CN101942494A - Method for preparing VB12 by using fermentation method - Google Patents
Method for preparing VB12 by using fermentation method Download PDFInfo
- Publication number
- CN101942494A CN101942494A CN 201010272408 CN201010272408A CN101942494A CN 101942494 A CN101942494 A CN 101942494A CN 201010272408 CN201010272408 CN 201010272408 CN 201010272408 A CN201010272408 A CN 201010272408A CN 101942494 A CN101942494 A CN 101942494A
- Authority
- CN
- China
- Prior art keywords
- steep liquor
- corn steep
- fermentation
- fermentation method
- prepares
- 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 relates to a method for preparing VB12 in the field of fermentation engineering, in particular to a method for preparing VB12 by using an anaerobic fermentation method. The method comprises the following steps of: purifying corn syrup; preparing a culture medium with the purified corn syrup and inoculating propionibaterium shermanii for anaerobic fermentation; and extracting and refining VB12 from the fermentation liquid through a general program to obtain a VB12 product. The method is adopted to improve a general anaerobic fermentation process of the propionibaterium shermanii and increase the fermentation titer.
Description
Technical field
The present invention relates to a kind of field of fermentation engineering that is used for and produce VB
12Method, especially a kind of Unareobic fermentation prepares VB
12Method.
Background technology
Vitamins B
12(VB
12) be a kind of important water-soluble vitamins, claim cobalami again, can be subdivided into again according to the substituent difference of molecule: cyanocobalamin, adenosylcobalamin, hydroxycobalamine, methyl cobalamin etc., VB
12Be mainly used in industries such as medicine, food, feed.At present, VB
12Adopt Production by Microorganism Fermentation, mainly contain anaerobic technique that adopts Xie Shi propionibacterium (Propionibacteria shermanni) and the aerobic process that adopts Pseuomonas denitrifican.Wherein, the fermentation unit height of aerobic process, cost are low, occupy an leading position in cyanocobalamin and feed grade product, and the quality of the adenosylcobalamin that anaerobic technique obtains are best.Bacterium acidi propionici becomes propionic acid, acetate and carbonic acid gas by anaerobically fermenting with glucose metabolism, and this basic biochemical route needs the catalysis of 2-methylmalonyl-CoA isomerase, this enzyme require VB
12As coenzyme, bacterium acidi propionici can excessively synthesize and store this coenzyme in cell, therefore can obtain VB through further separation and purification by cultivating bacterium acidi propionici and harvested cell
12Product.The minimum medium of fermentation adopts 1%～3% corn steep liquor and 5%～10% carbohydrate (glucose etc.), and wherein, the inner quality of corn steep liquor has a strong impact on fermentation level, the anaerobism VB that causes because of corn steep liquor
12The fluctuation of fermentation unit can reach 10%～30% amplitude.
Summary of the invention
Be to solve the deficiencies in the prior art, the invention provides and a kind ofly can improve anaerobism VB
12The fermentation method of fermentation unit prepare VB
12Method.
For achieving the above object, fermentation method of the present invention prepares VB
12Method, comprise the steps:
A carries out purifying treatment to corn steep liquor;
B adopts the corn steep liquor preparation substratum through above-mentioned processing, behind the inoculation Xie Shi propionibacterium, carries out anaerobically fermenting;
C, the extraction of the frequent rules preface of the fermented liquid of step b gained being carried out VB12 is refining, obtains VB
12Product.Concretely, refining according to the extraction that filtration, hydrolysis, resin absorption, parsing concentrate, conventional procedure such as crystallization carries out VB12 successively, obtain VB
12Product.
Concretely, above-mentioned steps a comprises the steps:
A1 dilutes common corn steep liquor;
A2 adds the compound that contains calcium ion in the corn steep liquor after dilution, regulate pH4.5～8.0.
Can comprise step behind the step a2:
A3, the corn steep liquor that adding is contained ionic calcium cpd carries out heat treated at 30 ℃～90 ℃, obtains containing the corn steep liquor of insoluble flocculent precipitate, heating, it is fast to make throw out form speed, and precipitation is many, and the ability that is adsorbed with harmful substances is strong, and filtration velocity is fast.
Further, comprise step behind the step a3:
A4 carries out liquid-solid separation with the corn steep liquor that contains insoluble flocculent precipitate of step a3 gained, removes throw out.
The conventional corn slurry is diluted to dry matter content 5%～20% (w/v) among the step a1.
Concrete, the compound that contains calcium ion is at least a in calcium hydroxide, calcium oxide, the calcium chloride.
When the compound that contains calcium ion was calcium hydroxide, its add-on was 5 grams～100 gram calcium hydroxides/kg corn slurry dry-matter.
Corn steep liquor dry matter content in the step b substratum after purifying treatment is 0.5%～4% (w/v).
Leavening temperature is 25～35 ℃ among the step b, and fermented incubation time is 48～120 hours.
Seed culture and fermentation culture pH value are 5.0～7.5 among the step b.
Adopt technical scheme of the present invention, because compound that contains calcium ion that adds in step a and the phytic acid in the corn steep liquor react, form insoluble flocculent precipitate, liquid-solid separation is the precipitation that forms for except that dereaction, and this throw out can adsorb some VB more when temperature is higher
12The supressor of fermentation, so liquid-solid generally speaking separation should be carried out behind step a3 while hot, wherein, it is most convenient that filtering mode is adopted in liquid-solid separation, also can adopt the mode of centrifugation.
Among the step a1, owing to depend on the concentration of corn steep liquor on the fine or not certain degree of reaction, in general, lower concentration is handled and is helped following VB
12Fermentation, but low excessively concentration is inconvenient to use, and therefore, corn steep liquor is diluted to 5%～20% (w/v) can obtains effect preferably.Can obtain cleaning effect preferably when corn steep liquor dilution back concentration is lower than 5%, but operational volume is bigger than normal a bit, but is higher than 20%, supressor is removed not enough, therefore, scope is placed on 5-20%.
In addition, seed culture and fermentation culture pH value are controlled at 5.0～7.5 among the step b, about fermentation was to 48 hours or residual sugar be about 3% to add precursor DBI (5,6-dimethyl benzene imipramine), then the effect of fermentation can be better.
In sum, adopt technique scheme, can improve VB from two aspects
12Fermentation: 1, because the purification of corn steep liquor descends the content of some supressor wherein, fermentation titer is improved.At this moment, because the minimizing of phytic acid is to not influence of fermentation, but it is influential to the filtration after the fermentation ends, phytic acid content is crossed low solid-liquid separation difficulty and is strengthened, so mutually adjusting of temperature and pH also is in order not only to remove supressor but also to protect phytic acid, both take into account mutually, 2, because the purification of corn steep liquor, the original microorganism of corn steep liquor bacterium (as the milk-acid bacteria etc.) quantity of mixing is descended, reduced the sterilization conditions needed, also reduced the microbiological contamination rate in the fermentation.
Embodiment
Below in conjunction with specific embodiment the present invention is done further to describe in detail:
Embodiment one
Step a, the purifying treatment of corn steep liquor
A1, with tap water conventional corn slurry being diluted to dry matter content is 10% concentration;
A2 gets dry matter content and is 10% corn steep liquor 1L, adds calcium hydroxide solid or emulsion, makes pH regulator to 5.5;
A3 is heated to 60 ℃ with the corn steep liquor of above-mentioned adding calcium hydroxide, keeps 10min, flocculation sediment occurs;
A4, vacuum filtration removes filter cake, obtains to purify corn steep liquor 0.94L.
Get 10 milliliters and purify corn steep liquor, dried 2 hours for 105 ℃, measuring its dry matter content is 9.2%, and it is standby that all the other purify corn steep liquors refrigeration.
Step b, fermentation
(1) bacterial strain: Xie Shi propionibacterium (Propionibacteria shermanni)
(2) solid culture based formulas (w/v): glucose 3%, corn steep liquor dry-matter 1%, potassium primary phosphate 0.1%, cobalt chloride 0.001%, agar 2%, pH6.5-7.2.
(3) first order seed and secondary seed medium prescription (w/v): glucose 5%, corn steep liquor dry-matter 1%, potassium primary phosphate 0.15%, cobalt chloride 0.0015%, pH7.0.
(4) fermentative medium formula (dry w/v): glucose 7.0%, corn steep liquor dry-matter 2%, potassium primary phosphate 0.15%, cobalt chloride 0.0015%, pH7.0
(5) sterilization: adopt sugar, slurry sterilization process respectively, untoward reaction is avoided in sterilization in 115 ℃, 25 minutes.
(6) zymotechnique: adopt 200 milliliters of fermentations of the bottled liquid of 250ml triangle, inoculum size is 15%.That is: one-level kind 200ml inserts refrigerator preservation seed 1ml, cultivated 48 hours, and 30 ℃ of culture temperature, per 8 hours with aseptic ammoniacal liquor accent pH7.0; Secondary seed 170ml inserts first order seed 30ml, cultivates 48 hours, and per 8 hours with aseptic ammoniacal liquor accent pH7.0, fermentation flask 170ml inserts secondary seed 30ml, and 30 ℃ of culture temperature were transferred pH7.0 with aseptic ammoniacal liquor in per 8 hours, added 15ppmDBI, cultivated 84 hours in the 48th hour.
(7) contrast design: the checking group adopts and purifies corn steep liquor, and control group adopts the conventional corn slurry.
(8) method of inspection: liquid phase method detects VB
12Fermentation titer.
(9) comparing result: purify corn steep liquor as can be seen from Table 1 fermentation unit is had a very significant increase.
Table 1 conventional corn slurry and purification corn steep liquor are to VB
12The influence of fermentation
| VB 12Fermentation titer mg/L | VB 12Relative value % | |
| Conventional corn slurry control canisters | 30.3 | 100％ |
| Purify the corn steep liquor test tank | 56.8 | 187.4％ |
(10) fermented liquid of gained, refining according to the extraction that filtration, hydrolysis, resin absorption, parsing concentrate, conventional procedure such as crystallization carries out VB12, obtain the VB12 product.
Embodiment two
Step a, the purifying treatment of corn steep liquor
A1 is that to be diluted to dry matter content be 12% concentration to 41.9% conventional corn slurry with tap water with dry matter content;
A2 adds calcium hydroxide, makes pH regulator to 6.0;
A3 with the corn steep liquor steam heating to 65 of above-mentioned adding calcium hydroxide ℃, kept 20 minutes;
A4, filter press obtains to purify corn steep liquor.
Step b, fermentation
Bacterial strain: Xie Shi propionibacterium (Propionibac teria shermanni)
(1) seed tank culture based formulas (w/v): glucose 5%, corn steep liquor 1%, potassium primary phosphate 0.15%, cobalt chloride 0.0015%, pH7.0.
(2) fermentative medium formula (dry w/v):
Control canisters (120 tons of jars, 100 tons of dress liquid): glucose 7.0%, conventional corn slurry 2%, potassium primary phosphate 0.15%, cobalt chloride 0.0015%, pH7.0
Experimental tank (120 tons of jars, 100 tons of dress liquid): glucose 7.0%, purification corn steep liquor 2%, potassium primary phosphate 0.15%, cobalt chloride 0.0015%, pH7.0
(3) zymotechnique:
Preservation bacterial classification----the female bottle of triangular flask activated seed-----first class seed pot (1 ton of jar), 30 ℃ of 60 hours-----10% that spread cultivation are inoculated into secondary seed jar (10 tons of jars), 30 ℃ of 48 hours-----10% that spread cultivation are inserted fermentor tank (100 tons), add DBI, cultivate altogether 86 hours in 48 hours.Per 8 hours ammoniacal liquor of seed culture process is regulated pH7.0, and per 4 hours ammoniacal liquor of fermenting process is regulated pH7.0.
(4) method of inspection: liquid phase method detects vitamins B
12Fermentation titer.
(5) experimental result: by table 2 as seen, under the situation that other compositions and operating process parameter all remain unchanged, purify the VB of corn steep liquor group
12Output is 164% of control group, when the present invention uses on 120 tons of fermentor tanks, and VB
12Increase rate also fairly obvious.
Table 2 purifies corn steep liquor at VB
12Experimental result in the fermentative production
| The fermentation titer mg/L of VB12 | VB12 relative value % | |
| Conventional corn slurry control canisters | 30.7 | 100％ |
| Purify the corn steep liquor test tank | 50.2 | 164％ |
(6) fermented liquid of gained, refining according to the extraction that filtration, hydrolysis, resin absorption, parsing concentrate, conventional procedure such as crystallization carries out VB12, obtain the VB12 product.
Claims (10)
1. a fermentation method prepares VB
12Method, it is characterized in that this method comprises the steps:
A carries out purifying treatment to corn steep liquor;
B adopts the corn steep liquor preparation substratum through above-mentioned processing, behind the inoculation Xie Shi propionibacterium, carries out anaerobically fermenting;
C, the extraction of the frequent rules preface of the fermented liquid of step b gained being carried out VB12 is refining, obtains VB
12Product.
2. fermentation method according to claim 1 prepares VB
12Method, it is characterized in that described step a comprises the steps:
A1 dilutes common corn steep liquor;
A2 adds the compound that contains calcium ion in the corn steep liquor after dilution, and regulates pH4.5-8.0.
3. fermentation method according to claim 2 prepares VB
12Method, it is characterized in that comprising behind the described step a2 step:
A3, the corn steep liquor that adding is contained ionic calcium cpd carries out heat treated at 30 ℃～90 ℃, obtains containing the corn steep liquor of insoluble flocculent precipitate.
4. fermentation method according to claim 3 prepares VB
12Method, it is characterized in that comprising behind the described step a3 step:
A4 carries out liquid-solid separation with the corn steep liquor that contains insoluble flocculent precipitate of step a3 gained, removes throw out.
5. prepare VB according to each described fermentation method among the claim 2-4
12Method, it is characterized in that: conventional corn slurry among the step a1 is diluted to dry matter content 5%-20% (w/v).
6. prepare VB according to each described fermentation method among the claim 2-4
12Method, it is characterized in that: the described compound that contains calcium ion is at least a in calcium hydroxide, calcium oxide, the calcium chloride.
7. fermentation method according to claim 6 prepares VB
12Method, it is characterized in that: the described compound that contains calcium ion is a calcium hydroxide, its add-on is 5 grams～100 gram calcium hydroxides/kg corn slurry dry-matteies.
8. prepare VB according to each described fermentation method among the claim 1-4
12Method, it is characterized in that: it is 0.5%-4% (w/v) that corn steep liquor dry-matter in the step b substratum after purifying treatment adds content.
9. prepare VB according to each described fermentation method among the claim 1-4
12Method, it is characterized in that: leavening temperature is 25-35 ℃ among the step b, fermented incubation time is 48～120 hours.
10. prepare VB according to each described fermentation method among the claim 1-4
12Method, it is characterized in that: seed culture and fermentation culture pH value are for 5.0-7.5 among the step b.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010272408 CN101942494A (en) | 2010-09-06 | 2010-09-06 | Method for preparing VB12 by using fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010272408 CN101942494A (en) | 2010-09-06 | 2010-09-06 | Method for preparing VB12 by using fermentation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101942494A true CN101942494A (en) | 2011-01-12 |
Family
ID=43434699
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010272408 Pending CN101942494A (en) | 2010-09-06 | 2010-09-06 | Method for preparing VB12 by using fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101942494A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358797A (en) * | 2019-08-13 | 2019-10-22 | 南京牧奇亚生物科技有限公司 | A kind of fermentation method production method of vitamin B12 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1032333A (en) * | 1964-02-18 | 1966-06-08 | Chinoin Gyogyszer Es Vegyeszet | Process for the production of compounds of the b-vitamin group |
-
2010
- 2010-09-06 CN CN 201010272408 patent/CN101942494A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1032333A (en) * | 1964-02-18 | 1966-06-08 | Chinoin Gyogyszer Es Vegyeszet | Process for the production of compounds of the b-vitamin group |
Non-Patent Citations (2)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库工程科技I辑》 20100515 马蕙等 发酵法生产丙酸的工艺研究 B016-3 , 第05期 2 * |
| 《生物工程学报》 20080625 马蕙等 维生素B12的生物合成、发酵生产与应用 第927-932页 第24卷, 第6期 2 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358797A (en) * | 2019-08-13 | 2019-10-22 | 南京牧奇亚生物科技有限公司 | A kind of fermentation method production method of vitamin B12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Anastassiadis et al. | Citric acid production patent review | |
| CN102268490B (en) | Clean technique for co-producing xylose, xylitol and arabinose from agricultural waste and forest waste | |
| CN102174449B (en) | Method for producing high-yield gamma-propalanine and application thereof | |
| US20240102058A1 (en) | Caproate-producing bacterium with multiple substrate utilization capabilities and its applications | |
| CN103695489B (en) | A kind of arginine process for refining | |
| CN101705253B (en) | Method for treating xylose mother solution | |
| CN108251476B (en) | Method for extracting vitamin B12 from enzyme preparation wastewater | |
| CN110904163A (en) | Method for improving lactic acid content of corn steep liquor | |
| CN113321580A (en) | Method for producing malic acid | |
| CN103693779B (en) | A kind for the treatment of process of lysine fermentation liquor Waste water concentrating liquid and the method for fermentative production Methionin | |
| CN104531810A (en) | Method for preparing maltonic acid through efficient microbial conversion | |
| CN102533890A (en) | Production method of lysine | |
| CN100497611C (en) | Method for preparing nuclease P1 by ferment process | |
| CN108977482B (en) | Preparation method of polymyxin B sulfate | |
| CN103695490A (en) | High-purity arginine production process | |
| CN101475970A (en) | Method for producing crystal D-ribose | |
| CN101942494A (en) | Method for preparing VB12 by using fermentation method | |
| CN103667367A (en) | Method for producing mannitol by taking brown sugar as carbon source through fermentation of leukonid | |
| CN102181490B (en) | Method for producing citric acid | |
| CN103992964A (en) | High pH value tolerant bacterial strain and novel fermentation method for producing lysine | |
| Chin et al. | Effect of recycled laboratory backset on fermentation of wheat mashes | |
| CN105189766A (en) | Fermentation based on hydrolyzed corn and/or sugar cane mash to produce propionic acid | |
| CN1074242A (en) | The method that using solid distillers ' grains, yellow water by fermentation are produced lactic acid | |
| CN103667383B (en) | The preparation method of L-glutaminate | |
| CN101974500A (en) | Production method of high-purity and intermediate-temperate alpha-amylase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110112 |