CN102181504A - Applications of companion fungus activity extracellular fluid preparation in two-step fermentation of Vc - Google Patents
Applications of companion fungus activity extracellular fluid preparation in two-step fermentation of Vc Download PDFInfo
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- CN102181504A CN102181504A CN2011100624336A CN201110062433A CN102181504A CN 102181504 A CN102181504 A CN 102181504A CN 2011100624336 A CN2011100624336 A CN 2011100624336A CN 201110062433 A CN201110062433 A CN 201110062433A CN 102181504 A CN102181504 A CN 102181504A
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Abstract
The invention relates to the field of second-step fermentation of vitamin C, particularly discloses applications of a companion fungus activity extracellular liquid preparation in two-step fermentation of Vc, comprising the following applications: (1) selecting a companion fungus in two-step fermentation of Vc; inoculating the companion fungus into a beef extract peptone culture medium and the like; carrying out shaking culture at the temperature of 33 DEG C until the growth stage is stable; collecting fermentation mash; filtering abandoned mycelium by a 0.45 mu m filter membrane; ultrafiltering filtrate liquor by the filter membrane the cut-off molecular weight of which is 3-5 Dalton to concentrate into 10-20mg/ml protein, so as to prepare the companion fungus activity extracellular liquid preparation; carrying out sterile separation packing, and preserving at the low temperature; and (2) in the two-step fermentation of Vc, when the fermentation enters middle stage or later stage, respectively injecting 10-12L/100M3 activity extracellular liquid preparation into the fermentation tank; and simultaneously replenishing a sorbose aqueous solution until the final concentration of the sorbose can reach 10-11wt%. By utilizing the invention, the problems that the controllability of the fermentation process is poor, the high-low fluctuation of the fermentation level is excessive, the production is unstable, and the production efficiency is seriously influenced and the like can be solved.
Description
Technical field:
The present invention relates to vitamin C bio fermentation field, be specially the regulate and control method of fermentation level in a kind of Vc two stage fermentation, promptly the active extracellular fluid preparation of concomitance bacterium produces and the application in the Vc two stage fermentation.
Background technology:
Vitamins C second step fermentation is the mixed fermentation process of two kinds of bacterium, is class medium dependent form fermentation.Concomitance bacterium produces sour release of measuring with its medium and changes by the product acid (acid of Gu dragon) of release medium (active protein) promotion acid-producing bacteria in the fermentation.Fermentation and transform in the biologically active of the medium that relies on own, its release and its specific rule that played a role: mixed fungus fermentation initial stage, concomitance bacterium (as bacillus megaterium) is cultivated and was entered logarithmic phase in 4-6 hour, entered stationary phase to 24 hour in 12 hours, change decline phase afterwards over to, cell rupture, gemma disengages, no longer grow and metabolic process, also no longer including medium discharges, because the activity of medium can keep 12 hours during the fermentation, so concomitance bacterium efficiently promotes acid-producing bacteria to produce the window of opportunity of acid when being 12-40 hour.
The restriction that discharged by the bacillus megaterium biological activity, acid-producing bacteria (being the oxidizing glucose acidfast bacilli) primary growth is slower, produces acid seldom, is in accurate idle state; Enter ferment middle, secrete in a large number with the concomitance bacterium active protein, the acid-producing bacteria growth is quickened, and acid production rate significantly improves; Enter the fermentation later stage, replenish because of no longer including new medium, the acid-producing bacteria activity weakens gradually to termination, and ferment after 40 hours it, to produce sour measuring only be preceding 24 hours 25%.For improving fermentation efficiency, increasing output, need fully to excavate the ferment middle and the fermentation potential in later stage.
Summary of the invention:
The object of the present invention is to provide the application of the active extracellular fluid preparation of a kind of concomitance bacterium in the Vc two stage fermentation, solve in the prior art fermenting process poor controllability, fermentation level just fluctuate excessive, produce unstablely, production efficiency is difficult to problems such as further raising.
Technical scheme of the present invention is:
The application of the active extracellular fluid preparation of a kind of concomitance bacterium in the Vc two stage fermentation comprises the steps:
(1) select Vc two stage fermentation concomitance bacterium to insert in the beef-protein medium, 33 ℃ of concussions are cultured to the stationary phase of growing, collect fermentation liquid, abandon thalline with 0.45 μ m membrane filtration, filtrate is 10-20mg/ml with molecular weight cut-off 3-10 ten thousand daltonian ultra-filtration membrane ultrafiltration and concentration to protein contents, make active extracellular fluid preparation, aseptic subpackaged, cryopreservation;
(2) in the Vc two stage fermentation, when fermentation enters mid-term and/or later stage, inject active extracellular fluid preparation 10-12L/100M to fermentor tank respectively
3, add the sorbose aqueous solution to whole sorbose concentration simultaneously and reach 10-11wt%.
The application of the active extracellular fluid preparation of described concomitance bacterium in the Vc two stage fermentation, concomitance bacterium is bacillus megaterium, Bacillus licheniformis, bacillus cereus, shadow yeast, class alopecia candiyeast, bacillus thuringiensis or saccharomyces cerevisiae etc.
The application of the active extracellular fluid preparation of described concomitance bacterium in the Vc two stage fermentation, growth stationary phase of concomitance bacterium is for cultivating 20-30 hour.
The application of the active extracellular fluid preparation of described concomitance bacterium in the Vc two stage fermentation, the bacteria-free filtrate molecular weight cut-off is that 3-10 ten thousand daltonian ultra-filtration membrane ultrafiltration and concentration are to including 10-20mg protein/ml, promptly make active extracellular fluid preparation, but aseptic subpackaged, preservation 3-5 day below 20 ℃, but about 4 ℃ of preservation 10-20 days.
The application of the active extracellular fluid preparation of described concomitance bacterium in the Vc two stage fermentation, the Vc two stage fermentation enters mid-term and later stage (35-50 hour), adds active extracellular fluid preparation 10-12L/100M in fermentor tank
3, should add the fermentation sorbose aqueous solution of 10-20wt% simultaneously.
The present invention has following advantage:
1, the Vc two stage fermentation is the mixed fermentation of two kinds of bacterium, this mixed fungus fermentation, on producing, used more than 50 year, but because the interaction that mixes between bacterium is not investigated thoroughly fully, many problems have been exposed in the actual production, mainly comprise process poor controllability, fermentation level just fluctuate excessive, produce unstablely, have a strong impact on the further raising of production efficiency etc.The present invention can fundamentally solve fermentation level height, the excessive difficult problem of fluctuation, has stablized production, makes it really step into the track of high yield, stable yields.
2, the present invention develops the active extracellular fluid preparation of concomitance bacterium according to bioactive release of concomitance bacterium-bacillus megaterium and activity change; According to acid-producing bacteria, promptly the fermentation character of oxidizing glucose acidfast bacilli adds active extracellular fluid preparation in right amount in the specified phase of fermentation, supplies the disappearance of active media when the time comes, keeps the high-efficient operation of fermentation.
3, in the Vc two stage fermentation, when fermentation enters mid-term and later stage, add active born of the same parents' external preparation and sorb liquid glucose in right amount, can effectively regulate fermentation and develop towards predetermined direction, fermentation substrate sorbose concentration improves 20-40%, and fermentation yield improves 2-4 percentage point.
Embodiment:
The application of the active extracellular fluid preparation of concomitance bacterium of the present invention in the Vc two stage fermentation comprises the steps:
(1) select Vc two stage fermentation concomitance bacterium (bacillus megaterium, Bacillus licheniformis, bacillus cereus, shadow yeast, class alopecia candiyeast, bacillus thuringiensis or saccharomyces cerevisiae etc.) to insert in the beef-protein medium, 33 ℃ of concussions are cultured to the stationary phase (20-30 hour) of growing, collect fermentation liquid, abandon thalline with 0.45 μ m membrane filtration, filtrate is 10-20mg/ml with molecular weight cut-off 3-5 ten thousand daltonian ultra-filtration membrane ultrafiltration and concentration to protein contents, make active extracellular fluid preparation, aseptic subpackaged, cryopreservation;
(2) in the Vc two stage fermentation, when fermentation enter mid-term (fermenting 35-40 hour) and/or later stage (fermenting 41-50 hour), inject active extracellular fluid preparation 10-12L/100M to fermentor tank respectively
3, the sorbose aqueous solution to the whole sorbose concentration of adding concentration simultaneously and be 10-20wt% reaches 10-11wt%, fermentation period 50 hours, and fermentation conversion rate reaches 92%.
Embodiment 1:
Select Vc fermentation combination bacterium G.o+B2980, the number ratio of G.o and B2980 is about 1000: 1 in the combination bacterium, inserts 250ml triangular flask (including beef-protein medium 20ml), 29 ℃, concussion (180 rev/mins) was cultivated 35 hours, add active extracellular fluid preparation 0.2ml, sorbose 0.4g, continue to cultivate 8 hours, add active extracellular fluid preparation 0.2ml again, sorbose 0.2g, secondary fermentation in 6 hours finishes, and fermentation yield improves 3%, and fermentation substrate concentration improves 37%.
Wherein, G.o (Gluconobacter oxydans) is an acid-producing bacteria, i.e. oxidizing glucose acidfast bacilli, and B2980 is concomitance bacterium, i.e. bacillus megaterium.
Embodiment 2:
Choosing combination bacterium G.o+B2980, the number ratio of G.o and B2980 is about 1000: 1 in the combination bacterium, insert 250ml triangular flask (including beef-protein medium 20ml), 29 ℃, concussion (180 rev/mins) was cultivated 42 hours, added active extracellular fluid preparation 0.2ml, sorbose 0.4g, continue fermentation 6 hours, fermentation ends, fermentation yield improves 2.7%, and fermentation substrate concentration improves 25%.
Embodiment 3-6:
Difference from Example 1 is (table 1):
Table 1
In the table 1, in the G.o+B2980 combination bacterium, the number ratio of G.o and B2980 is about 1000: 1.
Application examples 1:
4M
3Fermentor tank injects 2.8 tons of beef-protein mediums, inserts 0.3 ton of G.o+B2980 kind liquid, and conventional fermentation 40 hours adds active extracellular fluid preparation 0.5L, ferments 4 hours, improves ancient imperial sour yield 3.0%; Fermenting reached terminal point in 6 hours, and contrast finished fermentation in 4 hours ahead of time.
Application examples 2:
4M
3Fermentor tank, inject 2.8 tons of beef-protein mediums, insert 0.3 ton of G.o+B2980 kind liquid, conventional fermentation 35 hours adds active extracellular fluid preparation 0.4L, sorbose 56kg, continues fermentation after 8 hours, add active extracellular fluid preparation 0.5L again, secondary fermentation in 6 hours finishes, and improves ancient imperial sour yield 4.6%, and sorbose concentration has increased by 25%.
Claims (5)
1. the application of the active extracellular fluid preparation of concomitance bacterium in the Vc two stage fermentation is characterized in that, comprises the steps:
(1) select Vc two stage fermentation concomitance bacterium to insert in the beef-protein medium, 33 ℃ of concussions are cultured to the stationary phase of growing, collect fermentation liquid, abandon thalline with 0.45 μ m membrane filtration, filtrate is 10-20mg/ml with molecular weight cut-off 3-10 ten thousand daltonian ultra-filtration membrane ultrafiltration and concentration to protein contents again, make active extracellular fluid preparation, aseptic subpackaged, cryopreservation;
(2) in the Vc two stage fermentation, when fermentation enters mid-term and/or later stage, inject active extracellular fluid preparation 10-12L/100M to fermentor tank respectively
3, add the sorbose aqueous solution to whole sorbose concentration simultaneously and reach 10-11wt%.
2. according to the application of the active extracellular fluid preparation of the described concomitance bacterium of claim 1 in the Vc two stage fermentation, it is characterized in that concomitance bacterium is bacillus megaterium, Bacillus licheniformis, bacillus cereus, shadow yeast, class alopecia candiyeast, bacillus thuringiensis or saccharomyces cerevisiae etc.
3. according to the application of the active extracellular fluid preparation of the described concomitance bacterium of claim 1 in the Vc two stage fermentation, it is characterized in that growth stationary phase of concomitance bacterium is for cultivating 20-30 hour.
4. according to the application of the active extracellular fluid preparation of the described concomitance bacterium of claim 1 in the Vc two stage fermentation, it is characterized in that, filtrate is that 3-10 ten thousand daltonian ultra-filtration membrane ultrafiltration and concentration are to including 10-20mg protein/ml with molecular weight cut-off, active extracellular fluid preparation, aseptic subpackaged, preservation 3-5 day below 20 ℃, 4 ℃ of preservation 10-20 days.
5. according to the application of the active extracellular fluid preparation of the described concomitance bacterium of claim 1 in the Vc two stage fermentation, it is characterized in that, 35-50 hour ferment middle of Vc two stage fermentation and fermentation later stage, in fermentor tank, add active extracellular fluid preparation 10-12L/100M
3, adding concentration simultaneously is the sorbose aqueous solution of 10-20wt%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634562A (en) * | 2012-04-13 | 2012-08-15 | 天津大学 | Method for detecting nutrition environment change in subculture process of vitamin C production strain |
| CN102978273A (en) * | 2012-11-13 | 2013-03-20 | 中国科学院沈阳应用生态研究所 | Method using three-bacterium mixed fermentation to convert sorbose into 2-keto-L-gulonic acid |
| CN105420222A (en) * | 2015-12-04 | 2016-03-23 | 帝斯曼江山制药（江苏）有限公司 | Mutation screening method of ketogulonigenium vulgare |
| CN110295206A (en) * | 2019-07-11 | 2019-10-01 | 沈阳中科微道生物科技有限公司 | A kind of method and its application using the fermented abandoned mash preparation association preparation of Vc |
-
2011
- 2011-03-15 CN CN2011100624336A patent/CN102181504A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634562A (en) * | 2012-04-13 | 2012-08-15 | 天津大学 | Method for detecting nutrition environment change in subculture process of vitamin C production strain |
| CN102978273A (en) * | 2012-11-13 | 2013-03-20 | 中国科学院沈阳应用生态研究所 | Method using three-bacterium mixed fermentation to convert sorbose into 2-keto-L-gulonic acid |
| CN102978273B (en) * | 2012-11-13 | 2014-01-08 | 中国科学院沈阳应用生态研究所 | Method using three-bacterium mixed fermentation to convert sorbose into 2-keto-L-gulonic acid |
| CN105420222A (en) * | 2015-12-04 | 2016-03-23 | 帝斯曼江山制药（江苏）有限公司 | Mutation screening method of ketogulonigenium vulgare |
| CN110295206A (en) * | 2019-07-11 | 2019-10-01 | 沈阳中科微道生物科技有限公司 | A kind of method and its application using the fermented abandoned mash preparation association preparation of Vc |
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Application publication date: 20110914 |