CN107674131A - A kind of method of the extraction purification hyaluronic acid from zymotic fluid - Google Patents
A kind of method of the extraction purification hyaluronic acid from zymotic fluid Download PDFInfo
- Publication number
- CN107674131A CN107674131A CN201710969336.2A CN201710969336A CN107674131A CN 107674131 A CN107674131 A CN 107674131A CN 201710969336 A CN201710969336 A CN 201710969336A CN 107674131 A CN107674131 A CN 107674131A
- Authority
- CN
- China
- Prior art keywords
- adds
- zymotic fluid
- hyaluronic acid
- added
- precipitated
- 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
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
Abstract
The invention belongs to technical field of microbial fermentation, is related to a kind of method of the extraction purification hyaluronic acid from zymotic fluid, that includes following steps：Step 1) zymotic fluid dilutes centrifuging and taking supernatant, step 2) adds liquor potassic permanganate reaction after being adjusted to appropriate pH, step 3) adds charcoal absorption, step 4) filters and takes filtrate, step 5) ultrafiltration takes trapped fluid, step 6) is precipitated with ethanol, is dried in vacuo after step 7) washing precipitation.Extracting method of the present invention has high income, easy to operate, low-cost, it is environmentally safe the advantages that.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to a kind of extraction purification hyaluronic acid from zymotic fluid
Method
Background technology
Hyaluronic acid (hyaluronic acid, HA) be by glucuronic acid and 2-Acetamido-2-deoxy-D-glucose respectively with β-
1,3 and β -1,4 glycosidic bonds repeat the biopolymer with linear chain structure being alternately formed by connecting.The hyaluronic acid of separate sources
The degree of polymerization be that there were significant differences for molecular weight, molecular weight ranges are from 104To 107Da.In animal body, hyaluronic acid generally with
Hyaluronic acid salt form is present, and hyaluronic acid concentration is higher in skin, umbilical cord and vitreum.In addition, hyaluronic acid
It is present in a small number of microorganism kinds such as A groups and C group of streptococcus.
Hyaluronic acid has unique molecular structure and rheological properties and important physiologic function.It is excellent due to possessing
Good moisturizing water retention property and viscous-elastic behaviour, and non-immunogenicity and toxicity, hyaluronic acid are widely used in cosmetics, life
The field such as thing medicine and food service industry.
Production hyaluronic acid is occurred using microbial fermentation, has widened HA source significantly.Microbial fermentation production HA is not only
HA production cost is reduced, and there is higher quality and good quality.From broth extraction HA technical process meeting
Directly affect HA yield and production cost.
Due to the high viscosity degree of hyaluronic acid fermentation liquor, and contain substantial amounts of mycoprotein, nucleic acid, pigment, metal ion
And other impurities by different methods, it is necessary to isolate and purify HA.Extraction purification is so set certain difficulty to be present, product purity is not
The problems such as high, to extraction purification technology, it is necessary to optimize.Conventional HA extracting methods in the prevalence of cost is high, recovery rate not
The shortcomings of high, with low quality and quality is unstable, environmental pollution, it is difficult to adapt to the needs of industrialized production and practical application.
Charcoal absorption is a kind of conventional method, pigment, the foreign protein in zymotic fluid can be effectively removed, so as to improve
HA quality.As disclosed in Publication No. CN105859911A《A kind of method of separation purifying hyaluronic acid》Individually use 1%-
10% (w/v) charcoal absorption is decolourized, and the activated carbon not being used only is more, and de- charcoal effect is poor, can block milipore filter fenestra.Such as
Disclosed in Publication No. 103304684A《A kind of production method of extraction purification hyaluronic acid》In, it is not by thickening
Alcohol precipitation is carried out, the dosage of ethanol can be greatly increased, causes to waste and the problems such as environmental pollution.
The content of the invention
To overcome problem above, the invention provides a kind of method of the extraction purification hyaluronic acid from zymotic fluid, not only
Can be separated well with thalline, remove protein, pigment, the impurity such as metal ion, and solve cost is high, yield is low,
The problems such as with low quality, environmental pollution, make product quality more stable, make it that medicine, cosmetic field be applied even more extensively.This
Invent the technical scheme for the proposition that solves the above problems：
A kind of method of the extraction purification hyaluronic acid from zymotic fluid, it is characterised in that using following steps：
Zymotic fluid is diluted 5 times by step 1), 4000rpm/min centrifugation 20min, takes supernatant；
The pH of supernatant obtained by step 1) is adjusted to 3.5-4.5 by step 2), is added 1% potassium permanganate (w/v), is used water-bath
40-50 DEG C is heated to, stirring carries out oxidation reaction 0.5-1h；
Step 3) adds activated carbon, and 2 must not be less than by putting into the amount of activated carbon and the ratio of potassium permanganate:1, continue in 40-
50 DEG C of stirred in water bath, react 0.5-1h；
Step 4) filters while hot
The clear filtrate obtained by step 4) is concentrated by ultrafiltration in step 5), is rinsed in ultra-filtration process with pure water
3% (w/v) sodium chloride is added in the trapped fluid that step 6) obtains in step 5), 2-3 times of absolute ethyl alcohol is added and enters
Row precipitation, places a night
Step 7) is with the multiple washing step 6 of absolute ethyl alcohol) in obtained flocculent deposit, vacuum drying, drying temperature 40-
60℃。
Beneficial effects of the present invention：Dilution bactofugation body used in the present invention, thorough, the Er Qiecao that not only thalline removes
Make simple, there is no the side effect degraded to the molecular weight of hyaluronic acid.Decolourized, removed using the combination of potassium permanganate and activated carbon
Protein, pigment removal effect is obvious, and simple to operate.With ultrafiltration, the small molecular protein of residual is removed, heavy metal ion,
The operational sequence of Sodium Hyaluronate is prepared from zymotic fluid, it is not only easy to use in the lab, and carry out industrialized production.
Embodiment
By the following specific examples further illustrate the invention, and detail is only used for explaining the present invention, but invents
It is not limited to the following example.
Embodiment 1
A kind of method of the extraction purification hyaluronic acid from zymotic fluid, it comprises the following steps：
Step 1) takes 1L zymotic fluids, adds pure water, is diluted to 5 times, 4000rpm centrifugation 20min, takes supernatant；
The pH of gained supernatant is adjusted to 4.0 by step 2), is added 0.1% (w/v) potassium permanganate, is put into 50 DEG C of water-baths
In, stir 0.5h；
Step 3) adds 0.2% (w/v) activated carbon, continues, in 50 DEG C of stirred in water bath, to react 1h；
Perlite is first pumped into pre- laying by step 4), is filtered；
Step 5) filters the supernatant liquid of gained, pours into ultrafiltration apparatus and is concentrated by ultrafiltration, the molecule of milipore filter retention
Measure as 20000MW, be rinsed in ultra-filtration process with pure water；
After step 6) adds 3% sodium chloride stirring and dissolving in the trapped fluid obtained by ultrafiltration, the anhydrous second of 2 times of volumes is added
Alcohol, it should be noted that be slowly added into, and be continuously agitated；
After precipitation that step 7) obtains stands 12h, then repeatedly washed with absolute ethyl alcohol, obtained white flock precipitate, put
Enter vacuum desiccator, drying temperature is 50 DEG C, drying time 8h.
Testing result：Extract yield is 90.8%, protein content 0.018, and protein removal rate is 94%.
Embodiment 2
Step 1) takes 500ml zymotic fluids, adds pure water, is diluted to 10 times, pours into ceramic membrane and filter, membrane aperture is
200nm, take filtrate；
Step 2) adds 1% activated carbon in filtrate, stirs 1h；
Step 3) pads 3 layers of filter paper on a buchner funnel, after completing the pre- laying of perlite, filters to clarification；
Step 4) pours into filtrate in ultrafiltration apparatus, carries out ultrafiltration, membrane aperture 20000MW, and with pure water rinsing, takes section
Stay liquid；
3% sodium chloride is added in step 5) trapped fluid, the absolute ethyl alcohol of 2 times of volumes is added after dissolving, while stirring
To flocculent deposit；
After step 6) stands 12h, precipitation is repeatedly washed with absolute ethyl alcohol, is put into vacuum desiccator and is dried, dried
6h。
Testing result：Extract yield is 65.1%, protein content 0.078%, and protein removal rate is 91%.
Embodiment 3
Step 1) takes 1L zymotic fluids, is diluted to 10 times, adds 3% perlite, carries out plate-frame filtering；
Step 2) takes the filtrate obtained in step 1), imports in ultrafiltration apparatus, progress ultrafiltration, membrane aperture 20000MW, and
With pure water rinsing, trapped fluid is taken；
3% sodium chloride is added in step 3) trapped fluid, the absolute ethyl alcohol of 2 times of volumes is added after dissolving, while stirring
To flocculent deposit；
After step 4) stands 12h, precipitation is repeatedly washed with absolute ethyl alcohol, is put into vacuum desiccator and is dried, dried
6h。
Testing result：Extract yield is 72.3%, protein content 0.086%, and protein removal rate is 86%.Finally
It should be noted that above example enters the technical elements rather than limiting the scope of the invention for illustrating the present invention,
It will be understood by those of skill in the art that technical scheme can be appropriately modified or equivalent substitution, but not
The spirit and scope of technical solution of the present invention can be departed from.
Claims (7)
1. a kind of method of the extraction purification hyaluronic acid from zymotic fluid, it comprises the following steps：
Step 1) zymotic fluid dilutes centrifuging and taking supernatant, and step 2) adds liquor potassic permanganate reaction, step after being adjusted to appropriate pH
3) charcoal absorption is added, step 4) filters and takes filtrate, and step 5) ultrafiltration takes trapped fluid, and step 6) is precipitated with ethanol, step
It is dried in vacuo after rapid 7) washing precipitation.
2. according to the method for claim 1, it is characterised in that the method for extraction and purification comprises the following steps：
Zymotic fluid is diluted 4-6 times by step 1), centrifuging and taking supernatant；
The pH of supernatant obtained by step 1) is adjusted to 3.5-4.5 by step 2), potassium permanganate is added, with heating water bath to 40-50
DEG C, stirring carries out oxidation reaction 0.5-1h；
Step 3) adds activated carbon, and 2 must not be less than by putting into the amount of activated carbon and the ratio of potassium permanganate:1, continue at 40-50 DEG C
Stirred in water bath, react 0.5-1h；
Step 4) filters while hot；
The clear filtrate obtained by step 4) is concentrated by ultrafiltration in step 5), is rinsed in ultra-filtration process with pure water；
Sodium chloride is added in the trapped fluid that step 6) obtains in step 5), 2-3 times of absolute ethyl alcohol is added and is precipitated；
Step 7) is with the multiple washing step 6 of absolute ethyl alcohol) in obtained flocculent deposit, vacuum drying, drying temperature 40-60
℃。
3. according to the method for claim 2, it is characterised in that step 1) dilutes 5 times, centrifugation rate 4000rpm/min,
Time is 20min.
4. according to the method for claim 2, it is characterised in that the ratio that step 2) adds potassium permanganate is 0.1% (w/v).
5. method for extraction and purification according to claim 2, it is characterised in that step 4) adds the pre- laying of perlite, and carries out
Filter while hot.
6. according to the method for claim 2, it is characterised in that the molecular cut off of step 5) milipore filter is 20000MW, and
It is rinsed with pure water.
7. according to the method for claim 2, it is characterised in that step 6) adds 3% (w/v) sodium chloride, after dissolving, while stirring
Mix side to be precipitated, gained is precipitated as flocculent deposit in small, broken bits.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710969336.2A CN107674131A (en) | 2017-10-18 | 2017-10-18 | A kind of method of the extraction purification hyaluronic acid from zymotic fluid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710969336.2A CN107674131A (en) | 2017-10-18 | 2017-10-18 | A kind of method of the extraction purification hyaluronic acid from zymotic fluid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107674131A true CN107674131A (en) | 2018-02-09 |
Family
ID=61140750
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710969336.2A Pending CN107674131A (en) | 2017-10-18 | 2017-10-18 | A kind of method of the extraction purification hyaluronic acid from zymotic fluid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107674131A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452944A (en) * | 2019-09-21 | 2019-11-15 | 冯世红 | A method of hyaluronic acid is prepared using salt alcohol method |
| CN112851833A (en) * | 2021-02-04 | 2021-05-28 | 合肥博思科创医药科技有限公司 | Purification method for reducing protein content in sodium hyaluronate and derivatives thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550199A (en) * | 2009-05-09 | 2009-10-07 | 山东众山生物科技有限公司 | Method for preparing sodium hyaluronate from hyaluronic acid zymotic fluid |
| CN101935362A (en) * | 2009-06-30 | 2011-01-05 | 上海佰加壹医药有限公司 | Method for purifying hyaluronic acid by pre-laying filter aid |
| CN104356254A (en) * | 2014-10-23 | 2015-02-18 | 深圳先进技术研究院 | Modified hyaluronicacid and preparation method thereof |
-
2017
- 2017-10-18 CN CN201710969336.2A patent/CN107674131A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550199A (en) * | 2009-05-09 | 2009-10-07 | 山东众山生物科技有限公司 | Method for preparing sodium hyaluronate from hyaluronic acid zymotic fluid |
| CN101935362A (en) * | 2009-06-30 | 2011-01-05 | 上海佰加壹医药有限公司 | Method for purifying hyaluronic acid by pre-laying filter aid |
| CN104356254A (en) * | 2014-10-23 | 2015-02-18 | 深圳先进技术研究院 | Modified hyaluronicacid and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 姚日生等: "《药用高分子材料》", 30 April 2008, 化学工业出版社 * |
| 谢艳等: "《骨伤科临床用药指南》", 31 December 2014, 郑州大学出版社 * |
| 郭腊梅: "《纺织结构成型学3：纺织品染整》", 31 March 2016, 东华大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452944A (en) * | 2019-09-21 | 2019-11-15 | 冯世红 | A method of hyaluronic acid is prepared using salt alcohol method |
| CN112851833A (en) * | 2021-02-04 | 2021-05-28 | 合肥博思科创医药科技有限公司 | Purification method for reducing protein content in sodium hyaluronate and derivatives thereof |
| CN112851833B (en) * | 2021-02-04 | 2022-06-28 | 合肥博思科创医药科技有限公司 | Purification method for reducing protein content in sodium hyaluronate and derivatives thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101550199B (en) | Method for preparing sodium hyaluronate from hyaluronic acid zymotic fluid | |
| JPH07504928A (en) | High-molecular glucuronic acid compounds, their production methods and uses, especially as gelling agents, viscosity-imparting agents, moisture-imparting agents, stabilizers, chelating agents, or flocculants | |
| CN103805650B (en) | A kind of fermentation method for producing of non-pigment pulullan polysaccharide | |
| CA2732234C (en) | Process for the co-production of chitin, its derivatives and polymers containing glucose, mannose and/or galactose, by the fermentation of the yeast pichia pastoris | |
| CN1259345C (en) | Method of preparing transparent sodium protonate from transparent protonic acid fermentation liquid | |
| US20090162905A1 (en) | Method for Purification of Hyaluronic Acid Salt | |
| CN106397630A (en) | Method for extracting sodium hyaluronate based on membrane separation technology | |
| ITMI991465A1 (en) | PROCESS FOR THE PREPARATION OF K4 AND K5 POLYSACCHARIDES FROM ESCHERICHIACOLI | |
| CN107674131A (en) | A kind of method of the extraction purification hyaluronic acid from zymotic fluid | |
| KR19980080116A (en) | Purification Method of Sodium Hyaluronate | |
| CN110922506A (en) | High-clarity heparin sodium | |
| AU2003243186B2 (en) | Polysaccharide gum and process for its manufacture | |
| CN106434443B (en) | A kind of production technology of Sodium Hyaluronate | |
| CN105440163A (en) | Method for preparing and purifying enoxaparin sodium | |
| CN110863024A (en) | Method for preparing micromolecular hyaluronic acid by utilizing squid eyes | |
| CN116178577B (en) | High-purity microalgae polysaccharide and extraction method thereof | |
| KR100312638B1 (en) | Method for manufacturing high purity hyaluronic acid | |
| CN111825777B (en) | Method for preparing heparinoids from chondroitin | |
| CN109467621A (en) | A kind of production of Enoxaparin Sodium and purification process | |
| CN116715705A (en) | Process for preparing glucosamine by using N-acetylglucosamine fermentation broth | |
| CN117362476A (en) | Method for preparing refined heparin by using bovine lung and corresponding refined heparin | |
| CN117024622A (en) | Preparation process of low molecular weight fucoidin and prepared fucoidin | |
| CN113004436A (en) | Preparation method of dalteparin sodium and application of method in preparation of low-molecular-weight heparin sodium | |
| DE19712931B4 (en) | Process for the production of hyaluronic acid | |
| CN116179626A (en) | Method for fermenting and extracting low-acyl gellan gum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180209 |