CN103014104A - Method for producing glutathione by high-density fermentation - Google Patents
Method for producing glutathione by high-density fermentation Download PDFInfo
- Publication number
- CN103014104A CN103014104A CN2012105747784A CN201210574778A CN103014104A CN 103014104 A CN103014104 A CN 103014104A CN 2012105747784 A CN2012105747784 A CN 2012105747784A CN 201210574778 A CN201210574778 A CN 201210574778A CN 103014104 A CN103014104 A CN 103014104A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- gsh
- high density
- yeast
- described high
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for producing glutathione by high-density fermentation. The method comprises the following steps of: with pichia pastoris (Pichia.pastoris SL1220) as a fermentation strain, inoculating primarily-cultured and secondarily-cultured secondary seeds to a yeast culture medium for fermentation cultivation; supplementing precursor amino acid of glutathione to control the carbon source concentration in the fermentation liquor to 0.01-0.02mol/L after the OD (Optical Density) of the fermentation liquor reaches 100-200; and cultivating for 48-64 hours. The method provided by the invention can greatly improve the yield of the glutathione and effectively reduce the fermentation cost.
Description
Technical field
The present invention relates to the biological fermentation engineering field, be specifically related to the method that high density fermentation is produced gsh.
Background technology
Gsh (glutathione, GSH) is the tripeptides that is combined into by L-glutamic acid, halfcystine and glycine, and the sulfydryl on the halfcystine is its active group.Gsh extensively is present in most animals, plant and the microorganism as a kind of active three skins, is most important a kind of non-albumen coloured glaze based compound in the organism.Gsh is divided into two kinds of reduced glutathion (GSH) and Sleep-promoting factor B (GSSG), exist in a large number in vivo and that play a major role is GSH, it has participated in many physiological activities mainly as antioxidant, immunostimulant and toxinicide in cell, the chemical, biological defense system of living organism is played an important role.
The generation of organism glutathion inside is mainly by synthetic and reduce two approach: the gsh biosynthesizing relies on the enzymatic reactions of Triphosaden to consist of by two steps, at first L-glutamic acid and halfcystine synthesize gamma-glutamyl cysteine under gamma-glutamylcysteine synthetase (γ-glutamylcysteine synthetase) catalysis, synthetic raw material comes by the extracellular transhipment or by the glutamyltranspeptidase reaction, then under glutathione synthetase (glutathionesynthetase) effect, gamma-glutamyl cysteine and glycine synthesizing glutathion.And gsh reduction approach can be acted on GSSG (GSSG) and obtained by glutathione reductase, participates in the NADPH of reductive action by phosphopentose pathway.In the normal physiological situation, reaction is mainly carried out to the GSH direction, but these two kinds of forms of GSH and GSSG can transform mutually.
Since gsh was found, it not only was widely used in the research mensuration of biological chemistry, medical science, biology and chemistry as reagent, and has become a kind of extremely important biochemical pharmacology.According to the prediction of the external apparatus of information, gsh will be more and more wide as the application of a kind of multi-functional biologically active additives in food-processing industry, and its demand increases just day by day.Along with biochemical functions and the character of gsh are constantly studied discovery, people, also will constantly increase its demand growing its interest on medicine industry, foodstuffs industry, sports field and relevant biological study field.So gsh has extremely wide market outlook.
Summary of the invention
The object of the invention is to use high density fermentation to produce gsh, can increase substantially gsh output, and effectively reduce fermentation costs.
For solving the problems of the technologies described above, technical scheme provided by the invention is: a kind of high density fermentation is produced the method for gsh, it comprises take pichia pastoris phaff (Pichia.pastoris SL1220) as fermentation strain, be inoculated in the yeast culture base by the secondary seed after one-level cultivation and the secondary cultivation and carry out fermentation culture, after fermented liquid OD reaches 100～200, add the precusor amino acids of gsh, carbon source concentration in the controlled fermentation liquid is cultured to till 48～64h at 0.01～0.02mol/L.The present invention selects pichia pastoris phaff (Pichia.pastoris SL1220) to be optimized cultivation.Pichia pastoris phaff is that the class in the methyl alcohol nutritional type yeast can utilize methyl alcohol as the yeast of sole carbon source and the energy.The same with other yeast, mainly exist with the monoploid form in asexual vegetative period, when environmental nutrient limits, often induce 2 mating type haploid cell mating that the physiology type is different, be fused into amphiploid.Selected pichia pastoris phaff is characterized in that desired bacterial strain is that the high yield biotechnology is transformed bacterium.
Further: in the method for above-specified high density glutathion production by fermentation, described one-level is cultivated and the secondary cultivation refers to the thalline of picking pichia pastoris phaff (Pichia.pastoris SL1220) fermentation strain from the fresh inclined-plane, access is equipped with in the 250ml shaking flask of 50 ± 5ml nutrient solution, 100 ± 5rpm, 32 ± 2 ℃ of shaking culture 10～12h; With 10% inoculum size first order seed is accessed the secondary seed nutrient solution again, control pH value 6.5 ± 0.5, dissolved oxygen gets secondary seed after cultivating 12～14h more than or equal to 50%, 32 ± 2 ℃, and described I and II nutrient solution all is bacteria culture fluids that those skilled in the art commonly use.The inoculum size that secondary seed is linked into the yeast culture base is 0.5～5%.Preferred inoculum size is 1.5%.The ratio that inoculum size refers to move into the volume of seed liquor and inoculates rear nutrient solution volume, the size of inoculum size is decided by to produce the speed of bacterial classification growth and breeding in fermentor tank, adopt larger inoculum size can shorten the time that the mycelia breeding peaks in the fermentor tank, the formation of product is arrived in advance, and can reduce the growth machine meeting of miscellaneous bacteria.But inoculum size is excessive or too small, all can affect fermentation.Cross conference and cause that dissolved oxygen is not enough, it is synthetic to affect product; And can too much move into metabolic waste, also uneconomical; The too small meeting Extending culture time, the productivity of reduction fermentor tank.The carbon source of yeast culture base is at least a in glucose, methyl alcohol, molasses, wort, the sucrose, the nitrogenous source of yeast culture base is the carbon source molasses preferably of at least a yeast culture base in yeast powder, yeast extract paste, peptone, bean cake powder, the ammoniacal liquor, and the nitrogenous source of yeast culture base is peptone preferably.Fermentation culture pH in the yeast culture base is 5.0～8.0, and the fermentation culture temperature is 25～32 ℃.Fermentation culture pH optimum in the yeast culture base is 6.5, and optimum fermentation culture temperature is 30 ℃.The precusor amino acids of gsh is at least a in Pidolidone, Cys, the L-glycine, preferably Cys.
Compared with prior art, the present invention selects pichia pastoris phaff (Pichia.pastoris SL1220) to be optimized cultivation.Pichia pastoris phaff is that the class in the methyl alcohol nutritional type yeast can utilize methyl alcohol as the yeast of sole carbon source and the energy.The same with other yeast, mainly exist with the monoploid form in asexual vegetative period, when environmental nutrient limits, often induce 2 mating type haploid cell mating that the physiology type is different, be fused into amphiploid.Selected pichia pastoris phaff is characterized in that desired bacterial strain is that the high yield biotechnology is transformed bacterium.So present method can increase substantially gsh output, and effectively reduce fermentation costs.
Embodiment
For better understanding the present invention, the invention will be described further below in conjunction with embodiment, but need to prove, the scope of protection of the presently claimed invention is not limited to the scope that embodiment explains.
Example 1
After fermentation strain (Pichia.pastoris SL1220) is cultivated through one-level, secondary, with 1%(v/v) inoculum size is seeded in the fermention medium that contains molasses and peptone, uses 50 liters of fermentor tanks to carry out cultivation and fermentation, air quantity 0.5～3.0M
3, rotating speed 100～700rpm, pH7.0,32 ℃ of temperature are cultivated 63h, by the ALLOXAN(tetraoxypyrimidine) and derivatization method measures the glutathione content in the fermented liquid, and the gsh productive rate is 4.32g/L as a result.
Example 2
After fermentation strain (Pichia.pastoris SL1220) is cultivated through one-level, secondary, with 1.5%(v/v) inoculum size is seeded in the fermention medium that contains molasses and peptone, uses 50 liters of fermentor tanks to carry out cultivation and fermentation, air quantity 0.5～3.0M
3, rotating speed 100～700rpm, pH7.0,32 ℃ of temperature, the fermentation later stage adds Cys, L-glycine, cultivates 54h, by the ALLOXAN(tetraoxypyrimidine) and derivatization method measures the glutathione content in the fermented liquid, and the gsh productive rate is 4.81g/L as a result.
Example 3
After fermentation strain (Pichia.pastoris SL1220) is cultivated through one-level, secondary, with 2.5%(v/v) inoculum size is seeded in the fermention medium that contains molasses and peptone, uses 50 liters of fermentor tanks to carry out cultivation and fermentation, air quantity 0.5～3.0M
3, rotating speed 100～700rpm, pH7.0,32 ℃ of temperature, the fermentation later stage adds Cys, cultivates 52h, by the ALLOXAN(tetraoxypyrimidine) and derivatization method measures the glutathione content in the fermented liquid, and the gsh productive rate is 4.53g/L as a result.
Example 4
After fermentation strain (Pichia.pastoris SL1220) is cultivated through one-level, secondary, with 1.5%(v/v) inoculum size is seeded in the fermention medium that contains molasses and peptone, uses 50 liters of fermentor tanks to carry out cultivation and fermentation, air quantity 0.5～3.0M
3, rotating speed 100～700rpm, pH6.0,32 ℃ of temperature, the fermentation later stage adds Cys, L-glycine and glutamic acid, cultivates 55h, by the ALLOXAN(tetraoxypyrimidine) derivatization method measures the glutathione content in the fermented liquid, and the gsh productive rate is 5.43g/L as a result.
Example 5
After fermentation strain (Pichia.pastoris SL1220) is cultivated through one-level, secondary, with 1.5%(v/v) inoculum size is seeded in the fermention medium that contains molasses and peptone, uses 50 liters of fermentor tanks to carry out cultivation and fermentation, air quantity 0.5～3.0M
3, rotating speed 100～700rpm, pH6.5,32 ℃ of temperature, the fermentation later stage adds Cys, L-glycine and glutamic acid, cultivates 55h, by the ALLOXAN(tetraoxypyrimidine) derivatization method measures the glutathione content in the fermented liquid, and the gsh productive rate is 5.74g/L as a result.
Example 6
After fermentation strain (Pichia.pastoris SL1220) is cultivated through one-level, secondary, with 1.5%(v/v) inoculum size is seeded in the fermention medium that contains molasses and peptone, uses 50 liters of fermentor tanks to carry out cultivation and fermentation, air quantity 0.5～3.0M
3, rotating speed 100～700rpm, pH6.5,30 ℃ of temperature, the fermentation later stage adds Cys, L-glycine and glutamic acid, cultivates 53h, by the ALLOXAN(tetraoxypyrimidine) derivatization method measures the glutathione content in the fermented liquid, and the gsh productive rate is 6.33g/L as a result.
Example 7
After fermentation strain (Pichia.pastoris SL1220) is cultivated through one-level, secondary, with 1.5%(v/v) inoculum size is seeded in the fermention medium that contains molasses and peptone, uses 50 liters of fermentor tanks to carry out cultivation and fermentation, air quantity 0.5～3.0M
3, rotating speed 100～700rpm, pH6.5,25 ℃ of temperature, the fermentation later stage adds Cys, L-glycine and glutamic acid, cultivates 55h, by the ALLOXAN(tetraoxypyrimidine) derivatization method measures the glutathione content in the fermented liquid, and the gsh productive rate is 5.84g/L as a result.
Claims (10)
1. a high density fermentation is produced the method for gsh, it is characterized in that comprising take pichia pastoris phaff (Pichia.pastoris SL1220) as fermentation strain, be inoculated in the yeast culture base by the secondary seed after one-level cultivation and the secondary cultivation and carry out fermentation culture, after fermented liquid OD reaches 100～200, add the precusor amino acids of gsh, carbon source concentration in the controlled fermentation liquid is cultured to till 48～64h at 0.01～0.02mol/L.
2. high density fermentation according to claim 1 is produced the method for gsh, it is characterized in that: described one-level is cultivated and the secondary cultivation refers to the thalline of picking pichia pastoris phaff (Pichia.pastoris SL1220) fermentation strain from the fresh inclined-plane, access is equipped with in the 250ml shaking flask of 50 ± 5ml nutrient solution, 100 ± 5rpm, 32 ± 2 ℃ of shaking culture 10～12h; With 10% inoculum size first order seed is accessed the secondary seed nutrient solution again, control pH value 6.5 ± 0.5, dissolved oxygen is cultivated more than or equal to 50%, 32 ± 2 ℃ must secondary seed behind 12～14h.
3. the described high density fermentation method of producing gsh according to claim 2, it is characterized in that: the inoculum size that secondary seed is linked into the yeast culture base is 0.5～5%.
4. the described high density fermentation method of producing gsh according to claim 3, it is characterized in that: preferred inoculum size is 1.5%.
5. the described high density fermentation method of producing gsh according to claim 3, it is characterized in that: the carbon source of yeast culture base is at least a in glucose, methyl alcohol, molasses, wort, the sucrose, and the nitrogenous source of yeast culture base is at least a in yeast powder, yeast extract paste, peptone, bean cake powder, the ammoniacal liquor.
6. the described high density fermentation method of producing gsh according to claim 5, it is characterized in that: the carbon source of yeast culture base is molasses preferably, and the nitrogenous source of yeast culture base is peptone preferably.
7. the described high density fermentation method of producing gsh according to claim 3, it is characterized in that: the fermentation culture pH in the yeast culture base is 5.0～8.0, the fermentation culture temperature is 25～32 ℃.
8. the described high density fermentation method of producing gsh according to claim 7, it is characterized in that: the fermentation culture pH in the yeast culture base is 6.5, the fermentation culture temperature is 30 ℃.
9. the described high density fermentation method of producing gsh according to claim 3 is characterized in that: the precusor amino acids of gsh is at least a in Pidolidone, Cys, the L-glycine.
10. the described high density fermentation method of producing gsh according to claim 9, it is characterized in that: the precusor amino acids of gsh is Cys.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105747784A CN103014104A (en) | 2012-12-26 | 2012-12-26 | Method for producing glutathione by high-density fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105747784A CN103014104A (en) | 2012-12-26 | 2012-12-26 | Method for producing glutathione by high-density fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103014104A true CN103014104A (en) | 2013-04-03 |
Family
ID=47963219
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012105747784A Pending CN103014104A (en) | 2012-12-26 | 2012-12-26 | Method for producing glutathione by high-density fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103014104A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586814A (en) * | 2017-09-28 | 2018-01-16 | 正大天晴药业集团股份有限公司 | A kind of method of glutathion production by fermentation |
| CN108018325A (en) * | 2017-08-23 | 2018-05-11 | 江南大学 | The method for improving glutathione yield |
| CN110396511A (en) * | 2018-04-25 | 2019-11-01 | 生百兴业有限公司 | The preparation method of zearalenone hydrolase |
| CN110698536A (en) * | 2019-10-30 | 2020-01-17 | 江西诚志生物工程有限公司 | Novel method for producing glutathione by adopting fermentation method |
| CN110746484A (en) * | 2019-12-12 | 2020-02-04 | 湖北工业大学 | Method for synthesizing glutathione by high-density fermentation of molasses and feather hydrolyzed powder |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225402A (en) * | 2007-01-16 | 2008-07-23 | 上海医药工业研究院 | Pichia yeast genetic engineering bacteria for producing glutathione and recombinant plasmid used for constructing the same |
| CN101613707A (en) * | 2008-06-27 | 2009-12-30 | 中国科学院上海生命科学研究院 | A kind of method of producing gsh with metabolic engineering bacteria |
-
2012
- 2012-12-26 CN CN2012105747784A patent/CN103014104A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225402A (en) * | 2007-01-16 | 2008-07-23 | 上海医药工业研究院 | Pichia yeast genetic engineering bacteria for producing glutathione and recombinant plasmid used for constructing the same |
| CN101613707A (en) * | 2008-06-27 | 2009-12-30 | 中国科学院上海生命科学研究院 | A kind of method of producing gsh with metabolic engineering bacteria |
Non-Patent Citations (1)
| Title |
|---|
| 王晓菊: "重组巴斯德毕赤酵母(Pichia pastoris)生产还原型谷胱甘肽条件及其分离纯化的研究", 《新疆农业大学硕士学位论文》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018325A (en) * | 2017-08-23 | 2018-05-11 | 江南大学 | The method for improving glutathione yield |
| CN107586814A (en) * | 2017-09-28 | 2018-01-16 | 正大天晴药业集团股份有限公司 | A kind of method of glutathion production by fermentation |
| CN110396511A (en) * | 2018-04-25 | 2019-11-01 | 生百兴业有限公司 | The preparation method of zearalenone hydrolase |
| CN110396511B (en) * | 2018-04-25 | 2021-05-18 | 生百兴业有限公司 | Preparation method of zearalenone hydrolase |
| CN110698536A (en) * | 2019-10-30 | 2020-01-17 | 江西诚志生物工程有限公司 | Novel method for producing glutathione by adopting fermentation method |
| CN110746484A (en) * | 2019-12-12 | 2020-02-04 | 湖北工业大学 | Method for synthesizing glutathione by high-density fermentation of molasses and feather hydrolyzed powder |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5587795B2 (en) | Fermentation medium and process thereof | |
| CN102168115A (en) | Industrialized production method of coenzyme Q10 | |
| CN103014104A (en) | Method for producing glutathione by high-density fermentation | |
| CN102965416A (en) | Method for producing cordycepin through semi-continuous liquid fermentation of cordyceps militaris | |
| CN1837352A (en) | Method for culturing heterotrophic chlorella with high density | |
| Xu et al. | Economic process to co-produce poly (ε-l-lysine) and poly (l-diaminopropionic acid) by a pH and dissolved oxygen control strategy | |
| CN102978252A (en) | L-tryptophan fed-batch fermentation technology | |
| CN102690762A (en) | Method for producing L-alanine by bioconverting L-aspartic acid by free cell process | |
| CN108265096B (en) | Preparation of pneumocandin B by microbial fermentation0Method (2) | |
| CN104419657A (en) | D-lactic acid producing strain with high growth rate and acid producing velocity and application thereof | |
| CN109593801A (en) | A kind of technique of fermenting and producing L-Trp | |
| CN101153294B (en) | Immobilized cell single-tank high-strength continuous fermentation process for succinic acid | |
| CN101955978A (en) | Method for improving concentration and production strength of succinic acid by adding osmotic pressure protective agent | |
| CN101709318B (en) | Method for preparing glutathione by fed-batch fermentation of Candida utilis | |
| CN103911419A (en) | Method for producing L-valine by combined fermentation of double bacterial strains | |
| CN103333926A (en) | Method for accelerating synthesis of epsilon-polylysine | |
| CN103525881A (en) | Method for producing L-aspartic acid converted from fumaric acid through Escherichia coli undergoing fermentation culture in large pot with volume of 20m<3> | |
| CN1952164A (en) | Combined fermentation process for producing D-lactic acid | |
| CN110387387A (en) | A kind of recombination bacillus coli produces the zymotechnique of L-Trp | |
| CN102181509A (en) | Method for producing vernine | |
| CN104593306A (en) | High-density culture method of escherichia coli strains HY-05C | |
| CN109161507A (en) | Corynebacterium glutamicum capable of producing L-ornithine at high yield and application thereof | |
| CN105861344A (en) | Synchronous culture method for improving yeast biomass and intracellular trehalose content | |
| CN103667107A (en) | Enterococcus faecium strain capable of producing L-lactic acid | |
| CN104250622A (en) | Solid culture method for improving liquor flavor substance Bacillus sp. B-010 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130403 |