CN102559801A - Fermentation method of hyaluronic acid capable of overcoming glucose and lactic acid restraining - Google Patents
Fermentation method of hyaluronic acid capable of overcoming glucose and lactic acid restraining Download PDFInfo
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- CN102559801A CN102559801A CN2011104295997A CN201110429599A CN102559801A CN 102559801 A CN102559801 A CN 102559801A CN 2011104295997 A CN2011104295997 A CN 2011104295997A CN 201110429599 A CN201110429599 A CN 201110429599A CN 102559801 A CN102559801 A CN 102559801A
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Abstract
The invention provides a fermentation method of hyaluronic acid capable of overcoming glucose and lactic acid restraining, wherein streptococcus zooepidemicus is taken as a seed, and a fermentation technical process comprises primary seed cultivation, secondary seed cultivation and fermenting cultivation. The primary seed cultivation is carried out from seed transplantation in an inclined-surface test tube to a conical flask, wherein the cultivation temperature ranges from 33 DEG C to 37 DEG C, and the cultivation time is 18-24 hours. The secondary seed cultivation comprises the following steps of: firstly preparing a secondary seed culture medium, transplanting into primary seeds to be cultivated according to 8-12% of inoculation amount, wherein the cultivation temperature ranges from 33 DEG C to 37 DEG C, and the cultivation time is 20-24 hours; and transplanting into fermentation liquor when an OD660 value is increased to be 0.8-1.2. The fermenting cultivation comprises the following steps of: firstly preparing a fermentation culture medium, and transplanting into secondary seeds to be cultivated according to 5-10% of inoculation amount, wherein the cultivation temperature ranges from 33 DEG C to 37 DEG C, and the cultivation period is 30-35 hours.
Description
Technical field
The invention belongs to the microbial fermentation technology field, particularly a kind of hyaluronic acid fermentation method that overcomes glucose and lactic acid inhibition.
Background technology
Mucinase (Hyaluronic Acid is called for short HA) has another name called glass acid; Its molecular weight at hundreds of thousands of between millions of; Because structural characteristics determine it to have comparison special physicochemical character, its aqueous solution has good visco-elasticity and rheological; At food, all there is application cosmetic and medical aspect.The HA of different purposes has demands of different to molecular weight; The HA of small molecular weight is used in the healthcare products more; The HA of intermediate molecular weight is mainly used in makeup; And high-molecular weight HA can be used for ophthalmology viscosurgery, treatment joint disease, soft tissue repair and as pharmaceutical carrier etc., particularly in prevention with reduce after the surgical operation and have very high using value in the tissue adhesion.Therefore in the production of HA, HMW and high yield still are the major objectives of research.
It is ripe that China HA tissue extraction method research in recent years has been tending towards.But raw material is in short supply, complex process, and cost is high, can't satisfy the home market needs.Mucinase transfers Production by Microorganism Fermentation to by traditional animal organ's extraction method at present, and microbe fermentation method is the main method of preparation HA, and abroad to fermentation method research early, technology is also day by day ripe, but because of manufacturer is few, world demand still breach is big; Though the many scholars of China actively carry out the research of this respect in recent years, the difficult problem owing at aspects such as strain improvement, zymotechnique and extraction purifying makes China's progress in this respect slow.China should accelerate the hyaluronic paces of fermentative Production, and key is to improve productive rate, improves the technical elements of molecular weight product and separation and purification and tackles key problems, and catches up with advanced world levels, to reduce import volume.
In the streptococcus zooepidemicus fermenting process, hyaluronic output increases along with the increase of concentration of substrate within the specific limits, if concentration of substrate is too high, hyaluronic production is had restraining effect, and the mucinase relative molecular mass that obtains is also less.And when producing HA, also have lactic acid and generate; The growth of thalline and the generation of HA are all had restraining effect, in this invention, to the difference of fermentation substrate with generate what of product; Study the restraining effect that substrate and product are produced mucinase, and point out to remove the method for inhibition.
Summary of the invention
The objective of the invention is to overcome the prior art defective, a kind of hyaluronic acid fermentation method that overcomes glucose and lactic acid inhibition is provided.
For achieving the above object, scheme of the present invention is:
Hyaluronic zymotechnique is a seed with the streptococcus zooepidemicus, comprises the cultivation of one-level kind, and the secondary kind is cultivated and fermentation culture, each process substratum formulation by weight such as following table:
It is to change to plant from slant tube to Erlenmeyer flask, to carry out 33 ℃～37 ℃ of culture temperature, incubation time 18～24 hours that said one-level kind is cultivated;
The step that said secondary kind is cultivated is: at first prepare secondary seed medium, cultivate 33 ℃～37 ℃ of culture temperature, incubation time 20～24 hours, OD according to the inoculum size access first order seed of weight 8%～12%
660Value is increased at 0.8～1.2 o'clock and inserts in the fermented liquid;
The step of said fermentation culture is: at first prepare fermention medium, cultivate 33 ℃～37 ℃ of leavening temperatures, fermentation period 30～35 hours according to the inoculum size access secondary seed of weight 5%～10%.
The invention has the beneficial effects as follows: improved hyaluronic output, increased hyaluronic molecular weight; Remove the restraining effect of substrate and product, the present invention not only can improve hyaluronic output, and the hyaluronic molecular weight that obtains is big.
Embodiment
The present invention should pay attention to following technical essential in the specific implementation:
1. the inclined-plane of preparation must be fit to thalli growth and preservation thereof, can not be too soft, too wet, or too hard, the inclined-plane appearance is dry and cracked then can not be used.In per generation,, new slant preservation was in 4 ℃ in refrigerator, generally should not surpass for 3～4 weeks, and overlong time reduces viable count, influences hyaluronic acid volume of production.The passage number on inclined-plane should not surpass for the 6th generation, and spawn degeneration is lured in too much going down to posterity into, should avoid.
2. cultivate from the inclined-plane to the secondary kind, the inclined-plane should be in the usage period, and transfer amount is wanted suitably, can not be very little, otherwise seed growth is slowly or not long, should guarantee secondary kind bacteria concentration OD
660About 0.8.
3. cultivate fermentation culture from the secondary kind, inoculum size is 5%～10% (weight), and the revolution that stirs between yeast phase can not be excessive, descends otherwise cross ambassador's molecular weight of product because of shearing force.
4. (NH
4)
2SO
4Be made into the aqueous solution of 20% (weight), glucose is made into the aqueous solution of 50% (weight), after 112 ℃ of 20min sterilizations, adds in addition, and glucose and ammonium sulfate all are that mucinase synthesizes essential raw material is provided.
5. when the fermentation beginning, mainly be the quick growth of requirement thalline, the fermentative prodn mucinase so require first sugared concentration lower, answers the batch feeding mode to add glucose again, promotes that carbon source is converted into HA.
6. leavening temperature is too high, can accelerate the speed of thalli growth, but is unfavorable for the mucinase of synthetic macromolecule.
7. in the fermenting process, get into logarithmic growth in earlier stage at thalline, the control of pH should not be higher than 8.0; And in the logarithmic growth middle and later periods, pH should not be lower than 8.0, after adding ammonium sulfate, glucose; PH should be at 8.0～8.5; Until fermentation ends, pH helped the mucinase biosynthesizing at 8.0～8.5 o'clock.
8. there is lactic acid to generate during the fermentation, the pH of fermented liquid is reduced,, when adding alkali, should select the alkali lye of the alkalescence of suitable concentration, to reduce the aggregative growth of fermentating liquid volume, to improve the output of HA so need carry out repeatedly the adjusting of pH.
9. along with the growth of fermentation time, the dense output with HA of bacterium all increases gradually, gets into the fermentation growth later stage, and the self-dissolving speed of thalline strengthens, OD
660Value reduces.And the Unidasa that thalline produces has Decomposition to HA, so the mucinase accumulation volume is also descending.Therefore the hyaluronic fermentation period of fermentative prodn is about 30～35h.
Embodiment
With the fermentor tank of 10L, air flow is 0.7～1.0vvm, is seed with the streptococcus zooepidemicus, and culture temperature is 33～37 ℃.First order seed is inoculated in the secondary seed nutrient solution by the inoculum size about 9% (weight) before and after 33～37 ℃ of cultivation 20h.Secondary seed solution is with after shaking bottle concussion cultivation 20～24h; Be inoculated in the ferment tank liquid, inoculum size is 5%～10% (weight), and the fermentation initial stage adds a spot of glucose; Mend sugar once after fermentation for some time, fermenting adds remaining glucose again after stationary phase.NaHCO with 1mol/L
3Carried out the adjusting of 6 pH altogether, (NH
4)
2SO
4The aqueous solution adds when fermenting to logarithmic phase, and fermentation period is 30～35h.The hyaluronic acid volume of production that obtains has improved about 16%, and relative molecular mass reaches (2.55-3.36) * 10 approximately
6Dalton.
Each process substratum formulation by weight such as following table:
Claims (4)
1. a hyaluronic acid fermentation method that overcomes glucose and lactic acid inhibition is a seed with the streptococcus zooepidemicus, and fermentation technology process comprises the cultivation of one-level kind, and the secondary kind is cultivated and fermentation culture, it is characterized in that each process substratum formulation by weight such as following table:
2. the hyaluronic acid fermentation method that overcomes glucose and lactic acid inhibition as claimed in claim 1 is characterized in that it is to change to plant from slant tube to Erlenmeyer flask, to carry out 33 ℃～37 ℃ of culture temperature, incubation time 18～24 hours that the one-level kind is cultivated.
3. the hyaluronic acid fermentation method that overcomes glucose and lactic acid inhibition as claimed in claim 1; It is characterized in that the step that the secondary kind is cultivated is: at first prepare secondary seed medium; Inoculum size access first order seed according to weight 8%～12% is cultivated; 33 ℃～37 ℃ of culture temperature, incubation time 20～24 hours, OD
660Value is increased at 0.8～1.2 o'clock and inserts in the fermented liquid.
4. the hyaluronic acid fermentation method that overcomes glucose and lactic acid inhibition as claimed in claim 1; The step that it is characterized in that fermentation culture is: at first prepare fermention medium; Inoculum size access secondary seed according to weight 5%～10% is cultivated; 33 ℃～37 ℃ of leavening temperatures, fermentation period 30～35 hours.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592849A (en) * | 2020-12-14 | 2021-04-02 | 宁夏泰胜生物科技有限公司 | Culture medium and culture method for producing high molecular weight hyaluronic acid by fermentation of streptomyces zooepidemicus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619331A (en) * | 2009-07-20 | 2010-01-06 | 四川理工学院 | Method for producing hyaluronic acid (HA) by streptococcus zooepidemicus fermentation |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619331A (en) * | 2009-07-20 | 2010-01-06 | 四川理工学院 | Method for producing hyaluronic acid (HA) by streptococcus zooepidemicus fermentation |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| 佘永红等: "透明质酸发酵过程中产物抑制研究", 《安徽农业科学》 * |
| 佘永红等: "透明质酸发酵过程中底物抑制及解除抑制的方法研究", 《安徽农业科学》 * |
| 田毅红: "透明质酸的发酵调控研究", 《三峡大学学报（自然科学版）》 * |
| 罗瑞明: "透明质酸(HA)的国内外研究现状", 《宁夏农学院学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592849A (en) * | 2020-12-14 | 2021-04-02 | 宁夏泰胜生物科技有限公司 | Culture medium and culture method for producing high molecular weight hyaluronic acid by fermentation of streptomyces zooepidemicus |
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Application publication date: 20120711 |