CN101864471A - Microbial fermentation method for producing hyaluronic acid - Google Patents
Microbial fermentation method for producing hyaluronic acid Download PDFInfo
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- CN101864471A CN101864471A CN201010301064A CN201010301064A CN101864471A CN 101864471 A CN101864471 A CN 101864471A CN 201010301064 A CN201010301064 A CN 201010301064A CN 201010301064 A CN201010301064 A CN 201010301064A CN 101864471 A CN101864471 A CN 101864471A
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Abstract
The invention relates to a microbial fermentation method for producing hyaluronic acid, which comprises the following steps of: (1) preparing shaking flask seed liquid; (2) preparing seeding tank seed liquid; (3) performing fermentation culture to obtain fermentation liquor; (4) extracting and purifying the fermentation liquor, namely settling the fermentation liquor with ethanol, redissolving the sediment to obtain lysate, filtering the lysate with a secondary plate frame until the lysate is clear, filtering the lysate for three times again to obtain clear liquid, and spraying the clear liquid with the ethanol to obtain spray sediment; and (5) drying and pulverizing, namely dehydrating all the sediments obtained by the previous step with absolute ethyl alcohol, emulsifying and pulverizing with an emulsification pump to obtain powdery sediment, further dehydrating the powdery sediment with the absolute ethyl alcohol, and drying the dehydrated sediment under vacuum at the temperature of between 50 and 60 DEG C to obtain the hyaluronic acid. The production strain, which has large capsule and high hyaluronic acid yield and does not produce hyaluronidase, is screened by compound mutagenesis technology and has high adaptability to fermentation materials; the yield of the hyaluronic acid product is high and can reach 6 to 7g/L, the recovery rate reaches 92 percent, the fermentation time is only 24 to 26 hours, so that the energy consumption is effectively saved.
Description
Technical field
The invention belongs to the microbial fermentation field, the hyaluronic method of especially a kind of Production by Microorganism Fermentation.
Background technology
Hyaluronan molecule has good moisture preserving and viscoelasticity, thereby be widely used in various makeup, healthcare products ophthalmologic operation, joint injection, promotion wound healing, regulate the aspects such as permeability, medical shaping material and healthcare products of vessel wall, since mid-term 1990's, world market hyaluronic acid sales volume rises gradually, global medicinal hyaluronic acid total sales volume is hundred million dollars of 4.2-5.5 at present, is certain to above 1,000,000,000 dollars by 2010.Present domestic research level is low, and throughput only is 10 tons, can not satisfy the demand in market far away.
The technology fermentation period that the hyaluronic enterprise of present domestic most of Production by Microorganism Fermentation adopts is long, generally about 36-40, molecular weight of product is low, product printing opacity rate variance (generally about 90%), the protein content height, its finished product glucuronic acid content is generally 35-38%, part producer hyaluronic acid product form is fibrous, this kind hyaluronic acid dissolving difficulty, generally take about 2 days, and yield is lower than 75%, and in order to satisfy the demand market of domestic continuous expansion, hyaluronic production technique is demanded urgently improving.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of fermentation period weak point, product solvability height, the hyaluronic method of Production by Microorganism Fermentation that cost is low are provided.
The objective of the invention is to be achieved through the following technical solutions:
The hyaluronic method of a kind of Production by Microorganism Fermentation, its step comprise that (1) shake-flask seed liquid, (2) seeding tank seed liquor, (3) fermentation culture obtain fermented liquid, and be further comprising the steps of:
(4) purification by liquid extraction of fermented liquid: fermented liquid is got filtrate behind Plate Filtration, add 2-4 ethanolic soln precipitation doubly, the collecting precipitation thing adds and the isopyknic Nacl solution stirring dissolving of filtrate, carry out the secondary Plate Filtration to clarification, obtain post-defecation liquid, post-defecation liquid behind the filter membrane or filter plate filtration of 0.3-0.5 micron, is added acetate and regulates pH to 4.0-5.0, obtain three secondary clearing liquid after the acid treatment, spray three secondary clearing liquid with ethanolic soln again, precipitate again, obtain throw out;
(5) dry powder process: whole throw outs that above-mentioned steps is obtained carry out emulsification powder process with emulsification pump after the dehydrated alcohol dehydration, the powdery precipitation, further with after the dehydrated alcohol dehydration, after the 50-60 ℃ of vacuum-drying, moisture being controlled at below 10% promptly gets hyaluronic acid.
And the diatomaceous model that described Plate Filtration uses is No. 821 and No. 616.
And described No. 821 and No. 616 diatomaceous part by weight are 1: 1-5.
And the concentration of described ethanolic soln is: 95% (v/v).
And the concentration of described Nacl solution is: 0.1mol/L.
Advantage of the present invention and positively effect are:
1, to adopt the complex mutation technology screening to go out pod membrane big in the present invention, the hyaluronic acid productivity height, do not produce the production bacterial classification of the bacterial classification of Unidasa, this bacterial classification is strong to fermentation raw material adaptability, hyaluronic acid product productive rate height can reach 6-7g/L, and its rate of recovery is up to 92%, its fermentation time only is 24-26 hour, effectively saves energy consumption.
2, the present invention adopts the spray powder-making technique of original creation, makes the product structure form fine and smooth more, and use properties is more superior, and solvability is better, and its transmittance reaches 99%.
3, the purification process of product of the present invention adopts Plate Filtration and micropore filter plate filtering technique, substitutes complex processes such as charcoal absorption and CPC absorption, makes production operation easier.
4, the present invention adopts acidifying ethanol spray powder-making technique, and the product of making is Powdered, dissolves than cotton-shaped being easy to.
5, the present invention can be developed to hyaluronic acid food grade, cosmetics-stage and medical grade products.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The fermented bacterium that the present invention uses belongs to external bacterial classification, and bacterium classification is a streptococcus zooepidemicus.
The hyaluronic method of a kind of Production by Microorganism Fermentation, its technological process is as follows:
(1) shake-flask seed liquid: slant strains is inoculated in the 1L Erlenmeyer flask that sterile medium 400ml is housed, cultivated 12-16 hour for 37 ℃, the om observation thalli growth is good, and no living contaminants can be inoculated in the seeding tank;
(2) seeding tank seed liquor: press the formulated nutrient solution 60L of shake-flask seed liquid, add in the seeding tank, logical steam heating, sterilized 30 minutes for 121 ℃, be cooled to 37 ℃,, shake-flask seed liquid be inoculated in the seeding tank with the flame method inoculation, air flow 60L/min, mixing speed 120-140/min cultivated 12-16 hour for 37 ℃, and the om observation thalli growth is good, no living contaminants can be inoculated in the fermentor tank;
(3) fermentation culture: the prescription of pressing seeding tank, the consumption of sugar is brought up to 6%, preparation nutrient solution 600L, add in 1 ton the fermentor tank, logical steam heating, sterilized 30 minutes for 121 ℃, be cooled to 37 ℃, seed liquor is inoculated in the fermentor tank by the culture transferring pipeline, keep air flow 400-600L/min, mixing speed 140-160/min cultivated 24-26 hour for 37 ℃, in the fermenting process, below the density loss to 0.5% of sugar, PH descends very slow or when no longer descending, is fermentation termination, obtains containing hyaluronic acid fermentation liquid; (above 3 steps are in Ma Chaomei (Liuzhou Chemical Industry Group Co., Ltd), fermentative Production hyaluronic acid technical study, Guangxi Technical College's journal, 2007,18 (3): report is arranged among the 8-12, belong to prior art, the present patent application is just described in detail no longer more), the present patent application also comprises:
(4) purification by liquid extraction of fermented liquid: fermented liquid is got filtrate behind Plate Filtration, 95% ethanol sedimentation that adds 3 times, the collecting precipitation thing adds the Nacl solution stirring dissolving with the isopyknic 0.1mol/L of filtrate, carry out the secondary Plate Filtration to clarification, obtain post-defecation liquid; Post-defecation liquid after 0.45 micron filter membrane or filter plate filter, is added acetate and regulates pH to 4.0-5.0, obtain three secondary clearing liquid after the acid treatment, spray three secondary clearing liquid with 95% ethanol again, precipitate again, obtain throw out;
The diatomaceous model of Plate Filtration is No. 821 and No. 616, and two kinds of diatomite are mixed in proportion, and the sheet frame of packing into after obtaining filters, and No. 821 and No. 616 diatomaceous part by weight are 1: 1-5.
(5) dry powder process: whole throw outs that will obtain carry out emulsification powder process with emulsification pump after the dehydrated alcohol dehydration, get the powdery precipitation, and further with after the dehydrated alcohol dehydration, after the 50-60 ℃ of vacuum-drying, moisture being controlled at below 10% promptly gets hyaluronic acid.
Hyaluronic acid product productive rate height through the present invention obtains can reach 6-7g/L, and its rate of recovery is 92%.
Product performance index:
Proterties: white powder or fine particle;
Smell: odorless, tasteless;
pH(1g/L)： 6.5-7.0；
Clarity (1g/L): transmittance＞99%;
Weight loss on drying:＜10%;
Molecular weight:＞1.8 * 106;
Protein:＜0.05%;
Glucuronic acid: 〉=44%;
Plumbous:＜20mg/kg;
Arsenic:＜2mg/kg;
Total plate count:＜10/gram;
Excrement colibacillus group: must not detect;
Streptococcus aureus: must not detect;
Pseudomonas aeruginosa: must not detect.
Claims (5)
1. hyaluronic method of Production by Microorganism Fermentation, its step comprises that (1) shake-flask seed liquid, (2) seeding tank seed liquor, (3) fermentation culture obtain fermented liquid, is characterized in that: further comprising the steps of:
(4) purification by liquid extraction of fermented liquid: fermented liquid is got filtrate behind Plate Filtration, add 2-4 ethanolic soln precipitation doubly, the collecting precipitation thing adds and the isopyknic Nacl solution stirring dissolving of filtrate, carry out the secondary Plate Filtration to clarification, obtain post-defecation liquid, post-defecation liquid behind the filter membrane or filter plate filtration of 0.3-0.5 micron, is added acetate and regulates pH to 4.0-5.0, obtain three secondary clearing liquid after the acid treatment, spray three secondary clearing liquid with ethanolic soln again, precipitate again, obtain throw out;
(5) dry powder process: whole throw outs that above-mentioned steps is obtained carry out emulsification powder process with emulsification pump after the dehydrated alcohol dehydration, the powdery precipitation, further with after the dehydrated alcohol dehydration, after the 50-60 ℃ of vacuum-drying, moisture being controlled at below 10% promptly gets hyaluronic acid.
2. the hyaluronic method of a kind of Production by Microorganism Fermentation according to claim 1 is characterized in that: the diatomaceous model that described Plate Filtration uses is No. 821 and No. 616.
3. the hyaluronic method of a kind of Production by Microorganism Fermentation according to claim 2 is characterized in that: described No. 821 and No. 616 diatomaceous part by weight are 1: 1-5.
4. the hyaluronic method of a kind of Production by Microorganism Fermentation according to claim 1, it is characterized in that: the concentration of described ethanolic soln is: 95% (v/v).
5. the hyaluronic method of a kind of Production by Microorganism Fermentation according to claim 1, it is characterized in that: the concentration of described Nacl solution is: 0.1mol/L.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559801A (en) * | 2011-12-20 | 2012-07-11 | 宁夏大学 | Fermentation method of hyaluronic acid capable of overcoming glucose and lactic acid restraining |
| CN105434468A (en) * | 2015-12-15 | 2016-03-30 | 天津市康婷生物工程有限公司 | Preparation method of skin cell damage repairing reagent |
| CN106589164A (en) * | 2016-12-02 | 2017-04-26 | 天津市康婷生物工程有限公司 | Method for increasing yield of hyaluronic acid |
| CN106632728A (en) * | 2016-12-02 | 2017-05-10 | 天津市康婷生物工程有限公司 | Method for improving yield of hyaluronic acid with high molecular weight |
| CN107929209A (en) * | 2017-12-23 | 2018-04-20 | 卞国民 | A kind of Essence of loach facial mask |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5496726A (en) * | 1993-04-16 | 1996-03-05 | Lucky Limited | Streptococcus zooepidemicus medium and process for preparing hyaluronic acid |
| CN101020724A (en) * | 2006-02-14 | 2007-08-22 | 镇江东方生物工程设备技术有限责任公司 | Process of preparing low molecular weight sodium hyaluronate |
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2010
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559801A (en) * | 2011-12-20 | 2012-07-11 | 宁夏大学 | Fermentation method of hyaluronic acid capable of overcoming glucose and lactic acid restraining |
| CN105434468A (en) * | 2015-12-15 | 2016-03-30 | 天津市康婷生物工程有限公司 | Preparation method of skin cell damage repairing reagent |
| CN106589164A (en) * | 2016-12-02 | 2017-04-26 | 天津市康婷生物工程有限公司 | Method for increasing yield of hyaluronic acid |
| CN106632728A (en) * | 2016-12-02 | 2017-05-10 | 天津市康婷生物工程有限公司 | Method for improving yield of hyaluronic acid with high molecular weight |
| CN107929209A (en) * | 2017-12-23 | 2018-04-20 | 卞国民 | A kind of Essence of loach facial mask |
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| CN101864471B (en) | 2012-08-29 |
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Inventor after: Liu Xiaobing Inventor after: Li Aihong Inventor after: Zhou Ying Inventor before: Liu Xiaobing |
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Address after: No.9 Saida South Road, Xiqing Economic and Technological Development Zone, Tianjin 300200 Patentee after: Tianjin Kangting Bioengineering Group Co., Ltd. Address before: 300385 No. 5 Jianfu Road, Xiqing Economic Development Zone, Tianjin Patentee before: Tianjin Kangting Biotechnology Co., Ltd. |
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