CN101475971A - Method for producing D-ribose by microbial fermentation - Google Patents
Method for producing D-ribose by microbial fermentation Download PDFInfo
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- CN101475971A CN101475971A CNA2008100322828A CN200810032282A CN101475971A CN 101475971 A CN101475971 A CN 101475971A CN A2008100322828 A CNA2008100322828 A CN A2008100322828A CN 200810032282 A CN200810032282 A CN 200810032282A CN 101475971 A CN101475971 A CN 101475971A
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Abstract
The present invention provides a process for producing D-ribose by microorganism fermentation, including the following steps: taking glucose as raw material, producing D-ribose by microorganism fermentation in the fermentation medium, without adding calcium carbonate. By thoroughly resolving the dependence on the calcium carbonate for maintaining pH outside thalli cells in the fermentation process and combining with the sugar complementing technique, the invention has improved the D-ribose output to 80.0g/L and above, shortens the fermentation period to 38 - 45hr, and improves the sugar conversion rate to above 45.0%. As a result of not adding calcium carbonate in the fermentation liquor, the thalli in the fermentation liquor can be recycled for the producing the feeds, to further reduce production costs.
Description
Technical field
The present invention relates to the method for fermentative production D-ribose.
Background technology
D-ribose (D-ribose) is present among all animals, plant, the microorganism cells as the composition of genetic material Yeast Nucleic Acid, is a kind of pentose with important physiological significance.In food, medicine, chemical industry, has widespread use.D-ribose can extract from natural goods, also can be synthetic from material chemical conversions such as D-glucose, and that microbe fermentation method prepares D-ribose output is big, cost is low, the most suitable suitability for industrialized production.
The eighties, Kishimoto etc. are at patent JP 01/157, report utilizes bacillus pumilus transketolase deficient strain in 369, shake-flask culture 55hr under the situation of adding die aromatischen Aminosaeuren, the accumulation volume of D-ribose can reach 92.1g/L, nineteen ninety-five, seed selections such as Hoshino obtain tolerating D-ribose and the Hexose phosphate dehydrogenase and the high transketolase defective type bacillus pumilus of gluconokinase of high density, and the accumulation volume of shake flask fermentation D-ribose is up to 120.0g/L.De Wulf etc. are at J.Ferment.Bioeng., 82,1-7, report uses a strain transketolase defective type subtilis in 1996 documents, make D-ribose output reach 60.0g/L, and in depth studied the metabolism key enzyme system and the fermentation condition of D-ribose on this basis.The Deng Chong of Jiangsu institute of microbiology is bright to equal to utilize in 1997 ultraviolet light and chemomorphosis subtilis SM-18, seed selection obtains shikimic acid deficient strain JSIM-1018 high yield bacterium, shake flask fermentation D-ribose production peak can reach 92.0g/L, and mean yield is 64.0g/L in the fermentor tank.
In order to improve the output of D-ribose, mainly concentrate on the transformation of superior strain at present about the research of fermentative Production D-ribose, by traditional physics, chemomorphosis and biological domestication or advanced genetic engineering means, obtain the superior strain of transketolase defective type.
All rely on lime carbonate to keep the outer pH value of thalline born of the same parents wholly or in part in the existing report both at home and abroad, can not wholly replace fall lime carbonate.Because use lime carbonate can produce great amount of carbon dioxide during the fermentation, and also can produce great amount of carbon dioxide when when fermentation liquor treatment, calcium ion need be removed, increase the exhaust emissions amount.Also have because residual lime carbonate is arranged, thalline residual in the fermented liquid can not be advantageously used in producing feed.
Summary of the invention
Purpose of the present invention just is to provide a kind of method of Production by Microorganism Fermentation D-ribose, to overcome the above-mentioned defective that above technology exists.
The method of Production by Microorganism Fermentation D-ribose of the present invention comprises the steps:
With glucose is raw material, in fermention medium, does not add under the situation of lime carbonate and passes through producing D-ribose by microbial fermentation.
Concrete, comprise the steps:
(1) seed culture: will activate good bacterial classification and insert in the seed liquor substratum, and cultivate 12～16hr, obtain sophisticated seed for 36.0～37.0 ℃;
Said bacterial classification is selected from subtilis (Bacillus subtilis) and is bacterial classification well known in the art, existing report in US3970522;
The bacterial classification that said activation is good refers to, adopt? the method of bibliographical information activates in slant medium described bacterial classification;
(2) fermentation culture: with the sophisticated seed of step (1) is that 5.0～10.0% inoculum size inserts fermention medium by volume, and bubbling air carries out aerobic and cultivates;
Fermentation condition is as follows:
Leavening temperature is 35.5～37.5 ℃;
Fermented incubation time: 38～45hr;
Fermentation pressure is 0.02～0.08Mpa;
Mixing speed is 300～700r/min;
And, when control pH, begin to add glucose at different different suitable pH value and the dissolved oxygens of stage control;
Preferably, pH value, best dissolved oxygen scope and fermented incubation time are:
0～16 hour, the pH value was 6.5～7.5, and best dissolved oxygen scope is 45～50%;
16～45 hours, the pH value was 5.5～6.5, and best dissolved oxygen scope is 50～60%;
In the described method, said dissolved oxygen refers to by the measured dissolved oxygen relative value of plum Teller dissolved oxygen electrode, promptly uses the S-WAT suppressed zero, after fermentation initial calibration full scale, and the shown numerical value of dissolved oxygen electrode in the fermenting process.
In the described method, be to realize by the aqueous solution that stream adds alkaline matter in the different suitable pH value of different stage control;
Said alkaline matter includes but not limited to that weight concentration is 1～25% sodium hydroxide, potassium hydroxide or solution of ammonium hydroxide;
In the described method, the speed of adding glucose is 10.0～20.0mL/hr, and the glucose concn of adding is 50.0～80.0% (g/g);
Said slant medium is the aqueous solution that contains following proportion substance:
Sorbyl alcohol: 5.0g/L, peptone: 10.0g/L, yeast extract paste: 20.0g/L, K
2HPO
4: 2.0g/L, KH
2PO
4: 1.0g/L, NaCl:2.0g/L, agar: 20.0g/L, PH nature;
Said seed culture medium is the aqueous solution that contains following proportion substance:
Glucose: 20.0g/L, yeast extract paste: 2.0g/L, K
2HPO
4: 3.0g/L, KH
2PO
4: 1.0g/L, PH:7.0;
Said fermention medium is the aqueous solution that contains following proportion substance:
Glucose: 140.0～180.0g/L; Corn steep liquor: 10.0～30.0g/L; Yeast powder: 1.0～10.0g/L; KH
2PO
4: 1.0～10.0g/L; K
2HPO
4: 1.0～10.0g/L; (NH
4)
2SO
4: 1.0～10.0g/L; MnSO
4: 0～0.5g/L; MgSO
4: 0～0.5g/L; Polyethers defoamer: 0.01～10.0g/L;
Said polyethers defoamer is selected from polyoxyethylene polyoxypropylene glyceryl ether (GPE) or polyoxyethylene polyoxypropylene tetramethylolmethane ether (PPE).
Method of the present invention has solved in the fermenting process to keeping the dependence of the outer pH of thalline born of the same parents to lime carbonate by thorough, and the sugared technology of combination benefit is brought up to more than the 80.0g/L D-ribose output, fermentation period foreshortens to 38～45hr, and the sugar weight transformation efficiency is reached more than 45.0%.Because do not add lime carbonate in the fermented liquid, the thalline in the fermented liquid can reclaim and be used to produce feed, further reduces production costs.
Embodiment
Below in conjunction with specific examples the present invention is further described.It may be noted that following example only is used to describe the present invention, limits the scope of the invention and be not used in.
Embodiment 1
Subtilis (Bacillus subtilis) slant strains that activation is good is seeded to seed and shakes in the bottle, and the liquid amount that seed shakes bottle is 100/500mL, and the rotary type shaking table is cultivated 15hr for 36.5 ℃.
Seed culture medium consist of glucose 20.0g/L, yeast extract paste 2.0g/L, K
2HPO
43.0g/L, KH
2PO
41.0g/L.
Volume ratio inoculum size according to 10.0% inserts sophisticated seed liquor the 7L fermentation cylinder for fermentation that the 3.2L fermented liquid is housed.
Consisting of of fermented liquid: glucose (containing a part crystal water): 150.0g/L; Corn steep liquor 26.0g/L; Yeast powder: 3.0g/L; KH
2PO
4: 1.0g/L; K
2HPO
4: 3.0g/L; (NH
4)
2SO
4: 7.0g/L; MnSO
4: 0.05g/L; MgSO
4: 0.05g/L; Polyoxyethylene polyoxypropylene glyceryl ether (GPE): 1.0g/L, pH 7.8.
Controlled temperature is 36.0 ± 0.5 ℃ in the fermenting process; The tank pressure of fermentor tank: 0.04MPa.Stirring initial rotating speed is: 300r/min.
(0～16hr) dissolved oxygen is 50% to keep the thalli growth phase; Producing D-ribose phase (behind the 16hr) 60%, is 10% sodium hydroxide solution control pH with weight concentration, and the maintenance thalli growth phase, (0～16hr) pH was 6.5; Producing D-ribose phase (behind the 16hr) pH is 6.3, and dissolved oxygen is 50%, begins stream and add and mend sugar in control pH, and weight concentration is that the flow acceleration of 72.0% glucose is 10.0mL/hr, and mending sugared total amount is 200.0mL.Fermentation culture 40hr finishes, and surveying D-ribose content with the orcinol method is 81.3g/L, and the weight transformation efficiency is 45.2%.
Embodiment 2
With embodiment 1, wherein the polyethers defoamer is a polyoxyethylene polyoxypropylene tetramethylolmethane ether (PPE), and:
It is that 5% sodium hydroxide solution is regulated pH value that stream adds weight concentration, and the maintenance thalli growth phase, (0～16hr) pH was 6.4, dissolved oxygen 45%; It is 6.2 that thalline produces D-ribose phase (behind the 16hr) pH, and dissolved oxygen is 50%, and other condition is with embodiment 1, and fermented incubation time is 42hr, and surveying D-ribose content with the orcinol method is 78.6g/L, and the weight transformation efficiency is 43.6%.
Embodiment 3
With embodiment 1, wherein:
It is that 10% potassium hydroxide solution is regulated pH value that stream adds weight concentration, and the maintenance thalli growth phase, (0～16hr) pH was 6.6, dissolved oxygen 45%; It is 6.2 that thalline produces D-ribose phase (behind the 16hr) pH, and dissolved oxygen is 50%, and other condition is with embodiment 1, and fermented incubation time is 38hr, and surveying D-ribose content with the orcinol method is 84.5g/L, and the weight transformation efficiency is 46.9%.
Embodiment 4
With embodiment 1, wherein:
It is that 1% potassium hydroxide solution is regulated pH value that stream adds weight concentration, and the maintenance thalli growth phase, (0～16hr) pH was 6.6, dissolved oxygen 45%; It is 6.2 that thalline produces D-ribose phase (behind the 16hr) pH, and dissolved oxygen is 50%, and other condition is with embodiment 1, and fermented incubation time is 38hr, and surveying D-ribose content with the orcinol method is 71.7g/L, and the weight transformation efficiency is 39.8%.
Embodiment 5
With embodiment 1, wherein:
Stream adds saturated solution of ammonium hydroxide and regulates the pH value, and (0～16hr) control pH6.5, thalline produce D-ribose phase (behind the 16hr) control pH6.25, stop stream and add when the pH value is on the rise in earlier stage at thalli growth.Regulating rotating speed control thalli growth phase dissolved oxygen is 50%; It is 60% that thalline produces D-ribose phase (behind the 16hr) dissolved oxygen, and other condition is with embodiment 1, and fermentation culture 44hr is 86.9g/L with orcinol method survey D-ribose content, and the weight transformation efficiency is 48.3%.
Claims (8)
1. the method for a producing D-ribose by microbial fermentation is characterized in that, comprises the steps: with glucose to be raw material, in fermention medium, does not add under the situation of lime carbonate and passes through producing D-ribose by microbial fermentation.
2. method according to claim 1 is characterized in that, comprises the steps:
(1) seed culture: will activate good bacterial classification and insert in the seed liquor substratum, and cultivate 12～16hr, obtain sophisticated seed for 36.0～37.0 ℃;
Said bacterial classification is selected from subtilis (Bacillus subtilis);
(2) fermentation culture: with the sophisticated seed of step (1) is that 5.0～10.0% inoculum size inserts fermention medium by volume, and bubbling air carries out aerobic and cultivates;
And, when control pH, begin to add glucose at different different suitable pH value and the dissolved oxygens of stage control.
3. method according to claim 2 is characterized in that fermentation condition is as follows:
Leavening temperature is 35.5～37.5 ℃;
Fermented incubation time: 38～45hr;
Fermentation pressure is 0.02～0.08Mpa;
Mixing speed is 300～700r/min.
4. method according to claim 2 is characterized in that, said slant medium is the aqueous solution that contains following proportion substance:
Sorbyl alcohol: 5.0g/L, peptone: 10.0g/L, yeast extract paste: 20.0g/L, K
2HPO
4: 2.0g/L, KH
2PO
4: 1.0g/L, NaCl:2.0g/L, agar: 20.0g/L, PH nature;
Said seed culture medium is the aqueous solution that contains following proportion substance:
Glucose: 20.0g/L, yeast extract paste: 2.0g/L, K
2HPO
4: 3.0g/L, KH
2PO
4: 1.0g/L, PH:7.0;
Said fermention medium is the aqueous solution that contains following proportion substance:
Glucose: 140.0～180.0g/L; Corn steep liquor: 10.0～30.0g/L; Yeast powder: 1.0～10.0g/L; KH
2PO
4: 1.0～10.0g/L; K
2HPO
4: 1.0～10.0g/L; (NH
4)
2SO
4: 1.0～10.0g/L; MnSO
4: 0～0.5g/L; MgSO
4: 0～0.5g/L; Polyethers defoamer: 0.01～10.0g/L;
Said polyethers defoamer is selected from polyoxyethylene polyoxypropylene glyceryl ether (GPE) or polyoxyethylene polyoxypropylene tetramethylolmethane ether (PPE).
5. method according to claim 2 is characterized in that, described is to realize by the aqueous solution that stream adds alkaline matter in the different suitable pH value of different stage control.
6. method according to claim 4 is characterized in that, said alkaline matter includes but not limited to that weight concentration is 1%～25% sodium hydroxide, potassium hydroxide or ammonium hydroxide aqueous solution.
7. method according to claim 2 is characterized in that, the speed of adding glucose is 10.0～20.0mL/hr, and the glucose concn of adding is 50.0～80.0% (g/g).
8. method according to claim 2 is characterized in that, the pH of the different steps of fermentation and dissolved oxygen are: 0～16 hour, the pH value was 6.5～7.5, and dissolved oxygen is 45～50%; 16～45 hours, the pH value was 5.5～6.5, and dissolved oxygen is 50～60%.
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|---|---|---|---|
| CNA2008100322828A CN101475971A (en) | 2008-01-04 | 2008-01-04 | Method for producing D-ribose by microbial fermentation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103861330A (en) * | 2014-02-25 | 2014-06-18 | 苏州恒康新材料有限公司 | Antifoaming agent and preparation method thereof |
| CN113101254A (en) * | 2021-04-02 | 2021-07-13 | 仇俊鹏 | Plant-based fermented cosmetic raw material and preparation method thereof |
| CN113142542A (en) * | 2021-02-25 | 2021-07-23 | 仇俊鹏 | Fermented seasoning sauce using grains as raw materials, preparation method and application |
-
2008
- 2008-01-04 CN CNA2008100322828A patent/CN101475971A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103861330A (en) * | 2014-02-25 | 2014-06-18 | 苏州恒康新材料有限公司 | Antifoaming agent and preparation method thereof |
| CN113142542A (en) * | 2021-02-25 | 2021-07-23 | 仇俊鹏 | Fermented seasoning sauce using grains as raw materials, preparation method and application |
| CN113101254A (en) * | 2021-04-02 | 2021-07-13 | 仇俊鹏 | Plant-based fermented cosmetic raw material and preparation method thereof |
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Open date: 20090708 |