CN104531635A - Method for extracting hyaluronidase crude product - Google Patents
Method for extracting hyaluronidase crude product Download PDFInfo
- Publication number
- CN104531635A CN104531635A CN201410807817.XA CN201410807817A CN104531635A CN 104531635 A CN104531635 A CN 104531635A CN 201410807817 A CN201410807817 A CN 201410807817A CN 104531635 A CN104531635 A CN 104531635A
- Authority
- CN
- China
- Prior art keywords
- solution
- liquid
- hyaluronidase
- crude product
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
- Cosmetics (AREA)
Abstract
The invention provides a method for extracting a hyaluronidase crude product. The method comprises the following steps of extracting with an acid solution and precipitating with low-temperature ethanol to obtain a hyaluronidase crude product, and carrying out chromatographic separation, thermal reduction treatment and lyophilizing on the crude product to obtain a hyaluronidase refined product. Compared with the traditional method, by extracting with the acid solution, salting out with ammonium sulfate to prepare the hyaluronidase crude product and dialyzing and lyophilizing the crude product to obtain the hyaluronidase refined product, higher yield and potency are achieved.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the method extracting hyaluronidase from fresh animal testis.
Background technology
Hyaluronidase has another name called Unidasa, and in mammiferous testis, content is very abundant, can be hydrolyzed hyaluronic acid, its viscosity is obviously declined, is conducive to sperm at fertilization and enters ovum.Also be present in the lysosome of sperm, submaxillary gland, bee venom, snake venom, skin, spleen, leech and cell.
The stability of hyaluronidase is better, and 42 DEG C of heating, 60 minutes vigor do not lose; 100 DEG C are heated 5 minutes, and vigor can retain 80%.Can recuperation section vigor by the laggard row cooling of heat inactivation.Still more stable at below pH5 or more than pH8 enzyme.Comparatively easy in inactivation in low concentration aqueous solution, but 0.2% or 0.5% gum arabic or the protection of 0.2% gelatin can be added.Fe
2+, Cu
2+reversible inhibition is had, Pb to enzyme
2+, Hg
2+, Ni
2+do not have a significant effect Deng to enzymic activity.Chondroitin sulfate B (dermatan), heparitin sulfate, keratan sulfate, heparin and high density hyaluronidase all have restraining effect to enzyme, but can by 0.15mol/L sodium-chlor or protamine sulfate reverse.
Pharmaceutical glass acid enzyme is white or micro-yellow powder, and odorlessness is soluble in water, is insoluble to the organic solvents such as acetone, ether, ethanol.Biochemical pharmacy mainly with ox, Testis Caprae seu Ovis be raw material carry out extraction preparation.
Cold ethanol method be 1940 by the Edwin J.Cohn teaching inventive of medical college of Harvard University, be therefore also called " Kong Shi method ".Kong Shi method is used for Separation of Bovine serum albumin at first, is applied to human plasma subsequently and is separated.Nineteen forty-four, albumin products carries out clinical infusion first, for rescuing the gob of 7 serious burns in Japanese army's Attack On Pearl Harbor, and starts the suitability for industrialized production of plasma protein products thus.Ethanol, as a kind of protein precipitant, has many advantages: specific inductivity is low, with water easily miscible, under home and working conditions without explosion hazard, lower molecular weight, relative inertness chemically, low toxicity, inexpensive, be easy to get and bacteriostatic action.So far, extensive plasma proteins is separated and still substantially adopts cold ethanol partition method.
The external beginning of the fifties puts into production, and records in Britain and Japanese Pharmacopoeia, trade(brand)name Rondas.China formally puts into production nineteen sixty-five, extracts from Testis Caprae seu Ovis, takes in 1977 editions Chinese Pharmacopoeias.Wang Yan, Zhou Shumin etc. had once delivered the paper of a section " research of Unidasa extraction process " by name on " the Heilungkiang medicine " of 15 volumes (1) phase in 2002, and more detailed describes the traditional technology extracting hyaluronidase in industrial production from Testis Caprae seu Ovis and the novel process improved by them on this basis; Li Dan, Guo Yutao etc. had once delivered the paper of a section " in bull testis Unidasa Study on extraction " by name on " the application chemical industry " of volume the 8th phase August the 40th in 2011, the mixed solution describing use hydrochloric acid and acetic acid dissolves, ammonium sulfate analysis method, extracts the experimental study of hyaluronidase from bull testis.Domestic traditional technology extracts hyaluronidase crude product with acid fluid dissolves and ammonium sulfate precipitation, crude product obtains quality hyaluronidase product through dialysis, freeze-drying, this patent adopts acid solution to extract and chilled alcohol precipitation extracts hyaluronidase crude product, crude product obtains quality hyaluronidase product through chromatographic separation, pyrogen process, freeze-drying, still belong to pioneering at home, the documents and materials such as domestic correlative theses, patent do not have the related introduction that chilled alcohol precipitation legal system gets hyaluronidase crude product, chromatographic separation produces quality hyaluronidase product.
Summary of the invention
The invention provides a kind of preparation method of hyaluronidase crude product, adopt acid solution extraction and chilled alcohol precipitation to obtain hyaluronidase crude product, crude product obtains quality hyaluronidase product through chromatographic separation, pyrogen process, freeze-drying.Concrete steps are as follows:
1 extracts:
By Testis Caprae seu Ovis except interior exodermis and epididymitis, be twisted into rotten slurry.Take rotten slurry, in the ratio of every 1kg gruel slurry acid liquid 1.0-1.5L, acid liquid stirs, and leaches after 4 hours, is filtered by leach liquor, and filtrate adjusts pH3.2 ~ 4.0.
Get filter residue, start the ratio of the gruel slurry acid liquid 0.2-0.3L added in every 1kg, acid liquid is filtered after stirring 1 hour again, and filtrate adjusts pH3.2 ~ 4.0.
Described acid solution is Glacial acetic acid, hydrochloric acid, the mix acid liquor of sodium-chlor and water.
2 is rough:
Merge twice filtered liquid, under constantly stirring, add the dehydrated alcohol of precooling, leave standstill and make it produce precipitation in 2 hours, precipitation is suspended in the sodium chloride solution of 0.15mol/L, pH is adjusted to form precipitation, get supernatant liquor, supernatant liquor is adjusted pH3.2-4.0, add the dehydrated alcohol of precooling, leave standstill 2 hours, gained precipitation is hyaluronidase crude product.
Rough operation need be carried out in 2-6 DEG C of freezer, 50% of the filtered liquid merged when the addition of twice dehydrated alcohol is beginning.
3 chromatographic separation:
Setting column flow rate 25L/h, chromatography column 2 hours are balanced with balance liquid, crude product is dissolved in upper prop after a small amount of balance liquid, pillar is respectively cleaned 25 minutes respectively again with balance liquid and scavenging solution, then elution is used 30 minutes, collect solution, add 4 times and collect the long-pending alcohol settling centrifuge dehydration after 12 hours of liquid, rotating speed 3700r/min; Wherein: balance liquid is sodium-acetate 1090g, adds purified water 160L, the solution after pH3.6 is adjusted; Scavenging solution is sodium-acetate 220g, adds purified water 16L, adjusts the solution after pH3.6; Elutriant is sodium-chlor 440g, adds purified water 15L, adjusts the solution after pH3.6;
4 pyrogen process:
By above-mentioned solution centrifugal, filter, filtrate is put in ice bath and cools, add 15% tertiary sodium phosphate (Na
3pO
412H
2o) solution, under constantly stirring, slowly adds 20% calcium acetate (Ca (CH
3cOO)
2h
2o) solution, and adjust pH 8.5 with NaOH solution, centrifugal, collect supernatant liquor, pH 6.5 ~ 7.0 adjusted by supernatant liquor afterwards;
5 freeze-drying
By filtrate lyophilize, obtain pyrogen-free quality hyaluronidase product.
compared with prior art, advantage of the present invention and positively effect are:
Domestic prior art produces hyaluronidase crude product by the method for acid fluid dissolves, ammonium sulfate precipitation, and crude product obtains quality hyaluronidase product through dialysis, freeze-drying; And the technology of the present invention adopts acid solution extraction and chilled alcohol precipitation to produce hyaluronidase crude product, crude product obtains quality hyaluronidase product through chromatographic separation, pyrogen process, freeze-drying, the documents and materials such as domestic correlative theses, patent do not have chilled alcohol precipitation legal system to get related introduction that hyaluronidase crude product and chromatographic separation produce quality hyaluronidase product, production technique of the present invention is simple and easy, yield is high, is applicable to large-scale industrial production.This achievement has good economic benefit and social benefit, and it not only solves the demand of domestic and international pharmaceutical drugs, is also the comprehensive utilization developing Liao Xin road of cultured product, has positive effect to the development of aquaculture.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
1 extracts:
Take rotten slurry 6kg acid liquid 7.8L, leach liquor, after 4 hours, filters by agitation leach, and filtrate adjusts pH3.59.
Get filter residue acid liquid 1.4L, stir after 1 hour, again filter, filtrate adjusts pH3.62.
2 is rough:
Twice filtered liquid 10.2L is merged in 2-6 DEG C of freezer, under constantly stirring, add the dehydrated alcohol 5.1L of precooling, leave standstill and make it produce precipitation in 2 hours, precipitation is suspended in the sodium chloride solution of 0.15mol/L, pH is adjusted to form precipitation, get supernatant liquor, supernatant liquor is adjusted pH3.66, add the dehydrated alcohol 5.1L of precooling, leave standstill 2 hours, gained precipitation is hyaluronidase crude product 782.28g.
3 chromatographic separation
Setting column flow rate 25L/h, chromatography column 2 hours are balanced with balance liquid, crude product 782.28g is dissolved in upper prop after 78L balance liquid, pillar is respectively cleaned 25 minutes respectively again with balance liquid and scavenging solution, then elution is used 30 minutes, collect solution 12.7L, add the centrifuge dehydration after 12 hours of 50.8L alcohol settling, rotating speed 3700r/min; Wherein: balance liquid is sodium-acetate 1090g, adds purified water 160L, the solution after pH3.6 is adjusted; Scavenging solution is sodium-acetate 220g, adds purified water 16L, adjusts the solution after pH3.6; Elutriant is sodium-chlor 440g, adds purified water 15L, adjusts the solution after pH3.6;
4 pyrogen process:
By above-mentioned solution centrifugal, filter, filtrate is put in ice bath and cools, add 15% tertiary sodium phosphate (Na
3pO
412H
2o) solution 8mL, under constantly stirring, slowly adds 20% calcium acetate (Ca (CH
3cOO)
2h
2o) solution 4mL, and adjust pH 8.5 with NaOH solution, centrifugal, collect supernatant liquor, pH 6.5 ~ 7.0 adjusted by supernatant liquor afterwards;
5 freeze-drying
By filtrate lyophilize, obtain pyrogen-free quality hyaluronidase product 299.26g.
Embodiment 2
1 extracts:
Take rotten slurry 3kg acid liquid 3.3L, leach liquor, after 4 hours, filters by agitation leach, and filtrate adjusts pH3.73.
Get filter residue acid liquid 0.6L, stir after 1 hour, again filter, filtrate adjusts pH3.61.
2 is rough:
Twice filtered liquid 4.0L is merged in 2-6 DEG C of freezer, under constantly stirring, add the dehydrated alcohol 2L of precooling, leave standstill and make it produce precipitation in 2 hours, precipitation is suspended in the sodium chloride solution of 0.15mol/L, pH is adjusted to form precipitation, get supernatant liquor, supernatant liquor is adjusted pH3.58, add the dehydrated alcohol 2L of precooling, leave standstill 2 hours, gained precipitation is hyaluronidase crude product 391.84g.
3 chromatographic separation
Setting column flow rate 25L/h, chromatography column 2 hours are balanced with balance liquid, crude product 391.84g is dissolved in upper prop after 39L balance liquid, pillar is respectively cleaned 25 minutes respectively again with balance liquid and scavenging solution, then elution is used 30 minutes, collect solution 8.2L, add the centrifuge dehydration after 12 hours of 32.8L alcohol settling, rotating speed 3700r/min; Wherein: balance liquid is sodium-acetate 1090g, adds purified water 160L, the solution after pH3.6 is adjusted; Scavenging solution is sodium-acetate 220g, adds purified water 16L, adjusts the solution after pH3.6; Elutriant is sodium-chlor 440g, adds purified water 15L, adjusts the solution after pH3.6;
4 pyrogen process:
By above-mentioned solution centrifugal, filter, filtrate is put in ice bath and cools, add 15% tertiary sodium phosphate (Na
3pO
412H
2o) solution 4mL, under constantly stirring, slowly adds 20% calcium acetate (Ca (CH
3cOO)
2h
2o) solution 2mL, and adjust pH 8.5 with NaOH solution, centrifugal, collect supernatant liquor, pH 6.5 ~ 7.0 adjusted by supernatant liquor afterwards;
5 freeze-drying
By filtrate lyophilize, obtain pyrogen-free quality hyaluronidase product 151.21g.
Through measuring and calculating, in embodiment 1, quality hyaluronidase product is tired as 353.19iu/mg, yield 4.99%; In embodiment 2, quality hyaluronidase product is tired as 353.04iu/mg, yield 5.04%.
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (9)
1. the preparation method of a hyaluronidase crude product, it is characterized in that, acid solution extraction and chilled alcohol precipitation is adopted to obtain hyaluronidase crude product, crude product obtains quality hyaluronidase product through chromatographic separation, pyrogen process, freeze-drying, to be extracted by acid solution with traditional method and ammonium sulfate precipitation produces hyaluronidase crude product, crude product obtains quality hyaluronidase product compare through dialysis, freeze-drying, has higher yield and tires.
2. method according to claim 1, is characterized in that, comprises the following steps:
(1) extract:
By Testis Caprae seu Ovis except interior exodermis and epididymitis, be twisted into rotten slurry;
Take rotten slurry, in the ratio of every 1kg gruel slurry acid liquid 1.0-1.5L, acid liquid stirs, and leaches after 4 hours, is filtered by leach liquor, and filtrate adjusts pH3.2 ~ 4.0;
Get filter residue, start the ratio of the gruel slurry acid liquid 0.2-0.3L added in every 1kg, acid liquid is filtered after stirring 1 hour again, and filtrate adjusts pH3.2 ~ 4.0;
Described acid solution is Glacial acetic acid, hydrochloric acid, the mix acid liquor of sodium-chlor and water;
(2) rough:
Merge twice filtered liquid, under constantly stirring, add the dehydrated alcohol of precooling, leave standstill and make it produce precipitation in 2 hours, precipitation is suspended in the sodium chloride solution of 0.15mol/L, pH is adjusted to form precipitation, get supernatant liquor, supernatant liquor is adjusted pH3.2-4.0, add the dehydrated alcohol of precooling, leave standstill 2 hours, gained precipitation is hyaluronidase crude product;
Rough operation need be carried out in 2-6 DEG C of freezer, 50% of the filtered liquid merged when the addition of twice dehydrated alcohol is beginning;
(3) chromatographic separation:
Setting column flow rate 25L/h, chromatography column 2 hours are balanced with balance liquid, crude product is dissolved in upper prop after a small amount of balance liquid, pillar is respectively cleaned 25 minutes respectively again with balance liquid and scavenging solution, then elution is used 30 minutes, collect solution, add 4 times and collect the long-pending alcohol settling centrifuge dehydration after 12 hours of liquid, rotating speed 3700r/min; Wherein: balance liquid is sodium-acetate 1090g, adds purified water 160L, the solution after pH3.6 is adjusted; Scavenging solution is sodium-acetate 220g, adds purified water 16L, adjusts the solution after pH3.6; Elutriant is sodium-chlor 440g, adds purified water 15L, adjusts the solution after pH3.6;
(4) pyrogen process:
By above-mentioned solution centrifugal, filter, filtrate is put in ice bath and cools, add 15% tertiary sodium phosphate (Na
3pO
412H
2o) solution, under constantly stirring, slowly adds 20% calcium acetate (Ca (CH
3cOO)
2h
2o) solution, and adjust pH 8.5 with NaOH solution, centrifugal, collect supernatant liquor, pH 6.5 ~ 7.0 adjusted by supernatant liquor afterwards;
(5) freeze-drying
By filtrate lyophilize, obtain pyrogen-free quality hyaluronidase product.
3. the method described in claims 2, is characterized in that, in step (), first time every 1kg gruel slurry acid liquid 1.0-1.5L, second time acid solution still adds according to the rotten slurry amount of beginning, every 1kg gruel slurry acid liquid 0.2-0.3L.
4. the method described in claims 2, is characterized in that, in step (), acid solution regulates the pH value of filtrate to be 3.2 ~ 4.0 after extracting.
5. the method described in claims 2, is characterized in that, step (two) need be carried out in 2-6 DEG C of freezer.
6. the method described in claims 2, is characterized in that, 50% of the filtered liquid merged when the addition of twice dehydrated alcohol is beginning in step (two).
7. the method described in claims 2, is characterized in that, in step (three), column flow rate is 25L/h, and after obtaining collecting liquid, add 4 times and collect the long-pending alcohol settling centrifuge dehydration again after 12 hours of liquid, its rotating speed is 3700r/min.
8. the method described in claims 2, is characterized in that, in step (three), balance liquid is sodium-acetate 1090g, adds purified water 160L, adjusts the solution after pH3.6; Scavenging solution is sodium-acetate 220g, adds purified water 16L, adjusts the solution after pH3.6; Elutriant is sodium-chlor 440g, adds purified water 15L, adjusts the solution after pH3.6.
9. the method described in claims 2, is characterized in that, " above-mentioned solution " in step (four) before filtration and all will carry out centrifugal after adjust pH to 8-9, centrifugation time: 20 minutes ~ 30 minutes, rotating speed: 2700 ~ 3200r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410807817.XA CN104531635A (en) | 2014-12-23 | 2014-12-23 | Method for extracting hyaluronidase crude product |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410807817.XA CN104531635A (en) | 2014-12-23 | 2014-12-23 | Method for extracting hyaluronidase crude product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104531635A true CN104531635A (en) | 2015-04-22 |
Family
ID=52847269
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410807817.XA Pending CN104531635A (en) | 2014-12-23 | 2014-12-23 | Method for extracting hyaluronidase crude product |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531635A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667210A (en) * | 2013-11-27 | 2014-03-26 | 青岛康原药业有限公司 | Low temperature ethanol method for extracting hyaluronidase crude product |
| CN113528488A (en) * | 2021-08-17 | 2021-10-22 | 南昌大学 | Method for preparing high-quality crude hyaluronidase product by stirring and adsorbing resin |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060288A (en) * | 2012-12-30 | 2013-04-24 | 青岛九龙生物医药有限公司 | Method for extracting hyaluronidase from pig testis |
| CN103451166A (en) * | 2013-08-31 | 2013-12-18 | 青岛康原药业有限公司 | Method for extracting hyaluronidase from pig testicle |
| CN103667210A (en) * | 2013-11-27 | 2014-03-26 | 青岛康原药业有限公司 | Low temperature ethanol method for extracting hyaluronidase crude product |
| CN103740677A (en) * | 2013-11-30 | 2014-04-23 | 青岛康原药业有限公司 | Method for extraction of quality hyaluronidase product |
-
2014
- 2014-12-23 CN CN201410807817.XA patent/CN104531635A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060288A (en) * | 2012-12-30 | 2013-04-24 | 青岛九龙生物医药有限公司 | Method for extracting hyaluronidase from pig testis |
| CN103451166A (en) * | 2013-08-31 | 2013-12-18 | 青岛康原药业有限公司 | Method for extracting hyaluronidase from pig testicle |
| CN103667210A (en) * | 2013-11-27 | 2014-03-26 | 青岛康原药业有限公司 | Low temperature ethanol method for extracting hyaluronidase crude product |
| CN103740677A (en) * | 2013-11-30 | 2014-04-23 | 青岛康原药业有限公司 | Method for extraction of quality hyaluronidase product |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667210A (en) * | 2013-11-27 | 2014-03-26 | 青岛康原药业有限公司 | Low temperature ethanol method for extracting hyaluronidase crude product |
| CN113528488A (en) * | 2021-08-17 | 2021-10-22 | 南昌大学 | Method for preparing high-quality crude hyaluronidase product by stirring and adsorbing resin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chew et al. | Liquid biphasic flotation for the purification of C-phycocyanin from Spirulina platensis microalga | |
| CN103060288B (en) | Method for extracting hyaluronidase from pig testis | |
| CN101343310B (en) | Method for preparing high purity phycobiliprotein with primary column chromatography | |
| CN103992402A (en) | Preparation method of high-purity phycocyanin | |
| CN103102408B (en) | Method for extracting phycocyanin from spirulina | |
| CN109021096A (en) | A kind of separation and Extraction purifying process of spirulina polysaccharide and phycocyanin | |
| CN102532304A (en) | Preparation method of human serum albumin | |
| CN103060291B (en) | Extraction method for hyaluronidase | |
| CN100376684C (en) | Process for preparing bacteriostatic peptide by discarded tobacco leaf protein | |
| CN104151424A (en) | Phycocyanin extraction method | |
| CN102732586A (en) | Method for culturing mesenchymal stem cell secretin | |
| CN104130319A (en) | Extraction method of phycoerythrin | |
| CN104531635A (en) | Method for extracting hyaluronidase crude product | |
| CN103740677B (en) | A kind of method of extracting hyaluronidase fine work | |
| CN103451166A (en) | Method for extracting hyaluronidase from pig testicle | |
| CN106496321B (en) | Purification method of recombinant human follistatin protein | |
| CN101367865B (en) | Production process for high purity porcine blood albumin and uses thereof | |
| CN104498452A (en) | Method for extracting hyaluronidase crude product by low-temperature ethanol method | |
| CN103667210B (en) | A kind of cold ethanol method extracts the method for hyaluronidase crude product | |
| CN103725665A (en) | Method for extracting chymotrypsin from sheep pancreas | |
| CN103103170B (en) | Production process for cow or sheep hyaluronidase | |
| CN104744965A (en) | Method for reducing insoluble substances of capsanthin | |
| CN108101980B (en) | Preparation method of high-purity phycocyanin | |
| CN206396103U (en) | A kind of extraction element of cross-linking sodium hyaluronate gel | |
| CN102603926B (en) | New preparing process of high-titer heparin sodium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150422 |