CN106632728A - Method for improving yield of hyaluronic acid with high molecular weight - Google Patents
Method for improving yield of hyaluronic acid with high molecular weight Download PDFInfo
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- CN106632728A CN106632728A CN201611096388.5A CN201611096388A CN106632728A CN 106632728 A CN106632728 A CN 106632728A CN 201611096388 A CN201611096388 A CN 201611096388A CN 106632728 A CN106632728 A CN 106632728A
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- hyaluronic acid
- fermentation liquid
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- liquid
- filtrate
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- 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
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- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention relates to a method for improving yield of hyaluronic acid with a high molecular weight. The method comprises the following steps: (1) performing primary treatment on fermentation liquor; (2) purifying the fermentation liquor; and (3) drying hyaluronic acid. The purification method provided by the invention is excellent in purification effect, particularly high in protein removal rate, the yield of hyaluronic acid products is high and can reach 8-10g/L, the recovery rate can be 95%, the service performance is excellent, the solubility is high, and the light transmittance reaches 99%.
Description
Technical field
The invention belongs to field of microbial fermentation, especially a kind of method of high molecular weight hyaluronic acid yield.
Background technology
The technique fermentation period that at present enterprise of most domestic Production by Microorganism Fermentation hyaluronic acid adopts is long, typically
In 36-40 or so, molecular weight of product is low, product light transmittance poor (typically 90% or so), and protein content is high, its finished product Fructus Vitis viniferae
Glucuronic acid content is generally 35-38%, and part producer hyaluronic acid product form is threadiness, and this kind of hyaluronic acid dissolving is tired
Difficulty, typically takes 2 days or so, and yield is less than 75%, in order to meet the domestic market for constantly expanding, the production of hyaluronic acid
Technique urgently improves.
Mostly HA is produced using fermentation method at present, produce transparent using Streptococcal fermentation early in nineteen eighty-three Japan's Shiseido
Matter acid.Subsequently the research of research on producing hyaluronic acid by fermentation method is more and more extensive, such as Japan Patent No.58-566922, United States Patent (USP)
No.60-500997, Korean Patent No.10-250573 etc..Due to containing the things such as thalline, protein and nucleic acid in fermentation liquid
Matter, the method for HA is extracted from fermentation liquid mainly following several：A. Macrodilution membrane filter technique, because thalline etc. is miscellaneous in fermentation liquid
Matter is more, easily causes Pore Blocking, and Macrodilution easily to form operational volume excessive；B. supercentrifugal process, when removing thalline
Requirement to equipment is very high；C. chloroform method, carries more HA secretly in bacterial sediment, and the response rate of HA is low, and chloroform is dirty to environment
Dye；D. ethanol precipitation, is a kind of more conventional method, but due to when the method is operated device therefor and workshop must prevent
Quick-fried, this certainly will increased the dangerous and production cost of operation.
In existing method, the typically no method of micro content of hyaluronic acid albumen is removed it again, micro albumen
Content easily causes allergy (varying with each individual) reaction, and especially for use in cosmetic field, the content of trace albumin is still present
Larger harm.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided it is micro- that a kind of product purity is high, protein content is low
The method that biological fermentation process produces hyaluronic acid.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method for improving hyaluronic acid volume of production, its step includes：(1) the single treatment of fermentation liquid, fermentation liquid be once
Process the drying of the (2) purification of fermentation liquid, (3) hyaluronic acid；Detailed process is as follows：
(1) the single treatment of fermentation liquid
By fermentation liquid Jing after plate-and-frame filtration filtrate, add 2-4 times ethanol solution precipitation, collect precipitate, add and
The isopyknic Nacl solution stirring dissolving of filtrate, carries out plate-and-frame filtration to clarifying, and obtains secondary filter liquid；
By secondary filter liquid through one section of metallic conduit of outer wrap magnetizing coil, magnetization treatment is carried out to fermentation liquid,
Magnetic field intensity is the Gausses of 1-3 ten thousand, and after magnetization, hydrone diminishes, and improves the dissolubility of hyaluronic acid, then by filtrate with ultraviolet
The irradiation 10-30 seconds, the albumen in solution is condensed, obtain micro- building-up effect, then the filter membrane or filter plate mistake of Jing 0.3-0.5 microns
Filter, enables trace albumin to remove；, and the nanometer magnetic bead of 5wt% is added in the medium of plate-and-frame filtration；
(2) fermentation liquid is further purified
Filtrate after filtration is added into second acid for adjusting pH to 4.0-5.0, then is sprayed with ethanol solution, precipitated, obtained
To bright matter acid deposit；
(3) the drying of hyaluronic acid
2-3 dehydrated alcohol dehydration of whole precipitate Jing that above-mentioned steps are obtained, it is aqueous after 50-60 DEG C of vacuum drying
Control obtains final product hyaluronic acid below 10%.
And, activated carbon is added in the kieselguhr that the plate-and-frame filtration is used, for decolourizing, the addition of activated carbon is total
Diatomaceous 20wt%.
And, the concentration of the ethanol solution is：95-98% (v/v).
And, the concentration of the Nacl solution is：0.1-2mol/L.
Advantages of the present invention and good effect are：
1st, the purification process purification effect that the present invention is improved is good, and especially albumen clearance is high, hyaluronic acid product yield
Height, can reach 8-10g/L, and its response rate is up to 95%, and performance is more superior, and more preferably, its light transmittance reaches dissolubility
99%.
2nd, the present invention using original creation spray powder-making technique, make product structure form finer and smoother, 3, the present invention will fermentation
Liquid Jing secondary filters liquid carries out magnetization treatment through one section of metallic conduit of outer wrap magnetizing coil to fermentation liquid, and magnetic field is strong
Spend for the Gausses of 1-3 ten thousand, after magnetization, hydrone diminishes, improve the dissolubility of hyaluronic acid, then by filtrate ultra-vioket radiation
The 10-30 seconds, the albumen in solution is condensed, obtain micro- building-up effect, then the filter membrane or filter plates of Jing 0.3-0.5 microns, made
Trace albumin is removed；Hyaluronic acid can be developed into without allergy level cosmetics-stage and medical grade products.
3rd, after the present invention increases magnetic bead in the sheet frame for filtering, the albumen one after magnetization can be improved removings, one improves
Reduce foreign protein content, it is ensured that the purity of hyaluronic acid, reduce anaphylaxiss probability.
Specific embodiment
With reference to embodiment, the present invention is further described, and following embodiments are illustrative, be not it is determinate,
Protection scope of the present invention can not be limited with following embodiments.
The fermented bacterium that the present invention is used belongs to external strain, and bacterium classification is streptococcus zooepidemicus.The side that the present invention is provided
In method, the acquisition of fermentation liquid adopts following steps：
(1) shake-flask seed liquid：Slant strains are inoculated in the 1L conical flasks equipped with sterile medium 400ml, 37 DEG C of cultures
12-16 hours, om observation thalli growth is good, without living contaminantses, you can in being inoculated in seed tank；
(2) seed tank seed liquor：Culture fluid 60L is prepared by the formula of shake-flask seed liquid, in adding seed tank, logical steam adds
Heat, 121 DEG C sterilize 30 minutes, are cooled to 37 DEG C, are inoculated with flame method, shake-flask seed liquid are inoculated in seed tank, ventilation
60L/min, speed of agitator 120-140/min, 37 DEG C of culture 12-16 hours, om observation thalli growth is good, dirty without miscellaneous bacteria
Dye, you can in being inoculated in fermentation tank；
(3) fermentation culture：By the formula of seed tank, the consumption of sugar is brought up to 6%, prepare culture fluid 600L, add 1 ton
Fermentation tank in, lead to steam heating, 121 DEG C sterilize 30 minutes, are cooled to 37 DEG C, and seed liquor is inoculated in by subcultivation pipeline
In fermentation tank, ventilation 400-600L/min, speed of agitator 140-160/min, 37 DEG C of culture 24-26 hours, sweat are maintained
In, the lowering of concentration of sugar to less than 0.5%, PH declines very slow or when no longer declining, as fermentation termination, obtains containing hyalomitome
Acid fermentation liquid；(above 3 steps are in Ma Chaomei (Liuzhou Chemical Industry Group Co., Ltd), fermentative Production hyalomitome
Sour technical study, Guangxi Technical College's journal, 2007,18 (3):Have been reported that in 8-12, belong to prior art, the present patent application is just not
It is described in more detail again).
The method for improving hyaluronic acid volume of production according to the present invention, its step includes：(1) the single treatment of fermentation liquid, fermentation
The drying of the single treatment of the liquid (2) purification of fermentation liquid, (3) hyaluronic acid；Detailed process is as follows：
(1) the single treatment of fermentation liquid
By fermentation liquid Jing after plate-and-frame filtration filtrate, add 2-4 times ethanol solution precipitation, collect precipitate, add and
The isopyknic Nacl solution stirring dissolving of filtrate, carries out plate-and-frame filtration to clarifying, and obtains secondary filter liquid；
By secondary filter liquid through one section of metallic conduit of outer wrap magnetizing coil, magnetization treatment is carried out to fermentation liquid,
Magnetic field intensity is the Gausses of 1-3 ten thousand, and after magnetization, hydrone diminishes, and improves the dissolubility of hyaluronic acid, then by filtrate with ultraviolet
The irradiation 10-30 seconds, the albumen in solution is condensed, obtain micro- building-up effect, then the filter membrane or filter plate mistake of Jing 0.3-0.5 microns
Filter, enables trace albumin to remove；
The nanometer magnetic bead of 5wt% is added in the medium of plate-and-frame filtration, to be added and can increase foreign protein after nanometer magnetic bead
Clearance, after magnetization, micro- poly- foreign protein can produce adsorption to whole solution with magnetic bead, and then be removed.
The diatomaceous model of plate-and-frame filtration No. 821 and No. 616, two kinds of kieselguhr are mixed in proportion, and are filled after obtaining
Enter sheet frame to be filtered, No. 821 and No. 616 diatomaceous part by weight are 1：1-5.The nanometer magnetic bead of addition is evenly distributed on plate
Inframe.
(2) fermentation liquid is further purified
Filtrate after filtration is added into second acid for adjusting pH to 4.0-5.0, then is sprayed with ethanol solution, precipitated, obtained
To bright matter acid deposit；
(3) the drying of hyaluronic acid
2-3 dehydrated alcohol dehydration of whole precipitate Jing that above-mentioned steps are obtained, it is aqueous after 50-60 DEG C of vacuum drying
Control obtains final product hyaluronic acid below 10%.
(4) the purification by liquid extraction of fermentation liquid：Fermentation liquid is obtained into filtrate Jing after plate-and-frame filtration, 3 times of 95% ethanol precipitation is added,
Precipitate is collected, is added and is dissolved with the Nacl solution stirring of the isopyknic 0.1mol/L of filtrate, carry out secondary plate-and-frame filtration to clear
Clearly, post-defecation liquid is obtained；By post-defecation liquid Jing 0.45 micron filter membrane or filter plates after, add second acid for adjusting pH extremely
4.0-5.0, obtains three secondary clearing liquid after acid treatment, then sprays three secondary clearing liquid with 95% ethanol, then is precipitated, and is sunk
Starch；
The diatomaceous model of plate-and-frame filtration No. 821 and No. 616, two kinds of kieselguhr are mixed in proportion, and are filled after obtaining
Enter sheet frame to be filtered, No. 821 and No. 616 diatomaceous part by weight are 1：1-5.
(5) powder processed is dried：By the whole precipitate for obtaining Jing after dehydrated alcohol dehydration, emulsifying powder is carried out with emulsification pump, obtained
Powdery precipitates, further with after dehydrated alcohol dehydration, after 50-60 DEG C of vacuum drying, containing water management below 10%, obtain final product transparent
Matter acid.
The hyaluronic acid product yield that the Jing present invention is obtained is high, can reach 8-10g/L, and its response rate is 95%.
Product performance index：
Character：White powder or fine particle；
Abnormal smells from the patient：Odorless, tasteless；
pH(1g/L)：6.9-7.0；
Clarity (1g/L)：Light transmittance>99%；
Loss on drying：<8%；
Molecular weight：>1.8×106；
Protein：<0.03%；
Glucuronic acid：>=44.5%；
Sensitivity reduces by 80%.
Claims (4)
1. a kind of method of high molecular weight hyaluronic acid yield, it is characterised in that：Its step includes：
(1) the single treatment of the single treatment of fermentation liquid, fermentation liquid；(2) the purification of fermentation liquid；(3) the drying of hyaluronic acid；Specifically
Process is as follows：
(1) the single treatment of fermentation liquid
Fermentation liquid is obtained into filtrate Jing after plate-and-frame filtration, 2-4 times of ethanol solution precipitation is added, precipitate is collected, is added and filtrate
Isopyknic Nacl solution stirring dissolving, carries out plate-and-frame filtration to clarifying, and obtains secondary filter liquid；
By secondary filter liquid through one section of metallic conduit of outer wrap magnetizing coil, magnetization treatment, magnetic field are carried out to fermentation liquid
Intensity is the Gausses of 1-3 ten thousand, after magnetization, then by filtrate with the ultra-vioket radiation 10-30 seconds, then Jing 0.3-0.5 microns filter membrane or
Filter plates, and the nanometer magnetic bead of 5wt% is added in the medium of plate-and-frame filtration；
(2) fermentation liquid is further purified
Filtrate after filtration is added into second acid for adjusting pH to 4.0-5.0, then is sprayed with ethanol solution, precipitated, obtain bright
Matter acid deposit；
(3) the drying of hyaluronic acid
2-3 dehydrated alcohol dehydration of whole precipitate Jing that above-mentioned steps are obtained, after 50-60 DEG C of vacuum drying, containing water management
Below 10%, hyaluronic acid is obtained final product.
2. it is according to claim 1 improve hyaluronic acid volume of production method, it is characterised in that：What the plate-and-frame filtration was used
Activated carbon is added in kieselguhr.
3. it is according to claim 1 improve hyaluronic acid volume of production method, it is characterised in that：The concentration of the ethanol solution
For：95-98%v/v.
4. it is according to claim 1 improve hyaluronic acid volume of production method, it is characterised in that：The concentration of the Nacl solution
For：0.1-2mol/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410927A (en) * | 2017-12-09 | 2018-08-17 | 新疆阜丰生物科技有限公司 | A kind of method of fermentation method production high molecular weight hyaluronic acid |
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| US20040167243A1 (en) * | 2003-02-20 | 2004-08-26 | Seikagaku Corporation | Method for producing low molecular weight glycosaminoglycan by ultraviolet ray irradiation |
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2016
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|---|---|---|---|---|
| US20040167243A1 (en) * | 2003-02-20 | 2004-08-26 | Seikagaku Corporation | Method for producing low molecular weight glycosaminoglycan by ultraviolet ray irradiation |
| CN1869246A (en) * | 2006-06-29 | 2006-11-29 | 北京大学 | Method of filtering transparent protonic acid fermentation liquid using magnetic field fortified membrane |
| CN101864471A (en) * | 2010-02-02 | 2010-10-20 | 天津市康婷生物工程有限公司 | Microbial fermentation method for producing hyaluronic acid |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410927A (en) * | 2017-12-09 | 2018-08-17 | 新疆阜丰生物科技有限公司 | A kind of method of fermentation method production high molecular weight hyaluronic acid |
| CN108410927B (en) * | 2017-12-09 | 2020-10-09 | 新疆阜丰生物科技有限公司 | Method for producing high molecular weight hyaluronic acid by fermentation method |
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Application publication date: 20170510 |