CN102086445A - Method for increasing Gluconobacter oxydans biomass by fermenting with oxygen-carrier-added culture medium - Google Patents
Method for increasing Gluconobacter oxydans biomass by fermenting with oxygen-carrier-added culture medium Download PDFInfo
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Abstract
The invention relates to a method for increasing Gluconobacter oxydans biomass by fermenting with an oxygen-carrier-added culture medium. An appropriate oxygen carrier is added into a culture medium to provide an oxygen carrier fermenting system, so that the concentration of dissolved oxygen in the culture medium is greatly increased without adding other substances under the condition of no extra energy consumption, thereby greatly increasing the fermentation biomass.
Description
Technical field
The invention belongs to the biological medicine technology field, be specifically related to a kind of method of utilizing the substratum fermentation that adds oxygen carrier to improve the bacillus of oxidizing glucose biomass.
Technical background
Miglitol (migliton) is the antidiabetic thing of Beyer Co., Ltd in the listing of 1997 years.It is a kind of novel intestinal alpha-glucosidase inhibitors of finding from the bacillus broth culture, is the parent modified outcome of 1-S-GI, belongs to N-substituted-1-deoxynojirimycin type, and structure is similar to glucose.Although the method with chemosynthesis can prepare miglitol, it is more that chemical complete synthesizing process prepares the step of miglitol, and four chiral centres that contain in the miglitol molecule have also brought very big difficulty to chemosynthesis.Industrial scale adopts biosynthesizing to prepare miglitol mostly at present.The biological synthesis process of miglitol roughly has two routes according to the literature: the one, and produce bacterium by nojirimycin or 1-S-GI earlier and ferment, prepare nojirimycin or 1-S-GI, and then obtaining miglitol through chemical synthesis process, this is primary approach; The 2nd, to use oxidizing glucose acid bacterium and transform preparation miglitol key intermediate N replacement-1-S-GI, it mainly prepares by the method for chemosynthesis-bio-transformation-chemosynthesis, and operational path is seen Figure of description.
US4266025, EP0477160, USP2001/0019837 have announced the production method of other miglitol, i.e. chemosynthesis is in conjunction with the method for biocatalysis.But because the thalline vigor is not high, cause the biocatalysis overlong time, cause the production cost height, the production cycle is long, brings very big obstacle to suitability for industrialized production.
Because the efficient specificity of microbial enzyme, omitted in the chemosynthesis process to reaching and transformed adding that certain purpose group must carry out and the step that removes blocking group single-mindedly, simplified preparation process greatly, improved reaction yield.The oxidizing glucose acidfast bacilli is used to transform that specific substrates prepares the 1-S-GI and derivative has lot of advantages: 1) conversion fluid need not separation and purification and goes out intermediate through the centrifugal thalline of removing, and just can carry out that next step is synthetic; 2) need not radical protection, cost reduces greatly, and avoids because of removing the rate of recovery decline that protective material causes; 3) intermediate 6-(substituted amido)-6-deoxidation-α-L-sorb furanose has higher solubleness and stability, is difficult for being degraded.But also not for the report of transformation mechanism, can be certainly, contain the biological enzyme or the enzyme system that participate in this conversion reaction in the bacillus of oxidizing glucose, the quantity and the vigor of the biological enzyme system that the catalytic capability of bacillus of oxidizing glucose contains with it have direct relation.
At present, though miglitol has been realized suitability for industrialized production, because spawn activity is low, transformation efficiency is low when utilizing microorganism that the 1-hydroxyethylamine-1-deoxy-D-sorbierite is converted into glucofuranose amine, and transformation time is long, and labour intensity is big, the production cost height.The tradition strain improvement mainly adopts the method for selection by mutation, and the method for mutagenesis is with a long history, and the method that the past relies on mutagenesis always improves output, and present most of biological fermentation industrial production are still in these methods of use.But this method has sizable randomness, and bacterial classification is if the life-time service mutagenic compound are handled, and its vigor can descend gradually.
At present, also do not utilize the substratum fermentation that adds oxygen carrier to improve the pertinent literature report of the method for bacillus of oxidizing glucose biomass.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing the substratum fermentation that adds oxygen carrier to improve the bacillus of oxidizing glucose biomass, by in substratum, adding suitable oxygen carrier, a kind of oxygen carrier fermentation system is provided, do not adding other material, do not have under the condition of additional energy source consumption, significantly improve the dissolved oxygen concentration in the substratum, thereby fermentation biomass is increased substantially, and can't influence the catalytic capability of this bacterial classification.
It is microbial strains that the present invention adopts bacillus of oxidizing glucose, obtains mycelium through spawn culture, fermentation culture centrifugation, it is characterized in that adding in the fermention medium oxygen carrier, tensio-active agent, and its concrete steps are as follows:
Concrete steps and method are as follows:
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 20-30 hour in the 150-220rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 20-30 ℃ to pH.
Wherein, bacterium culture medium contains: sorbyl alcohol 50-80g/L, yeast extract paste 10-40g/L, potassium primary phosphate 0.5-2g/L.
(2) fermentation culture
With to be inoculated into the pH that adds oxygen carrier, tensio-active agent be in 7.0 the fermention medium through cultivating the seed culture fluid obtain,, cultivate after 36-48 hour centrifugal collection thalline in the 150-220rpm constant-temperature shaking culture case at 20-30 ℃.Wherein, fermention medium contains: sorbyl alcohol 50-80g/L, yeast extract paste 10-40g/L, potassium primary phosphate 0.5-2g/L;
Oxygen carrier is normal hexane or n-dodecane in the substratum, and the volume ratio of oxygen carrier and substratum is 5-50: 1000.Normal hexane or n-dodecane here are a kind of and water does not dissolve mutually, the oxygen carrier than the high-solubility oxygen ability is not poisoned, had to microorganism; Simultaneously in order to reduce the viscosity of substratum, further improve the level of dissolved oxygen, basal culture medium adding tensio-active agent is one or more in tween-80, stearic acid, the Sodium dodecylbenzene sulfonate, contains tensio-active agent 0.2-2g in every liter of substratum, preferred tween-80.
Centrifugal employing high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm.
Resulting mycelium is carried out the biocatalysis checking:
It is 6.0 6% N-hydroxyethyl glucosamine that the mycelium that obtains in the step (2) is joined pH with 1: 1 ratio, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dry up colour developing observation, analytical results in iodine flask.
In the said process, the adjusting of seed culture medium and fermention medium pH all adopts 5% sodium hydroxide solution commonly used to regulate; Because catalytic substrate N-hydroxyethyl glucosamine is an alkalescence, need regulate about pH to 6.0 with concentrated hydrochloric acid.
The present invention is by adding oxygen carrier and tensio-active agent, it is fast to make fermentation system have the oxygen transmission speed, having foam generates few, advantages such as shearing force is little, can significantly improve the biomass of bacillus of oxidizing glucose, high-biomass can reach and not add the oxygen carrier fermentation and obtain 2 times of biomass, and the biocatalysis ability of thalline is enhanced.
Embodiment
Embodiment 1
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 25 hours in the 150rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 25 ℃ to pH.The composition of seed culture medium and content are: sorbyl alcohol 50g/L, yeast extract paste 20g/L, potassium primary phosphate 0.5g/L.
(2) fermentation culture
To be inoculated in the fermention medium through cultivating the seed culture fluid that obtains, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 50g/L, and yeast extract paste 20g/L, potassium primary phosphate 1g/L, pH are 7.0.The fermentation culture mode adopts shake-flask culture, promptly adopts 500 milliliters of triangular flasks, and liquid amount is 50 milliliters, at 25 ℃, cultivate after 40 hours in the 200rpm constant-temperature shaking culture case, use high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm collects thalline and obtains thalline 4.8 grams.
(3) biocatalysis
Take by weighing the centrifugal mycelium that obtains 2 grams, take by weighing N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 and add 33 milliliters of purified water with concentrated hydrochloric acid, making concentration is about 6%, puts into 500 milliliters of triangular flasks, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dries up, and develops the color in iodine flask, the result shows that transformation efficiency is about 70%.
Embodiment 2
(1) spawn culture
Is that 7.0 seed is supported in the base with the bacillus of oxidizing glucose bacterial classification inoculation to pH, at 25 ℃, cultivates 25 hours in the 150rpm constant-temperature shaking culture case.The composition of seed culture medium and content are: sorbyl alcohol 50g/L, yeast extract paste 20g/L, potassium primary phosphate 0.5g/L.
(2) fermentation culture
To be inoculated in the fermention medium through cultivating the seed culture fluid that obtains, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 50g/L, yeast extract paste 20g/L, potassium primary phosphate 1g/L, 20ml/ tween-80 L is through the normal hexane of filtration sterilization, and pH is 7.0.The fermentation culture mode adopts shake-flask culture, promptly adopt 500 milliliters of triangular flasks, liquid amount is 50 milliliters, at 25 ℃, cultivate after 40 hours in the 200rpm constant-temperature shaking culture case, use high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 7.2 grams, Duo 50% than thalline 4.8 grams that the substratum fermentation that does not add oxygen carrier is collected.
(3) biocatalysis
Take by weighing the centrifugal mycelium that obtains 2 grams, take by weighing N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 and add 33 milliliters of purified water with concentrated hydrochloric acid, making concentration is about 6%, puts into 500 milliliters of triangular flasks, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dries up, and develops the color in iodine flask, the result shows that transformation efficiency is about 75%.
Embodiment 3
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 20 hours in the 200rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 30 ℃ to pH.The composition of seed culture medium and content are: sorbyl alcohol 80g/L, yeast extract paste 20g/L, potassium primary phosphate 2g/L.
(2) fermentation culture
To be inoculated in the fermention medium through cultivating the seed culture fluid that obtains, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 60g/L, and yeast extract paste 20g/L, potassium primary phosphate 1g/L, 30ml/L is through the normal hexane of filtration sterilization, and pH is 7.0.The fermentation culture mode adopts shake-flask culture, promptly adopt 500 milliliters of triangular flasks, liquid amount is 50 milliliters, at 30 ℃, cultivate after 48 hours in the 220rpm constant-temperature shaking culture case, use high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 7.8 grams, Duo 62.5% than thalline 4.8 grams that the substratum fermentation that does not add oxygen carrier is collected.
(3) biocatalysis
Take by weighing the centrifugal mycelium that obtains 2 grams, take by weighing N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 and add 33 milliliters of purified water with concentrated hydrochloric acid, making concentration is about 6%, puts into 500 milliliters of triangular flasks, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dries up, and develops the color in iodine flask, the result shows that transformation efficiency is about 65%.
Embodiment 4
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 20 hours in the 200rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 30 ℃ to pH.The composition of seed culture medium and content are: sorbyl alcohol 60g/L, yeast extract paste 15g/L, potassium primary phosphate 1.5g/L.
(2) fermentation culture
To be inoculated in the fermention medium through cultivating the seed culture fluid that obtains, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 60g/L, yeast extract paste 15g/L, potassium primary phosphate 1.5g/L, the tween-80 of 0.5g/L, 40ml/L is through the normal hexane of filtration sterilization, and pH is 7.0.The fermentation culture mode adopts shake-flask culture, promptly adopt 500 milliliters of triangular flasks, liquid amount is 50 milliliters, at 22 ℃, cultivate after 38 hours in the 220rpm constant-temperature shaking culture case, use high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 8.1 grams, Duo 68.8% than thalline 4.8 grams that the substratum fermentation that does not add oxygen carrier is collected.
(3) biocatalysis
Take by weighing the centrifugal mycelium that obtains 2 grams, take by weighing N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 and add 33 milliliters of purified water with concentrated hydrochloric acid, making concentration is about 6%, puts into 500 milliliters of triangular flasks, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dries up, and develops the color in iodine flask, the result shows that transformation efficiency is about 70%.
Embodiment 5
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 20 hours in the 200rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 30 ℃ to pH.The composition of seed culture medium and content are: sorbyl alcohol 50g/L, yeast extract paste 10g/L, potassium primary phosphate 0.5g/L.
(2) fermentation culture
To be inoculated in the fermention medium through cultivating the seed culture fluid that obtains, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 50g/L, yeast extract paste 10g/L, potassium primary phosphate 0.5g/L, the tween-80 of 1g/L, 50ml/L is through the normal hexane of filtration sterilization, and pH is 7.0.The fermentation culture mode adopts shake-flask culture, promptly adopt 500 milliliters of triangular flasks, liquid amount is 50 milliliters, at 22 ℃, cultivate after 38 hours in the 220rpm constant-temperature shaking culture case, use high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 6.9 grams, Duo 44% than thalline 4.8 grams that the substratum fermentation that does not add oxygen carrier is collected.
(3) biocatalysis
Take by weighing the centrifugal mycelium that obtains 2 grams, take by weighing N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 and add 33 milliliters of purified water with concentrated hydrochloric acid, making concentration is about 6%, puts into 500 milliliters of triangular flasks, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dries up, and develops the color in iodine flask, the result shows that transformation efficiency is about 60%.
Embodiment 6
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 20 hours in the 200rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 30 ℃ to pH.The composition of seed culture medium and content are: sorbyl alcohol 80g/L, yeast extract paste 20g/L, potassium primary phosphate 2g/L.
(2) fermentation culture
To be inoculated in the fermention medium through cultivating the seed culture fluid that obtains, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 80g/L, yeast extract paste 20g/L, potassium primary phosphate 2g/L, the tween-80 of 1.5g/L, 40ml/L is through the normal hexane of filtration sterilization, pH7.0.The fermentation culture mode adopts shake-flask culture, promptly adopt 500 milliliters of triangular flasks, liquid amount is 50 milliliters, at 22 ℃, cultivate after 38 hours in the 220rpm constant-temperature shaking culture case, use high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 9.9 grams, Duo 106% than thalline 4.8 grams that the substratum fermentation that does not add oxygen carrier is collected.
(3) biocatalysis
Take by weighing the centrifugal mycelium that obtains 2 grams, take by weighing N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 and add 33 milliliters of purified water with concentrated hydrochloric acid, making concentration is about 6%, puts into 500 milliliters of triangular flasks, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dries up, and develops the color in iodine flask, the result shows that transformation efficiency is about 75%.
Embodiment 7
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 20 hours in the 200rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 30 ℃ to pH.The composition of seed culture medium and content are: sorbyl alcohol 70g/L, yeast extract paste 20g/L, potassium primary phosphate 1.5g/L.
(2) fermentation culture
To be inoculated in the fermention medium through cultivating the seed culture fluid that obtains, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 70g/L, yeast extract paste 20g/L, potassium primary phosphate 1.5g/L, the tween-80 of 2.0g/L, 50ml/L is through the normal hexane of filtration sterilization, and pH is 7.0.The fermentation culture mode adopts shake-flask culture, promptly adopt 500 milliliters of triangular flasks, liquid amount is 50 milliliters, at 28 ℃, cultivate after 45 hours in the 200rpm constant-temperature shaking culture case, use high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 9.4 grams, Duo 95.8% than thalline 4.8 grams that the substratum fermentation that does not add oxygen carrier is collected.
(3) biocatalysis
Take by weighing the centrifugal mycelium that obtains 2 grams, take by weighing N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 and add 33 milliliters of purified water with concentrated hydrochloric acid, making concentration is about 6%, puts into 500 milliliters of triangular flasks, at 20 ℃, transform 15 hours in the 200rpm constant-temperature shaking culture case, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: the ammoniacal liquor volume ratio is a chromatography in 4: 3: 3 the chromatographic solution, dries up, and develops the color in iodine flask, the result shows that transformation efficiency is about 75%.
Description of drawings:
Accompanying drawing 1 is used the process route chart that oxidizing glucose acid bacterium transforms preparation miglitol key intermediate N replacement-1-S-GI, wherein (I) is the 1-hydroxyethylamine-1-deoxy-D-sorbierite, (II) being glucofuranose amine, (III) is miglitol.
Claims (8)
1. fermentation process that improves the bacillus of oxidizing glucose biomass, with the glucose oxidation and bacillus is microbial strains, obtain mycelium through spawn culture, fermentation culture centrifugation, it is characterized in that adding in the fermention medium oxygen carrier, tensio-active agent, its concrete steps are as follows:
(1) spawn culture
Be in 7.0 the seed culture medium,, to cultivate 20-30 hour in the 150-220rpm constant-temperature shaking culture case the bacillus of oxidizing glucose bacterial classification inoculation at 20-30 ℃ to pH;
(2) fermentation culture
It is in 7.0 the fermention medium that the seed culture fluid that obtains in the step (1) is inoculated into the pH that adds oxygen carrier, tensio-active agent, at 20～30 ℃, cultivate 36～48 hours in 150～220rpm constant-temperature shaking culture case after, centrifugal collection thalline.
2. method according to claim 1 is characterized in that oxygen carrier is normal hexane or n-dodecane.
3. method according to claim 1, the volume ratio that it is characterized in that oxygen carrier and substratum is 5-50: 1000.
4. according to the described method of claim 1 step (1), it is characterized in that: bacterium culture medium contains: sorbyl alcohol 50-80g/L, yeast extract paste 10-40g/L, potassium primary phosphate 0.5-2g/L.
5. according to the described method of claim 1 step (2), it is characterized in that: fermention medium contains sorbyl alcohol 50-80g/L, yeast extract paste 10-40g/L, potassium primary phosphate 0.5-2g/L.
6. according to the described method of claim 1 step (2), it is characterized in that adding in the fermention medium tensio-active agent and be in tween-80, stearic acid, the Sodium dodecylbenzene sulfonate one or more.
7. according to the described method of claim 1 step (2), it is characterized in that containing in every liter of substratum tensio-active agent 0.2-2g.
8. according to the described method of claim 1 step (2), it is characterized in that adding tensio-active agent in the fermention medium is tween-80.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215317A (en) * | 2013-05-09 | 2013-07-24 | 南京林业大学 | Method for producing xylosic acid through xylose whole-cell catalysis by direct oxygen introduction and pressurization |
| CN103333921A (en) * | 2013-06-28 | 2013-10-02 | 华南农业大学 | Method for promoting high-density fermentation of Pichia pastoris by using oxygen carriers |
| CN111226771A (en) * | 2020-03-10 | 2020-06-05 | 温州芳植生物科技有限公司 | Deep liquid flow culture method for soilless culture oxygen carrier |
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2009
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215317A (en) * | 2013-05-09 | 2013-07-24 | 南京林业大学 | Method for producing xylosic acid through xylose whole-cell catalysis by direct oxygen introduction and pressurization |
| CN103215317B (en) * | 2013-05-09 | 2014-08-27 | 南京林业大学 | Method for producing xylosic acid through xylose whole-cell catalysis by direct oxygen introduction and pressurization |
| CN103333921A (en) * | 2013-06-28 | 2013-10-02 | 华南农业大学 | Method for promoting high-density fermentation of Pichia pastoris by using oxygen carriers |
| CN111226771A (en) * | 2020-03-10 | 2020-06-05 | 温州芳植生物科技有限公司 | Deep liquid flow culture method for soilless culture oxygen carrier |
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Effective date of registration: 20161229 Address after: 276005 Hongqi Road, Shandong, Linyi, No. 209 Patentee after: Lunan Pharmaceutical Group Co., Ltd. Address before: 273400 Shandong city of Linyi province Feixian County North Ring Road No. 1 Patentee before: Shangdong New Time Pharmaceutical Co., Ltd. |