CN102286564A - Method for culturing halophilic microorganism to produce ectoin and hydroxyl ectoin - Google Patents
Method for culturing halophilic microorganism to produce ectoin and hydroxyl ectoin Download PDFInfo
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- CN102286564A CN102286564A CN2011102560945A CN201110256094A CN102286564A CN 102286564 A CN102286564 A CN 102286564A CN 2011102560945 A CN2011102560945 A CN 2011102560945A CN 201110256094 A CN201110256094 A CN 201110256094A CN 102286564 A CN102286564 A CN 102286564A
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Abstract
The invention aims to provide a method for culturing halophilic microorganism to produce ectoin and hydroxyl ectoin, which can improve the synthetic efficiency of ectoin and hydroxyl ectoin by reasonable cost. The method for culturing halophilic microorganism to produce ectoin and hydroxyl ectoin comprises the following steps of: (1) activating a strain; (2) culturing seeds; (3) fermenting; (4) quickly improving the salinity of a culture medium to 3-3.5M by feed supplement, continuously culturing for 3-8h to greatly improve the synthetic amount of ectoin; and (5) improving culture temperature to higher temperature from the optimal growth temperature, and continuously culturing for 3-10h to obtain higher hydroxyl ectoin synthetic mount. In the fermentation process, an inoculating fermentation strain is cultivated with an osmotic pressure shock and thermal shock method, and the synthetic rate of producing ectoin and hydroxyl ectoin is greatly improved by the reasonable cost.
Description
Technical field
The present invention relates to the production method of a kind of tetrahydropyrimidine and hydroxy tetrahydro pyrimidine, especially a kind of method of cultivating halophilic microorganism production tetrahydropyrimidine and hydroxy tetrahydro pyrimidine.
Background technology
Tetrahydropyrimidine be a kind of halophilic microorganism for a kind of compatible solute of synthetic in the body of resisting extraneous high salt concentration, and derivative-hydroxy tetrahydro pyrimidine that some halophilic microorganisms can the tetrahydrobiopterin synthesis pyrimidine.Tetrahydropyrimidine is compared with the known compatible solute of hydroxy tetrahydro pyrimidine and other, and its pair cell, nucleic acid, enzyme, albumen etc. have the better protection effect under extreme conditions such as ray, drying, freezing, high temperature, high salt.
Because its special structure, so far can't synthetic tetrahydropyrimidine or hydroxy tetrahydro pyrimidine, can only be by synthetic acquisition the in the extracellular microbial.According to open source literature, the synthetic of tetrahydropyrimidine mainly is to adopt the bacterium method of milking to produce, and carries out high density fermentation at present.
The common fermentation method is: for the production of tetrahydropyrimidine, seed culture is with above-mentioned 1,2 step, and 150ml kind daughter bacteria liquid is inoculated into the 3L fermention medium then, 30 ℃ of cultivations, and pH is controlled at 7.0-7.2.Sodium chloride concentration is 3M in the fermention medium, and other is the same.Cultivate the centrifugal collection thalline of 42h, the alcoholic extraction tetrahydropyrimidine concentrates, membrane filtration, and crystallization obtains product.For the production of hydroxy tetrahydro pyrimidine, seed culture is with above-mentioned 1,2 step, and 150ml kind daughter bacteria liquid is inoculated into the 3L fermention medium then, 42 ℃ of cultivations behind 30 ℃ of cultivation 20h, and pH is controlled at 7.0-7.2.Sodium chloride concentration is controlled at 3M in the fermention medium, centrifugal collection thalline behind the continuation cultivation 31h, and alcoholic extraction tetrahydropyrimidine and hydroxy tetrahydro pyrimidine concentrate, membrane filtration, crystallization obtains product.
Because the salt concn of the suitableeest growth of halophilic bacterium and can to reach the salt concn of high yield tetrahydropyrimidine inconsistent, influenced the combined coefficient of tetrahydropyrimidine, moderate halophilic bacterium Halomonas ventosae DL7 for example, its the suitableeest growth salt concn is 1M, but the salt concn of optimum tetrahydrobiopterin synthesis pyrimidine is 3M.It syntheticly is subjected to inducing of thermal stimulus to the hydroxy tetrahydro pyrimidine as the derivative of tetrahydropyrimidine, under the optimal growth temperature of halophilic bacterium, have only a small amount of synthetic, usually to improve synthetic that growth temperature can more amount, for example Halomonas ventosae DL7 shaking table in the nutritional medium test tube is cultivated, culture temperature is brought up to 37 ℃ or 42 ℃ from 30 ℃, but its growth that improved the growth temperature severe inhibition, cause the serious decline of tetrahydropyrimidine resultant quantity again, thereby also influenced the output of hydroxy tetrahydro pyrimidine.
Summary of the invention
The purpose of this invention is to provide the method that a kind of cultivation halophilic microorganism of improving the combined coefficient of tetrahydropyrimidine and hydroxy tetrahydro pyrimidine with reasonable cost produces tetrahydropyrimidine and hydroxy tetrahydro pyrimidine.
Realize that the cultivation halophilic microorganism of one of the object of the invention produces the method for tetrahydropyrimidine, comprises the steps:
(1) actication of culture is put into the test tube that fills nutritive medium with the halophilic bacterium kind, cultivates activation on shaking table;
(2) seed culture is put into the triangular flask that fills nutrient solution and halophilic bacterium kind with the bacterial classification after the activation, cultivates on shaking table;
(3) fermentation stage, fermentation is cultivated under the optimal growth salinity of halophilic bacterium, and maximum concentration is arrived in cell proliferation;
(4) by feed supplement the salt concn of substratum is brought up to 3～3.5M rapidly, continue then to cultivate 3～8h, the resultant quantity of tetrahydropyrimidine is improved greatly.
Actication of culture in the described step (1) is 30 ℃ of temperature, and shaking table is cultivated 18～24h.
Seed culture in the described step (2) is 30 ℃ of temperature, and shaking table is cultivated 18～24h.
Optimal growth salinity in the described step (3) is 1～2M NaCl, cultivates 18～24h, reaches maximum concentration.
The time that feed supplement in the described step (4) improves salt concn is 1～2h.
Realize the method that two the cultivation halophilic microorganism of the object of the invention produces the hydroxy tetrahydro pyrimidine, comprise the steps:
(1) actication of culture is put into the test tube that fills nutritive medium with the halophilic bacterium kind, cultivates activation on shaking table;
(2) seed culture is put into the triangular flask that fills nutrient solution and halophilic bacterium kind with the bacterial classification after the activation, cultivates on shaking table;
(3) fermentation stage, fermentation is cultivated under the optimal growth salinity of halophilic bacterium, and maximum concentration is arrived in cell proliferation;
(4) by feed supplement the salt concn of substratum is brought up to 3～3.5M rapidly, continue then to cultivate 3-8h, the resultant quantity of tetrahydropyrimidine is improved greatly;
(5) culture temperature is brought up to comparatively high temps from the optimal growth temperature, continue to cultivate 3～10h, obtain higher hydroxy tetrahydro pyrimidine resultant quantity.
Actication of culture in the described step (1) is 30 ℃ of temperature, and shaking table is cultivated 18～24h; Seed culture in the described step (2) is 30 ℃ of temperature, and shaking table is cultivated 18～24h.
Optimal growth salinity in the described step (3) is 1～2M NaCl, cultivates 18～24h, reaches maximum concentration.
The time that feed supplement in the described step (4) improves salt concn is 1～2h.
Optimal growth temperature in the described step (5) is 30～32 ℃; The time of improving temperature is 1～2h; Described comparatively high temps is 37～42 ℃.
The beneficial effect of the method for cultivation halophilic microorganism production tetrahydropyrimidine of the present invention and hydroxy tetrahydro pyrimidine is as follows:
The method that cultivation halophilic microorganism of the present invention produces tetrahydropyrimidine and hydroxy tetrahydro pyrimidine, impacting by osmotic pressure during the fermentation (is to pass through to improve rapidly environment osmotic pressure within a short period of time, for example in the 1h mistake na concn is brought up to 3M from 1M, the mycetocyte external penetration is pressed rapidly improve.) and the thermal shocking method (promptly improve envrionment temperature at short notice rapidly, for example culture temperature brought up to 42 ℃ from 30 ℃ in the 1h, the outside hot pressing of mycetocyte is improved rapidly.) cultivate the fermentation inoculating strain, improved production tetrahydropyrimidine and hydroxy tetrahydro pyrimidine synthetic ratio greatly with reasonable cost.
Embodiment
Embodiment 1
1, getting a transfering loop receives Halomonas ventosae DL7 bacterial strain in the test tube that contains the 6ml liquid nutrient media in the shaking table of 240rpm 30 ℃ and cultivates the 20h activation down on Bechtop.
2, in the 500ml triangular flask of 50ml seed culture medium is housed, insert the above-mentioned bacterium liquid of activatory 2.5ml, on the shaking table of 240rpm, cultivate 24h for 30 ℃, parallel cultivation is 3 bottles altogether.Seed culture medium (1L): glucose 10g, sodium-chlor 58.4g, ammonium chloride 2g, sal epsom 2g, dipotassium hydrogen phosphate 0.6g, yeast extract paste 1g, peptone 1g.
3, in being housed, the 5L fermentor tank of 3L fermention medium inserts 150ml kind daughter bacteria liquid, 800 rev/mins, pH is controlled at 7.0-7.2, air flow is 0.2-0.4v/v.min (volume ratio of the logical sterile air of the every volume substratum of per minute), begin feed supplement behind the fermentation 10h, glucose concn is maintained about 20g/l, cultivate 18h down at 30 ℃, add NaCl to 3M behind the 18h in the 1h, continue to cultivate 8h.If it is main producing tetrahydropyrimidine, fermentation leaves it at that, and centrifugal subsequently recovery thalline discharges tetrahydropyrimidine product in the born of the same parents, concentrate then, and membrane filtration, crystallization obtains the tetrahydropyrimidine product.Produce the hydroxy tetrahydro pyrimidine and change step 4 over to.Fermention medium (1L): glucose 20g, sodium-chlor 58.4g, ammonium chloride 2g, sal epsom 2g, dipotassium hydrogen phosphate 0.6g, ferrous sulfate 0.01g, yeast extract paste 1g, peptone 1g, Sodium Glutamate 5g.Supplemented medium (1L): glucose 500g, sodium-chlor fluctuates as required, ammonium chloride 20g, sal epsom 20g, dipotassium hydrogen phosphate 6g, ferrous sulfate 0.02g, yeast extract paste 20g, peptone 20g.
4, with improving culture temperature to 42 ℃ in the fermented liquid 1h of above-mentioned cultivation 24h, continue to cultivate 8h, keep pH in the scope of 7.0-7.2 by the NaOH that replenishes 3M in the culturing process.Cultivate and finish the back by centrifugal recovery thalline, alcoholic extraction discharges tetrahydropyrimidine and hydroxy tetrahydro pyrimidine product in the born of the same parents, concentrate then, and membrane filtration, crystallization obtains tetrahydropyrimidine and hydroxy tetrahydro pyrimidine.
The fermentation process of present embodiment and the productive rate of fermentation process commonly used are compared as follows:
Embodiment 2
1, getting a transfering loop receives Halomonas ventosae DL7 bacterial strain in the test tube that contains the 6ml liquid nutrient media in the shaking table of 240rpm 30 ℃ and cultivates the 18h activation down on Bechtop.
2, in the 500ml triangular flask of 50ml seed culture medium is housed, insert the above-mentioned bacterium liquid of activatory 2.5ml, on the shaking table of 240rpm, cultivate 18h for 30 ℃, parallel cultivation is 3 bottles altogether.Seed culture medium (1L): glucose 10g, sodium-chlor 58.4g, ammonium chloride 2g, sal epsom 2g, dipotassium hydrogen phosphate 0.6g, yeast extract paste 1g, peptone 1g.
3, in being housed, the 5L fermentor tank of 3L fermention medium inserts 150ml kind daughter bacteria liquid, 800 rev/mins, pH is controlled at 7.0-7.2, air flow is 0.2-0.4v/v.min (volume ratio of the logical sterile air of the every volume substratum of per minute), begin feed supplement behind the fermentation 10h, glucose concn is maintained about 20g/l, cultivate 20h down at 30 ℃, add NaCl to 3.5M behind the 20h in the 2h, continue to cultivate 3h.If it is main producing tetrahydropyrimidine, fermentation leaves it at that, and centrifugal subsequently recovery thalline discharges tetrahydropyrimidine product in the born of the same parents, concentrate then, and membrane filtration, crystallization obtains the tetrahydropyrimidine product.Produce the hydroxy tetrahydro pyrimidine and change step 4 over to.Fermention medium (1L): glucose 20g, sodium-chlor 58.4g, ammonium chloride 2g, sal epsom 2g, dipotassium hydrogen phosphate 0.6g, ferrous sulfate 0.01g, yeast extract paste 1g, peptone 1g, Sodium Glutamate 5g.Supplemented medium (1L): glucose 500g, sodium-chlor fluctuates as required, ammonium chloride 20g, sal epsom 20g, dipotassium hydrogen phosphate 6g, ferrous sulfate 0.02g, yeast extract paste 20g, peptone 20g.
4, with improving culture temperature to 37 ℃ in the fermented liquid 2h of above-mentioned cultivation, continue to cultivate 10h, keep pH in the scope of 7.0-7.2 by the NaOH that replenishes 3M in the culturing process.Cultivate and finish the back by centrifugal recovery thalline, alcoholic extraction discharges tetrahydropyrimidine and hydroxy tetrahydro pyrimidine product in the born of the same parents, concentrate then, and membrane filtration, crystallization obtains tetrahydropyrimidine and hydroxy tetrahydro pyrimidine.
The fermentation process of present embodiment and the productive rate of fermentation process commonly used are compared as follows:
Embodiment 3
1, getting a transfering loop receives Halomonas ventosae DL7 bacterial strain in the test tube that contains the 6ml liquid nutrient media in the shaking table of 240rpm 30 ℃ and cultivates the 24h activation down on Bechtop.
2, in the 500ml triangular flask of 50ml seed culture medium is housed, insert the above-mentioned bacterium liquid of activatory 2.5ml, on the shaking table of 240rpm, cultivate 20h for 30 ℃, parallel cultivation is 3 bottles altogether.Seed culture medium (1L): glucose 10g, sodium-chlor 58.4g, ammonium chloride 2g, sal epsom 2g, dipotassium hydrogen phosphate 0.6g, yeast extract paste 1g, peptone 1g.
3, in being housed, the 5L fermentor tank of 3L fermention medium inserts 150ml kind daughter bacteria liquid, 800 rev/mins, pH is controlled at 7.0-7.2, air flow is 0.2-0.4v/v.min (volume ratio of the logical sterile air of the every volume substratum of per minute), begin feed supplement behind the fermentation 10h, glucose concn is maintained about 20g/l, cultivate 24h down at 30 ℃, add NaCl to 3.3M behind the 24h in the 1.5h, continue to cultivate 5h.If it is main producing tetrahydropyrimidine, fermentation leaves it at that, and centrifugal subsequently recovery thalline discharges tetrahydropyrimidine product in the born of the same parents, concentrate then, and membrane filtration, crystallization obtains the tetrahydropyrimidine product.Produce the hydroxy tetrahydro pyrimidine and change step 4 over to.Fermention medium (1L): glucose 20g, sodium-chlor 58.4g, ammonium chloride 2g, sal epsom 2g, dipotassium hydrogen phosphate 0.6g, ferrous sulfate 0.01g, yeast extract paste 1g, peptone 1g, Sodium Glutamate 5g.Supplemented medium (1L): glucose 500g, sodium-chlor fluctuates as required, ammonium chloride 20g, sal epsom 20g, dipotassium hydrogen phosphate 6g, ferrous sulfate 0.02g, yeast extract paste 20g, peptone 20g.
4, with improving culture temperature to 40 ℃ in the fermented liquid 1.5h of above-mentioned cultivation, continue to cultivate 3h, keep pH in the scope of 7.0-7.2 by the NaOH that replenishes 3.3M in the culturing process.Cultivate and finish the back by centrifugal recovery thalline, alcoholic extraction discharges tetrahydropyrimidine and hydroxy tetrahydro pyrimidine product in the born of the same parents, concentrate then, and membrane filtration, crystallization obtains tetrahydropyrimidine and hydroxy tetrahydro pyrimidine.
The fermentation process of present embodiment and the productive rate of fermentation process commonly used are compared as follows:
Claims (10)
1. cultivate the method that halophilic microorganism produces tetrahydropyrimidine for one kind, comprise the steps:
(1) actication of culture is put into the test tube that fills nutritive medium with the halophilic bacterium kind, cultivates activation on shaking table;
(2) seed culture is put into the triangular flask that fills nutrient solution and halophilic bacterium kind with the bacterial classification after the activation, cultivates on shaking table;
(3) fermentation stage, fermentation is cultivated under the optimal growth salinity of halophilic bacterium, and maximum concentration is arrived in cell proliferation;
(4) by feed supplement the salt concn of substratum is brought up to 3～3.5M rapidly, continue to cultivate 3～8h then.
2. the method that cultivation halophilic microorganism according to claim 1 produces tetrahydropyrimidine, it is characterized in that: the actication of culture in the described step (1) is 30 ℃ of temperature, shaking table is cultivated 18～24h.
3. the method that cultivation halophilic microorganism according to claim 1 produces tetrahydropyrimidine, it is characterized in that: the seed culture in the described step (2) is 30 ℃ of temperature, shaking table is cultivated 18～24h.
4. the method that cultivation halophilic microorganism according to claim 1 produces tetrahydropyrimidine, it is characterized in that: the optimal growth salinity in the described step (3) is 1～2M NaCl, cultivates 18～24h, reaches maximum concentration.
5. the method that cultivation halophilic microorganism according to claim 1 produces tetrahydropyrimidine, it is characterized in that: the time that the feed supplement in the described step (4) improves salt concn is 1～2h.
6. cultivate the method that halophilic microorganism produces the hydroxy tetrahydro pyrimidine for one kind, comprise the steps:
(1) actication of culture is put into the test tube that fills nutritive medium with the halophilic bacterium kind, cultivates activation on shaking table;
(2) seed culture is put into the triangular flask that fills nutrient solution and halophilic bacterium kind with the bacterial classification after the activation, cultivates on shaking table;
(3) fermentation stage, fermentation is cultivated under the optimal growth salinity of halophilic bacterium, and maximum concentration is arrived in cell proliferation;
(4) by feed supplement the salt concn of substratum is brought up to 3～3.5M rapidly, continue to cultivate 3-8h then;
(5) culture temperature is brought up to comparatively high temps from the optimal growth temperature, continue to cultivate 3～10h.
7. the method that cultivation halophilic microorganism according to claim 6 produces the hydroxy tetrahydro pyrimidine, it is characterized in that: the actication of culture in the described step (1) is 30 ℃ of temperature, shaking table is cultivated 18～24h; Seed culture in the described step (2) is 30 ℃ of temperature, and shaking table is cultivated 18～24h.
8. the method that cultivation halophilic microorganism according to claim 6 produces the hydroxy tetrahydro pyrimidine, it is characterized in that: the optimal growth salinity in the described step (3) is 1～2M NaCl, cultivates 18～24h, reaches maximum concentration.
9. the method that cultivation halophilic microorganism according to claim 6 produces the hydroxy tetrahydro pyrimidine, it is characterized in that: the time that the feed supplement in the described step (4) improves salt concn is 1～2h.
10. the method that cultivation halophilic microorganism according to claim 6 produces the hydroxy tetrahydro pyrimidine, it is characterized in that: the optimal growth temperature in the described step (5) is 30～32 ℃; The time of improving temperature is 1～2h; Described comparatively high temps is 37～42 ℃.
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| CN103805528A (en) * | 2012-11-08 | 2014-05-21 | 中国科学院微生物研究所 | Novel deep-sea bacterial strain and its application |
| CN104273131A (en) * | 2013-07-01 | 2015-01-14 | 镇江拜因诺生物科技有限公司 | Melon and fruit yield increasing agent |
| CN104513835A (en) * | 2013-10-08 | 2015-04-15 | 镇江拜因诺生物科技有限公司 | Method for preparing fuel ethanol by using brewed lees |
| CN104560915A (en) * | 2013-10-11 | 2015-04-29 | 镇江拜因诺生物科技有限公司 | A high-density fermentation production method of alkaline xylanase |
| CN104560901A (en) * | 2013-10-10 | 2015-04-29 | 镇江拜因诺生物科技有限公司 | Production of tetrahydropyridine hydroxylase through marine microorganism fermentation |
| CN104560902A (en) * | 2013-10-16 | 2015-04-29 | 镇江拜因诺生物科技有限公司 | Method for producing ectoine hydroxylase by waste molasses through fermentation |
| CN105018403A (en) * | 2015-07-14 | 2015-11-04 | 天津科技大学 | Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof |
| CN105177078A (en) * | 2015-09-30 | 2015-12-23 | 天津科技大学 | Preparation method of hydroxyectoine |
| CN109053587A (en) * | 2018-08-31 | 2018-12-21 | 山东福田药业有限公司 | A method of the separation and Extraction tetrahydropyrimidine from halophilic microorganism fermentation liquid |
| CN116200297A (en) * | 2022-12-27 | 2023-06-02 | 山东丰金美业科技有限公司 | High-yield tetrahydropyrimidine salt-addicted single island bacillus cereus and culture method and application thereof |
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| CN103805528A (en) * | 2012-11-08 | 2014-05-21 | 中国科学院微生物研究所 | Novel deep-sea bacterial strain and its application |
| CN104273131A (en) * | 2013-07-01 | 2015-01-14 | 镇江拜因诺生物科技有限公司 | Melon and fruit yield increasing agent |
| CN104513835A (en) * | 2013-10-08 | 2015-04-15 | 镇江拜因诺生物科技有限公司 | Method for preparing fuel ethanol by using brewed lees |
| CN104560901A (en) * | 2013-10-10 | 2015-04-29 | 镇江拜因诺生物科技有限公司 | Production of tetrahydropyridine hydroxylase through marine microorganism fermentation |
| CN104560915A (en) * | 2013-10-11 | 2015-04-29 | 镇江拜因诺生物科技有限公司 | A high-density fermentation production method of alkaline xylanase |
| CN104560902A (en) * | 2013-10-16 | 2015-04-29 | 镇江拜因诺生物科技有限公司 | Method for producing ectoine hydroxylase by waste molasses through fermentation |
| CN105018403A (en) * | 2015-07-14 | 2015-11-04 | 天津科技大学 | Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof |
| CN105018403B (en) * | 2015-07-14 | 2018-01-09 | 天津科技大学 | A kind of genetic engineering bacterium for producing tetrahydropyrimidine and its construction method and application |
| CN105177078A (en) * | 2015-09-30 | 2015-12-23 | 天津科技大学 | Preparation method of hydroxyectoine |
| CN109053587A (en) * | 2018-08-31 | 2018-12-21 | 山东福田药业有限公司 | A method of the separation and Extraction tetrahydropyrimidine from halophilic microorganism fermentation liquid |
| CN116200297A (en) * | 2022-12-27 | 2023-06-02 | 山东丰金美业科技有限公司 | High-yield tetrahydropyrimidine salt-addicted single island bacillus cereus and culture method and application thereof |
| CN116200297B (en) * | 2022-12-27 | 2023-10-03 | 山东丰金美业科技有限公司 | High-yield tetrahydropyrimidine salt-addicted single island bacillus cereus and culture method and application thereof |
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Application publication date: 20111221 |