering, and washing the solid with hot water at 40 ℃ for 3 times to obtain precursor resin;
(2) And adding the precursor resin into dichloromethane, then adding 1, 4-bis (chloromethoxy) -butane and anhydrous aluminum chloride, heating to 45 ℃, reacting for 3 hours, cooling to room temperature, adding octa-aminophenyl-POSS, heating to 45 ℃, continuously reacting for 24 hours, removing the dichloromethane, and washing with deionized water to obtain the modified macroporous adsorption resin.
Further, the oil phase comprises the following components in parts by weight: 30-40 parts of N-isopropyl acrylamide, 30-50 parts of divinylbenzene, 0.05-0.1 part of benzophenone peroxide and 200-300 parts of methylene dichloride.
Further, the aqueous phase comprises the following components in parts by weight: 0.05-0.1 part of hydroxymethyl cellulose and 500-600 parts of deionized water; the weight ratio of the oil phase to the water phase is 1-1.5:3.
Further, the feeding weight ratio of the precursor resin, methylene dichloride, 1, 4-bis (chloromethoxy) -butane, anhydrous aluminum chloride and octap-aminophenyl-POSS in the step (2) is 50-80:300-350:20-25:2-5:8-10.
Further, the organic solvent in the step S1 is one of normal hexane and petroleum ether with the boiling range of 30-60 ℃.
Further, the solid-to-liquid ratio (w/v) of the selenium-enriched rice flour to the organic solvent in the step S1 is 1 to 1.5:5.
Further, the weight ratio of the selenoprotein-enriched extracting solution to the immobilized enzyme in the enzymolysis reaction is 150-250:1.
Further, the immobilized enzyme is one or a combination of more of neutral protease, papain, bromelain, ficin and flavourzyme.
Further, the enzymolysis reaction time is 30-90min, and the temperature is 30-60 ℃.
Further, the flow rate of the adsorption process is 5-15BV/h, and the temperature is 25-35 ℃.
Further, the flow rate of the elution process is 10-20BV/h, and the temperature is 35-45 ℃.
Further, the eluent comprises the following components in percentage by volume: 80-85% of ethyl acetate, 10-15% of ethanol and the balance of water.
The beneficial effects of the invention are as follows:
(1) The invention provides a method for extracting selenium polypeptide from selenium-enriched rice, which comprises the steps of degreasing the selenium-enriched rice, leaching, enzymolysis, resin adsorption and desorption, wherein the temperature of the extraction process is mild, the enzymolysis extraction has high specificity, the condition is controllable, the reaction speed is high, the loss of organic selenium caused by the traditional alkali extraction and acid precipitation technology is avoided, the extraction efficiency of the selenium polypeptide is high, and the obtained selenium polypeptide has high quality and small molecular weight;
(2) According to the invention, the precursor resin is synthesized by the N-isopropyl acrylamide and the divinylbenzene, wherein the N-isopropyl acrylamide has a temperature-sensitive effect, so that the N-isopropyl acrylamide is introduced, and the adsorption and desorption of the selenium polypeptide can be promoted through the temperature regulation, so that the modified macroporous adsorption resin has a higher adsorption and desorption rate, and the extraction efficiency of the selenium polypeptide is improved;
(3) According to the invention, a chloromethyl group is introduced on a benzene ring of a precursor resin through 1, 4-bis (chloromethoxy) -butane and anhydrous aluminum chloride, and then the chloromethyl group reacts with octap-aminophenyl-POSS to obtain the modified macroporous adsorption resin, the octap-aminophenyl-POSS has a cage structure, the specific surface area of the resin is increased by an inner porous pore canal, so that the contact area with polypeptide molecules is increased, the POSS group also has stability and organic solvent resistance, so that the modified macroporous adsorption resin can perform adsorption and desorption for more times, and unreacted amino groups on the octap-aminophenyl-POSS can also form hydrogen bonds with selenium polypeptide molecules, thereby promoting the adsorption of the modified macroporous adsorption resin to the selenium polypeptide.
Drawings
FIG. 1 is a flow chart of a method of extracting selenium polypeptide from selenium-enriched rice in accordance with the present invention;
FIG. 2 shows the effect of selenium polypeptide extraction in various examples and comparative examples of the present invention;
FIG. 3 shows selenium content of polypeptides of examples and comparative examples of the present invention;
FIG. 4 shows the recovery of organic selenium for each of the examples and comparative examples of the present invention;
FIG. 5 shows the DPPH radical scavenging ability and the hydroxyl radical scavenging ability of selenium polypeptide products extracted in the examples and the comparative examples of the present invention;
FIG. 6 is an SDS-PAGE gel electrophoresis of selenium polypeptide products extracted in the examples and comparative examples of the present invention.
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without making any inventive effort are within the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the application.
The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.
Example 1
Method for extracting selenium polypeptide from selenium-enriched rice
According to fig. 1, the method comprises the steps of:
S1, degreasing: drying selenium-enriched rice for 40 hours at 50 ℃ by using a vacuum drying oven, crushing, sieving to obtain selenium-enriched rice flour, soaking and degreasing by using an organic solvent, timely removing upper-layer grease of the solution and replacing the solvent until no obvious grease exists on the upper layer of the solution, filtering to obtain solid, and drying and ventilating for 12 hours at room temperature to obtain defatted rice flour;
S2, leaching: homogenizing the defatted rice flour and deionized water, leaching for 2 hours at 2 ℃, repeatedly leaching for 2 times, and combining the filtrates to obtain selenium-rich protein extract;
S3, enzymolysis: adding the selenium-enriched protein extract into an immobilized fermentation tank, performing enzymolysis reaction, and performing centrifugal separation to obtain a supernatant;
s4, adsorption and desorption: and (3) adsorbing the supernatant by using modified macroporous adsorption resin, eluting after the adsorption is finished, collecting eluent, concentrating and drying to obtain a selenium polypeptide product.
The modified macroporous adsorption resin is prepared by the following steps:
(1) Adding N-isopropyl acrylamide, divinylbenzene and benzophenone peroxide into methylene dichloride to obtain an oil phase, dissolving hydroxymethyl cellulose into deionized water to obtain a water phase, adding the oil phase into the water phase, heating to 60 ℃, carrying out suspension polymerization, reacting for 4 hours, filtering, and washing the solid with hot water at 40 ℃ for 3 times to obtain precursor resin;
(2) And adding the precursor resin into dichloromethane, then adding 1, 4-bis (chloromethoxy) -butane and anhydrous aluminum chloride, heating to 45 ℃, reacting for 3 hours, cooling to room temperature, adding octa-aminophenyl-POSS, heating to 45 ℃, continuously reacting for 24 hours, removing the dichloromethane, and washing with deionized water to obtain the modified macroporous adsorption resin.
The oil phase comprises the following components in parts by weight: 30 parts of N-isopropyl acrylamide, 30 parts of divinylbenzene, 0.05 part of benzophenone peroxide and 200 parts of methylene dichloride; the water phase comprises the following components in parts by weight: 0.05 parts of hydroxymethyl cellulose and 500 parts of deionized water; the weight ratio of the oil phase to the water phase is 1:3, a step of; the weight ratio of the precursor resin, methylene dichloride, 1, 4-bis (chloromethoxy) -butane, anhydrous aluminum chloride and octap-aminophenyl-POSS in the step (2) is 50:300:20:2:8.
The organic solvent in the step S1 is n-hexane.
The solid-to-liquid ratio (w/v) of the selenium-enriched rice flour to the organic solvent in the step S1 is 1:5.
The weight ratio of the selenoprotein-enriched extracting solution to the immobilized enzyme in the enzymolysis reaction is 150:1.
The immobilized enzymes are neutral protease, papain, bromelain and flavourzyme according to the following weight ratio of 2:1:1:1 weight ratio.
The enzymolysis reaction time is 30min, and the temperature is 30 ℃.
The flow rate of the adsorption process is 5BV/h, and the temperature is 25 ℃.
The flow rate in the elution process is 10BV/h, and the temperature is 35 ℃; the eluent comprises the following components in percentage by volume: 80% of ethyl acetate, 10% of ethanol and the balance of water.
Example 2
Method for extracting selenium polypeptide from selenium-enriched rice
The method comprises the same steps as in example 1 as the steps involved in the preparation of the modified macroporous adsorbent resin.
The oil phase comprises the following components in parts by weight: 40 parts of N-isopropyl acrylamide, 50 parts of divinylbenzene, 0.1 part of benzophenone peroxide and 300 parts of methylene dichloride; the water phase comprises the following components in parts by weight: 0.1 part of hydroxymethyl cellulose and 600 parts of deionized water; the weight ratio of the oil phase to the water phase is 1.5:3, a step of; the weight ratio of the precursor resin, methylene dichloride, 1, 4-bis (chloromethoxy) -butane, anhydrous aluminum chloride and octap-aminophenyl-POSS in the step (2) is 80:350:25:5:10.
The organic solvent in the step S1 is petroleum ether with the boiling range of 30-60 ℃.
The solid-to-liquid ratio (w/v) of the selenium-enriched rice flour to the organic solvent in the step S1 is 1.5:5.
The weight ratio of the selenoprotein-enriched extracting solution to the immobilized enzyme in the enzymolysis reaction is 250:1.
The immobilized enzymes are neutral protease, papain, bromelain, ficin and flavourzyme according to the following weight ratio of 2:1:0.5:0.5:0.5 weight ratio.
The enzymolysis reaction time is 90min, and the temperature is 60 ℃.
The flow rate of the adsorption process is 15BV/h, and the temperature is 35 ℃.
The flow rate in the elution process is 20BV/h, and the temperature is 45 ℃; the eluent comprises the following components in percentage by volume: 85% of ethyl acetate, 10% of ethanol and the balance of water.
Example 3
Method for extracting selenium polypeptide from selenium-enriched rice
The method comprises the same steps as in example 1 as the steps involved in the preparation of the modified macroporous adsorbent resin.
The oil phase comprises the following components in parts by weight: 35 parts of N-isopropyl acrylamide, 40 parts of divinylbenzene, 0.08 part of benzophenone peroxide and 250 parts of methylene dichloride; the water phase comprises the following components in parts by weight: 0.06 parts of hydroxymethyl cellulose and 550 parts of deionized water; the weight ratio of the oil phase to the water phase is 1.2:3, a step of; the weight ratio of the precursor resin, methylene dichloride, 1, 4-bis (chloromethoxy) -butane, anhydrous aluminum chloride and octap-aminophenyl-POSS in the step (2) is 70:320:24:3:9.
The organic solvent in the step S1 is n-hexane.
The solid-to-liquid ratio (w/v) of the selenium-enriched rice flour to the organic solvent in the step S1 is 1.2:5.
The weight ratio of the selenoprotein-enriched extracting solution to the immobilized enzyme in the enzymolysis reaction is 200:1.
The immobilized enzymes are neutral protease, papain and bromelain according to the following 2:1:1, and a combination of weight ratios.
The enzymolysis reaction time is 60min, and the temperature is 40 ℃.
The flow rate of the adsorption process is 10BV/h, and the temperature is 30 ℃.
The flow rate in the elution process is 15BV/h, and the temperature is 40 ℃; the eluent comprises the following components in percentage by volume: 80% of ethyl acetate, 15% of ethanol and the balance of water.
Comparative example 1
Method for extracting selenium polypeptide from selenium-enriched rice
The modified macroporous adsorption resin prepared in this comparative example does not contain POSS groups, i.e. differs from example 1 in that the precursor resin is used to adsorb and desorb selenium polypeptides instead of the modified macroporous adsorption resin, and the rest of the components, the component contents, and the preparation process include the same steps as example 1.
Comparative example 2
Method for extracting selenium polypeptide from selenium-enriched rice
The modified macroporous adsorbent resin prepared in this comparative example does not contain N-isopropylacrylamide, i.e., differs from example 1 in that the preparation of the modified macroporous adsorbent resin is performed by using a polydivinylbenzene resin instead of the precursor resin, and the remaining components, component contents, and preparation process include the same steps as example 1.
Comparative example 3
Method for extracting selenium polypeptide from selenium-enriched rice
The comparative example adopts a traditional alkali extraction and acid precipitation method to extract selenium polypeptide in selenium-enriched rice, and the method comprises the following steps:
S1, drying selenium-enriched rice for 40 hours at 55 ℃ by using a vacuum drying oven, crushing, sieving to obtain selenium-enriched rice powder, soaking and degreasing by using normal hexane, timely removing upper-layer grease of the solution, replacing the solvent until the upper layer of the solution has no obvious grease, filtering, and drying and ventilating and drying for 12 hours at room temperature to obtain defatted rice flour;
s2, adding water into the defatted rice flour, adjusting pH=10.5 by using a 0.1mol/L sodium hydroxide solution, extracting for 4 hours at the extraction temperature of 50 ℃, and centrifuging to extract supernatant, wherein the centrifugation speed is 4500rpm, and the centrifugation time is 20min, so as to obtain supernatant;
S3, adjusting the pH value of the supernatant to be 4.5 by using 0.1mol/L of hydrogen chloride solution, centrifuging to extract precipitate, wherein the centrifuging time is20 min at 4500rpm, and obtaining the precipitate;
S4, freeze-drying the precipitate at the freezing temperature of-40 ℃ for 8 hours to obtain the selenium polypeptide product.
Analysis of results
The extraction effect, the selenium content in the polypeptide and the organic selenium recovery rate of the selenium polypeptide products extracted by the examples and the comparative examples are calculated, and the results are shown in fig. 2,3 and 4. As can be seen from fig. 2-4, the extraction effect, the selenium content in the polypeptide and the organic selenium recovery rate of the selenium polypeptide products extracted by the comparative examples are higher than those of the comparative examples, and are far higher than the detection result of the comparative example 3 extracted by the traditional method.
DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of selenium polypeptide products extracted in each of the examples and comparative examples of the present invention were measured, and the results are shown in FIG. 5.
The DPPH radical scavenging ability was measured as follows: weighing 0.0039gDPPH in a beaker, dissolving with a small amount of absolute ethyl alcohol, transferring to a 100mL brown volumetric flask, fixing the volume with absolute ethyl alcohol, shaking uniformly, placing in a dark place, taking selenium polypeptide product solution with the concentration of 1.0mg/mL and equivalent 0.1mmol/LDPPH solution in a test tube, mixing uniformly, reacting at room temperature in a dark place for 30min, and measuring absorbance value A1 with an enzyme-labeled instrument under the condition of 517nm wavelength; simultaneously measuring the absorbance value A2 of the protein solution with different concentrations and the absorbance value A0 of the anhydrous alcohol and the DPPH solution with the same amount, repeating each group of experiments for three times, taking an average value, taking ascorbic acid as a control group, and adopting a DPPH free radical clearance calculation formula as follows:
DPPH radical clearance% = [ a0- (a1+a2)/a0 ] ×100%.
The method for measuring the scavenging ability of the hydroxyl radical is as follows: taking selenium polypeptide product solution with the degree of 1.0mg/mL, sequentially adding equal amounts of 1mmol/L FeSO 4 solution and 0.3% H 2O2 solution into a test tube, uniformly mixing, standing at room temperature for 10min for reaction, adding equal amounts of 1mmol/L salicylic acid solution, uniformly mixing, carrying out water bath for 30min at 37 ℃, measuring an absorbance value A1 by using an enzyme-labeling instrument at the wavelength of 510nm, wherein a reagent blank group is equal amounts of 1.0mg/mL selenium polypeptide product solution, 1mmol/L FeSO 4 solution, deionized water and 1mmol/L salicylic acid solution absorbance value A2, a sample blank group is equal amounts of deionized water, 1mmol/L FeSO 4 solution, 0.3% H 2O2 solution and 1mmol/L salicylic acid solution absorbance value A0, repeating each experiment three times, taking an average value, taking ascorbic acid as a control group, and calculating a hydroxyl radical clearance formula as follows:
The clearance of hydroxyl radical is%A0- (A1+A2)/A0 ]. Times.100%.
The selenium polypeptide products extracted by the examples and the comparative examples of the present invention were analyzed by SDS-PAGE gel electrophoresis using DYCZ-24DN gel electrophoresis apparatus, and the analysis results are shown in FIG. 6.
As can be seen from FIG. 5, the DPPH radical scavenging ability and the hydroxyl radical scavenging ability of the selenium polypeptide products extracted from the comparative examples of the present invention are better than those of the comparative examples, especially higher than that of comparative example 3; as can be seen from FIG. 6, the molecular weight of the selenium polypeptide product extracted from each comparative example is smaller than that of each comparative example, and the smaller molecular weight brings about improvement of antioxidant capacity, which corresponds to the detection result of FIG. 5.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The invention and its embodiments have been described above with no limitation, and the invention is illustrated in the figures of the accompanying drawings as one of its embodiments, without limitation in practice. In summary, those skilled in the art, having benefit of this disclosure, will appreciate that the invention can be practiced without the specific details disclosed herein.
Claims (10)
1. A method for extracting selenium polypeptide from selenium-enriched rice, which is characterized by comprising the following steps: the method comprises the following steps:
S1, degreasing: drying selenium-enriched rice for 40 hours at 50 ℃ by using a vacuum drying oven, crushing, sieving to obtain selenium-enriched rice flour, soaking and degreasing by using an organic solvent, timely removing upper-layer grease of the solution and replacing the solvent until no obvious grease exists on the upper layer of the solution, filtering to obtain solid, and drying and ventilating for 12 hours at room temperature to obtain defatted rice flour;
S2, leaching: homogenizing the defatted rice flour and deionized water, leaching for 2 hours at 2 ℃, repeatedly leaching for 2 times, and combining the filtrates to obtain selenium-rich protein extract;
S3, enzymolysis: adding the selenium-enriched protein extract into an immobilized fermentation tank, performing enzymolysis reaction, and performing centrifugal separation to obtain a supernatant;
s4, adsorption and desorption: and (3) adsorbing the supernatant by using modified macroporous adsorption resin, eluting after the adsorption is finished, collecting eluent, concentrating and drying to obtain a selenium polypeptide product.
2. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 1, wherein the step of: the modified macroporous adsorption resin is prepared by the following steps:
(1) Adding N-isopropyl acrylamide, divinylbenzene and benzophenone peroxide into methylene dichloride to obtain an oil phase, dissolving hydroxymethyl cellulose into deionized water to obtain a water phase, adding the oil phase into the water phase, heating to 60 ℃, carrying out suspension polymerization, reacting for 4 hours, filtering, and washing the solid with hot water at 40 ℃ for 3 times to obtain precursor resin;
(2) And adding the precursor resin into dichloromethane, then adding 1, 4-bis (chloromethoxy) -butane and anhydrous aluminum chloride, heating to 45 ℃, reacting for 3 hours, cooling to room temperature, adding octa-aminophenyl-POSS, heating to 45 ℃, continuously reacting for 24 hours, removing the dichloromethane, and washing with deionized water to obtain the modified macroporous adsorption resin.
3. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 2, wherein the step of extracting the selenium polypeptide comprises: the oil phase comprises the following components in parts by weight: 30-40 parts of N-isopropyl acrylamide, 30-50 parts of divinylbenzene, 0.05-0.1 part of benzophenone peroxide and 200-300 parts of methylene dichloride; the water phase comprises the following components in parts by weight: 0.05-0.1 part of hydroxymethyl cellulose and 500-600 parts of deionized water; the weight ratio of the oil phase to the water phase is 1-1.5:3, a step of; the weight ratio of the precursor resin to the dichloromethane to the 1, 4-bis (chloromethoxy) -butane to the anhydrous aluminum chloride to the octap-aminophenyl-POSS in the step (2) is 50-80:300-350:20-25:2-5:8-10.
4. A method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 3, wherein: the organic solvent in the step S1 is one of normal hexane and petroleum ether with the boiling range of 30-60 ℃.
5. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 4, wherein the step of extracting comprises: the solid-to-liquid ratio (w/v) of the selenium-enriched rice flour to the organic solvent in the step S1 is 1-1.5:5.
6. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 5, wherein the step of extracting comprises: the weight ratio of the selenoprotein-enriched extracting solution to the immobilized enzyme in the enzymolysis reaction is 150-250:1.
7. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 6, wherein the step of: the immobilized enzyme is one or a combination of more of neutral protease, papain, bromelain, ficin and flavourzyme.
8. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 7, wherein the step of extracting the selenium polypeptide comprises: the enzymolysis reaction time is 30-90min, and the temperature is 30-60 ℃.
9. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 8, wherein the step of: the flow rate of the adsorption process is 5-15BV/h, and the temperature is 25-35 ℃.
10. The method for extracting selenium polypeptide from selenium-enriched rice as recited in claim 9, wherein the step of: the flow rate in the elution process is 10-20BV/h, and the temperature is 35-45 ℃; the eluent comprises the following components in percentage by volume: 80-85% of ethyl acetate, 10-15% of ethanol and the balance of water.
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