CN102086445B - 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 biomedicine technical field, be specifically related to a kind of method that utilization adds the substratum fermentation raising bacillus of oxidizing glucose biomass of oxygen carrier.
Technical background
Miglitol (migliton) is the antidiabetic thing of Beyer Co., Ltd in the listing of 1997 years.It is the novel intestinal alpha-glucosidase inhibitors of one found from bacillus broth culture, is the parent modified outcome of 1-DNJ, and belong to N-substituted-1-deoxynojirimycin type, structure is similar to glucose.Although can prepare miglitol by the method for chemosynthesis, the step that chemical complete synthesizing process prepares miglitol is more, and four chiral centres contained in miglitol molecule also bring very large difficulty to chemosynthesis.Current industrial scale adopts biosynthesizing to prepare miglitol mostly.The biological synthesis process of miglitol roughly has two routes according to the literature: one is first fermented by nojirimycin or 1-DNJ producing strains, prepare nojirimycin or 1-DNJ, and then obtaining miglitol through chemical synthesis process, this is the most original approach; Two is that miglitol key intermediate N replacement-1-DNJ is prepared in the conversion of application Gluconobacter oxydans, and prepared by its method mainly through chemosynthesis-bio-transformation-chemosynthesis, operational path is shown in Figure of description.
US4266025, EP0477160, USP2001/0019837 disclose the production method of other miglitol, and namely chemosynthesis is in conjunction with the method for biocatalysis.But because thalline vigor is not high, cause biocatalysis overlong time, cause production cost high, the production cycle is long, brings very large obstacle to suitability for industrialized production.
Due to the efficient specificity of microbial enzyme, eliminate in chemosynthesis process for reach exclusively transform that certain object group must carry out add the step with deprotection group, enormously simplify preparation process, improve reaction yield.Gluconobacter oxvdans is for transforming specific substrates and prepare 1-DNJ and derivative having lot of advantages: 1) conversion fluid is through centrifugal removing thalline, goes out intermediate without the need to separation and purification, just can carry out next step synthesis; 2) without the need to radical protection, cost reduces greatly, and avoids the rate of recovery caused because removing protective material to decline; 3) intermediate 6-(substituted amido)-6-deoxidation-α-L-sorb furanose has higher solubleness and stability, is not easily degraded.But also not for the report of transformation mechanism, can certainly, containing the biological enzyme or the enzyme system that participate in this conversion reaction in bacillus of oxidizing glucose, the quantity of the biological enzyme system that the catalytic capability of bacillus of oxidizing glucose contains with it and vigor have direct relation.
At present, although miglitol realizes suitability for industrialized production, because spawn activity is low, when utilizing microorganism 1-hydroxyethylamine-1-deoxy-D-sorbierite to be converted into glucofuranose amine, transformation efficiency is low, and transformation time is long, and labour intensity is large, and production cost is high.Tradition strain improvement mainly adopts the method for selection by mutation, and the method for mutagenesis is with a long history, and the past relies on the method for mutagenesis to improve output always, and current most of biological fermentation industrial production is still in these methods of use.But this method has sizable randomness, and if the process of bacterial classification life-time service mutagenic compound, its vigor can decline gradually.
At present, the substratum fermentation adding oxygen carrier is not also utilized to improve the pertinent literature report of the method for bacillus of oxidizing glucose biomass.
Summary of the invention
The object of this invention is to provide a kind of method that utilization adds the substratum fermentation raising bacillus of oxidizing glucose biomass of oxygen carrier, by adding suitable oxygen carrier in the medium, a kind of oxygen carrier fermentation system is provided, do not adding other material, under the condition not having additional energy source to consume, significantly improve the dissolved oxygen concentration in substratum, thus fermentation biomass is increased substantially, and the catalytic capability of this bacterial classification can't be affected.
The present invention adopts bacillus of oxidizing glucose to be microbial strains, obtains mycelium through spawn culture, fermentation culture centrifugation, and it is characterized in that adding oxygen carrier, tensio-active agent in fermention medium, its concrete steps are as follows:
Concrete steps and method as follows:
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, in 20-30 DEG C, 150-220rpm constant-temperature shaking incubator, cultivate 20-30 hour.
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
By through cultivation the seed culture fluid that obtains be inoculated into add oxygen carrier, the pH of tensio-active agent is in the fermention medium of 7.0, cultivate 36-48 hour in 20-30 DEG C, 150-220rpm constant-temperature shaking incubator after, collected by centrifugation thalline.Wherein, fermention medium contains: sorbyl alcohol 50-80g/L, yeast extract paste 10-40g/L, potassium primary phosphate 0.5-2g/L;
In substratum, oxygen carrier is normal hexane or n-dodecane, and the volume ratio of oxygen carrier and substratum is 5-50: 1000.Normal hexane or n-dodecane are here a kind ofly not dissolve mutually with water, do not poison microorganism, have oxygen carrier compared with high-solubility oxygen ability; Simultaneously in order to reduce the viscosity of substratum, the level of further raising dissolved oxygen, it is one or more in tween-80, stearic acid, Sodium dodecylbenzene sulfonate that basal culture medium adds tensio-active agent, containing tensio-active agent 0.2-2g in often liter of substratum, and preferred tween-80.
Centrifugal employing high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm.
Biocatalysis checking is carried out to obtained mycelium:
By the mycelium obtained in step (2) with 1: 1 ratio join the N-hydroxyethyl glucosamine that pH is 6.0 6%, at 20 DEG C, 15 hours are transformed in 200rpm constant-temperature shaking incubator, sampling, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dry up, colour developing observation, analytical results in iodine flask.
In said process, the adjustment of seed culture medium and fermention medium pH all adopts 5% conventional sodium hydroxide solution to regulate; Substrate N-hydroxyethyl glucosamine due to catalysis is alkalescence, needs to regulate about pH to 6.0 with concentrated hydrochloric acid.
The present invention is by adding oxygen carrier and tensio-active agent, fermentation system is made to have oxygen transmission speed fast, having foam generates few, the advantages such as shearing force is little, the biomass of bacillus of oxidizing glucose can be significantly improved, most high-biomass can reach and not add 2 times that oxygen carrier fermentation obtains biomass, and the biocatalysis ability of thalline is enhanced.
Embodiment
Embodiment 1
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, at 25 DEG C, cultivate 25 hours in 150rpm constant-temperature shaking incubator.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 fermention medium through cultivating the seed culture fluid obtained, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 50g/L, yeast extract paste 20g/L, and potassium primary phosphate 1g/L, pH are 7.0.Fermentation culture mode adopts shake-flask culture, and namely adopt 500 milliliters of triangular flasks, liquid amount is 50 milliliters, at 25 DEG C, cultivate in 200rpm constant-temperature shaking incubator after 40 hours, with high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collects thalline and obtains thalline 4.8 grams.
(3) biocatalysis
Take the centrifugal mycelium obtained 2 grams, take N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 with concentrated hydrochloric acid and add purified water 33 milliliters, make concentration be about 6%, put into 500 milliliters of triangular flasks, at 20 DEG C, 15 hours are transformed, sampling in 200rpm constant-temperature shaking incubator, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dries up, and develops the color in iodine flask, result shows, and transformation efficiency is about 70%.
Embodiment 2
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, at 25 DEG C, cultivate 25 hours in 150rpm constant-temperature shaking incubator.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 fermention medium through cultivating the seed culture fluid obtained, 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.Fermentation culture mode adopts shake-flask culture, namely 500 milliliters of triangular flasks are adopted, liquid amount is 50 milliliters, at 25 DEG C, cultivate in 200rpm constant-temperature shaking incubator after 40 hours, with high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 7.2 grams, the thalline collected than the substratum fermentation not adding oxygen carrier is more than 4.8 grams 50%.
(3) biocatalysis
Take the centrifugal mycelium obtained 2 grams, take N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 with concentrated hydrochloric acid and add purified water 33 milliliters, make concentration be about 6%, put into 500 milliliters of triangular flasks, at 20 DEG C, 15 hours are transformed, sampling in 200rpm constant-temperature shaking incubator, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dries up, and develops the color in iodine flask, result shows, and transformation efficiency is about 75%.
Embodiment 3
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, at 30 DEG C, cultivate 20 hours in 200rpm constant-temperature shaking incubator.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 fermention medium through cultivating the seed culture fluid obtained, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 60g/L, yeast extract paste 20g/L, and potassium primary phosphate 1g/L, 30ml/L are through the normal hexane of filtration sterilization, and pH is 7.0.Fermentation culture mode adopts shake-flask culture, namely 500 milliliters of triangular flasks are adopted, liquid amount is 50 milliliters, at 30 DEG C, cultivate in 220rpm constant-temperature shaking incubator after 48 hours, with high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 7.8 grams, the thalline collected than the substratum fermentation not adding oxygen carrier is more than 4.8 grams 62.5%.
(3) biocatalysis
Take the centrifugal mycelium obtained 2 grams, take N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 with concentrated hydrochloric acid and add purified water 33 milliliters, make concentration be about 6%, put into 500 milliliters of triangular flasks, at 20 DEG C, 15 hours are transformed, sampling in 200rpm constant-temperature shaking incubator, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dries up, and develops the color in iodine flask, result shows, and transformation efficiency is about 65%.
Embodiment 4
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, at 30 DEG C, cultivate 20 hours in 200rpm constant-temperature shaking incubator.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 fermention medium through cultivating the seed culture fluid obtained, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 60g/L, yeast extract paste 15g/L, the tween-80 of potassium primary phosphate 1.5g/L, 0.5g/L, 40ml/L is through the normal hexane of filtration sterilization, and pH is 7.0.Fermentation culture mode adopts shake-flask culture, namely 500 milliliters of triangular flasks are adopted, liquid amount is 50 milliliters, at 22 DEG C, cultivate in 220rpm constant-temperature shaking incubator after 38 hours, with high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 8.1 grams, the thalline collected than the substratum fermentation not adding oxygen carrier is more than 4.8 grams 68.8%.
(3) biocatalysis
Take the centrifugal mycelium obtained 2 grams, take N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 with concentrated hydrochloric acid and add purified water 33 milliliters, make concentration be about 6%, put into 500 milliliters of triangular flasks, at 20 DEG C, 15 hours are transformed, sampling in 200rpm constant-temperature shaking incubator, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dries up, and develops the color in iodine flask, result shows, and transformation efficiency is about 70%.
Embodiment 5
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, at 30 DEG C, cultivate 20 hours in 200rpm constant-temperature shaking incubator.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 fermention medium through cultivating the seed culture fluid obtained, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 50g/L, yeast extract paste 10g/L, the tween-80 of potassium primary phosphate 0.5g/L, 1g/L, 50ml/L is through the normal hexane of filtration sterilization, and pH is 7.0.Fermentation culture mode adopts shake-flask culture, namely 500 milliliters of triangular flasks are adopted, liquid amount is 50 milliliters, at 22 DEG C, cultivate in 220rpm constant-temperature shaking incubator after 38 hours, with high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 6.9 grams, the thalline collected than the substratum fermentation not adding oxygen carrier is more than 4.8 grams 44%.
(3) biocatalysis
Take the centrifugal mycelium obtained 2 grams, take N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 with concentrated hydrochloric acid and add purified water 33 milliliters, make concentration be about 6%, put into 500 milliliters of triangular flasks, at 20 DEG C, 15 hours are transformed, sampling in 200rpm constant-temperature shaking incubator, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dries up, and develops the color in iodine flask, result shows, and transformation efficiency is about 60%.
Embodiment 6
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, at 30 DEG C, cultivate 20 hours in 200rpm constant-temperature shaking incubator.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 fermention medium through cultivating the seed culture fluid obtained, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 80g/L, yeast extract paste 20g/L, the tween-80 of potassium primary phosphate 2g/L, 1.5g/L, 40ml/L through the normal hexane of filtration sterilization, pH7.0.Fermentation culture mode adopts shake-flask culture, namely 500 milliliters of triangular flasks are adopted, liquid amount is 50 milliliters, at 22 DEG C, cultivate in 220rpm constant-temperature shaking incubator after 38 hours, with high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 9.9 grams, the thalline collected than the substratum fermentation not adding oxygen carrier is more than 4.8 grams 106%.
(3) biocatalysis
Take the centrifugal mycelium obtained 2 grams, take N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 with concentrated hydrochloric acid and add purified water 33 milliliters, make concentration be about 6%, put into 500 milliliters of triangular flasks, at 20 DEG C, 15 hours are transformed, sampling in 200rpm constant-temperature shaking incubator, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dries up, and develops the color in iodine flask, result shows, and transformation efficiency is about 75%.
Embodiment 7
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, at 30 DEG C, cultivate 20 hours in 200rpm constant-temperature shaking incubator.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 fermention medium through cultivating the seed culture fluid obtained, inoculum size is 5%, and the composition of fermention medium and content are: sorbyl alcohol 70g/L, yeast extract paste 20g/L, the tween-80 of potassium primary phosphate 1.5g/L, 2.0g/L, 50ml/L is through the normal hexane of filtration sterilization, and pH is 7.0.Fermentation culture mode adopts shake-flask culture, namely 500 milliliters of triangular flasks are adopted, liquid amount is 50 milliliters, at 28 DEG C, cultivate in 200rpm constant-temperature shaking incubator after 45 hours, with high speed freezing centrifuge, centrifugal 15 minutes of 10000rpm, collect thalline and obtain thalline 9.4 grams, the thalline collected than the substratum fermentation not adding oxygen carrier is more than 4.8 grams 95.8%.
(3) biocatalysis
Take the centrifugal mycelium obtained 2 grams, take N-hydroxyethyl glucosamine 2 grams, regulate pH to 6.0 with concentrated hydrochloric acid and add purified water 33 milliliters, make concentration be about 6%, put into 500 milliliters of triangular flasks, at 20 DEG C, 15 hours are transformed, sampling in 200rpm constant-temperature shaking incubator, centrifugal, supernatant liquor point silica-gel plate, at methyl alcohol: ethanol: ammoniacal liquor volume ratio is chromatography in the chromatographic solution of 4: 3: 3, dries up, and develops the color in iodine flask, result shows, and transformation efficiency is about 75%.
Accompanying drawing illustrates:
Accompanying drawing 1 is applied Gluconobacter oxydans and is transformed the process route chart preparing miglitol key intermediate N replacement-1-DNJ, wherein (I) is 1-hydroxyethylamine-1-deoxy-D-sorbierite, (II) be glucofuranose amine, (III) is miglitol.
Claims (3)
1. one kind is improved the fermentation process of bacillus of oxidizing glucose biomass, take glucose oxidation and bacillus as microbial strains, mycelium is obtained through spawn culture, fermentation culture centrifugation, it is characterized in that in fermention medium, adding oxygen carrier, tensio-active agent, described oxygen carrier is normal hexane, and the volume ratio of oxygen carrier and substratum is 5-50: 1000; Described tensio-active agent is tween-80, containing tensio-active agent 0.2-2g in often liter of substratum; Its concrete steps are as follows:
(1) spawn culture
Be in the seed culture medium of 7.0 to pH by bacillus of oxidizing glucose strain inoculation, in 20-30 DEG C, 150-220rpm constant-temperature shaking incubator, cultivate 20-30 hour;
(2) fermentation culture
The seed culture fluid obtained in step (1) is inoculated into add oxygen carrier, tensio-active agent pH be in the fermention medium of 7.0, at 20 ~ 30 DEG C, cultivate in 150 ~ 220rpm constant-temperature shaking incubator after 36 ~ 48 hours, collected by centrifugation thalline.
2. according to the method described in claim 1, it is characterized in that: described in step 1), seed culture medium contains: sorbyl alcohol 50-80g/L, yeast extract paste 10-40g/L, potassium primary phosphate 0.5-2g/L.
3., according to the method described in claim 1, it is characterized in that: step 2) described fermention medium contains: sorbyl alcohol 50-80g/L, yeast extract paste 10-40g/L, potassium primary phosphate 0.5-2g/L.
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| 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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| 杨秋艳等.氧载体(正己烷)对黄原胶发酵的影响.《食品科技》.2009,(第04期), * |
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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. |