CN105779532A - Method for producing chondroitin sulfate - Google Patents
Method for producing chondroitin sulfate Download PDFInfo
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- CN105779532A CN105779532A CN201610307883.XA CN201610307883A CN105779532A CN 105779532 A CN105779532 A CN 105779532A CN 201610307883 A CN201610307883 A CN 201610307883A CN 105779532 A CN105779532 A CN 105779532A
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- chondroitin sulfate
- sulfate
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- dihydrogen phosphate
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- 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 provides a method for producing chondroitin sulfate. The method comprises the following steps of: inoculating a seed culture medium with inoculating bacillus megatherium with the inoculum size of 4-6 percent; performing shaking culture to obtain a seed solution, inoculating the seed solution with the inoculum size of 4-6 percent into a fermentation culture medium, performing culture fermentation by virtue of a shaker so as to obtain a fermentation broth containing chondroitin sulfate; and purifying the fermentation broth containing chondroitin sulfate, thereby obtaining the chondroitin sulfate. The method is simple in step, wild in condition, relatively high in yield, and free from animal bone raw material shortage limitation for fermentation production raw materials; fermented products can be used for raw materials for pharmaceutical products and raw materials for health care products.
Description
Technical field
The invention belongs to field of microbial fermentation, relate to a kind of chondroitin sulfate, a kind of production sulphuric acid is soft specifically
Ossein fermentation liquid method.
Background technology
Chondroitin sulfate (Chondroitin Sulfate, CS) is the class being distributed widely in animal cartilage tissue
Acidic mucopolysaccharide.Within 2010, many fermentable are analyzed by Jolly, are found to have multiple-microorganism and also can produce sulfur
Aching and limp ossein.Such as p pestic (Pasteurella multocida), escherichia coli (Escherichia coli), bud
, there is chondroitin sulfate etc. in minority antibacterial in spore bacillus (Bacillus).Chondroitin sulfate molecular structure is by D-glucose
The polysaccharide of the repetition disaccharide unit composition that aldehydic acid and N-acetylamino galactosamine are constituted, chondroitin sulfate typically contains 50 ~ 70
Individual diglycosyl our unit, relative molecular mass is between 10 000 ~ 50 000.Different by the sulfate group position of esterification, sulphuric acid
Chrondroitin is divided into the polytypes such as A, B, C, D, E, F.It is mainly used in arthritis, omarthralgia, coronary heart diseases and angina pectoris etc. clinically
Auxiliary treatment, in recent years, chondroitin sulfate is more widely available in fields such as pharmaceutical preparation, medical material, cosmetics, health product should
With, the market demand becomes the biggest.China produces and export volume increases substantially year by year, and development prospect is wide.
At present, China mainly use enzymatic isolation method or alkali carry-enzymatic isolation method technique prepares chondroitin sulfate from animal bone.Seldom
There is the report using Production by Microorganism Fermentation chondroitin sulfate.And animal bone resource is poorer, anxious new biology to be developed
Resource and new technical matters produce chondroitin sulfate.
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of method producing chondroitin sulfate, institute
The method prior art to be solved of this production chondroitin sulfate stated produces in the method for chondroitin sulfate from animal bone,
There is lack of raw materials is difficult to scale up production-scale technical problem for bone.
The invention provides a kind of method producing chondroitin sulfate, comprise the steps:
1) bacillus megaterium is seeded on seed culture medium carry out concussion with inoculum concentration for 4-6% and cultivates 15-18h, obtain
Seed culture fluid, seed culture condition is: rotating speed 120-160r/m, and initial pH value is 7.0-7.2, and fermentation temperature is 28-35
℃；
2) by step 1) obtain seed liquor and be inoculated into equipped with in the container of fermentation medium with inoculum concentration for 4-6%, on shaking table
Cultivate 30-35h；I.e. can get sulfur acid chrondroitin fermentation liquid, condition of culture is: rotating speed 120-160r/m, and initial pH value is
7.0-7.2, temperature is 30-35 DEG C；
3) chondroitin sulfate is obtained after being purified by the chondroitin sulfate fermentation liquid obtained.
Further, described bacillus megaterium is bacillus megaterium CICC 10487.
Further, described seed culture medium by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate,
Calcium chloride, manganese sulfate and water composition, in every L culture medium, containing sucrose 15-20g, peptone 15-20g, yeast powder 15-20g, chlorine
Change sodium 0.5-1.0g, magnesium sulfate 0.5-1.0g, potassium dihydrogen phosphate 1.5-2.5g, described calcium chloride 0.2-0.5g, manganese sulfate
0.1-0.5g, surplus is water.
Further, described fermentation medium by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate,
Calcium chloride, manganese sulfate and water composition, described buffer is made up of disodium hydrogen phosphate, sodium dihydrogen phosphate；In every L culture medium, contain
Sucrose 20-50g, peptone 10-15g, yeast powder 10-15g, sodium chloride 0.5-2.0g, magnesium sulfate 1.5-2.5g, potassium dihydrogen phosphate
1.5-2.5g, calcium chloride 0.2-0.5g, manganese sulfate 0.05-0.15g, disodium hydrogen phosphate 1.0-4.0 g, sodium dihydrogen phosphate 0.3-
3.0g, surplus is water.
Concrete, use ethanol precipitation to be purified process and obtain chondroitin sulfate.
By the yield about 127mg/L of the chondroitin sulfate polysaccharide that the inventive method obtains.
The present invention compares with prior art, and its technological progress is significant.The method step of the present invention is simple, condition temperature
With, yield is higher, and by animal bone, there is lack of raw materials is limited for fermenting and producing raw material, and fermented product can be used for pharmaceutical raw material and health care
Product raw material.
Detailed description of the invention
Embodiment 1
The invention provides a kind of method producing chondroitin sulfate, comprise the steps:
(1) it is 5% to be seeded to bacillus megaterium CICC 10487 on seed culture medium to carry out concussion and cultivate with inoculum concentration
16h, obtains seed culture fluid.Seed culture condition is: rotating speed 120/m, and initial pH value is 7.0, and fermentation temperature is 30 DEG C, institute
State seed culture medium by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, manganese sulfate and water group
Become, in every L culture medium, containing sucrose 15-20g, peptone 15-20g, yeast powder 15-20g, sodium chloride 0.5-1.0g, magnesium sulfate
0.5-1.0g, potassium dihydrogen phosphate 1.5-2.5g, calcium chloride 0.2-0.5g, manganese sulfate 0.1-0.5g, surplus is water.
(2) (1) obtaining seed liquor with inoculum concentration is 5% to be inoculated into equipped with in fermentation medium triangular flask, on shaking table
Cultivate 30h；I.e. can get chondroitin sulfate fermentation liquid.Condition of culture is: rotating speed 120r/m, and initial pH value is 7.2, and temperature is
32℃.Described fermentation medium is by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sulphuric acid
Manganese and water composition, described buffer is made up of disodium hydrogen phosphate, sodium dihydrogen phosphate.In every L culture medium, containing sucrose 20-50g,
Peptone 10-15g, yeast powder 10-15g, sodium chloride 0.5-2.0g, magnesium sulfate 1.5-2.5g, potassium dihydrogen phosphate 1.5-2.5g, chlorine
Changing calcium 0.2-0.5g, manganese sulfate 0.05-0.15g, disodium hydrogen phosphate 1.0-4.0g, sodium dihydrogen phosphate 0.3-3.0g, surplus is
Water.
(3) fermentation liquid produced is purified process through ethanol precipitation commonly used in the art again, and i.e. to can get sulphuric acid soft
Ossein, the yield of polysaccharide about 112mg/L.
Embodiment 2
The invention provides a kind of method producing chondroitin sulfate, comprise the steps:
(1) it is 4% to be seeded to bacillus megaterium CICC 10487 on seed culture medium to carry out concussion and cultivate with inoculum concentration
18h, obtains seed culture fluid.Seed culture condition is: rotating speed 140r/m, and initial pH value is 7.1, and fermentation temperature is 28 DEG C,
Described seed culture medium is by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, manganese sulfate and water
Composition, in every L culture medium, containing sucrose 15-20g, peptone 15-20g, yeast powder 15-20g, sodium chloride 0.5-1.0g, sulphuric acid
Magnesium 0.5-1.0g, potassium dihydrogen phosphate 1.5-2.5g, calcium chloride 0.2-0.5g, manganese sulfate 0.1-0.5g, surplus is water.
(2) (1) obtaining seed liquor with inoculum concentration is 4% to be inoculated into equipped with in fermentation medium triangular flask, on shaking table
Cultivate 35h；I.e. can get chondroitin sulfate fermentation liquid.Condition of culture is: rotating speed 120r/m, and initial pH value is 7.2, and temperature is
30℃.Described fermentation medium is by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sulphuric acid
Manganese and water composition, described buffer is made up of disodium hydrogen phosphate, sodium dihydrogen phosphate；In every L culture medium, in every L culture medium, contain
Sucrose 20-50g, peptone 10-15g, yeast powder 10-15g, sodium chloride 0.5-2.0g, magnesium sulfate 1.5-2.5g, potassium dihydrogen phosphate
1.5-2.5g, calcium chloride 0.2-0.5g, manganese sulfate 0.05-0.15g, disodium hydrogen phosphate 1.0-4.0g, sodium dihydrogen phosphate 0.3-
3.0g, surplus is water.
(3) fermentation liquid produced is purified process through ethanol precipitation commonly used in the art again, and i.e. to can get sulphuric acid soft
Ossein, the yield of polysaccharide about 105mg/L.
Embodiment 3
The invention provides a kind of method producing chondroitin sulfate, comprise the steps:
(1) it is that 6% bacterium is seeded on seed culture medium to carry out concussion and cultivates 15h by bacillus cereus CICC 10487 with inoculum concentration,
Obtain seed culture fluid.Seed culture condition is: rotating speed 160r/m, and initial pH value is 7.0, and fermentation temperature is 32 DEG C, described
Seed culture medium is by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, manganese sulfate and water group
Become, in every L culture medium, containing sucrose 15-20g, peptone 15-20g, yeast powder 15-20g, sodium chloride 0.5-1.0g, magnesium sulfate
0.5-1.0g, potassium dihydrogen phosphate 1.5-2.5g, calcium chloride 0.2-0.5g, manganese sulfate 0.1-0.5g, surplus is water.
(2) (1) obtaining seed liquor with inoculum concentration is 6% to be inoculated into equipped with in fermentation medium triangular flask, on shaking table
Cultivate 32h；I.e. can get chondroitin sulfate fermentation liquid.Condition of culture is: rotating speed 160r/m, and initial pH value is 7.0, and temperature is
30℃.Described fermentation medium is by sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sulphuric acid
Manganese and water composition, described buffer is made up of disodium hydrogen phosphate, sodium dihydrogen phosphate；In every L culture medium, containing sucrose 20-50g,
Peptone 10-15g, yeast powder 10-15g, sodium chloride 0.5-2.0g, magnesium sulfate 1.5-2.5g, potassium dihydrogen phosphate 1.5-2.5g, chlorine
Changing calcium 0.2-0.5g, manganese sulfate 0.05-0.15g, disodium hydrogen phosphate 1.0-4.0g, sodium dihydrogen phosphate 0.3-3.0g, surplus is
Water.
(3) fermentation liquid produced is purified process through ethanol precipitation commonly used in the art again, and i.e. to can get sulphuric acid soft
Ossein, the yield of polysaccharide about 127mg/L.
Described content is only the basic explanation under present inventive concept, and according to technical scheme made any etc.
Effect conversion, all should belong to protection scope of the present invention.
Claims (4)
1. the method producing chondroitin sulfate, it is characterised in that comprise the steps:
1) bacillus megaterium is seeded on seed culture medium carry out concussion with inoculum concentration for 4-6% and cultivates 15-18h, obtain
Seed culture fluid, seed culture condition is: rotating speed 120-160r/m, and initial pH value is 7.0-7.2, and fermentation temperature is 28-35
℃；
2) by step 1) obtain seed liquor and be inoculated into equipped with in the container of fermentation medium with inoculum concentration for 4-6%, on shaking table
Cultivate 30-35h；I.e. can get sulfur acid chrondroitin fermentation liquid, condition of culture is: rotating speed 120-160r/m, and initial pH value is
7.0-7.2, temperature is 30-35 DEG C；
3) chondroitin sulfate is obtained after being purified by the chondroitin sulfate fermentation liquid obtained.
A kind of method producing chondroitin sulfate the most according to claim 1, it is characterised in that: described huge spore bar
Bacterium is bacillus megaterium CICC 10487.
A kind of method producing chondroitin sulfate the most according to claim 1, it is characterised in that: described seed culture medium by
Sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, manganese sulfate and water composition, every L culture medium
In, containing sucrose 15-20g, peptone 15-20g, yeast powder 15-20g, sodium chloride 0.5-1.0g, magnesium sulfate 0.5-1.0g, phosphorus
Acid dihydride potassium 1.5-2.5g, described calcium chloride 0.2-0.5g, manganese sulfate 0.1-0.5g, surplus is water.
A kind of method producing chondroitin sulfate the most according to claim 1, it is characterised in that: described fermentation medium by
Sucrose, peptone, yeast powder, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, manganese sulfate and water composition, described buffer
By disodium hydrogen phosphate, sodium dihydrogen phosphate forms；In every L culture medium, containing sucrose 20-50g, peptone 10-15g, yeast powder 10-
15g, sodium chloride 0.5-2.0g, magnesium sulfate 1.5-2.5g, potassium dihydrogen phosphate 1.5-2.5g, calcium chloride 0.2-0.5g, manganese sulfate
0.05-0.15g, disodium hydrogen phosphate 1.0-4.0 g, sodium dihydrogen phosphate 0.3-3.0g surplus is water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610307883.XA CN105779532A (en) | 2016-05-11 | 2016-05-11 | Method for producing chondroitin sulfate |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610307883.XA CN105779532A (en) | 2016-05-11 | 2016-05-11 | Method for producing chondroitin sulfate |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591262A (en) * | 2016-12-07 | 2017-04-26 | 江南大学 | Method for improving yield of cold-adapted beta-galactosidase |
| CN107151685A (en) * | 2017-06-23 | 2017-09-12 | 山东众山生物科技有限公司 | A kind of method of chondroitin sulfate produced by fermentation method |
| CN110777179A (en) * | 2019-05-10 | 2020-02-11 | 赤峰蒙广生物科技有限公司 | Method for producing chondroitin sulfate by fermentation |
-
2016
- 2016-05-11 CN CN201610307883.XA patent/CN105779532A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591262A (en) * | 2016-12-07 | 2017-04-26 | 江南大学 | Method for improving yield of cold-adapted beta-galactosidase |
| CN107151685A (en) * | 2017-06-23 | 2017-09-12 | 山东众山生物科技有限公司 | A kind of method of chondroitin sulfate produced by fermentation method |
| CN107151685B (en) * | 2017-06-23 | 2020-06-02 | 山东众山生物科技有限公司 | Method for producing chondroitin sulfate by fermentation method |
| CN110777179A (en) * | 2019-05-10 | 2020-02-11 | 赤峰蒙广生物科技有限公司 | Method for producing chondroitin sulfate by fermentation |
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Application publication date: 20160720 |