CN101736071B - Method for producing 2-keto-l-gulonic acid - Google Patents
Method for producing 2-keto-l-gulonic acid Download PDFInfo
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- CN101736071B CN101736071B CN200910260056XA CN200910260056A CN101736071B CN 101736071 B CN101736071 B CN 101736071B CN 200910260056X A CN200910260056X A CN 200910260056XA CN 200910260056 A CN200910260056 A CN 200910260056A CN 101736071 B CN101736071 B CN 101736071B
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Abstract
The invention provides a method for producing 2-keto-l-gulonic acid, including the following steps of: feeding nitrogen source nutrient solution from the 20th to the 26th hour in the fermentation process to compensate nutritional demands of microorganism growth in the fermentation process, finishing feeding solution in the 40th to the 45th hour, wherein the dissolved oxygen is controlled to be 30-50% in the nutrient solution feeding process. The invention utilizes a method of feeding nutrient solution at middle and later stages of fermentation, overcomes disadvantage that biomass grows and generates acid slowly, combines control of factors of temperature, pH, dissolved oxygen, low initial glucose, fed-batch glucose compensation and the like, and optimizes the growth relations of biomasses with different sizes in microbial mixed fermentation. The gulonic acid content of the invention is 105-115mg/ml, and the glucose acid convert ratio is 92-95%.
Description
Technical field
The present invention relates to the working method of the ancient dragon acid of a kind of 2-ketone group-L-, belong to the microorganisms producing field.
Background technology
The ancient dragon acid of the domestic production at present two-step fermentings that adopt more; What two-step fermenting adopted is mixed fungus fermentation, and two kinds of bacterium commonly used are a kind of to be that the oxidizing glucose acidfast bacilli is commonly called as little bacterium, and another kind is that bacillus megaterium is commonly called as big bacterium; Its medium and small bacterium is an acid-producing bacteria; Emiocytosis L-sorb ketooxidase can be oxidized to the ancient dragon acid of 2-ketone group-L-with sorbose, but the single culture poor growth, and acid producing ability is low; Big bacterium can not produce acid, but it can be used as the concomitance bacterium of little bacterium, can promote the growth of little bacterium and produce acid.
Confirm that by research big mycetocyte body fluid and extracellular fluid bacterium all can promote little bacteria growing, shorten the lag period of little bacteria growing.In the mixed bacterium culturing process of two stage fermentation; The interaction of two kinds of bacteria growings is key points, expect the acid of high density product, and its fermented liquid must drop into the sorbose of high density; And the sorbose of higher concentration has inhibition to microorganism growth; The producer of the ancient dragon acid of domestic production generally adopts the initial sorbose concentration of fermented liquid lower, adds the method for mending sugar through batch benefit sugar or stream, with the inhibition of minimizing high density sorbose to microorganism growth.This method makes that the initial loading liquid measure is lower; The fermentation later stage increases fermentating liquid volume along with mending sugar, and cell concentration descends, nutritive ingredient in the fermented liquid (mainly being nitrogenous source) deficiency; Microorganism growth is obstructed; It is sour low to cause the middle and later periods to be produced, and can not give full play to the high acid ability of this two stage fermentation, makes the product acid concentration low relatively.
Summary of the invention
The working method that the purpose of this invention is to provide the ancient dragon acid of a kind of 2-ketone group-L-, the method that it adopts middle and later periods stream Ensure Liquid liquid makes that the middle and later periods thalline keeps growing faster and producing acid.
In order to realize the object of the invention; The working method of the ancient dragon acid of a kind of 2-ketone group of the present invention-L-; It adopts to begin in 20～26 hours during the fermentation to flow and adds the nitrogenous source nutritive medium; To nutrient demand, stream added end in 40～45 hours with microorganism growth in the afterfermentation process, and nitrogenous source nutrition flow adds controls dissolved oxygen 30～50% in the process.
The content of each composition is in the said nitrogenous source nutritive medium: steeping water 100～110g/L, potassium primary phosphate 5～7g/L, sal epsom 1～2g/L, urea 10～15g/L.
In the fermenting process in the fermention medium content of each composition be: steeping water 1～2%, potassium primary phosphate 0.1～0.2%, sal epsom 0.1～0.2%, urea 0.5～1%, sorbose 2～2.5%.
Initial sorbose concentration in the fermenting process is 20～25mg/ml, and stream adds in the process when fermented liquid remaining sugar concentration (sorbose concentration) when being lower than 20mg/ml, begins stream and adds sorbose, flows to add in the process that sorbose concentration is controlled at 15～20mg/ml in the fermented liquid.
The concentration that stream adds the sorbose liquid glucose is 200～250mg/L.
Initial pH is 6.4～6.7, is controlled at 6.7～7.0 in the fermenting process.
Starting temperature is 29 ℃, and temperature rose to 30 ℃ in 20～30 hours, and temperature rose to 31 ℃ in 30～36 hours, and temperature rises to 32 ℃ after 36 hours.
The present invention produces in the process of the ancient dragon acid of 2-ketone group-L-, and fermentation period is 46～50 hours.
The present invention adopts the method for middle and later periods stream Ensure Liquid liquid, solves the middle and later periods thalli growth and slowly produces the slow shortcoming of acid, and combine temperature, pH, dissolved oxygen, low sugar just and stream to add the control of mending factors such as sugar, optimizes the growth phase mutual relation of big or small bacterium in the mixed fungus fermentation.
Fermentation termination produces acid concentration raising 10～15% in the working method of the ancient dragon acid of 2-ketone group of the present invention-L-, reaches 105～115mg/ml, and glucose acid invert ratio improves 2～5%, reaches 92～95%.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Embodiment 1
Bacterial classification: oxidizing glucose acidfast bacilli (little bacterium) and bacillus megaterium (big bacterium).
Bottle inclined plane culture medium of eggplant: yeast extract paste 0.1%, peptone 0.2%, steeping water 1.5%, lime carbonate 0.15%, sal epsom 0.1%, urea 0.1%, glucose 0.1%, sorbose 2.0%, agar 2.0%.
Eggplant bottle inclined-plane loading amount 80ml gets 3 ring little bacterium and the big bacterium of semi-ring with transfering loop and evenly is coated onto eggplant bottle inclined-plane, 29 ℃ of cultivations 80 hours.
Shake-flask seed substratum: yeast extract paste 0.1%, steeping water 1.5%, lime carbonate 0.15%, sal epsom 0.1%, urea 0.1%, glucose 0.1%, sorbose 2.0%.
Specification is 100ml for the 1000ml seed shakes bottled amount, the inoculation of eggplant bottle inclined-plane seed, and each eggplant bottle inclined-plane seed connects the 8-10 bottle.200r/min, 30 ℃ of concussions were cultivated 20～22 hours.
The initial loading liquid measure is 9L in the 30L fermentor tank, and the initial sorbose concentration of fermented liquid is 25mg/ml, and inoculum size is 30% of an original volume.
The content of each composition is in the fermention medium: steeping water 1.5%, potassium primary phosphate 0.2%, sal epsom 0.1%, urea 0.5%, sorbose 2.5%.
When residual sorbose sugar was reduced to 20mg/ml, beginning stream, to add concentration be 200mg/L sorbose liquid glucose, and it is 19 ± 1mg/ml that stream adds process control sugar content; 20 hours begin to flow Ensure Liquid liquid, and the content of each composition is in the nutritive medium: steeping water 100g/L, potassium primary phosphate 5g/L, sal epsom 1g/L, urea 10g/L.Stopped stream in 40 hours and add, always flowing dosage is 0.5L, and it is 35 ± 5% that stream adds in the process control dissolved oxygen.
The initial pH of fermented liquid is 6.5 ± 0.1, is controlled at 6.8 ± 0.1 in the fermenting process.
The fermented liquid starting temperature is 29 ℃, and temperature rose to 30 ℃ in 20-30 hour, and temperature rose to 31 ℃ in 30～36 hours, and temperature rises to 32 ℃ after 36 hours.
The whole fermentation period of present embodiment is 46 hours, and the fermentation final volume is 24L, and ancient imperial acid content is 110.4mg/ml, and molar yield is 92.8%.
Embodiment 2
140m
3The initial loading liquid measure is 30m in the fermentor tank
3, initial sorbose concentration is 25mg/ml, inoculum size is 30% of an original volume.
The content of each composition is in the fermention medium: steeping water 1.5%, potassium primary phosphate 0.2%, sal epsom 0.1%, urea 0.5%, sorbose 2.5%.
When residual sorbose sugar was reduced to 20mg/ml, beginning stream, to add concentration be 220mg/L sorbose liquid glucose, and it is 19 ± 1mg/ml that stream adds process control sugar content; 20 hours begin to flow Ensure Liquid liquid, and the content of each composition is in the nutritive medium: steeping water 110g/L, and potassium primary phosphate 6g/L, sal epsom 1.5g/L, urea 15g/L stopped stream in 40 hours and adds, and always flowing dosage is 2.5m
3, it is 40 ± 5% that stream adds in the process control dissolved oxygen.
Initial pH is 6.5 ± 0.1, is controlled at 6.8 ± 0.1 in the fermenting process.
Starting temperature is 29 ℃, and temperature rose to 30 ℃ in 20-30 hour, and temperature rose to 31 ℃ in 30～36 hours, and temperature rises to 32 ℃ after 36 hours, and fermentation period is 44 hours, and the fermentation final volume is 110m
3, ancient imperial acid content is 112.8mg/ml, molar yield is 92.26%.
Embodiment 3
140m
3The initial loading liquid measure is 35m in the fermentor tank
3, initial sorbose concentration is 20mg/ml, inoculum size is 30% of an original volume.
The content of each composition is in the fermention medium: steeping water 2%, potassium primary phosphate 0.2%, sal epsom 0.2%, urea 1%, sorbose 2%.
When residual sorbose sugar was reduced to 15mg/ml, beginning stream, to add concentration be 240mg/L sorbose liquid glucose, and it is 16 ± 1mg/ml that stream adds process control sorbose content; 24 hours begin to flow Ensure Liquid liquid, and the content of each composition is in the nutritive medium: steeping water 100g/L, and potassium primary phosphate 5g/L, sal epsom 2g/L, urea 10g/L stopped stream in 45 hours and adds, and always flowing dosage is 2m
3, it is 35 ± 5% that stream adds in the process control dissolved oxygen.
Initial pH is 6.6 ± 0.1, is controlled at 6.8 ± 0.1 in the fermenting process.
Starting temperature is 29 ℃, and temperature rose to 30 ℃ in 20-30 hour, and temperature rose to 31 ℃ in 30～36 hours, and temperature rises to 32 ℃ after 36 hours, and fermentation period is 50 hours, and the fermentation final volume is 116m
3, ancient imperial acid content is 113.6mg/ml, molar yield is 93.79%.
Embodiment 4
140m
3The initial loading liquid measure is 35m in the fermentor tank
3, initial sorbose concentration is 25mg/ml, inoculum size is 30% of an original volume.
The content of each composition is in the fermention medium: steeping water 2%, potassium primary phosphate 0.15%, sal epsom 0.15%, urea 0.7%, sorbose 2.5%.
When sorbose concentration was reduced to 20mg/ml in the fermented liquid, beginning stream, to add concentration be 250mg/L sorbose liquid glucose, and it is 17 ± 1mg/ml that stream adds process control sugar content; 24 hours begin to flow Ensure Liquid liquid, and the content of each composition is in the nutritive medium: steeping water 110g/L, and potassium primary phosphate 7g/L, sal epsom 2g/L, urea 15g/L stopped stream in 44 hours and adds, and always flowing dosage is 2m
3, it is 40 ± 5% that stream adds in the process control dissolved oxygen.
Initial pH is 6.6 ± 0.1, is controlled at 6.8 ± 0.1 in the fermenting process.
Starting temperature is 29 ℃, and temperature rose to 30 ℃ in 20-30 hour, and temperature rose to 31 ℃ in 30～36 hours, and temperature rises to 32 ℃ after 36 hours, and fermentation period is 50 hours, and the fermentation final volume is 118m
3, ancient imperial acid content is 115.2mg/ml, molar yield is 94.08%.
Comparative Examples 1 existing production technique
70m
3The initial loading liquid measure is 35m in the fermentor tank
3, initial sorbose concentration is 60mg/ml, inoculum size is 20%.Sorbose concentration is reduced to 20mg/ml when following in the fermented liquid, and it is 250mg/L sorbose liquid glucose that the beginning intermittent flow adds concentration, and stream added once in per four hours, flows to add 4～6m at every turn
3, residual sorbose concentration in the fermented liquid is once surveyed in sampling in per four hours, when maximum liquid amount, stops to add sorbose.
The content of each composition is in the fermention medium: steeping water 1%, potassium primary phosphate 0.2%, sal epsom 0.15%, urea 0.6%, sorbose 6%, lime carbonate 0.2%.
Initial pH transfers to 6.7, and pH is controlled at 6.7 ± 0.1 in the fermenting process.
Temperature is controlled at 29 ± 0.5 ℃ in the fermenting process, and fermentation period is 50 hours, and the fermentation final volume is 65m
3Ancient imperial acid content is 90mg/ml, and molar yield is 90%.
This shows that the working method of ancient dragon acid of the present invention is with respect to existing technology, fermentation termination produces acid concentration and improves 10～15%, reaches 105～110mg/ml, and glucose acid invert ratio improves 2～5%, reaches 92～95%.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (7)
1. the working method of the ancient dragon acid of a 2-ketone group-L-is characterized in that, it adopts and began stream during the fermentation in 20～26 hours and add 40～45 hours stream of nitrogenous source nutritive medium and add end, and nitrogenous source nutrition flow adds in the process control dissolved oxygen 30～50%; The content of each composition is in the said nitrogenous source nutritive medium: steeping water 100～110g/L, potassium primary phosphate 5～7g/L, sal epsom 1～2g/L, urea 10～15g/L.
2. working method according to claim 1 is characterized in that, the initial sorbose concentration of fermented liquid is 20～25mg/ml.
3. working method according to claim 2 is characterized in that, stream adds in the nitrogenous source nutritive medium process, when the fermented liquid remaining sugar concentration is lower than 20mg/ml, begins stream and adds sorbose, and stream adds in the process that sorbose concentration is controlled at 15～20mg/ml in the fermented liquid.
4. working method according to claim 3 is characterized in that, the concentration that stream adds the sorbose liquid glucose is 200～250mg/L.
5. according to any described working method of claim 1～4, it is characterized in that the initial pH that ferments is 6.4～6.7, pH is controlled at 6.7～7.0 in the fermenting process.
6. according to any described working method of claim 1～4, it is characterized in that starting temperature is 29 ℃ during fermentation, temperature rose to 30 ℃ in 20～30 hours, and temperature rose to 31 ℃ in 30～36 hours, and temperature rises to 32 ℃ after 36 hours.
7. according to any described working method of claim 1～4, it is characterized in that fermentation time is 46～50 hours.
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| CN102242178A (en) * | 2011-06-21 | 2011-11-16 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of 2-keto-L-gulonic acid |
| CN103074406B (en) * | 2011-10-26 | 2014-07-23 | 中国科学院沈阳应用生态研究所 | Gulonic acid high-temperature fermentation method |
| CN102586381A (en) * | 2011-11-01 | 2012-07-18 | 江苏江山制药有限公司 | Production process for improving fermentative strength of 2-keto-L-gulonic acid |
| CN103509846B (en) * | 2013-10-25 | 2015-07-22 | 东北制药集团股份有限公司 | Method for preparing 2-keto-L-gulonic acid through adaptive regulation of companion fungus number |
| CN103614424A (en) * | 2013-11-21 | 2014-03-05 | 宁夏启元药业有限公司 | Method of improving fermentation unit of gulconic acid |
| CN103710398A (en) * | 2013-12-18 | 2014-04-09 | 江苏江山制药有限公司 | Method for reinforcing activity of fermentation strain of 2-keto-L-gulonic acid |
| CN104131043B (en) * | 2014-07-01 | 2017-03-01 | 安徽丰原发酵技术工程研究有限公司 | A kind of 2-KLG that improves produces sour fermentation process |
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| CN101392274A (en) * | 2008-11-18 | 2009-03-25 | 江苏江山制药有限公司 | 2-keto-L-gulonic acid high concentration fermentation production technology |
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| CN101392274A (en) * | 2008-11-18 | 2009-03-25 | 江苏江山制药有限公司 | 2-keto-L-gulonic acid high concentration fermentation production technology |
Non-Patent Citations (2)
| Title |
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| 杨丽宁 等.间歇流加L- 山梨糖发酵生产2- 酮基- L- 古龙酸研究.《河北化工》.2008,第31卷(第6期),33-35. * |
| 白秀峰 主编.第七章 发酵过程的控制.《发酵工艺学》.2003,122. * |
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