CN112322684A - Method for extracting bile acid by using high-efficiency enzyme of oxgall - Google Patents
Method for extracting bile acid by using high-efficiency enzyme of oxgall Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 44
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 44
- 239000003613 bile acid Substances 0.000 title claims abstract description 42
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- 238000003756 stirring Methods 0.000 claims abstract description 34
- 210000000941 bile Anatomy 0.000 claims abstract description 28
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000002360 preparation method Methods 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 239000002994 raw material Substances 0.000 claims abstract description 19
- 238000000926 separation method Methods 0.000 claims abstract description 15
- 239000000284 extract Substances 0.000 claims abstract description 14
- 239000000047 product Substances 0.000 claims abstract description 14
- 108010000231 Choloylglycine hydrolase Proteins 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 238000007127 saponification reaction Methods 0.000 claims abstract description 12
- 239000007787 solid Substances 0.000 claims abstract description 12
- 238000010438 heat treatment Methods 0.000 claims abstract description 11
- 238000005406 washing Methods 0.000 claims abstract description 11
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 10
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 230000000415 inactivating effect Effects 0.000 claims abstract description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 7
- 108091005508 Acid proteases Proteins 0.000 claims description 7
- 108090000145 Bacillolysin Proteins 0.000 claims description 7
- 108091005658 Basic proteases Proteins 0.000 claims description 7
- 102000004882 Lipase Human genes 0.000 claims description 7
- 108090001060 Lipase Proteins 0.000 claims description 7
- 239000004367 Lipase Substances 0.000 claims description 7
- 102000035092 Neutral proteases Human genes 0.000 claims description 7
- 108091005507 Neutral proteases Proteins 0.000 claims description 7
- 102000015439 Phospholipases Human genes 0.000 claims description 7
- 108010064785 Phospholipases Proteins 0.000 claims description 7
- 235000019421 lipase Nutrition 0.000 claims description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 6
- 108010007119 flavourzyme Proteins 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
- 230000009849 deactivation Effects 0.000 claims description 5
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 8
- 239000012535 impurity Substances 0.000 abstract description 7
- 238000000605 extraction Methods 0.000 abstract description 6
- 239000002253 acid Substances 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 5
- 231100000614 poison Toxicity 0.000 abstract description 3
- 239000003440 toxic substance Substances 0.000 abstract description 3
- 239000003513 alkali Substances 0.000 abstract description 2
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 4
- 206010004542 Bezoar Diseases 0.000 description 4
- 239000004380 Cholic acid Substances 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- WBWWGRHZICKQGZ-UHFFFAOYSA-N Taurocholic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCCS(O)(=O)=O)C)C1(C)C(O)C2 WBWWGRHZICKQGZ-UHFFFAOYSA-N 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 4
- 229960002471 cholic acid Drugs 0.000 description 4
- 235000019416 cholic acid Nutrition 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 230000020477 pH reduction Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 4
- WBWWGRHZICKQGZ-GIHLXUJPSA-N taurocholic acid Chemical compound C([C@@H]1C[C@H]2O)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@H](O)C1 WBWWGRHZICKQGZ-GIHLXUJPSA-N 0.000 description 4
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 3
- 229960003964 deoxycholic acid Drugs 0.000 description 3
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 3
- 239000000787 lecithin Substances 0.000 description 3
- 229940067606 lecithin Drugs 0.000 description 3
- 235000010445 lecithin Nutrition 0.000 description 3
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010015031 Glycochenodeoxycholic Acid Proteins 0.000 description 2
- 108010007979 Glycocholic Acid Proteins 0.000 description 2
- SMEROWZSTRWXGI-UHFFFAOYSA-N Lithocholsaeure Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 SMEROWZSTRWXGI-UHFFFAOYSA-N 0.000 description 2
- RFDAIACWWDREDC-UHFFFAOYSA-N Na salt-Glycocholic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)C(O)C2 RFDAIACWWDREDC-UHFFFAOYSA-N 0.000 description 2
- BHTRKEVKTKCXOH-UHFFFAOYSA-N Taurochenodesoxycholsaeure Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCCS(O)(=O)=O)C)C1(C)CC2 BHTRKEVKTKCXOH-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000003858 bile acid conjugate Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 2
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- GHCZAUBVMUEKKP-GYPHWSFCSA-N glycochenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)CC1 GHCZAUBVMUEKKP-GYPHWSFCSA-N 0.000 description 2
- RFDAIACWWDREDC-FRVQLJSFSA-N glycocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 RFDAIACWWDREDC-FRVQLJSFSA-N 0.000 description 2
- 229940099347 glycocholic acid Drugs 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- SMEROWZSTRWXGI-HVATVPOCSA-N lithocholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 SMEROWZSTRWXGI-HVATVPOCSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229960003080 taurine Drugs 0.000 description 2
- BHTRKEVKTKCXOH-AYSJQVDDSA-N taurochenodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)C1C2C2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)CC1 BHTRKEVKTKCXOH-AYSJQVDDSA-N 0.000 description 2
- GHCZAUBVMUEKKP-UHFFFAOYSA-N ursodeoxycholic acid glycine-conjugate Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)CC2 GHCZAUBVMUEKKP-UHFFFAOYSA-N 0.000 description 2
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000002082 anti-convulsion Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000008845 cholagoga Substances 0.000 description 1
- 229940124571 cholagogue Drugs 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000010235 enterohepatic circulation Effects 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 230000004377 improving vision Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- -1 or the like Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Classifications
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- 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
- C12P33/00—Preparation of steroids
- C12P33/005—Degradation of the lateral chains at position 17
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for extracting bile acid by using high-efficiency enzyme of oxgall. Belongs to the technical field of effective extraction of animal bile. The method comprises the following steps: mixing the raw materials and the complex enzyme preparation, adjusting pH, performing enzymolysis, stirring, and performing solid-liquid separation to obtain an extract; inactivating enzyme, adding bile salt hydrolase for enzymolysis, adjusting pH, heating, stirring for saponification, centrifuging, and adjusting pH to obtain supernatant; adding hydrogen peroxide, stirring and mixing uniformly, reacting at room temperature, and filtering to obtain a treatment solution; slowly adding butyl acetate and stirring, then adjusting the pH value of the solution, and carrying out solid-liquid separation to obtain a solid matter; centrifuging, washing with water, and drying until the water content is not higher than 1% to obtain bile acid extract. The invention adopts the enzyme preparation for extraction, improves the product purity, reduces impurities, improves the product yield, fundamentally avoids pollution caused by toxic substances, reduces the problem of environmental pollution caused by strong acid and strong alkali, and has simple process, less investment and low cost.
Description
Technical Field
The invention relates to the technical field of effective extraction of animal bile, in particular to a method for extracting bile acid by using high-efficiency enzyme of oxgall.
Background
The ox bile is bile of cattle or buffalo, and the fresh bile is greenish brown or dark brown, slightly transparent, slightly sticky, fishy smell, bitter taste, etc., and is one of the main raw materials for extracting CA and DCA. Bile is a mixture of multiple components, and mainly contains bile acid, cholesterol, fatty acid, lecithin, macroelements, microelements, vitamins, amino acids, inorganic salt ions and the like except a large amount of water, and is similar to body fluid; in addition, metabolic products such as urea and purine, thyroxine, sex hormones, immune proteins, and the like are also contained. The content of bile acid in the bile is 50-60%, and the bile acid is one of main components of the bile. Bile acids are a generic term for a class of cholanic acids present in bile. Bile acids are classified into free bile acids including Cholic Acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) and bound bile acids according to their structures; the combined bile acid is a product obtained by combining the bile acid with glycine and taurine respectively, and mainly comprises glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA).
More than 90% of bile acid exists in the animal body in a combined form, the combined bile acid is formed by combining glycine and taurine through peptide bonds, such as glycobile acid and taurocholic acid, and the taurocholic acid is the bile acid which is common in animal bile, but the free bile acid content in the animal bile is very little or almost none.
The application of ox bile in China has long been known, and according to the record of the compendium of materia medica, the ox bile has the functions of clearing liver and improving vision, benefiting gallbladder and relaxing bowel, detoxifying and reducing swelling and the like. CA is a main raw material for synthesizing artificial bezoar and also is a main substance for clinical application of bezoar. Because the natural bezoar mainly comes from ox bile, and modern medical research has proved that the bezoar has the functions of tranquilizing, anti-convulsion, cholagogue and anti-inflammation.
At present, bile of cattle, sheep and pigs is generally separated and extracted at home and abroad, and the following three extraction methods are mainly adopted: ethanol crystallization, chloroform, ethyl acetate separation; chloroform method, ethanol crystallization method, and ethyl acetate separation method, however, the above methods all involve toxic substances (chloroform, ethyl acetate), and in order to reduce impurities, protein denaturation by ethanol, strong alkali, strong acid, or the like, fatty acid saponification, and the like are required, and the methods have the disadvantages of large reagent consumption, high cost, low yield, low purity, and high impurity content.
If the microbial treatment is adopted, as the bile acid belongs to the raw material of enterohepatic circulation in the animal body, the ileum and the large intestine content of the animal are extracted to culture and extract the microorganisms in the animal body, and the combined bile acid is hydrolyzed into free bile acid, so that the method is not suitable for industrial production, the process is complicated, and the cost is increased.
Therefore, how to provide a method for efficiently extracting bile acid by using oxgall is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention provides a method for extracting bile acid by using oxgall high-efficiency enzyme. Simple operation, mild condition, low reagent consumption, high purity bile acid extracted from ox bile, high yield and suitability for large-scale industrial production.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting bile acid by using oxgall high-efficiency enzyme comprises the following steps:
(1) mixing the raw materials and the complex enzyme preparation, adjusting pH, performing enzymolysis, stirring, and performing solid-liquid separation to obtain an extract;
(2) inactivating enzyme of the extractive solution, adding bile salt hydrolase for enzymolysis, adjusting pH, heating, stirring for saponification, centrifuging, and adjusting pH to obtain supernatant;
(3) adding hydrogen peroxide into the supernatant, stirring and mixing uniformly, reacting at room temperature, and filtering to obtain a treatment solution;
(4) slowly adding butyl acetate into the treatment solution, stirring, adjusting the pH value of the solution, and carrying out solid-liquid separation to obtain a solid matter;
(5) centrifuging the solid, washing with water, and drying until the water content is not higher than 1% to obtain bile acid extract product.
Has the advantages that: degrading protein, cholesterol, lecithin and the like in the oxgall into small molecular substances, and facilitating subsequent separation and extraction;
and (2) adding bile salt hydrolase for enzymolysis to enable the conjugated bile acid of the oxgall to be free bile acid.
Further: the equipment used in the step (1) is a reaction kettle.
Preferably: the raw material in the step (1) is fresh oxgall or a mixture of bile paste and water, wherein the mass ratio of the bile paste to the water is 1 (5-9).
Preferably: the complex enzyme preparation in the step (1) comprises the following components in percentage by mass: 20-35% of alkaline protease, 10-20% of neutral protease, 5-10% of acid protease, 5-10% of flavourzyme, 1-5% of lipase, 1-5% of phospholipase, 5% of glycerol, 5% of polyethylene glycol and 5-48% of water.
Further, the complex enzyme preparation in the step (1) optimally comprises the following components in percentage by mass: 20% of alkaline protease, 10% of neutral protease, 5% of acid protease, 5% of flavourzyme, 1% of lipase, 1% of phospholipase, 5% of glycerol, 5% of polyethylene glycol and 48% of water.
Preferably: in the step (1), the mass percentage of the addition amount of the complex enzyme preparation is 0.01-0.1% by mass based on the mass of the raw materials.
Preferably: adjusting the pH value to 5.0-9.0; the enzymolysis temperature is 30-50 ℃, and the enzymolysis time is 30-120 min; the stirring speed is 100-150 rpm.
Preferably: the enzyme deactivation in the step (2) is carried out for 30-60 min at the temperature of 80-100 ℃;
preferably: in the step (2), the mass of the bile salt hydrolase added is 0.1-1.0% of the weight of the extracting solution.
Preferably: step (2) enzymolysis: adjusting the pH value to 3.0-8.0; the temperature is 30-50 ℃; adjusting the pH value to 9-11; heating to 100 ℃; and adjusting the pH value to 7-10; the stirring and saponification time is 1-3 hours.
Further: adjusting the pH value to 9-11, and using a sodium hydroxide solution; adjusting the pH value to 7-10 by using hydrochloric acid;
preferably: the mass volume ratio of the supernatant to the hydrogen peroxide in the step (3) is 10: (1-5) kg/L.
Further: putting the supernatant in the step (3) into a decoloring tank; the reaction time is 24 hours at room temperature; introducing the treatment solution into an acidification tank; the volume fraction of butyl acetate in the step (4) is 1%, the pH value of the solution is adjusted to 3, and hydrochloric acid is used for adjusting; the water washing in the step (5) is stopped until the pH value of the separated water phase is neutral (the beneficial effect is that water is continuously added for washing in the centrifugal process to remove the water-soluble impurities attached to the surface of the white solid matter); the drying temperature was 100 ℃.
The invention also provides a bile acid finished product prepared by the method.
According to the technical scheme, compared with the prior art, the invention discloses a method for extracting bile acid by using oxgall high-efficiency enzyme, and the technical effects are as follows:
the enzyme preparation is adopted for extraction, so that the product purity is improved, impurities are reduced, the product yield is improved, the pollution brought by toxic substances participating in the process is fundamentally avoided, the problem of environmental pollution caused by strong acid and strong base is reduced, and the method is simple in process, low in investment and low in cost;
hydrolyzing the conjugated bile acid into free bile acid by using bile salt hydrolase at an optimum reaction temperature of 30-50 ℃ and an optimum pH of 3.0-8.0;
by using the complex enzyme preparation (containing alkaline protease, neutral protease, acid protease, lipase and phospholipase), protein, lecithin and other impurities in the bile can be degraded under mild conditions.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a method for extracting bile acid by using high-efficiency ox bile enzyme.
Example 1
A method for extracting bile acid by using oxgall high-efficiency enzyme comprises the following steps:
(1) mixing the raw materials and the complex enzyme preparation, adjusting pH, performing enzymolysis, stirring, and performing solid-liquid separation to obtain an extract;
(2) inactivating enzyme of the extractive solution, adding bile salt hydrolase for enzymolysis, adjusting pH, heating, stirring for saponification, centrifuging, and adjusting pH to obtain supernatant;
(3) adding hydrogen peroxide into the supernatant, stirring and mixing uniformly, reacting at room temperature, and filtering to obtain a treatment solution;
(4) slowly adding butyl acetate into the treatment solution, stirring, adjusting the pH value of the solution, and carrying out solid-liquid separation to obtain a solid matter;
(5) centrifuging the solid, washing with water, and drying until the water content is not higher than 1% to obtain bile acid extract product.
In order to further optimize the technical scheme: the equipment used in the step (1) is a reaction kettle.
In order to further optimize the technical scheme: the raw material in the step (1) is fresh oxgall or a mixture of bile paste and water, wherein the mass ratio of the bile paste to the water is 1: 5.
In order to further optimize the technical scheme: the complex enzyme preparation in the step (1) comprises the following components in percentage by mass: 20% of alkaline protease, 10% of neutral protease, 5% of acid protease, 5% of flavourzyme, 1% of lipase, 1% of phospholipase, 5% of glycerol, 5% of polyethylene glycol and 48% of water.
In order to further optimize the technical scheme: in the step (1), the adding amount of the complex enzyme preparation is 0.01 percent by mass based on the mass of the raw materials.
In order to further optimize the technical scheme: adjusting the pH value to 5.0; the enzymolysis temperature is 30 ℃, and the enzymolysis time is 30 min; the stirring speed was 100 rpm.
In order to further optimize the technical scheme: the enzyme deactivation in the step (2) is carried out for 30min at the temperature of 80 ℃;
in order to further optimize the technical scheme: in the step (2), the mass of the bile salt hydrolase added is 0.1 percent based on the weight of the extracting solution.
In order to further optimize the technical scheme: step (2) enzymolysis: adjusting the pH to 3.0; the temperature is 30 ℃; then adjusting the pH value to 9; heating to 100 ℃; and adjusting the pH to 7; the time for stirring saponification was 1 hour.
In order to further optimize the technical scheme: adjusting the pH value to 9 by using a sodium hydroxide solution; adjusting the pH to 7 using hydrochloric acid;
in order to further optimize the technical scheme: the mass volume ratio of the supernatant to the hydrogen peroxide in the step (3) is 10: 1 kg/L.
In order to further optimize the technical scheme: putting the supernatant in the step (3) into a decoloring tank; the reaction time is 24 hours at room temperature; introducing the treatment solution into an acidification tank; the volume fraction of butyl acetate in the step (4) is 1%, the pH value of the solution is adjusted to 3, and hydrochloric acid is used for adjusting; the water washing in the step (5) is stopped until the pH value of the separated water phase is neutral; the drying temperature was 100 ℃.
Example 2
A method for extracting bile acid by using oxgall high-efficiency enzyme comprises the following steps:
(1) mixing the raw materials and the complex enzyme preparation, adjusting pH, performing enzymolysis, stirring, and performing solid-liquid separation to obtain an extract;
(2) inactivating enzyme of the extractive solution, adding bile salt hydrolase for enzymolysis, adjusting pH, heating, stirring for saponification, centrifuging, and adjusting pH to obtain supernatant;
(3) adding hydrogen peroxide into the supernatant, stirring and mixing uniformly, reacting at room temperature, and filtering to obtain a treatment solution;
(4) slowly adding butyl acetate into the treatment solution, stirring, adjusting the pH value of the solution, and carrying out solid-liquid separation to obtain a solid matter;
(5) centrifuging the solid, washing with water, and drying until the water content is not higher than 1% to obtain bile acid extract product.
In order to further optimize the technical scheme: the equipment used in the step (1) is a reaction kettle.
In order to further optimize the technical scheme: the raw material in the step (1) is fresh oxgall or a mixture of bile paste and water, wherein the mass ratio of the bile paste to the water is 1: 8.
In order to further optimize the technical scheme: the complex enzyme preparation in the step (1) optimally comprises the following components in percentage by mass: 20% of alkaline protease, 10% of neutral protease, 5% of acid protease, 5% of flavourzyme, 1% of lipase, 1% of phospholipase, 5% of glycerol, 5% of polyethylene glycol and 48% of water.
In order to further optimize the technical scheme: in the step (1), the adding amount of the complex enzyme preparation is 0.05% by mass based on the mass of the raw materials.
In order to further optimize the technical scheme: adjusting the pH value to 7; the enzymolysis temperature is 40 ℃, and the enzymolysis time is 80 min; the stirring speed was 120 rpm.
In order to further optimize the technical scheme: the enzyme deactivation in the step (2) is carried out for 45min at 90 ℃;
in order to further optimize the technical scheme: in the step (2), the mass of the bile salt hydrolase added is 0.5 percent based on the weight of the extracting solution.
In order to further optimize the technical scheme: step (2) enzymolysis: adjusting the pH to 6; the temperature is 40 ℃; then adjusting the pH value to 10; heating to 100 ℃; and adjusting the pH to 8; the time for stirring saponification was 2 hours.
In order to further optimize the technical scheme: adjusting the pH value to 10 by using a sodium hydroxide solution; adjusting the pH to 8 using hydrochloric acid;
in order to further optimize the technical scheme: the mass volume ratio of the supernatant to the hydrogen peroxide in the step (3) is 10: 3 kg/L.
In order to further optimize the technical scheme: putting the supernatant in the step (3) into a decoloring tank; the reaction time is 24 hours at room temperature; introducing the treatment solution into an acidification tank; the volume fraction of butyl acetate in the step (4) is 1%, the pH value of the solution is adjusted to 3, and hydrochloric acid is used for adjusting; the water washing in the step (5) is stopped until the pH value of the separated water phase is neutral; the drying temperature was 100 ℃.
Example 3
A method for extracting bile acid by using oxgall high-efficiency enzyme comprises the following steps:
(1) mixing the raw materials and the complex enzyme preparation, adjusting pH, performing enzymolysis, stirring, and performing solid-liquid separation to obtain an extract;
(2) inactivating enzyme of the extractive solution, adding bile salt hydrolase for enzymolysis, adjusting pH, heating, stirring for saponification, centrifuging, and adjusting pH to obtain supernatant;
(3) adding hydrogen peroxide into the supernatant, stirring and mixing uniformly, reacting at room temperature, and filtering to obtain a treatment solution;
(4) slowly adding butyl acetate into the treatment solution, stirring, adjusting the pH value of the solution, and carrying out solid-liquid separation to obtain a solid matter;
(5) centrifuging the solid, washing with water, and drying until the water content is not higher than 1% to obtain bile acid extract product.
In order to further optimize the technical scheme: the equipment used in the step (1) is a reaction kettle.
In order to further optimize the technical scheme: the raw material in the step (1) is fresh oxgall or a mixture of bile paste and water, wherein the mass ratio of the bile paste to the water is 1: 9.
In order to further optimize the technical scheme: the complex enzyme preparation in the step (1) comprises the following components in percentage by mass: 35% of alkaline protease, 20% of neutral protease, 10% of acid protease, 10% of flavourzyme, 5% of lipase, 5% of phospholipase, 5% of glycerol, 5% of polyethylene glycol and 5% of water.
In order to further optimize the technical scheme: in the step (1), the adding amount of the complex enzyme preparation is 0.1% by mass based on the mass of the raw materials.
In order to further optimize the technical scheme: adjusting the pH value to 9.0; the enzymolysis temperature is 50 ℃, and the enzymolysis time is 120 min; the stirring speed was 150 rpm.
In order to further optimize the technical scheme: the enzyme deactivation in the step (2) is carried out for 30min at 100 ℃;
in order to further optimize the technical scheme: in the step (2), the mass of the bile salt hydrolase added is 1.0 percent based on the weight of the extracting solution.
In order to further optimize the technical scheme: step (2) enzymolysis: adjusting the pH to 8.0; the temperature is 50 ℃; then adjusting the pH value to 11; heating to 100 ℃; and adjusting the pH to 10; the time for stirring saponification was 3 hours.
In order to further optimize the technical scheme: adjusting the pH value to 11 by using a sodium hydroxide solution; adjusting the pH to 10 using hydrochloric acid;
in order to further optimize the technical scheme: the mass volume ratio of the supernatant to the hydrogen peroxide in the step (3) is 10: 5 kg/L.
In order to further optimize the technical scheme: putting the supernatant in the step (3) into a decoloring tank; the reaction time is 24 hours at room temperature; introducing the treatment solution into an acidification tank; the volume fraction of butyl acetate in the step (4) is 1%, the pH value of the solution is adjusted to 3, and hydrochloric acid is used for adjusting; the water washing in the step (5) is stopped until the pH value of the separated water phase is neutral; the drying temperature was 100 ℃.
The bile acids prepared in examples 1 to 3 were hydrolyzed into free bile acids with high purity, low impurities and high product yield by using an enzyme preparation.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for extracting bile acid by using oxgall high-efficiency enzyme is characterized by comprising the following steps:
(1) mixing the raw materials and the complex enzyme preparation, adjusting pH, performing enzymolysis, stirring, and performing solid-liquid separation to obtain an extract;
(2) inactivating enzyme of the extractive solution, adding bile salt hydrolase for enzymolysis, adjusting pH, heating, stirring for saponification, centrifuging, and adjusting pH to obtain supernatant;
(3) adding hydrogen peroxide into the supernatant, stirring and mixing uniformly, reacting at room temperature, and filtering to obtain a treatment solution;
(4) slowly adding butyl acetate into the treatment solution, stirring, adjusting the pH value of the solution, and carrying out solid-liquid separation to obtain a solid matter;
(5) centrifuging the solid, washing with water, and drying until the water content is not higher than 1% to obtain bile acid extract product.
2. The method according to claim 1, wherein the raw material in the step (1) is fresh oxgall or a mixture of bile paste and water, wherein the mass ratio of the bile paste to the water is 1 (5-9).
3. The method according to claim 1, wherein the complex enzyme preparation in the step (1) comprises the following components in percentage by mass: 20-35% of alkaline protease, 10-20% of neutral protease, 5-10% of acid protease, 5-10% of flavourzyme, 1-5% of lipase, 1-5% of phospholipase, 5% of glycerol, 5% of polyethylene glycol and 5-48% of water.
4. The method according to claim 1, characterized in that in the step (1), the adding amount of the complex enzyme preparation is 0.01-0.1% by mass based on the mass of the raw material.
5. The method according to claim 1, wherein the pH is adjusted to 5.0-9.0 in step (1); the temperature of enzymolysis is 30-50 ℃, and the enzymolysis time is 30-120 min; the stirring speed is 100-150 rpm.
6. The method according to claim 1, wherein the enzyme deactivation in the step (2) is carried out at 80-100 ℃ for 30-60 min.
7. The method as claimed in claim 1, wherein the bile salt hydrolase added in step (2) is present in an amount of 0.1 to 1.0% by weight based on the weight of the extract.
8. The method of claim 1, wherein the step (2) of enzymatic hydrolysis: adjusting the pH value to 3.0-8.0 and the temperature to 30-50 ℃; adjusting the pH value to 9-11; heating to 100 ℃; adjusting the pH value to 7-10; the stirring saponification time is 1-3 hours.
9. The method according to claim 1, wherein the mass-to-volume ratio of the supernatant to the hydrogen peroxide in the step (3) is 10: (1-5) kg/L.
10. A finished bile acid product prepared by the method of any of claims 1 to 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113999886A (en) * | 2021-12-06 | 2022-02-01 | 江苏省农业科学院 | Method for extracting chicken bile acid by one-pot enzymolysis |
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| CN111334553A (en) * | 2020-03-26 | 2020-06-26 | 山东中京生物科技有限公司 | Process for producing bile paste by enzyme method |
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| CN101948496A (en) * | 2010-09-21 | 2011-01-19 | 湖南凯勒迪化学科技有限公司 | Method for extracting chenodeoxycholic acid from bile of fowl |
| CN107312054A (en) * | 2017-07-25 | 2017-11-03 | 贵州慧静生物科技有限公司 | A kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile |
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| CN113999886A (en) * | 2021-12-06 | 2022-02-01 | 江苏省农业科学院 | Method for extracting chicken bile acid by one-pot enzymolysis |
| CN113999886B (en) * | 2021-12-06 | 2023-07-04 | 江苏省农业科学院 | Method for extracting chicken bile acid by one-pot enzymolysis |
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Effective date of registration: 20210524 Address after: 415400 south of Xujiahu Road, Jinshi hi tech Zone, Changde City, Hunan Province Applicant after: Hunan Xiasheng Enzyme Technology Co.,Ltd. Applicant after: Cangzhou Xiasheng Enzyme Biotechnology Co.,Ltd. Applicant after: NINGXIA SUNSON INDUSTRY GROUP Co.,Ltd. Address before: 415400 south of Xujiahu Road, Jinshi hi tech Zone, Changde City, Hunan Province Applicant before: Hunan Xiasheng Enzyme Technology Co.,Ltd. |
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