CN102154389A - Process for producing beta-polymalic acid by feed-batch fermentation - Google Patents
Process for producing beta-polymalic acid by feed-batch fermentation Download PDFInfo
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Abstract
The invention discloses a feed-batch fermentation process improving the yield of beta-polymalic acid, which is implemented by the following scheme: culturing a strain aureobasidium pullulans ipe-1 in a culture medium containing a carbon source, a nitrogen source, inorganic salt and trace elements while controlling the pH value and dissolved oxygen; and based on a batch fermentation production process, adopting glucose as the initial sugar, and 36-48 hours later, starting to continuously add the feed-batch solution of which the carbon source is glucose, sucrose, a mixed solution of glucose and fructose or a mixed solution of glucose and sucrose, and the inorganic salt contains sodium nitrate, calcium chloride and zinc sulfate. The yield of beta-polymalic acid is improved by 20-50% in comparison with the batch fermentation process. Through the invention, the original production method is changed, the yield of the beta-polymalic acid is obviously improved, the cost is lowered, and the process disclosed by the invention can be used for industrial large-scale fermentation production of beta-polymalic acid.
Description
Technical field
Technical scheme of the present invention belongs to the fermentation engineering field, is specifically related to the method that a kind of employing supplying technics improves a strain Aureobasidium pullulans (A.pulluluns ipe-1) fermentative production Beta-polymalic acid.
Background technology
Polymalic acid is a kind of special aliphatic polyester, is only monomer with the oxysuccinic acid, forms by the ester bond connection mutually.Different with many other natural heteropolymers, many free carboxyl groups and asymmetric c atom are arranged in the polymalic acid molecule, these free carboxyl groups have been given its many special character, it also has two significant advantages except having good water-solubility, biodegradable, biocompatibility and Bioabsorbable: (1) is metabolic easily.Because L MALIC ACID is the intermediate of organism Nei Keleibaishi tricarboxylic acid cycle, Beta-polymalic acid is removed by normal tricarboxylic acid cycle pathways metabolism easily in vivo; (2) easy modification property.Polymalic acid has pendant carboxy group, easily makes the polymalic acid derivative with other functional group reactions, or introducing functional group or small-molecule drug, thereby makes many products with specific function.Therefore, polymalic acid and derivative thereof can be used as operating sutures, tissue engineering bracket material, pharmaceutical carrier or primary medicine, controlled drug delivery system, packaging material for food, makeup etc., obtain important use in fields such as biological medicine, food, makeup.
Up to the present, the patent of liquid submerged fermentation production polymalic acid is less, known NakajimaToshiaki etc. are at Japanese publication patent (patent name: the production method of poly--L MALIC ACID, application number: JP7284395A) be the immobilized cell of using Aureobasidium pullulans genetic engineering bacterium or its genetic engineering bacterium, under special conditions, use the cell that is in stable state as enzyme catalyst, utilize glycogen material and ferric ion solutions to produce poly--L MALIC ACID.
Song Cunjiang etc. apply for a patent (patent name: the preparation method who obtains the Beta-polymalic acid of by product Propiram simultaneously, application number: be to adopt ethanol fractional precipitation Propiram, melanochrome and Beta-polymalic acid 200710058396), realize separating of Beta-polymalic acid and by product Propiram.
Wan Yinhua etc. apply for a patent (patent name: the preparation method of Beta-polymalic acid and salt thereof, application number: 200910078227) be the Beta-polymalic acid that adopts film and fermentation coupling technique, the fermentation of ion-exchange purification Aureobasidium pullulans to produce.
Wan Yinhua etc. apply for a patent (patent name: new process for fermenting Beta-polymalic acid, application number: be that fermentating controling process by pH, dissolved oxygen improves the fermenting Beta-polymalic acid productive rate 200910236646).
In present polymalic acid zymotechnique, report is not arranged about the technology of fed-batch fermentation.
Summary of the invention
Main purpose of the present invention provides the novel method that a kind of supplying technics is produced Beta-polymalic acid, it is glucose, sucrose, glucose and fructose mixed solution or glucose and sucrose mixed solution that this method adds carbon source by stream, inorganic salt comprise that the feed solutions of calcium chloride, zinc sulfate and SODIUMNITRATE is achieved, solve the lower problem of Beta-polymalic acid productive rate in the batch fermentation, significantly improve the productive rate of Beta-polymalic acid, reduce production costs.
The present invention solves this technical problem the technical scheme that is adopted:
1. fed-batch fermentation is produced the technology of Beta-polymalic acid, it is characterized in that:
To be inoculated in the fermention medium after the activated and seed culture of Aureobasidium pullulans ipe-1, control pH6.0 and dissolved oxygen concentration greater than 70% condition under, in the substratum of carbonaceous sources, nitrogenous source, inorganic salt and trace element, cultivate Aureobasidium pullulans ipe-1; Just sugar adopts glucose, and fermentation 36-48h begins flow feeding solution, fermentation 60-120h.
Illustrate:
The activation medium that (1) the 1st step was adopted: potato glucose substratum, 121 ℃ of sterilization 15min;
The seed culture medium (w/v) that (2) the 1st steps were adopted: 8% glucose, 0.2% SODIUMNITRATE, 0.01% potassium primary phosphate, 0.02% magnesium sulfate heptahydrate, 0.05% Repone K, 0.1% peptone, pH4.0～4.5,121 ℃ sterilization 15min.
The fermention medium (w/v) that (3) the 1st steps were adopted: 100-300g/L glucose, the 6g/L SODIUMNITRATE, 0.5g/L potassium primary phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.5g/L Repone K, the 15g/L peptone, the 5g/L yeast powder, 0.005-1.0g/L Zinc Sulphate Heptahydrate, 0.04-1.0g/L calcium chloride, pH4.0-7.0,121 ℃ of sterilization 15min;
(4) the 1st step actication of culture methods
Adopt activation medium to carry out activation culture 48-96 hour Aureobasidium pullulans bacterial strain (Aureobasidium pullulans ipe-1) at 20-28 ℃;
The seed culture method that (5) the 1st steps adopted
Activatory inoculum on the inclined-plane is inoculated in the seed culture medium, at 20-28 ℃, under the 200r/min condition shaking culture 48-96h hour.
2. according to the technology of the described fed-batch fermentation production of step 1 Beta-polymalic acid, it is characterized in that: just sugar adopts glucose, and carbon source is glucose, sucrose, glucose and fructose mixed solution or glucose and sucrose mixed solution in the feed solutions.
3. according to the technology of the described fed-batch fermentation production of step 1 Beta-polymalic acid, it is characterized in that: inorganic salt comprise zinc sulfate, calcium chloride, SODIUMNITRATE in the feed solutions.
4. produce the technology of Beta-polymalic acid according to the described fed-batch fermentation of step 2, it is characterized in that: initial glucose concentration is 100-300g/L, glucose concn is 300-800g/L in the feed solutions, and adding carbon source by stream is the yield that the feed solutions of glucose can effectively improve Beta-polymalic acid in the fermentation period.
5. produce the technology of Beta-polymalic acid according to the described fed-batch fermentation of step 2, it is characterized in that: initial glucose concentration is 100-300g/L, sucrose concentration is 300-600g/L in the feed solutions, and adding carbon source by stream is yield and the productive rate that the feed solutions of sucrose can effectively improve Beta-polymalic acid in the fermentation period.
6. produce the technology of Beta-polymalic acid according to the described fed-batch fermentation of step 2, it is characterized in that: initial glucose concentration is 100-300g/L, fructose or sucrose concentration are 1/10th to 1/20th of glucose concn in glucose and fructose mixed solution or glucose and the sucrose mixed solution, glucose concn is 300-800g/L, and the beneficial effect that adopts the carbon source mixed solution is the cost that reduces feed solutions.
7. according to the technology of the described fed-batch fermentation production of step 3 Beta-polymalic acid, it is characterized in that: zinc sulfate concentration is that 0.005-0.1g/L, calcium chloride concentration are that 0.04-1.0g/L, sodium nitrate concentration are 2.0-8.0g/L in the feed solutions
The present invention compared with prior art, beneficial effect is: improved the fermentation production rate of Beta-polymalic acid by flow feeding solution, efficiency of pcr product in the fermentation period has reduced cost.
Embodiment
The present invention will be further described below in conjunction with embodiment, and main protection domain involved in the present invention is not limited only to these embodiment.
Embodiment 1
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
1. activation medium: potato glucose substratum, 121 ℃ of sterilization 15min;
2. the preparation of seed culture medium (w/v): 8% glucose, 0.2% SODIUMNITRATE, 0.01% potassium primary phosphate, 0.02% magnesium sulfate heptahydrate, 0.05% Repone K, 0.1% peptone, pH 4.0～4.5,121 ℃ of sterilization 15min.
3. the preparation of fermention medium (w/v): 100-300g/L glucose, the 6g/L SODIUMNITRATE, 0.1-0.6g/L potassium primary phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.5g/L Repone K, the 15g/L peptone, the 5g/L yeast powder, 0.005-0.1g/L Zinc Sulphate Heptahydrate, 0.04-1.0g/L calcium chloride, pH4.0-7.0,121 ℃ of sterilization 15min.
Second step, actication of culture
With the Aureobasidium pullulans bacterial strain (Aureobasidium pullulans ipe-1) of 4 ℃ of preservations, transfer on the activation medium inclined-plane, carried out activation culture 72-96 hour at 25 ℃.
The 3rd step, seed culture
Activatory inoculum on the inclined-plane is inoculated in the 500mL triangular flask of dress 100mL seed culture medium, at 25 ℃, shaking culture 48h under the 200r/min condition.
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure are at 0.01-0.1Mpa, and 16h control dissolved oxygen concentration is greater than 70% before the fermentation, control dissolved oxygen concentration afterwards 70%, cultivate 72h.Initial glucose concentration is respectively 100g/L or 300g/L, and fermentation period is respectively 48h and 72h, and Beta-polymalic acid content is respectively 25.0g/L and 32.0g/L in the fermented liquid, and dry cell weight is respectively 55.9g/L and 60.2g/L.
Embodiment 2
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure are at 0.01-0.1Mpa, and 16h control dissolved oxygen concentration is greater than 70% before the fermentation, control dissolved oxygen concentration afterwards 70%, cultivate 72h.Just sugar is 190g/L glucose or 190g/L sucrose, and fermentation period is respectively 60h and 72h, and Beta-polymalic acid content is respectively 43.8g/L and 42.3g/L in the fermented liquid, and dry cell weight is respectively 68.9g/L and 70.2g/L.
Embodiment 3
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure is at 0.01-0.1Mpa, and 16h control dissolved oxygen concentration is controlled dissolved oxygen concentration 70% afterwards greater than 70% before the fermentation.Just sugar is 190g/L glucose, fermentation 36h begins to contain 300g/L or 800g/L glucose in the feed solutions that stream adds, fermentation period is 72h, flow feeding solution 1L, Beta-polymalic acid content is respectively 38.6g/L and 45.3g/L in the fermentation ends fermented liquid, and dry cell weight is respectively 59.7g/L and 72.0g/L.
Embodiment 4
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure is at 0.01-0.1Mpa, and 16h control dissolved oxygen concentration is controlled dissolved oxygen concentration 70% afterwards greater than 70% before the fermentation.Just sugar is 190g/L glucose, fermentation 36h begins that glucose concn is 500g/L in the feed solutions that stream adds, sodium nitrate concentration is respectively 2g/L or 8g/L, fermentation period is 72h, flow feeding solution 1L, Beta-polymalic acid content is respectively 45.3g/L and 46.0g/L in the fermentation ends fermented liquid, and dry cell weight is respectively 70.1g/L and 71.5g/L.
Embodiment 5
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure is at 0.01-0.1Mpa, and 16h control dissolved oxygen concentration is controlled dissolved oxygen concentration 70% afterwards greater than 70% before the fermentation.Just sugar is 190g/L glucose, fermentation 36h begins stream and adds 500g/L glucose, 6.0g/L SODIUMNITRATE, calcium chloride concentration is respectively the feed solutions of 0.04g/L or 1.0g/L, fermentation period is 72h, flow feeding solution 1L, Beta-polymalic acid content is respectively 52.0g/L and 53.3g/L in the fermentation ends fermented liquid, and dry cell weight is respectively 73.1g/L and 72.5g/L.
Embodiment 6
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure is at 0.01-0.1Mpa, and 16h control dissolved oxygen concentration is controlled dissolved oxygen concentration 70% afterwards greater than 70% before the fermentation.Just sugar is 190g/L glucose, fermentation 36h begins stream and adds and contain 500g/L glucose, 0.1g/L calcium chloride, the 6g/L SODIUMNITRATE, zinc sulfate concentration is respectively the feed solutions of 0.005g/L and 0.1g/L, and fermentation period shortens to 60h, flow feeding solution 1L, Beta-polymalic acid content is respectively 52.5g/L and 51.8g/L in the fermentation ends fermented liquid, and dry cell weight is respectively 74.0g/L and 72.5g/L.
Embodiment 7
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure are at 0.01-0.1Mpa, and 16h control dissolved oxygen concentration is greater than 70% before the fermentation, control dissolved oxygen concentration afterwards 70%, cultivate 72h.Just sugar is 190g/L glucose, fermentation 36h begins stream and adds and contain 300g/L or 600g/L sucrose, 0.1g/L calcium chloride, the 6g/L SODIUMNITRATE, 0.005g/L the feed solutions of zinc sulfate, fermentation period are 72h, flow feeding solution 1L, Beta-polymalic acid content is respectively 42.6g/L and 62.3g/L in the fermentation ends fermented liquid, and dry cell weight is respectively 71.1g/L and 73.5g/L.
Embodiment 8
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure is at 0.01-0.1Mpa, 16h control dissolved oxygen concentration is controlled dissolved oxygen concentration 70% afterwards greater than 70% before the fermentation, cultivates 72h, just sugar is 190g/L glucose, fermentation 36h begins stream and adds and contain 500g/L glucose, 0.1g/L calcium chloride, 6g/L SODIUMNITRATE, 0.005g/L the feed solutions of zinc sulfate, fructose concentration is respectively 50g/L and 25g/L, fermentation 72h, flow feeding solution 1L, during fermentation ends in the fermented liquid Beta-polymalic acid content be respectively 63.4g/L and 62.8g/L, dry cell weight is 73.5g/L and 72.5g/L.
Embodiment 9
Fermentative production Beta-polymalic acid as follows:
The first step, the substratum preparation
With embodiment 1
Second step, actication of culture
With embodiment 1
The 3rd step, seed culture
With embodiment 1
The 4th step, inoculation fermentation
The 3rd culture that make of step is inoculated in the 7L fermentor tank of dress 4L fermention medium with 10% inoculum size, pH 6.0,25 ℃ of temperature, initial speed 800r/min, air flow 0.75vvm, tank pressure is at 0.01-0.1Mpa, 16h control dissolved oxygen concentration is greater than 70% before the fermentation, control dissolved oxygen concentration afterwards 70%, just sugar is 190g/L glucose, and the 36h that ferments begins to contain SODIUMNITRATE 6g/L in the feed solutions that stream adds, calcium chloride 0.1g/L, zinc sulfate 0.005g/L, glucose 500g/L, sucrose concentration are respectively 50g/L and 25g/L, fermentation 72h, flow feeding solution 1L, during fermentation ends in the fermented liquid Beta-polymalic acid content be respectively 64.1g/L and 62.0g/L, dry cell weight is 74.6g/L and 73.5g/L.
Claims (7)
1. the technology of fed-batch fermentation production Beta-polymalic acid is characterized in that:
To be inoculated in the fermention medium after the activated and seed culture of Aureobasidium pullulans ipe-1, under control pH and dissolved oxygen conditions, in the substratum of carbonaceous sources, nitrogenous source, inorganic salt and trace element, cultivate Aureobasidium pullulans ipe-1; First sugar-fermenting 36-48h begins stream and adds the feed solutions of being made up of carbon source and inorganic salt, fermentation 60-120h.
2. fed-batch fermentation according to claim 1 is produced the technology of Beta-polymalic acid, it is characterized in that: just sugar adopts glucose, and carbon source is glucose, sucrose, glucose and fructose mixed solution or glucose and sucrose mixed solution in the feed solutions.
3. fed-batch fermentation according to claim 1 is produced the technology of Beta-polymalic acid, and it is characterized in that: inorganic salt comprise zinc sulfate, calcium chloride, SODIUMNITRATE in the feed solutions.
4. fed-batch fermentation according to claim 2 is produced the technology of Beta-polymalic acid, and it is characterized in that: initial glucose concentration is 100-300g/L, and glucose concn is 300-800g/L in the feed solutions.
5. fed-batch fermentation according to claim 2 is produced the technology of Beta-polymalic acid, and it is characterized in that: initial glucose concentration is 100-300g/L, and sucrose concentration is 300-600g/L in the feed solutions.
6. fed-batch fermentation according to claim 2 is produced the technology of Beta-polymalic acid, it is characterized in that: initial glucose concentration is 100-300g/L, fructose or sucrose concentration are 1/10th to 1/20th of glucose concn in glucose and fructose mixed solution or glucose and the sucrose mixed solution, and glucose concn is 300-800g/L.
7. fed-batch fermentation according to claim 3 is produced the technology of Beta-polymalic acid, and it is characterized in that: zinc sulfate concentration is that 0.005-0.1g/L, calcium chloride concentration are that 0.04-1.0g/L, sodium nitrate concentration are 2.0-8.0g/L in the feed solutions.
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Cited By (5)
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| CN102492740A (en) * | 2011-12-16 | 2012-06-13 | 天津北洋百川生物技术有限公司 | Method for producing poly(malic acid) and pullulan together by using Aureobasidium pullulans |
| CN103045662A (en) * | 2012-12-31 | 2013-04-17 | 天津北洋百川生物技术有限公司 | Fermentation medium for improving output and purity of beta-polymalic acid produced by zymotechnics |
| CN103740774A (en) * | 2013-12-25 | 2014-04-23 | 天津北洋百川生物技术有限公司 | Method for high-density fermentation production of polymalic acid |
| CN104561143A (en) * | 2014-12-29 | 2015-04-29 | 天津北洋百川生物技术有限公司 | Method for improving yield of beta-polymalic acid by fed-batch fermentation |
| CN114790429A (en) * | 2021-01-26 | 2022-07-26 | 苏州乐卡食品有限公司 | High-density fermentation method for composite probiotics |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492740A (en) * | 2011-12-16 | 2012-06-13 | 天津北洋百川生物技术有限公司 | Method for producing poly(malic acid) and pullulan together by using Aureobasidium pullulans |
| CN102492740B (en) * | 2011-12-16 | 2013-08-07 | 天津北洋百川生物技术有限公司 | Method for producing poly(malic acid) and pullulan together by using Aureobasidium pullulans |
| CN103045662A (en) * | 2012-12-31 | 2013-04-17 | 天津北洋百川生物技术有限公司 | Fermentation medium for improving output and purity of beta-polymalic acid produced by zymotechnics |
| CN103740774A (en) * | 2013-12-25 | 2014-04-23 | 天津北洋百川生物技术有限公司 | Method for high-density fermentation production of polymalic acid |
| CN103740774B (en) * | 2013-12-25 | 2016-03-02 | 天津北洋百川生物技术有限公司 | The method of high-density fermentation production of polymalic acid |
| CN104561143A (en) * | 2014-12-29 | 2015-04-29 | 天津北洋百川生物技术有限公司 | Method for improving yield of beta-polymalic acid by fed-batch fermentation |
| CN114790429A (en) * | 2021-01-26 | 2022-07-26 | 苏州乐卡食品有限公司 | High-density fermentation method for composite probiotics |
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Application publication date: 20110817 |