CN105624233A - Method for increasing yield of pullulan - Google Patents
Method for increasing yield of pullulan Download PDFInfo
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- CN105624233A CN105624233A CN201510904313.4A CN201510904313A CN105624233A CN 105624233 A CN105624233 A CN 105624233A CN 201510904313 A CN201510904313 A CN 201510904313A CN 105624233 A CN105624233 A CN 105624233A
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Abstract
The invention discloses a method for increasing the yield of pullulan. The method comprises the following steps: after activating aureobasidium pullulans, inoculating a seed medium; after OD600 is greater than 0.3, inoculating seeds into a fermentation medium; monitoring OD600 of a fermentation liquid in real time; when OD600 is greater than 0.2 and less than 0.3, adding a compounding agent I which accounts for 0.02-0.03% of the volume of the fermentation liquid, so as to greatly increase the bacterium growth velocity and remarkably shorten the logarithmic growth phase; when OD600 is greater than or equal to 0.6, adding a compounding agent II which accounts for 0.03-0.05% of the volume of the fermentation liquid. The substrate conversion speed can be accelerated, the polysaccharide synthesis performance can be improved, and the yield can be greatly increased.
Description
Technical field
The present invention relates to the technical field of fermentable, specifically a kind of method improving pulullan yield.
Background technology
Pulullan polysaccharide, as a kind of favorable solubility, the hydrophilic colloid that degree of adhesion is excellent, has, in fields such as food, medicine, chemical industry, the scope that uses widely. But the pullulan fermentation time is long, yield is more difficult control also. Most of aminoacid, the somatomedin such as vitamin has facilitation for thalli growth. And aminoacid is different from the interpolation kind of vitamin and addition, the facilitation for thalli growth also has bigger difference.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method improving pulullan yield.
The present invention solves that the technical problem existed in known technology is adopted the technical scheme that:
The method of the raising pulullan yield of the present invention, comprises the following steps:
(1), after being activated by Aureobasidium pullulans, seed culture medium, OD are accessed600Reach more than 0.3, then seed is accessed in fermentation medium;
(2) monitor in real time fermentation liquid OD600, as 0.2 < OD600< 0.3, adds fermentating liquid volume 0.02-0.03% built agent I; Work as OD600>=0.6, add the built agent II of fermentating liquid volume 0.03-0.05%;
Wherein built agent I includes glutamic acid, methionine and vitamin B2;
Built agent II includes serine and uridylate.
The present invention can also adopt techniques below measure:
In seed culture medium: sucrose 90g/L, yeast powder 4g/L, sodium chloride 3g/L, anhydrous slufuric acid ammonium 1g/L, MgSO4 7H2O0.5g/L��K2HPO43g/L��FeSO4��7H2O0.05g/L, pH6.5; In fermentation medium: sucrose 150g/L, peptone 7g/L, sodium chloride 2.5g/L, MgSO4��7H2O0.4g/L��K2HPO46g/L��FeSO4��7H2O0.05g/L, initial pH6.0.
Aureobasidium pullulans Aureobasidiumpullulans, China Committee for Culture Collection of Microorganisms's common micro-organisms center within 25th, it is preserved in December in 2012, bacterium numbering CGMCCNO.7055, preservation address: biological study institute of the Datun Road, Chaoyang District, Beijing City Chinese Academy of Sciences.
In step (1), Aureobasidium pullulans bacterial strain is activated 6��8h, access in seed culture medium, in 28��32 DEG C, 180rpm, cultivates 28��32h; OD600During > 0.3, being accessed in fermentation medium, condition of culture is OD600During < 0.5, temperature controls at 32 DEG C, OD600When >=0.5, temperature controls at 28 DEG C, ventilation 1.0 (V/V), and rotating speed is 300rpm.
In built agent I, in mass ratio, glutamic acid: methionine: vitamin B2=8:6:1; In built agent II, in mass ratio, serine: uridylate=1:1.
The present invention has the advantage that and has the benefit effect that
In the method for the raising pulullan yield of the present invention, at lag phase (the 0.2 < OD of fermentation culture600< 0.3) add built agent I, biomass growth rate substantially increases, and reaches the exponential phase time and substantially shortens. Owing to its Glutamic Acid can resolve into ��-ketoglutaric acid in cell, methionine is decomposed into and enters central metabolic pathway into succinate coenzyme A, strengthen central metabolites, and vitamin B2 generates flavine-adenine dinucleotide through dehydrogenation reaction, flavin mononucleotide (FMN), strengthens the electron transmission of oxygen respiratory chain, and three cooperates, improve production capacity in born of the same parents, promote thalli growth. Exponential phase (OD in fermentation culture600>=0.6) adding built agent II, substrate conversion rate is accelerated, and polysaccharide synthesis capability strengthens, and yield substantially increases. Owing to the serine in built agent II can produce the secondary metabolite of polysaccharide synthesis, and uridylate is the important as precursors thing used by synthesis of pulullan polysaccharide synthesis precursor uridine diphosphoglucose, therefore pulullan yield has and substantially increases, substrate conversion efficiency improves.
Detailed description of the invention
By the following examples technical scheme is described in detail.
The method of the raising pulullan yield of the present invention, comprises the following steps:
(1), after being activated by Aureobasidium pullulans, seed culture medium, OD are accessed600Reach more than 0.3, then seed is accessed in fermentation medium;
(2) monitor in real time fermentation liquid OD600, as 0.2 < OD600< 0.3, adds fermentating liquid volume 0.02-0.03% built agent I; Work as OD600>=0.6, add the built agent II of fermentating liquid volume 0.03-0.05%;
Wherein built agent I includes glutamic acid, methionine and vitamin B2;
Built agent II includes serine and uridylate.
In built agent I, in mass ratio, glutamic acid: methionine: vitamin B2=8:6:1; In built agent II, in mass ratio, serine: uridylate=1:1.
Aureobasidium pullulans Aureobasidiumpullulans, China Committee for Culture Collection of Microorganisms's common micro-organisms center within 25th, it is preserved in December in 2012, bacterium numbering CGMCCNO.7055, preservation address: biological study institute of the Datun Road, Chaoyang District, Beijing City Chinese Academy of Sciences.
Embodiment 1:
(1), seed culture medium: sucrose 90g/L, yeast powder 4g/L, sodium chloride 3g/L, anhydrous slufuric acid ammonium 1g/L, MgSO4 7H2O0.5g/L��K2HPO43g/L��FeSO4��7H2O0.05g/L, pH6.5; Fermentation medium components: sucrose 150g/L, peptone 7g/L, sodium chloride 2.5g/L, MgSO4��7H2O0.4g/L��K2HPO46g/L��FeSO4��7H2O0.05g/L, initial pH6.0.
(2), Aureobasidium pullulans bacterial strain is activated 6h, access in the 500mL baffle plate bottle that liquid amount is 100mL, in 30 DEG C, 180rpm, cultivates 29h, records OD600It is 0.318. Being accessed in the 5L fermentation tank that liquid amount is 3.5L, condition of culture is OD600< when 0.5, temperature controls at 32 DEG C, OD600When >=0.5, temperature controls at 28 DEG C, ventilation 1.0 (V/V), and rotating speed is 300rpm.
(3), fermentation liquid OD is monitored in real time600, OD600The built agent I of 0.03% is added when being 0.219, after adding 16h, OD600Reaching 0.609, add the built agent II of 0.05%, fermentation reaches 60h total time, and residual sugar is substantially zeroed, and 78h terminates fermentation.
(4), taking that fermentation liquid is centrifugal removes thalline, supernatant is with the long-pending ethanol precipitate with ethanol of triploid, and after sucking filtration, 105 DEG C of constant temperature dryings are to constant weight. Recording thick pulullan yield is 81.45g/L.
Embodiment 2:
(1), seed culture medium: sucrose 90g/L, yeast powder 4g/L, sodium chloride 3g/L, anhydrous slufuric acid ammonium 1g/L, MgSO4 7H2O0.5g/L��K2HPO43g/L��FeSO4��7H2O0.05g/L, pH6.5; Fermentation medium components: sucrose 150g/L, peptone 7g/L, sodium chloride 2.5g/L, MgSO4��7H2O0.4g/L��K2HPO46g/L��FeSO4��7H2O0.05g/L, initial pH6.0.
(2), being activated after 7h by Aureobasidium pullulans bacterial strain, access in seed culture medium, 28 DEG C, 180rpm cultivates 32h, records OD600 when being 0.301, by the seed inoculum concentration access liquid amount with 3% be 18L 30L fermentation tank in, condition of culture is OD600< when 0.5, temperature controls at 32 DEG C, OD600When >=0.5, temperature controls at 28 DEG C, ventilation 1.0 (V/V), and rotating speed is 300rpm.
(3) fermentation liquid OD is monitored in real time600, OD600The built agent I of 0.02% is added when being 0.223, after adding 16h, OD600Reaching 0.605, add the built agent II of 0.05%, fermentation reaches 58h total time, and residual sugar is substantially zeroed, and 80h terminates fermentation.
(4), taking that fermentation liquid is centrifugal removes thalline, supernatant is with the long-pending ethanol precipitate with ethanol of triploid, and after sucking filtration, 105 DEG C of constant temperature dryings are to constant weight. Recording thick pulullan yield is 88.79g/L.
Embodiment 3:
(1), seed culture medium: sucrose 90g/L, yeast powder 4g/L, sodium chloride 3g/L, anhydrous slufuric acid ammonium 1g/L, MgSO4 7H2O0.5g/L��K2HPO43g/L��FeSO4��7H2O0.05g/L, pH6.5; Fermentation medium components: sucrose 150g/L, peptone 7g/L, sodium chloride 2.5g/L, MgSO4��7H2O0.4g/L��K2HPO46g/L��FeSO4��7H2O0.05g/L, initial pH6.0.
(2), by after Aureobasidium pullulans bacterial strain activation 8h, accessing in first order seed, 32 DEG C, 180rpm cultivates 28h, and with 3% inoculum concentration, first order seed is accessed secondary seed, 28 DEG C, ventilation 1.0 (V/V), and rotating speed is that 300rpm cultivates 16h, records OD600Be 0.323, by its with 3% inoculum concentration access liquid amount be 150L 200L fermentation tank in, condition of culture is OD600< when 0.5, temperature controls at 32 DEG C, OD600When >=0.5, temperature controls at 28 DEG C, ventilation 1.0 (V/V), and rotating speed is 300rpm.
(3) fermentation liquid OD is monitored in real time600, OD600The built agent I of 0.02% is added when being 0.257, after adding 18h, OD600Reaching 0.623, add the built agent II of 0.04%, fermentation reaches 60h total time, and residual sugar is substantially zeroed, and 75h terminates fermentation.
(4), taking that fermentation liquid is centrifugal removes thalline, supernatant is with the long-pending ethanol precipitate with ethanol of triploid, and after sucking filtration, 105 DEG C of constant temperature dryings are to constant weight. Recording thick pulullan yield is 92.79g/L.
The above, it it is only presently preferred embodiments of the present invention, not the present invention is done any pro forma restriction, although the present invention is with preferred embodiment openly as above, but, it is not limited to the present invention, any those skilled in the art, without departing within the scope of technical solution of the present invention, certainly the technology contents of announcement can be utilized to make a little change or modification, become the Equivalent embodiments of equivalent variations, in every case it is the content without departing from technical solution of the present invention, according to any simple modification that above example is made by the technical spirit of the present invention, equivalent variations and modification, belong in the scope of technical solution of the present invention.
Claims (5)
1. the method improving pulullan yield, comprises the following steps:
(1), after being activated by Aureobasidium pullulans, seed culture medium, OD are accessed600Reach more than 0.3, then seed is accessed in fermentation medium;
(2) monitor in real time fermentation liquid OD600, as 0.2 < OD600< 0.3, adds fermentating liquid volume 0.02-0.03% built agent I; Work as OD600>=0.6, add the built agent II of fermentating liquid volume 0.03-0.05%;
Wherein built agent I includes glutamic acid, methionine and vitamin B2;
Built agent II includes serine and uridylate.
2. the method for raising pulullan yield according to claim 1, it is characterised in that: in seed culture medium: sucrose 90g/L, yeast powder 4g/L, sodium chloride 3g/L, anhydrous slufuric acid ammonium 1g/L, MgSO4 7H2O0.5g/L��K2HPO43g/L��FeSO4?7H2O0.05g/L, pH6.5; In fermentation medium: sucrose 150g/L, peptone 7g/L, sodium chloride 2.5g/L, MgSO4��7H2O0.4g/L��K2HPO46g/L��FeSO4?7H2O0.05g/L, initial pH6.0.
3. the method for raising pulullan yield according to claim 1 and 2, it is characterized in that: Aureobasidium pullulans Aureobasidiumpullulans, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium numbering CGMCCNO.7055.
4. the method for raising pulullan yield according to claim 3, it is characterised in that: in step (1), Aureobasidium pullulans bacterial strain is activated 6��8h, access in seed culture medium, in 28��32 DEG C, 180rpm, cultivates 28��32h; OD600During > 0.3, being accessed in fermentation medium, condition of culture is OD600During < 0.5, temperature controls at 32 DEG C, OD600When >=0.5, temperature controls at 28 DEG C, ventilation 1.0(V/V), rotating speed is 300rpm.
5. the method for raising pulullan yield according to claim 4, it is characterised in that: in built agent I, in mass ratio, glutamic acid: methionine: vitamin B2=8:6:1; In built agent II, in mass ratio, serine: uridylate=1:1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107699500A (en) * | 2017-11-17 | 2018-02-16 | 山东省农业科学院农产品研究所 | The method of aureobasidium pullulans and its fermenting and producing pulullan polysaccharide |
| CN108715791A (en) * | 2018-05-29 | 2018-10-30 | 李伟达 | A kind of production method of apple vinegar beverage |
| CN109182421A (en) * | 2018-11-09 | 2019-01-11 | 盐城工学院 | A kind of method that solid state fermentation brewex's grains prepare feruloylated oligosaccharides and dietary fiber |
| CN111587893A (en) * | 2019-02-21 | 2020-08-28 | 财团法人食品工业发展研究所 | Aureobasidium pullulans fermentation product and preparation method thereof, microbial preparation for reducing fungal diseases and method for reducing fungal diseases of agricultural products |
| CN112760231A (en) * | 2021-01-15 | 2021-05-07 | 天津科技大学 | Culture system for preparing low-molecular-weight pullulan and production process |
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| CN103695500A (en) * | 2013-12-27 | 2014-04-02 | 天津北洋百川生物技术有限公司 | Method for increasing pulullan yield |
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| CN103805650A (en) * | 2012-11-09 | 2014-05-21 | 山东福瑞达生物科技有限公司 | Fermentation production method for pigment-free pullulan |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699500A (en) * | 2017-11-17 | 2018-02-16 | 山东省农业科学院农产品研究所 | The method of aureobasidium pullulans and its fermenting and producing pulullan polysaccharide |
| CN107699500B (en) * | 2017-11-17 | 2021-05-04 | 山东省农业科学院农产品研究所 | Aureobasidium pullulans and method for producing pullulan by fermenting same |
| CN108715791A (en) * | 2018-05-29 | 2018-10-30 | 李伟达 | A kind of production method of apple vinegar beverage |
| CN109182421A (en) * | 2018-11-09 | 2019-01-11 | 盐城工学院 | A kind of method that solid state fermentation brewex's grains prepare feruloylated oligosaccharides and dietary fiber |
| CN111587893A (en) * | 2019-02-21 | 2020-08-28 | 财团法人食品工业发展研究所 | Aureobasidium pullulans fermentation product and preparation method thereof, microbial preparation for reducing fungal diseases and method for reducing fungal diseases of agricultural products |
| CN111587893B (en) * | 2019-02-21 | 2022-03-29 | 财团法人食品工业发展研究所 | Aureobasidium pullulans fermentation product and preparation method thereof, microbial preparation for reducing fungal diseases and method for reducing fungal diseases of agricultural products |
| CN112760231A (en) * | 2021-01-15 | 2021-05-07 | 天津科技大学 | Culture system for preparing low-molecular-weight pullulan and production process |
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