CN101294180B - Method for preparing small-molecular weight hyaluronic acid by adding hydrogen phosphide and ascorbic acid in course of fermentation - Google Patents
Method for preparing small-molecular weight hyaluronic acid by adding hydrogen phosphide and ascorbic acid in course of fermentation Download PDFInfo
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- CN101294180B CN101294180B CN2008101233963A CN200810123396A CN101294180B CN 101294180 B CN101294180 B CN 101294180B CN 2008101233963 A CN2008101233963 A CN 2008101233963A CN 200810123396 A CN200810123396 A CN 200810123396A CN 101294180 B CN101294180 B CN 101294180B
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- hydrogen peroxide
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Abstract
The invention discloses a method for producing hyaluronic acid with small molecular weight by adding hydrogen peroxide and ascorbic acid in fermentation process, which belongs to the technology field of the application of oxidative-reductive degradation reaction in optimization of fermentation process. In the method, hydrogen peroxide (0.1 to 1.0 mmol/g hyaluronic acid) and ascorbic acid (0.05 to 0.5 mmol/g hyaluronic acid) are added in the fermentation process (8 h) to degrade hyaluronic acid, so that the viscosity of a fermentation broth decreases from reference 300 cp (without hyaluronidase) to 100 to 200 cp, the dissolved oxygen concentration is improved from reference 0 to 1% to 5 to 10%, the output of hyaluronic acid is improved from reference 5.0 g/L to 5.5 to 6.4 g/L, and the molecular weight of the hyaluronic acid decreases from reference 1,300 kDa to 80 to 200 kDa. The inventive method is simple and efficient, and has important industrial application value. The decrease of the viscosity of the fermentation broth results in the improvement of downstream purification efficiency and the reduction of production cost. The hyaluronic acid with small molecular weight produced by the method can be applied to the fields of medicines and healthy food.
Description
Technical field
A kind of hydrogen peroxide and hyaluronic method of xitix production small molecular weight of adding during the fermentation belongs to the technical field of redox DeR in the optimizing fermentation application facet.
Background technology
Mucinase is the straight chain line style negatively charged ion mucopolysaccharide that is connected into β-1,3 and β-1,4 glycosidic link by glucuronic acid and N-acetylglucosamine, is separated from bovine vitreous body by Meyer and Palmer in 1934.Mucinase is the important component in extracellular matrix, knuckle synovia, cartilaginous tissue, the umbilical cord, and the important physical function is arranged in human body, like the chemical constitution of regulating extracellular fluid, lubricated and promotion wound healing; Regulate the activation of adhesion, growth, motion and specific cell etc.
The size of hyaluronan molecule amount and its physiological function are closely related, and for example, mucinase stimulates the effect and the inversely proportional relation of hyaluronic molecular weight of chondrocyte and lymphoblast differentiation: molecular weight is 2 * 10
5～4 * 10
5Mucinase hormesis in the Da scope is the strongest, and molecular weight is greater than 10
6Mucinase then have no stimulation; High molecular weight hyaluronic acid can suppress the phagocytic activity of scavenger cell, and low-molecular-weight hyaluronic acid can impel scavenger cell to express the factor of some and inflammation-related.Compare with the high-molecular weight mucinase, low-molecular-weight mucinase can show different biological functions: low-molecular-weight hyaluronic acid can stimulate blood vessel hyperplasia, induces the expression of inflammation regulatory factor, the propagation of inhibition tumour and anti-microbial effect etc.In addition, low-molecular-weight mucinase also is widely used aspect protective foods, in Japan and China Taiwan the listing of mucinase aesthetic health care food is arranged all, and therefore, the preparation of research low-molecular-weight hyaluronic acid has important use and is worth and medical value.
Meanwhile, the Production by Microorganism Fermentation mucinase belongs to high viscosity fermentation, and oxyty is the bottleneck that hyaluronic acid volume of production improves.The factor that influences fermentation broth viscosity is a lot, and wherein hyaluronic molecular weight influence is very big, and molecular weight is big more, and viscosity is high more.Therefore,, then can reduce viscosity and improve dissolved oxygen, finally improve hyaluronic output if can degrade hyaluronic molecular weight during the fermentation.The present invention improves dissolved oxygen and then improves hyaluronic acid volume of production through adding hydrogen peroxide and degrade ascorbic acid mucinase during the fermentation, does not see bibliographical information as yet.
Summary of the invention
The purpose of this invention is to provide a kind of strategy that adds hydrogen peroxide and xitix during the fermentation and produce the hyaluronic method of small molecular weight, utilize the present invention can produce the mucinase of molecular weight between 80～200kDa.
Technical scheme of the present invention: a kind of hydrogen peroxide and hyaluronic method of xitix production small molecular weight of adding during the fermentation; Use hydrogen peroxide and Ascorbic Acid Oxidation degraded macromolecular amount mucinase during the fermentation; Reduce fermentation broth viscosity; Improve dissolved oxygen, and then improve hyaluronic acid volume of production and downstream purification efficient, the hyaluronic molecular weight of the small molecular weight of being produced is between 80～200kDa.
When fermentation 8h, add hydrogen peroxide (0.1～1.0mmol/g mucinase) and xitix (0.05～0.5mmol/g mucinase) degraded mucinase, improve dissolved oxygen and hyaluronic acid volume of production.
Beneficial effect of the present invention: the present invention adopts (8h) interpolation hydrogen peroxide (0.1～1.0mmol/g mucinase) and xitix (0.05～0.5mmol/g mucinase) degraded mucinase during the fermentation; The viscosity of fermented liquid drops to 100～200cp by the 300cp of contrast (not adding hydrogen peroxide and xitix); Oxyty brings up to 5%～10% by 0～1% of contrast; Hyaluronic acid volume of production also is increased to 5.5～6.5g/L by the 5.0g/L of contrast; Hyaluronic molecular weight is by 1 of contrast, and 300kDa drops to 80～200kDa.The inventive method is simple, efficient, has important industrial application value, because fermentation broth viscosity reduces, the purification efficiency in downstream improves, and production cost reduces.The small molecular weight mucinase that the inventive method is produced can be applied to medicine and field of health care food.In addition, the optimizing fermentation of degradable polymer for other has certain guidance and edifies meaning.
Embodiment
Control Example: the used bacterial classification of the present invention is streptococcus zooepidemicus Streptococcus zooepidemicusWSH-24; (or being designated as H-24) (Wen Qi, Liu Dengru, Chen Jian, stifled state become " add tensio-active agent and promote streptococcus zooepidemicus high yield mucinase "; " chemical industry progress " 2006; 25 (9), 1089～1094; Chinese patent 200610098195.3 is open), change the inclined-plane earlier and in 37 ℃ of incubators, cultivated 12 hours, carried out shake flask fermentation then 14 hours; 37 ℃ of temperature; Rotating speed 200rpm fermented in the 7L fermentor tank 16 hours at last, and fermentation condition is: pH 7.0; Mixing speed 200rpm, Ventilation Rate 1.0vvm.Fermention medium is (g/L): sucrose: 70; Yeast powder: 25; MgSO
47H
2O:2.0; Na
2HPO
4: 6.3; K
2SO
4: 7.0.Hyaluronic molecular weight is 1,300,000, and output is 5.0g/L.
Embodiment 1: ferment tank adopts hydrogen peroxide and xitix to add strategy, the same control Example of all the other conditions.When fermentation 8h, add hydrogen peroxide (0.1mmol/g mucinase) and xitix (0.05mmol/g mucinase).Hyaluronic acid volume of production is brought up to 5.5g/L during fermentation ends, and molecular weight is 200kDa.
Embodiment 2: ferment tank adopts hydrogen peroxide and xitix to add strategy, the same control Example of all the other conditions.When fermentation 8h, add hydrogen peroxide (0.6mmol/g mucinase) and xitix (0.3mmol/g mucinase).Hyaluronic acid volume of production is brought up to 5.7g/L during fermentation ends, and molecular weight is 120kDa.
Embodiment 3: ferment tank adopts hydrogen peroxide and xitix to add strategy, the same control Example of all the other conditions.When fermentation 8h, add hydrogen peroxide (1.0mmol/g mucinase) and xitix (0.5mmol/g mucinase).Hyaluronic acid volume of production is brought up to 6.5g/L during fermentation ends, and molecular weight is 80kDa.
Claims (1)
1. one kind is added hydrogen peroxide and the hyaluronic method of xitix production molecular weight between 80～200kDa during the fermentation; It is characterized in that used during the fermentation bacterial classification is streptococcus zooepidemicus (Streptococcus zooepidemicus) WSH-24; Fermentation condition is: pH 7.0; Mixing speed 200rpm, Ventilation Rate 1.0vvm; Fermention medium is: 70g/L sucrose; The 25g/L yeast powder; 2.0g/LMgSO
47H
2O; 6.3g/L Na
2HPO
47.0g/L K
2SO
4Through when fermenting 8h, adding hydrogen peroxide and degrade ascorbic acid mucinase; Addition is that every g mucinase adds hydrogen peroxidase 10 .1～1.0mmol and xitix 0.05～0.5mmol; Realized the reduction fermentation broth viscosity; Improve dissolved oxygen, and then improve hyaluronic acid volume of production and downstream purification efficient, hyaluronic molecular weight is dropped between 80～200kDa by the 1300kDa of contrast.
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|---|---|---|---|
| CN2008101233963A CN101294180B (en) | 2008-05-26 | 2008-05-26 | Method for preparing small-molecular weight hyaluronic acid by adding hydrogen phosphide and ascorbic acid in course of fermentation |
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|---|---|---|---|
| CN2008101233963A CN101294180B (en) | 2008-05-26 | 2008-05-26 | Method for preparing small-molecular weight hyaluronic acid by adding hydrogen phosphide and ascorbic acid in course of fermentation |
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| CN101294180A CN101294180A (en) | 2008-10-29 |
| CN101294180B true CN101294180B (en) | 2012-04-18 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102012009593B4 (en) | 2012-05-07 | 2019-03-07 | Innovent E.V. | Process for the degradation of polysaccharides selected from the substance groups of the glycosaminoglycans and their derivatives and the alginates |
| CN103397062B (en) * | 2013-08-13 | 2016-01-20 | 安阳九州药业有限责任公司 | A kind of method improving fermentation yield of hyaluronic acid |
| CN103993022B (en) * | 2013-12-20 | 2017-03-08 | 天津科技大学 | The preparation method of low-molecular-weight hyaluronic acid and engineering bacteria |
| CN110452944A (en) * | 2019-09-21 | 2019-11-15 | 冯世红 | A method of hyaluronic acid is prepared using salt alcohol method |
| CN117535363A (en) * | 2023-12-27 | 2024-02-09 | 常州加源医药科技研发有限公司 | Production method of functional hyaluronic acid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587387A (en) * | 2004-07-26 | 2005-03-02 | 清华大学 | Method for producing hyaluronic acid and its special strain |
| CN1978658A (en) * | 2006-12-08 | 2007-06-13 | 江南大学 | Method for improving fermentation-produced hyaluronicacid yield using intermittent high pH tolerant strategy |
| 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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2008
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587387A (en) * | 2004-07-26 | 2005-03-02 | 清华大学 | Method for producing hyaluronic acid and its special strain |
| 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 |
| CN1978658A (en) * | 2006-12-08 | 2007-06-13 | 江南大学 | Method for improving fermentation-produced hyaluronicacid yield using intermittent high pH tolerant strategy |
Non-Patent Citations (1)
| Title |
|---|
| SOLTES,L.等.Degradation of high-molar-mass hyaluronan and characterization of fragments.《BIOMACROMOLECULES》.2007,第8卷(第9期),2697－2705. * |
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Assignee: Jiangsu Haihua Biotech Co., Ltd. Assignor: Jiangnan University Contract record no.: 2012320000667 Denomination of invention: Method for preparing small-molecular weight hyaluronic acid by adding hydrogen phosphide and ascorbic acid in course of fermentation Granted publication date: 20120418 License type: Exclusive License Open date: 20081029 Record date: 20120528 |
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