CN106434443A - Production process of sodium hyaluronate - Google Patents
Production process of sodium hyaluronate Download PDFInfo
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- CN106434443A CN106434443A CN201610844974.7A CN201610844974A CN106434443A CN 106434443 A CN106434443 A CN 106434443A CN 201610844974 A CN201610844974 A CN 201610844974A CN 106434443 A CN106434443 A CN 106434443A
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- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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Abstract
The invention discloses a production process of sodium hyaluronate. A strain high in hyaluronic acid yield is screened out, improved fermentation technology is combined, production period is short, and the production process is simple; raw and auxiliary materials are common and easy to get, production is easy to control, impurity content is lower, the production process is suitable for industrial large-scale production requirements and high in yield, and production cost is lowered greatly.
Description
Technical field
The present invention relates to hyaluronic acid production technical field, more particularly to a kind of production technology of hyaluronate sodium.
Background technology
The technique fermentation period that Production by Microorganism Fermentation hyaluronic acid is adopted is long, and fermentation technology can not be according to strain properties
Carry out, cause bacterial activity be excited, hyaluronic acid productivity is low, poor product quality.Contain thalline, protein in fermentation liquid
And the material such as nucleic acid, extract extraction ratio that hyaluronic acid not only has high demands and its molecule does not occur depolymerization, and have high demands
Product purity, and from fermentation liquid extract hyaluronic acid technical process can directly affect hyaluronic acid Yield and quality and
Production cost.
The new fermentation strain of exploitation, meanwhile, improve fermentation technology and extraction process according to strain properties transparent to improve
The quality and yield of matter acid sodium, is the technical problem to be solved in the invention.
Content of the invention
The purpose of the present invention is aiming at above-mentioned defect and provides a kind of production technology of hyaluronate sodium.This
Bright filter out the high strain of hyaluronic acid productivity, in conjunction with improved fermentation technique, with short production cycle, simple production process, former auxiliary
Material is common to be easy to get, and production is easily controllable, and impurity content is lower, and suitable industrialization large-scale production is required, high income, and significantly
Reduce production cost.
A kind of production Technology scheme of hyaluronate sodium of the present invention is to comprise the following steps：
（1）Malian drainage（Streptococcus equi subsp. zooepidemicus）DCS005 shaking flask kind
Sub- liquid culture；
（2）Strain expanded culture；
（3）Fermentation, is first passed through the fermentation of aseptic oxygen-containing air, then is passed through nitrogen continuation fermentation；
（4）Fermentation liquid is diluted with water to 6-8 times of fermentating liquid volume, addition kieselguhr and activated carbon in diluent, 40 DEG C
Filter press after insulated and stirred, collects filtrate；Ultrafiltration is carried out to filtrate, removes the coloring matter of metal ion and small molecule；
（5）Solution after ultrafiltration is added the Sodium Chloride of 0.6mol/L, adds ethanol until completely dissolved, precipitate wine to terminal
Precision number is 60%-65%, is sufficiently stirred for, staticly settles, and takes out precipitate hyaluronate sodium；By supernatant sucking filtration, Ran Houliu
Plus the dehydrated alcohol of 0.3-2 times of volume, collected after centrifugation precipitation；
(6) washing dehydration dries：To be vacuum dried after hyaluronate sodium precipitates washed with EtOH, dehydration, obtain final product hyalomitome
Sour sodium finished product.
Malian drainage used by the present invention（Streptococcus equi subsp. zooepidemicus）
DCS005, the Malian drainage is in China Committee for Culture Collection of Microorganisms's common micro-organisms center guarantor at present
Hide, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101, its deposit number is：CGMCC No.12782,
The Latin title of strain isStreptococcus equi subsp. zooepidemicus, join the microorganism of evidence（Strain）：
DCS005, preservation date is on July 14th, 2016.
Step（1）、（2）、（3）In culture fluid include starch 1% by weight percentage, sucrose 0.5%, Carnis Bovis seu Bubali cream
0.5%th, yeast powder 0.5%, dipotassium hydrogen phosphate 0.02%, sodium hydroxide 0.05%, balance of water, pH7.0-7.3.
Step（1）In, condition of culture is：38.5 DEG C culture 12-16 hour.
Step（2）In, it is 12-16 hour that condition of culture is 38.5 DEG C of temperature, pH7.0-7.3, incubation time, tank pressure
0.05MPa.
Step（3）Specifically, after being passed through aseptic oxygen-containing air 37-40 DEG C fermentation 18-32 hour, being passed through 39-45 DEG C of nitrogen
Continue fermentation, be passed through the nitrogen stage be simultaneously introduced concentration for 800g/L glucose 300mL, fermentation liquid pH fall per minute
Terminate fermentation during less than 0.01, fermentation broth viscosity is detected, fermentation liquid is pressed in settling tank, wherein, fermentation tank tank pressure 0.05MP.
Kieselguhr addition is the 2% of fermentating liquid volume, and activated carbon addition is the 2% of fermentating liquid volume.
Ultrafiltration retaining molecular weight used by ultrafiltration is 30,000-50,000, and ultrafiltration is carried out in below 0.5MPa.
Beneficial effects of the present invention are：
The present invention filters out the high strain of hyaluronic acid productivity, in conjunction with improved fermentation technique, with short production cycle, production technology letter
Single, supplementary material is common to be easy to get, and production is easily controllable, and impurity content is lower, and suitable industrialization large-scale production is required, high income,
And greatly reduce production cost.Highly purified hyaluronate sodium can be obtained inexpensive, hyaluronic acid and its purity salt are up to
99.6%, glucuronic acid purity is more than 45%, and loss of molecular weight rate is less than 11%, and protein content is less than 0.04%.
Specific embodiment：
For a better understanding of the present invention, technical scheme is described in detail with instantiation below, but the present invention
It is not limited thereto.
Embodiment 1
Malian drainage used by the present invention（Streptococcus equi subsp. zooepidemicus）
DCS005, the Malian drainage is in China Committee for Culture Collection of Microorganisms's common micro-organisms center guarantor at present
Hide, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101, its deposit number is：CGMCC No.12782,
The Latin title of strain isStreptococcus equi subsp. zooepidemicus, join the microorganism of evidence（Strain）：
DCS005, preservation date is on July 14th, 2016.
A kind of production technology of hyaluronate sodium, comprises the following steps：
（1）Malian drainage（Streptococcus equi subsp. zooepidemicus）DCS005 shaking flask kind
Sub- liquid culture, condition of culture is：38.5 DEG C culture 12-16 hour；
（2）Strain expanded culture, it is 12-16 hour that condition of culture is 38.5 DEG C of temperature, pH7.0-7.3, incubation time, tank pressure
0.05MPa；
（3）Fermentation, after being passed through aseptic oxygen-containing air 37-40 DEG C fermentation 18-32 hour, it is passed through 39-45 DEG C of nitrogen and continues fermentation,
Being passed through the nitrogen stage is simultaneously introduced glucose 300mL, fermentation liquid pH per minute fall of the concentration for 800g/L less than 0.01
When terminate fermentation, detect fermentation broth viscosity, by fermentation liquid be pressed into settling tank in, wherein, fermentation tank tank pressure 0.05MP；
（4）Fermentation liquid is diluted with water to 6-8 times of fermentating liquid volume, and the kieselguhr of fermentating liquid volume 2% is added in diluent
With the activated carbon of fermentating liquid volume 2%, filter press after 40 DEG C of insulated and stirred, filtrate is collected；In below 0.5MPa retention point
Son amount carries out ultrafiltration for 30,000-50,000 ultrafilter membrane to filtrate, removes the coloring matter of metal ion and small molecule；
（5）Solution after ultrafiltration is added the Sodium Chloride of 0.6mol/L, adds ethanol until completely dissolved, precipitate wine to terminal
Precision number is 60%-65%, is sufficiently stirred for, staticly settles, and takes out precipitate hyaluronate sodium；By supernatant sucking filtration, Ran Houliu
Plus the dehydrated alcohol of 0.3-2 times of volume, collected after centrifugation precipitation；
(6) washing dehydration dries：To be vacuum dried after hyaluronate sodium precipitates washed with EtOH, dehydration, obtain final product hyalomitome
Sour sodium finished product.
Step（1）、（2）、（3）In culture fluid include starch 1% by weight percentage, sucrose 0.5%, Carnis Bovis seu Bubali cream
0.5%th, yeast powder 0.5%, dipotassium hydrogen phosphate 0.02%, sodium hydroxide 0.05%, balance of water, pH7.0-7.3.
The present invention inexpensive can be obtained highly purified hyaluronate sodium, and hyaluronic acid and its purity salt are up to 99.6%, Fructus Vitis viniferae
Alduronic acid purity is more than 45%, and loss of molecular weight rate is less than 11%, and protein content is less than 0.04%.
Claims (8)
1. a kind of production technology of hyaluronate sodium, it is characterised in that comprise the following steps：
（1）Malian drainage（Streptococcus equi subsp. zooepidemicus）DCS005 shaking flask kind
Sub- liquid culture；
（2）Strain expanded culture；
（3）Fermentation, is first passed through the fermentation of aseptic oxygen-containing air, then is passed through nitrogen continuation fermentation；
（4）Fermentation liquid is diluted with water to 6-8 times of fermentating liquid volume, addition kieselguhr and activated carbon in diluent, 40 DEG C
Filter press after insulated and stirred, collects filtrate；Ultrafiltration is carried out to filtrate；
（5）Solution after ultrafiltration is added the Sodium Chloride of 0.6mol/L, adds ethanol until completely dissolved, precipitate wine to terminal
Precision number is 60%-65%, is sufficiently stirred for, staticly settles, and takes out precipitate hyaluronate sodium；By supernatant sucking filtration, Ran Houliu
Plus the dehydrated alcohol of 0.3-2 times of volume, collected after centrifugation precipitation；
(6) washing dehydration dries：To be vacuum dried after hyaluronate sodium precipitates washed with EtOH, dehydration, obtain final product hyaluronic acid
Sodium finished product.
2. a kind of production technology of hyaluronate sodium according to claim 1, it is characterised in that described streptococcus equi beast
Epidemic disease subspecies（Streptococcus equi subsp. zooepidemicus）DCS005, which is in Chinese microorganism strain preservation
Administration committee's common micro-organisms center preservation, its deposit number is：CGMCC No.12782, preservation date is in July, 2016
14 days.
3. a kind of production technology of hyaluronate sodium according to claim 1, it is characterised in that step（1）、（2）、（3）
In culture fluid include starch 1% by weight percentage, sucrose 0.5%, Carnis Bovis seu Bubali cream 0.5%, yeast powder 0.5%, dipotassium hydrogen phosphate
0.02%th, sodium hydroxide 0.05%, balance of water, pH7.0-7.3.
4. a kind of production technology of hyaluronate sodium according to claim 1, it is characterised in that step（1）In, cultivate bar
Part is：38.5 DEG C of culture 12-16 hours.
5. a kind of production technology of hyaluronate sodium according to claim 1, it is characterised in that step（2）In, cultivate bar
Part is 38.5 DEG C of temperature, pH7.0-7.3, and incubation time is 12-16 hour, tank pressure 0.05MPa.
6. a kind of production technology of hyaluronate sodium according to claim 1, it is characterised in that step（3）Specifically, logical
After entering aseptic oxygen-containing air 37-40 DEG C fermentation 18-32 hour, be passed through 39-45 DEG C of nitrogen and continue fermentation, be passed through the nitrogen stage while
Terminate fermentation when adding concentration for glucose 300mL, the fermentation liquid pH fall per minute of 800g/L less than 0.01, detection is sent out
Zymotic fluid viscosity, fermentation liquid is pressed in settling tank, wherein, fermentation tank tank pressure 0.05MP.
7. the production technology of a kind of hyaluronate sodium according to claim 1, it is characterised in that kieselguhr addition is for sending out
The 2% of zymotic fluid volume, activated carbon addition is the 2% of fermentating liquid volume.
8. the production technology of a kind of hyaluronate sodium according to claim 1, it is characterised in that used by ultrafiltration, ultrafilter membrane cuts
Stay molecular weight for 30,000-50,000, ultrafiltration is carried out in below 0.5MPa.
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| CN201610844974.7A CN106434443B (en) | 2016-09-23 | 2016-09-23 | A kind of production technology of Sodium Hyaluronate |
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| CN201610844974.7A CN106434443B (en) | 2016-09-23 | 2016-09-23 | A kind of production technology of Sodium Hyaluronate |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536550A (en) * | 2018-12-06 | 2019-03-29 | 上海景峰制药有限公司 | A kind of Sodium Hyaluronate and preparation method thereof |
| CN109913518A (en) * | 2018-08-17 | 2019-06-21 | 上海昊海生物科技股份有限公司 | A kind of pipeline preparation method of Sodium Hyaluronate |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1102435A (en) * | 1994-08-17 | 1995-05-10 | 桂平县生物技术开发公司 | Method for producing sodium hyaluronate by microbial fermentation |
| CN101643760A (en) * | 2009-09-04 | 2010-02-10 | 四川理工学院 | Method for increasing the yield of hyaluronic acid |
| CN103173507A (en) * | 2011-12-23 | 2013-06-26 | 宁波林叶生物科技有限公司 | Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium |
-
2016
- 2016-09-23 CN CN201610844974.7A patent/CN106434443B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1102435A (en) * | 1994-08-17 | 1995-05-10 | 桂平县生物技术开发公司 | Method for producing sodium hyaluronate by microbial fermentation |
| CN101643760A (en) * | 2009-09-04 | 2010-02-10 | 四川理工学院 | Method for increasing the yield of hyaluronic acid |
| CN103173507A (en) * | 2011-12-23 | 2013-06-26 | 宁波林叶生物科技有限公司 | Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913518A (en) * | 2018-08-17 | 2019-06-21 | 上海昊海生物科技股份有限公司 | A kind of pipeline preparation method of Sodium Hyaluronate |
| CN109536550A (en) * | 2018-12-06 | 2019-03-29 | 上海景峰制药有限公司 | A kind of Sodium Hyaluronate and preparation method thereof |
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| Publication number | Publication date |
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| CN106434443B (en) | 2018-05-15 |
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Effective date of registration: 20200826 Address after: 257000 No.75 Huaihe Road, Dongying Economic Development Zone, Shandong Province Patentee after: DONGYING FOSTER BIOLOGICAL ENGINEERING Co.,Ltd. Address before: 257000, Shandong, Dongying, Kenli Town, West Tuo (Yong Xin Road, No. 98) Co-patentee before: DONGYING FOSTER BIOLOGICAL ENGINEERING Co.,Ltd. Patentee before: CHINA EASTAR HOLDINGS GROUP Co.,Ltd. |
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