CN103805651A - Production method for pullulan based on cell metabolism regulation strategies - Google Patents
Production method for pullulan based on cell metabolism regulation strategies Download PDFInfo
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- CN103805651A CN103805651A CN201210443770.4A CN201210443770A CN103805651A CN 103805651 A CN103805651 A CN 103805651A CN 201210443770 A CN201210443770 A CN 201210443770A CN 103805651 A CN103805651 A CN 103805651A
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- pulullan polysaccharide
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Abstract
The invention relates to a novel method for producing pullulan by using cell metabolism regulation technology to improve cell permeability. 0.02% to 2.14% of a polyether substance and 0.01% to 1.35% of a surfactant substance are added into a fermentation medium, thereby effectively increasing the yield of pullulan, improving the conversion efficiency of a substrate, shortening a fermentation cycle and reducing formation of melanin.
Description
Technical field
The present invention relates to a kind of pulullan polysaccharide production method based on cellular metabolism regulating strategy, belong to bioengineering field.
Background technology
Pulullan polysaccharide is a kind of water-soluble macromolecule exocellular polysaccharide that utilizes the fermentation of the small molecules such as sucrose, glucose carbohydrate to produce by Aureobasidium pullulans, because it has splendid film-forming properties, plasticity-, gas barrier property, be widely used in food fresh keeping, medical material, cosmetic field.The production method of pulullan polysaccharide is mainly biological fermentation process, but in its production process, exist many problems always, wherein in Aureobasidium pullulans fermenting process cycle low conversion rate long, that secrete a large amount of melanochrome and substrate sugar cause yielding poorly, separation and purification process complexity, greatly increase production cost, reduce product quality, become the key issue of restriction Propiram suitability for industrialized production, therefore, the domestic temporary report without pulullan polysaccharide suitability for industrialized production at present.
For these problems, the method generally adopting at present is both at home and abroad by screening and the few bacterial strain of mutagenic obtained product pigment, but the strain fermentation of these methods acquisitions yields poorly, and the cycle is still long, but also is to have a small amount of pigment formation and be difficult to remove.The present invention passes through to improve fermention medium, the generation of the angle control polysaccharide regulating and controlling from cellular metabolism and the secretion of pigment, and method is simple, and effect is remarkable.
Summary of the invention
For solving the gordian technique difficult problem in pulullan polysaccharide fermentation production process, the object of the invention is to propose a kind of pulullan polysaccharide production method based on cellular metabolism regulating strategy.
The present invention for the technical scheme that a technical solution difficult problem adopts is:
A kind of pulullan polysaccharide production method based on cellular metabolism regulating strategy mainly comprises the following steps:
(1) seed preparation.The bacterial classification that glycerine pipe is preserved carries out plate streaking separation and Culture 2 ~ 3 d, single bacterium colony access seed bottle that picking has been grown, and 28 ~ 30 ℃, 24 ~ 36 h are cultivated in 250 ~ 280 r/m shaking table concussions, to bacterium liquid counting, as reach 10
7individual/mL explanation bacterium liquid activation is intact;
(2) fermentation culture.Seed liquor is inoculated in fermention medium according to 2% ~ 10% inoculum size, and 28 ~ 30 ℃, 50 ~ 60 h are cultivated in 250 ~ 280 r/m shaking tables concussions, and when the content of surveying to pulullan polysaccharide no longer increases, and the concentration of residual sugar is reduced to 1 ~ 5 g/L when following, fermentation ends;
(3) extraction and purification.Pulullan polysaccharide aftertreatment extraction and purification comprises filtration sterilization, activated carbon decolorizing, ethanol desalination, dehydration, vacuum-drying.
Illustrate: the seed culture medium that step (1) adopts is: sucrose 5% ~ 8%, yeast extract paste 0.2% ~ 0.5%, dipotassium hydrogen phosphate 0.5% ~ 0.8%, magnesium sulfate 0.02 ~ 0.05%, ammonium sulfate 0.05% ~ 0.08%, sodium-chlor 0.05 ~ 0.15%; The fermention medium that step (2) adopts is: sucrose 10% ~ 15%, yeast extract paste 0.2% ~ 0.5%, dipotassium hydrogen phosphate 0.5% ~ 0.8%, magnesium sulfate 0.02 ~ 0.05%, ammonium sulfate 0.05% ~ 0.08%, sodium-chlor 0.05 ~ 0.15%, polyethers 0.05% ~ 5.0%, tensio-active agent 0.01% ~ 0.1 %.
This beneficial effect of the invention is: by adding during the fermentation polyether substance and surfactant-based material, strengthen Aureobasidium pullulans cell membrane permeability, improve the speed of the inside and outside substance transportation of cell, reduce intracellular polyse and accumulate the feedback inhibition to cell, cell metabolism speed is improved, fermentation period foreshortens to 50-55h, and substrate conversion efficiency reaches more than 70%, and output and the substrate conversion efficiency of pulullan polysaccharide are improved significantly.And the pigment producing is few and easily go out, plant that product colour is good, quality is high most.
To other products, particularly output, substrate conversion efficiency and the production intensity of intracellular product application cell metabolism regulation technology raising fermentation have certain help to this beneficial effect of the invention.
Embodiment
reference examples
(1) seed culture medium preparation and sterilizing: according to following formulated fermention medium
Sucrose 5%, yeast extract paste 0.2%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate 0.02%, ammonium sulfate 0.06%, sodium-chlor 0.1%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(2) seed preparation; The bacterial classification that glycerine pipe is preserved carries out plate streaking separation and Culture 3 d, single bacterium colony access seed bottle that picking has been grown, and 28.5 ℃, 30 h are cultivated in 260 r/m shaking table concussions;
(3) fermention medium preparation sterilizing: according to following formulated fermention medium
Sucrose 10%, yeast extract paste 0.2%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.02%, ammonium sulfate 0.06%, sodium-chlor 0.1%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(4) fermentation culture; Seed liquor is inoculated in fermention medium according to 5% inoculum size, and 28.5 ℃, 72 h are cultivated in 280 r/m shaking table concussions, and the concentration of residual sugar is 3 g/L fermentation ends, and the content that detects pulullan polysaccharide in fermented liquid is 45g/L, and substrate conversion efficiency is 45%;
(5) extraction and purification: pulullan polysaccharide aftertreatment extraction and purification comprises filtration sterilization, activated carbon decolorizing, alcohol desalination, dehydration, vacuum-drying, obtains yellow pulullan polysaccharide product, and product purity is 85%.
embodiment 1
(1) seed culture medium preparation and sterilizing: according to following formulated fermention medium
Sucrose 5%, yeast extract paste 0.2%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate 0.02%, ammonium sulfate 0.06%, sodium-chlor 0.1%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(2) seed preparation; The bacterial classification that glycerine pipe is preserved carries out plate streaking separation and Culture 3 d, single bacterium colony access seed bottle that picking has been grown, and 28.5 ℃, 30 h are cultivated in 260 r/m shaking table concussions;
(3) fermention medium preparation sterilizing: according to following formulated fermention medium
Sucrose 10%, yeast extract paste 0.2%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.02%, ammonium sulfate 0.06%, sodium-chlor 0.1%, polyethenoxy ether 1%, tween 20 0.05%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(4) fermentation culture; Seed liquor is inoculated in fermention medium according to 5% inoculum size, and 28.5 ℃, 55 h are cultivated in 280 r/m shaking table concussions, and the concentration of residual sugar is 3.2 g/L fermentation ends, and the content that detects pulullan polysaccharide in fermented liquid is 71g/L, and substrate conversion efficiency is 71%;
(5) extraction and purification: pulullan polysaccharide aftertreatment extraction and purification comprises filtration sterilization, activated carbon decolorizing, alcohol desalination, dehydration, vacuum-drying, obtains white pulullan polysaccharide product, and product purity is 98%.
embodiment 2
(1) seed culture medium preparation and sterilizing: according to following formulated fermention medium
Sucrose 6%, yeast extract paste 0.3%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.03%, ammonium sulfate 0.05%, sodium-chlor 0.05%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(2) seed preparation; The bacterial classification that glycerine pipe is preserved carries out plate streaking separation and Culture 3 d, single bacterium colony access seed bottle that picking has been grown, and 28.5 ℃, 25 h are cultivated in 260 r/m shaking table concussions;
(3) fermention medium preparation sterilizing: according to following formulated fermention medium
Sucrose 12%, yeast extract paste 0.3%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.05%, ammonium sulfate 0.06%, sodium-chlor 0.1%, polyoxyethylene poly-oxygen propylene aether 2%, tween-80 0.1%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(4) fermentation culture; Seed liquor is inoculated in fermention medium according to 5% inoculum size, and 28.5 ℃, 55 h are cultivated in 280 r/m shaking table concussions, and the concentration of residual sugar is 3 g/L fermentation ends, and the content that detects pulullan polysaccharide in fermented liquid is 91g/L, and substrate conversion efficiency is 75.8%;
(5) extraction and purification: pulullan polysaccharide aftertreatment extraction and purification comprises filtration sterilization, activated carbon decolorizing, alcohol desalination, dehydration, vacuum-drying, obtains white pulullan polysaccharide product, and product purity is 98.5%.
embodiment 3
(1) seed culture medium preparation and sterilizing: according to following formulated fermention medium
Sucrose 8%, yeast extract paste 0.5%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate 0.02%, ammonium sulfate 0.06%, sodium-chlor 0.05%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(2) seed preparation; The bacterial classification that glycerine pipe is preserved carries out plate streaking separation and Culture 3 d, single bacterium colony access seed bottle that picking has been grown, and 28.5 ℃, 24 h are cultivated in 260 r/m shaking table concussions;
(3) fermention medium preparation sterilizing: according to following formulated fermention medium
Sucrose 15%, yeast extract paste 0.3%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate 0.05%, ammonium sulfate 0.08%, sodium-chlor 0.1%, polyvingl ether 3%, tween-80 0.1%, after preparation, packing, utilizes 121 ℃ of sterilizing 20 min of high-pressure steam sterilizing pan;
(4) fermentation culture; Seed liquor is inoculated in fermention medium according to 5% inoculum size, and 28.5 ℃, 52 h are cultivated in 280 r/m shaking table concussions, and the concentration of residual sugar is 3 g/L fermentation ends, and the content that detects pulullan polysaccharide in fermented liquid is 118g/L, and substrate conversion efficiency is 78.6%;
(5) extraction and purification: pulullan polysaccharide aftertreatment extraction and purification comprises filtration sterilization, activated carbon decolorizing, alcohol desalination, dehydration, vacuum-drying, obtains white pulullan polysaccharide product, and product purity is 98.6%.
Claims (3)
1. the pulullan polysaccharide production method based on cellular metabolism regulating strategy, comprise the fermentation culture of Aureobasidium pullulans and the separation and purification of pulullan polysaccharide, it is characterized in that adding polyether substance and surfactant-based material in fermention medium, after fermentation culture 50-60h, carry out fermented liquid aftertreatment and extract purifying pulullan polysaccharide.
2. a kind of pulullan polysaccharide production method based on cellular metabolism regulating strategy according to claim 1, is characterized in that: detailed step is as follows:
Seed preparation: the bacterial classification that glycerine pipe is preserved carries out plate streaking separation and Culture 2 ~ 3 d, single bacterium colony access seed bottle that picking has been grown, 28 ~ 30 ℃, 24 ~ 36 h are cultivated in 250 ~ 280 r/m shaking table concussions, to bacterium liquid counting, as reach 10
7individual/mL explanation bacterium liquid activation is intact;
Fermentation culture: seed liquor is inoculated in fermention medium according to 2% ~ 10% inoculum size, 28 ~ 30 ℃, 50 ~ 60 h are cultivated in 250 ~ 280 r/m shaking table concussions, when the content of surveying to pulullan polysaccharide no longer increases, and when the concentration of residual sugar is reduced to below 1 ~ 5 g/L, fermentation ends;
Extraction and purification: pulullan polysaccharide aftertreatment extraction and purification comprises filtration sterilization, activated carbon decolorizing, the desalination of ethanol precipitation, dehydration, vacuum-drying.
3. according to claim 1, a kind of pulullan polysaccharide production method based on cellular metabolism regulating strategy described in 2, it is characterized in that: described fermention medium consists of: sucrose 10% ~ 15%, yeast extract paste 0.2% ~ 0.5%, dipotassium hydrogen phosphate 0.2% ~ 0.8%, magnesium sulfate 0.02 ~ 0.1%, ammonium sulfate 0.01% ~ 0.1%, sodium-chlor 0.01 ~ 0.1%, polyethers 0.02% ~ 2.14%, tensio-active agent 0.01% ~ 1.35%, wherein polyether substance is one or several in following material, these materials include but not limited to: polyethenoxy ether, polyoxyethylene poly-oxygen propylene aether, polyvingl ether, laureth, polyether glycol etc., surfactant-based material is one or several in following several material, these materials include but not limited to: tween 20, Tween-40, Tween-60, tween-80 etc.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450827A (en) * | 2014-12-16 | 2015-03-25 | 苏州大学 | Method for improving yield of pulullan biosynthesized by resting cells of aureobasidium pullulans |
| CN107760608A (en) * | 2017-06-08 | 2018-03-06 | 中国科学院青岛生物能源与过程研究所 | A kind of mutagenic strain of efficiently production low molecule pulullan polysaccharide and its application |
| CN108624630A (en) * | 2018-05-15 | 2018-10-09 | 日照金禾博源生化有限公司 | A method of shortening the citric acid fermentation period |
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
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| CN101215592A (en) * | 2008-01-15 | 2008-07-09 | 天津市工业微生物研究所 | Fermentation method for producing pullulan polysaccharide |
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| CN101215592A (en) * | 2008-01-15 | 2008-07-09 | 天津市工业微生物研究所 | Fermentation method for producing pullulan polysaccharide |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| CN104450827A (en) * | 2014-12-16 | 2015-03-25 | 苏州大学 | Method for improving yield of pulullan biosynthesized by resting cells of aureobasidium pullulans |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
| CN107760608A (en) * | 2017-06-08 | 2018-03-06 | 中国科学院青岛生物能源与过程研究所 | A kind of mutagenic strain of efficiently production low molecule pulullan polysaccharide and its application |
| CN107760608B (en) * | 2017-06-08 | 2018-06-26 | 中国科学院青岛生物能源与过程研究所 | A kind of mutagenic strain of efficient production low molecule pulullan polysaccharide and its application |
| CN108624630A (en) * | 2018-05-15 | 2018-10-09 | 日照金禾博源生化有限公司 | A method of shortening the citric acid fermentation period |
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