CN102586381A - Production process for improving fermentative strength of 2-keto-L-gulonic acid - Google Patents
Production process for improving fermentative strength of 2-keto-L-gulonic acid Download PDFInfo
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- CN102586381A CN102586381A CN2011103382818A CN201110338281A CN102586381A CN 102586381 A CN102586381 A CN 102586381A CN 2011103382818 A CN2011103382818 A CN 2011103382818A CN 201110338281 A CN201110338281 A CN 201110338281A CN 102586381 A CN102586381 A CN 102586381A
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Abstract
The invention discloses a production process for improving the fermentative strength of 2-keto-L-gulonic acid. The production process comprises the following steps of: controlling the temperature to be 28 to 33 DEG C during culture for 0 to 32 hours in the process of preparing the 2-keto-L-gulonic acid by fermenting mixed bacteria of Ketogulonigenium vulgare and Bacillus megaterium; culturing for 32 hours until the fermentation is finished, and controlling the temperature to be 34 to 38 DEG C; supplementing 0.2 to 0.5 percent of corn steep liquor during culture for 16 to 20 hours; and supplementing 0.1 to 0.35 percent of urea during culture for 28 to 32 hours. The physiological relationship of double bacteria in the fermentation of the2-keto-L-gulonic acid is adjusted through the research and development of stage-by-stage and fed-batch culture, so that environments and nutritional conditions are more suitable for the growth of mixed strain cells; therefore, the biosynthesis rate of a mixed strain is effectively improved; and strains are unchanged, and the process is easy to operate and popularize, low in cost, energy-saving and environment-friendly.
Description
Technical field:
The present invention relates to ascorbic preparation method, belong to the microbial fermentation technology field, particularly relate to the novel process of a kind of 2-of raising ketone group-L-gulonic acid ferment production efficiency.
Background technology:
The ancient dragon acid of 2-ketone group-L-(hereinafter to be referred as 2-KLG) is the important integral part of synthesise vitamins C.Present domestic main vitamins C manufacturer all adopts " two-step fermenting " production technique, and this technological committed step is its second step fermenting process: the L-sorbose is 2-KLG by bio-oxidation under the mixing fungus strain effect of ancient imperial sour bacterium (being commonly called as little bacterium in the industry) of ordinary student ketone group and bacillus megaterium (being commonly called as big bacterium in the industry) composition.Domestic vitamins C " two-step fermenting " is compared with external " Lai Shi method ", has simplified technology, reduces cost; Reduced pollution; But because present academia's big or small bacterium physiological relation in second step of the thorough fermenting process fully also, in actual production, still there is the technical problem of long, the high restriction production efficiency of energy consumption of cycle, therefore; Improve original fermentation manufacturing technique, ketone group-L-gulonic acid ferment production efficiency is very important to improve 2-.
Summary of the invention:
The present invention is through adjust two bacterium physiological relations in 2-ketone group-L-gulonic acid ferment with the research and development of feed supplement cultivation stage by stage; Make environment and nutritional condition help promoting bacillus megaterium and the ancient imperial sour bacterium of ordinary student ketone group to mix the growth of fungus strain cell; Thereby effectively improve the speed of mixing fungus strain biosynthesizing 2-KLG, reach the purpose that improves fermentation production efficiency.
A kind of novel process that improves 2-ketone group-L-gulonic acid ferment production efficiency, it is characterized in that: procedure of processing is following
1) selection of .. bacterial classification
Ancient imperial sour bacterium of ordinary student ketone group and bacillus megaterium;
2). substratum, following with weight unit per-cent proportioning,
Solid medium: sorbose 1.5, yeast extract paste 0.2, steeping water 0.5, urea 0.2 MgSO
47H
2O 0.03, KH
2PO
40.02, CaCO
30.5, agar 2.5, pH 7.0;
Liquid seed culture medium: sorbose 1.5, yeast extract paste 0.3, steeping water 0.5, urea 0.2, MgSO
47H
2O 0.01, KH
2PO
40.02, CaCO
30.5 pH 7.0;
Fermentation initial medium: sorbose initial content 2, steeping water 0.8～1.1, urea 0.1～0.2, MgSO
47H
2O 0.02, KH
2PO
40.05, initial pH6.7～7.0, wherein stream adds sorbose concentration 25%, and initial sorbose separates sterilization with other substratum;
3), shake-flask seed is cultivated
29 ℃ of culture temperature, incubation time are 24 hours, shaking speed 190rpm;
4), fermentor tank feed supplement and CONTROL PROCESS stage by stage
Cultivate during 0～32 hour 28～33 ℃ of temperature controls; Cultivate 32 hours to fermentation ends, 34～38 ℃ of temperature controls; During being cultured to 16～20 hours, add 0.2～0.5% steeping water; During being cultured to 28～32 hours, add 0.1～0.35% urea.
The present invention controls and supplying technics through fermentor tank stage by stage, and the former working method of biosynthesizing speed ratio of 2-KLG has improved 22.17% in the fermenting process, can effectively improve the 2-KLG fermentation production efficiency, saves production cost.Simplify technology, reduced pollution, overcome the defective that the production cycle is long, energy consumption is high.
Embodiment:
Embodiment 1:
Control group: with 1 liter of the mixed bacteria liquid of ancient imperial sour bacterium of cultured ordinary student ketone group and bacillus megaterium; Be linked in 3.5 liters of fermention mediums that contain 2% sorbose, 1.1% steeping water, 0.2% urea, 0.02% sal epsom, 0.05% potassium primary phosphate, fermenting container adopts 10 liters of automatic fermentor tanks, mixing speed 400rpm; 1:1vvm is compared in ventilation; Substratum initial p H6.7 cultivates and began in 8 hours with 25% sodium carbonate solution control fermented liquid PH7.0, cultivates to begin to flow that to add concentration be 25% sorbose solution in 10 hours; The final total sugar concentration 10.2% of control fermentation, 30 ℃ of temperature controls; The ancient imperial acid content 99.26mg/ml of fermented liquid terminal point 2-ketone group-L-, fermentation period 52 hours, rate of producing acid 1.91mg/mlh.
Experimental group:, be linked in 3.5 liters of fermention mediums that contain 2% sorbose, 0.8% steeping water, 0.1% urea, 0.02% sal epsom, 0.05% potassium primary phosphate 10 liters of automatic fermentor tanks of fermenting container employing with 1 liter of the mixed bacteria liquid of ancient imperial sour bacterium of cultured ordinary student ketone group and bacillus megaterium; Mixing speed 400rpm; Ventilation is than 1:1vvm, and substratum initial p H6.7 cultivates and began in 8 hours with 25% sodium carbonate solution control fermented liquid PH7.0; Cultivate and began to flow that to add concentration be 25% sorbose solution in 10 hours; The final total sugar concentration 10.2% of control fermentation was cultivated during 0～32 hour, 30 ℃ of temperature controls; Cultivate after 32 hours 34 ℃ of temperature controls; Be cultured to 20 hours, add 0.25% steeping water, be cultured to 32 hours, add 0.15% urea; The ancient imperial acid content 100.18mg/ml of fermented liquid terminal point 2-ketone group-L-, fermentation period 44 hours, rate of producing acid 2.28mg/mlh improves 19.37% than the control group rate of producing acid.
Embodiment 2:
Control group: with 10 liters of the mixed bacteria liquids of ancient imperial sour bacterium of cultured ordinary student ketone group and bacillus megaterium; Be linked in 35 liters of fermention mediums that contain 2% sorbose, 1.1% steeping water, 0.2% urea, 0.02% sal epsom, 0.05% potassium primary phosphate, fermenting container adopts 100 liters of automatic fermentor tanks, mixing speed 280rpm; Ventilation is than 1:0.8 (vvm); Substratum initial p H6.7 cultivates and began in 8 hours with 25% sodium carbonate solution control fermented liquid PH7.0, cultivates to begin to flow that to add concentration be 25% sorbose solution in 10 hours; The final total sugar concentration 10.5% of control fermentation, 31 ℃ of temperature controls; The ancient imperial acid content 101.46mg/ml of fermented liquid terminal point 2-ketone group-L-, fermentation period 50 hours, rate of producing acid 2.03 mg/mlh.
Experimental group:, be linked in 35 liters of fermention mediums that contain 2% sorbose, 0.8% steeping water, 0.1% urea, 0.02% sal epsom, 0.05% potassium primary phosphate 10 liters of automatic fermentor tanks of fermenting container employing with 10 liters of the mixed bacteria liquids of ancient imperial sour bacterium of cultured ordinary student ketone group and bacillus megaterium; Mixing speed 400rpm; Ventilation is than 1:0.8 (vvm), and substratum initial p H6.7 cultivates and began in 8 hours with 25% sodium carbonate solution control fermented liquid PH7.0; Cultivate and began to flow that to add concentration be 25% sorbose solution in 10 hours; The final total sugar concentration 10.5% of control fermentation was cultivated during 0～32 hour, 31 ℃ of temperature controls; Cultivate after 32 hours 35 ℃ of temperature controls; Be cultured to 18 hours, add 0.3% steeping water, be cultured to 30 hours, add 0.25% urea.The ancient imperial acid content 102.52mg/ml of fermented liquid terminal point 2-ketone group-L-, fermentation period 42.5 hours, rate of producing acid 2.48 mg/mlh improve 22.17% than the control group rate of producing acid.
Embodiment 3:
Control group: with mixed bacteria liquid 6 m of ancient imperial sour bacterium of cultured ordinary student ketone group and bacillus megaterium
3, be linked into 18m
3Contain in the fermention medium of 2% sorbose, 1.1% steeping water, 0.2% urea, 0.02% sal epsom, 0.05% potassium primary phosphate, fermenting container adopts 50m
3The standard fermentor tank; Mixing speed 150rpm ventilates than 1:0.4 (vvm) substratum initial p H6.7; Cultivate and began in 8 hours with 25% sodium carbonate solution control fermented liquid PH7.0; Cultivate and began to flow that to add concentration be 25% sorbose solution in 10 hours, the final total sugar concentration 10.8% of control fermentation, 30 ℃ of temperature controls; The ancient imperial acid content 102.19mg/ml of fermented liquid terminal point 2-ketone group-L-, fermentation period 49 hours, rate of producing acid 2.09 mg/mlh.
Experimental group: with mixed bacteria liquid 6 m of ancient imperial sour bacterium of cultured ordinary student ketone group and bacillus megaterium
3, be linked into 18m
3Contain in the fermention medium of 2% sorbose, 1.1% steeping water, 0.2% urea, 0.02% sal epsom, 0.05% potassium primary phosphate, fermenting container adopts 50m
3The standard fermentor tank, mixing speed 150rpm, ventilation is than 1:0.4 (vvm); Substratum initial p H6.7; Cultivate and began in 8 hours, cultivate and began to flow that to add concentration be 25% sorbose solution in 10 hours, the control final total sugar concentration 10.8% of fermenting with 25% sodium carbonate solution control fermented liquid PH7.0; Cultivate during 0～32 hour 32 ℃ of temperature controls; Cultivate after 32 hours 36 ℃ of temperature controls; Be cultured to 16 hours, add 0.3% steeping water, be cultured to 28 hours, add 0.3% urea.The ancient imperial acid content 104.86mg/ml of fermented liquid terminal point 2-ketone group-L-, fermentation period 42 hours, rate of producing acid 2.50 mg/mlh improve 19.62% than the control group rate of producing acid.
Claims (1)
1. production technique that improves 2-ketone group-L-gulonic acid ferment intensity, it is characterized in that: procedure of processing is following
1). the selection of bacterial classification
Ancient imperial sour bacterium of ordinary student ketone group and bacillus megaterium;
2). substratum, following with weight unit per-cent proportioning,
Solid medium: sorbose 1.5, yeast extract paste 0.2, steeping water 0.5, urea 0.2 MgSO
47H
2O 0.03, KH
2PO
40.02, CaCO
30.5, agar 2.5, pH 7.0;
Liquid seed culture medium: sorbose 1.5, yeast extract paste 0.3, steeping water 0.5, urea 0.2, MgSO
47H
2O 0.01, KH
2PO
40.02, CaCO
30.5 pH 7.0;
Fermentation initial medium: sorbose initial content 2, steeping water 0.8～1.1, urea 0.1～0.2, MgSO
47H
2O 0.02, KH
2PO
40.05, initial pH6.7～7.0, wherein stream adds sorbose concentration 25%, and initial sorbose separates sterilization with other substratum;
3), shake-flask seed is cultivated
29 ℃ of culture temperature, incubation time are 24 hours, shaking speed 190rpm;
4), fermentor tank feed supplement and CONTROL PROCESS stage by stage
Cultivate during 0～32 hour 28～33 ℃ of temperature controls; Cultivate 32 hours to fermentation ends, 34～38 ℃ of temperature controls; During being cultured to 16～20 hours, add 0.2～0.5% steeping water; During being cultured to 28～32 hours, add 0.1～0.35% urea.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851348A (en) * | 2012-08-30 | 2013-01-02 | 郑州拓洋实业有限公司 | Method for promoting high efficiency production of 2-keto-L-gulonic acid |
| CN102936615A (en) * | 2012-12-10 | 2013-02-20 | 石药集团维生药业（石家庄）有限公司 | Method for promoting L-sorbose to convert to generate 2-keto-L-gulonic acid |
| 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 |
| CN103627775A (en) * | 2013-12-18 | 2014-03-12 | 江苏江山制药有限公司 | Method for improving fermentation production efficiency of 2-keto-L-gulonic 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 |
| CN106434830A (en) * | 2016-12-20 | 2017-02-22 | 宁夏启元药业有限公司 | Method for improving 2-keto-L-gluconic acid fermentation efficiency |
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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 |
| CN101736071A (en) * | 2009-12-24 | 2010-06-16 | 安徽丰原发酵技术工程研究有限公司 | Method for producing 2-keto-l-gulonic acid |
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2011
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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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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851348A (en) * | 2012-08-30 | 2013-01-02 | 郑州拓洋实业有限公司 | Method for promoting high efficiency production of 2-keto-L-gulonic acid |
| 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 |
| CN102936615A (en) * | 2012-12-10 | 2013-02-20 | 石药集团维生药业（石家庄）有限公司 | Method for promoting L-sorbose to convert to generate 2-keto-L-gulonic acid |
| CN102936615B (en) * | 2012-12-10 | 2014-09-17 | 石药集团维生药业（石家庄）有限公司 | Method for promoting L-sorbose to convert to generate 2-keto-L-gulonic acid |
| CN103627775A (en) * | 2013-12-18 | 2014-03-12 | 江苏江山制药有限公司 | Method for improving fermentation production efficiency of 2-keto-L-gulonic 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 |
| CN105274154A (en) * | 2013-12-18 | 2016-01-27 | 帝斯曼江山制药（江苏）有限公司 | Method for improving activity of 2-keto-L-gulonic acid fermentation strains |
| CN103627775B (en) * | 2013-12-18 | 2016-05-25 | 帝斯曼江山制药（江苏）有限公司 | Improve the method for KGA fermentation production efficiency |
| CN106434830A (en) * | 2016-12-20 | 2017-02-22 | 宁夏启元药业有限公司 | Method for improving 2-keto-L-gluconic acid fermentation efficiency |
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Application publication date: 20120718 |