CN103695500A - Method for increasing pulullan yield - Google Patents
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Abstract
The invention discloses a method for increasing pulullan yield by sub-sectional adding of growth factors in a fermentation process, and belongs to the technical field of biological fermentation engineering. The method comprises the following steps: producing pulullan from aureobasidium pullulans CGMCC NO.7055 in a fermentation manner; adding 0.003-0.005% of vitamin B1 when bacteria growth is at the beginning of the logarithmic phase; and adding 0.002-0.004% of uridine monophosphate when the bacteria growth is at the early stage of a stable stage, so as to induce and facilitate accumulation of secondary metabolite, namely the pulullan. The growth factors are specifically added according to the characteristics of sugar production in different growth periods and fermentation processes, so that the method is convenient and fast to operate and has an obvious effect; the fermentation period is greatly shortened, the transformation rate of a substrate is increased, and the cost of the pulullan is reduced.
Description
Technical field:
The present invention relates to a kind of method of microorganism fermentative production pulullan polysaccharide, be specifically related to segmentation in a kind of fermenting process and add the method that somatomedin improves pulullan polysaccharide output.Belong to bio-fermentation engineering field.
Background technology:
Pulullan polysaccharide is a kind of water miscible unsetting dextran as the pattern polysaccharide of water-soluble polysaccharide, and it has been widely used in a plurality of fields such as food, pharmacy, oil, chemical industry as emulsifying agent, suspension agent, thickening material, stablizer, jelling agent, membrane-forming agent and lubricant etc.Pulullan polysaccharide, by being an important development direction of novel fermentation engineering from now on, is more and more subject to the attention of domestic enterprise.
Somatomedin refers in microorganism growth process necessary, the nutritive ingredient that must directly be provided by the external world, and common somatomedin has VITAMIN, amino acid, purine pyrimidine etc.Somatomedin can provide the component of the important chemical substance of microorganism, cofactor (coenzyme and prothetic group) and participate in metabolism.Pulullan polysaccharide belongs to secondary metabolite, at thalline, in the logarithmic phase later stage, in earlier stage starts stationary phase to produce.
In the middle of document, about pulullan polysaccharide output, improving is at present mainly to optimize by induction mutation of bacterium, medium component, and often effect is not too obvious, and fermentation period is long, and the transformation efficiency of substrate is not high, causes pulullan polysaccharide cost high.Application number is mutagenic strain and the cultural method > > thereof that 201210594876.4 Chinese invention patent < < produces pulullan polysaccharide in a large number, the Aureobasidium pullulans CGMCC NO.7055 of one strain product pulullan polysaccharide is disclosed, and disclose the method for utilizing this bacterial strain to produce pulullan polysaccharide, and fermention medium forms.Aureobasidium pullulans (Aureobasidium pullulans) BCSWGHPL101 is 96g/L in fermention medium fermentative production pulullan polysaccharide output, compared with the output of original strain 68 ± 5.2g/L, improved 41.2%, fermentation condition is 28 ℃, under 200 ± 20rpm, cultivates 3d.But still exist fermentation period longer, and substrate conversion efficiency is lower, and the problem that substrate utilization ratio is not high, causes pulullan polysaccharide cost higher, is unfavorable in enormous quantities, large-scale production.
Summary of the invention:
In order to solve the deficiency in pulullan polysaccharide tradition optimizing process, improve targetedly pulullan polysaccharide output, shorten pulullan polysaccharide fermentation period, improve substrate conversion efficiency, the invention provides a kind of segmentation and add the method that somatomedin improves pulullan polysaccharide output, not only easy to operate, and when improving output, also reduced the generation of pigment.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
1. a method that improves pulullan polysaccharide output, comprises the following steps:
1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 28-32h.
Described seed culture medium forms: sucrose 8g, and yeast soaks powder 0.2g, sodium-chlor 0.15g, dipotassium hydrogen phosphate 0.1g, ammonium sulfate 0.1g, magnesium sulfate 0.04g, ferrous sulfate 0.05g, distilled water is settled to 100mL.
2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate bottle, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is that 400rpm cultivates.Thalli growth during the initial stage, adds the vitamins B of 0.03-0.05 ‰ in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add the uridylate of 0.02-0.04 ‰, cultivate 57-63h, thereby reach guiding, promote that secondary metabolite is the accumulation of pulullan polysaccharide, improve substrate conversion efficiency, shorten fermentation period.
Described fermention medium forms: sucrose 10g, and peptone 0.4g, sodium-chlor 0.3g, dipotassium hydrogen phosphate 0.7g, magnesium sulfate 0.04g, ferrous sulfate 0.05g, distilled water is settled to 100mL, initial pH5.5-6.5.
Described vitamins B
1and then uridylate is prepared by the aseptic membrane filtration of 0.45um with the solution that deionized water is mixed with concentration 0.01g/L.
3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product.
Described Aureobasidium pullulans BCSWGHPL101(Aureobasidium pullulans) be by a strain of laboratory preservation, to produce the few Aureobasidium pullulans TKPM10017 of amount of pigment to obtain through Screening, Mutation, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.Preservation date on December 25th, 2012, deposit number is CGMCC NO.7055.
The more stable about 50-60g/L of described Aureobasidium pullulans TKPM10017 output, pigment formation is less, but fermentation period is longer, and substrate conversion efficiency is lower.
Described mutagenic processes comprises the steps:
1. open ultraviolet lamp preheating 30min.Get 5mL spore suspension, add in the 50mm culture dish that is placed with aseptic rotor, be placed on magnetic stirring apparatus, be put in to wavelength 254nm, in the ultraviolet mutagenesis case of power 30w, the distance of adjusting culture dish and ultraviolet lamp is 30cm.Open lid, open magnetic stirring apparatus constant speed and stir, under ultraviolet lamp, irradiate respectively 0s, 60s, 120s, 180s, 240s, 300s, 360s.The equal lucifuge of all operations is carried out, and postradiation bacteria suspension is done to gradient dilution, coats 28 ℃ of constant temperature lucifuges on PDA solid medium and cultivates 72h.
2. cultivating 72 hours through 28 ℃ of the spore suspensions of mutagenesis on PDA substratum will be coated, utilize vernier callipers to measure the diameter of single bacterium colony, and observe color, the variant of picking Aureobasidium pullulans (colony diameter is large, surface wettability, of light color or white).
3. by the single bacterium colony obtaining by dull and stereotyped primary dcreening operation, the single colony inoculation of picking is in fermention medium (100mL/500mL), and 28 ℃, 180r/min, utilize shaking table shaking culture 96h, measures fermented liquid color and measures pulullan polysaccharide output.
4. the bacterial strain filtering out was again transferred for 15 generations continuously on inclined-plane, in the picking third generation, the 6th generation, the 9th generation, the 12 generation and the 15 generation, carried out fermenting experiment, measure its color and measure pulullan polysaccharide output, select stable yield and pigment formation few, fermentation period is short, the bacterial strain that substrate conversion efficiency is high carries out preservation, obtains pulullan polysaccharide superior strain CGMCC NO.7055.
Described Aureobasidium pullulans CGMCC NO.7055 characteristic is as follows:
In fermented liquid, mainly present yeast sample form, oval and big or small homogeneous, the modes of reproduction of bacterial strain is similar to the gemmation mode of yeast; Bacterium colony is rounded, central elevation, and smooth surface, moistening, be difficult for picking, the growth later stage has mycelium to produce.Utilizable carbon source: glucose, sucrose, maltose, fructose, Zulkovsky starch, dextrin one of them.Utilizable nitrogenous source: extractum carnis, peptone, corn steep liquor, SODIUMNITRATE, yeast extract paste, ammonium sulfate, ammonium nitrate etc., above-mentioned nitrogenous source can mix use, also can singlely use.
Described interpolation somatomedin optimizing process:
Pulullan polysaccharide belongs to secondary metabolite, has confirmed in earlier stage to start to produce in microorganism logarithmic phase later stage, stationary phase; In order to shorten fermentation period, improve substrate conversion efficiency, take to add somatomedin and promote thalline Fast Growth, enter rapidly and produce the sugar stage, in the product sugar stage, by screening, add certain somatomedin, thereby promote that thalline utilizes substrate conversion for polysaccharide.
Laboratory lacks the ability of synthetic vitamin B group to finding Aureobasidium pullulans in the fermentation research of Aureobasidium pullulans, and vitamin B group deficiency restricts the Aureobasidium pullulans production output of pulullan polysaccharide and the reason place of productive rate just.Large quantity research has been carried out in laboratory to this, vitamin B complex element is screened, and consumption is optimized, and result is as table 1:
Table 1: the impact of different B vitamin different amounts on fermentation period
By table 1, can draw and add VB
1obviously shortened the time in thalline arrival logarithmic phase later stage.
Table 2: different concns VB
1impact on fermentation period
| VB 1(VitB1) concentration (‰) | Thalline enters logarithmic phase later stage (OD 620=0.5) time (h) |
| 0.01 | 18 |
| 0.02 | 17 |
| 0.03 | 14 |
| 0.04 | 13 |
| 0.05 | 13 |
By table 2, can draw the VB that adds 0.03-0.05 ‰
1the growth of thalline is all had to obvious promoter action, and the time shorten that thalline is entered to the logarithmic phase later stage is 13-14h.
The acellular enzyme liquid that the report Aureobasidium pullulans cells such as Taguchi in 1973 are made after fragmentation can utilize ATP that UDPG can be synthesized to pulullan polysaccharide through transglycosylation; Screening and optimizing has been carried out to nucleotide base in this laboratory: the impact of table 3 nucleotide base on pulullan polysaccharide fermentation
By table 3, can show that uridylic can significantly improve the whole output of pulullan polysaccharide, improve the transformation efficiency of substrate.
Table 4: the impact of the uridylic of different concns on pulullan polysaccharide fermentation
By table 4, can show that the uridylic that adds 0.02-0.04 ‰ has obvious promoter action to the generation of pulullan polysaccharide, improve whole output and transformation efficiency, shorten fermentation required time.
Beneficial effect
The present invention is directed in Aureobasidium pullulans CGMCC NO.7055 fermentative production pulullan polysaccharide process and have the defect that the cycle is long, substrate conversion efficiency is low, through large quantity research, determined that reason is that Aureobasidium pullulans self lacks the ability of synthesise vitamins, and vitamin B group is the important component part that forms coenzyme, therefore, at the logarithmic phase initial stage, add appropriate vitamin B targetedly
1promote thalli growth, thereby greatly shortened the time that thalline arrives stationary phase, thalline is accumulated in a large number, for follow-up pulullan polysaccharide synthetic provides assurance.The present invention adds somatomedin according to producing sugared feature in different growth periods and fermenting process targetedly, simple operation, and successful, has greatly shortened fermentation period, has improved the transformation efficiency of substrate, has reduced the cost of pulullan polysaccharide.Compare with the invention of quoting in background technology, although output is lower, its transformation efficiency obviously improves, and has improved the utilising efficiency of substrate, and fermentation period is shorter, greatly reduces the cost of pulullan polysaccharide.
Embodiment:
Embodiment 1
(1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 28h.
(2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is that 400rpm cultivates.Thalli growth during the initial stage, adds 0.03 ‰ vitamins B in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add 0.04 ‰ uridylate to carry out the accumulation that secondary metabolite is pulullan polysaccharide.
(3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product 76.3g/L.
Fermentation period is 58h, and does not add somatomedin fermentation period and needs 3d to compare, and has shortened 12h; Substrate conversion efficiency reaches 75%.
Embodiment 2
(1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 30h.
(2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate bottle, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is that 400rpm cultivates.Thalli growth during the initial stage, adds 0.04 ‰ vitamins B in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add 0.03 ‰ uridylate to carry out the accumulation that secondary metabolite is pulullan polysaccharide.
(3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product 76.4g/L.
Fermentation period is 59h, and does not add somatomedin fermentation period and needs 3d to compare, and has shortened 13h; Substrate conversion efficiency reaches 74%.
Embodiment 3
(1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 32h.
(2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate bottle, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is 400rpm, cultivates.Thalli growth during the initial stage, adds 0.05 ‰ vitamins B in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add 0.02 ‰ uridylate to carry out the accumulation that secondary metabolite is pulullan polysaccharide.
(3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product 75.6g/L.
Fermentation period is 61h, and does not add somatomedin fermentation period and needs 3d to compare, and has shortened 11h; Substrate conversion efficiency reaches 73%.
Claims (9)
1. a method that improves pulullan polysaccharide output, comprises the following steps:
(1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 28-32h;
(2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate bottle, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is 400rpm, cultivates 57-63h;
(3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product;
It is characterized in that, thalli growth during the initial stage, adds the vitamins B of 0.03-0.05 ‰ in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add the uridylate of 0.02-0.04 ‰;
Described Aureobasidium pullulans BCSWGHPL101(Aureobasidium pullulans) deposit number is CGMCC NO.7055.
2. improve according to claim 1 the method for pulullan polysaccharide output, it is characterized in that, described vitamins B
1and uridylate is through the aseptic membrane filtration of 0.45um.
3. improve according to claim 1 the method for pulullan polysaccharide output, it is characterized in that, described seed culture, substratum consists of: sucrose 8g, yeast soaks powder 0.2g, sodium-chlor 0.15g, dipotassium hydrogen phosphate 0.1g, ammonium sulfate 0.1g, magnesium sulfate 0.04g, ferrous sulfate 0.05g, distilled water is settled to 100mL.
4. improve according to claim 1 the method for pulullan polysaccharide output, it is characterized in that, described fermentation culture, substratum consists of: sucrose 10g, peptone 0.4g, sodium-chlor 0.3g, dipotassium hydrogen phosphate 0.7g, magnesium sulfate 0.04g, ferrous sulfate 0.05g, distilled water is settled to 100mL, initial pH5.5-6.5.
5. improve according to claim 1 the method for pulullan polysaccharide output, comprise the steps:
(1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 28h;
(2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is that 400rpm cultivates 58h; Thalli growth during the initial stage, adds 0.03 ‰ vitamins B in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add 0.04 ‰ uridylate;
(3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product 76.3g/L.
6. improve according to claim 1 the method for pulullan polysaccharide output, comprise the steps:
(1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 30h;
(2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate bottle, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is that 400rpm cultivates 59h; Thalli growth during the initial stage, adds 0.04 ‰ vitamins B in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add 0.03 ‰ uridylate;
(3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product 76.4g/L.
7. improve according to claim 1 the method for pulullan polysaccharide output, comprise the steps:
(1) seed culture
After being activated, Aureobasidium pullulans bacterial strain chooses a ring transition in 500ml baffle plate bottle, substratum liquid amount 100ml, and culture temperature is 32 ℃, and shaking speed is 180rpm, and incubation time is 32h;
(2) fermentation culture
The bottled liquid measure 100mL of 500mL baffle plate bottle, inoculum size is 4% (V/V), 28 ℃ of temperature, shaking speed is 400rpm, cultivates 61h; Thalli growth during the initial stage, adds 0.05 ‰ vitamins B in logarithmic phase
1, when thalli growth is in when early stage stationary phase, add 0.02 ‰ uridylate;
(3) separation of pulullan polysaccharide
After fermented liquid filtering thalline, add three times of ethanol, stir, 4 ℃ of standing over night, centrifugal after, re-use dehydrated alcohol and rinse 2 times, dryly to obtain white pulullan polysaccharide crude product 75.6g/L.
8. the application in pulullan polysaccharide is produced according to the method for the arbitrary described raising pulullan polysaccharide output of claim 1-7.
9. the pulullan polysaccharide of preparing according to the method for the arbitrary described raising pulullan polysaccharide output of claim 1-7.
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Cited By (11)
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|---|---|---|---|---|
| CN104711374A (en) * | 2014-12-26 | 2015-06-17 | 天津北洋百川生物技术有限公司 | Method for increasing pulullan output by controling temperature at two stage and feeding tween 60 |
| CN105624233A (en) * | 2015-12-09 | 2016-06-01 | 天津北洋百川生物技术有限公司 | Method for increasing yield of pullulan |
| 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 |
| CN108118003A (en) * | 2016-11-30 | 2018-06-05 | 财团法人食品工业发展研究所 | Black yeast, culture medium and method for producing β -glucan, and black yeast culture and composition |
| CN108795777A (en) * | 2018-05-31 | 2018-11-13 | 浙江理工大学 | Aureobasidium pullulans RM 1603 and its application |
| 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 |
| CN111971395A (en) * | 2018-04-04 | 2020-11-20 | 科莱恩国际有限公司 | Fermentation method of ascomycetes |
| 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 |
| CN113652456A (en) * | 2021-08-02 | 2021-11-16 | 浙江科技学院 | Method for producing pullulan polysaccharide by cell recycling and application |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104711374A (en) * | 2014-12-26 | 2015-06-17 | 天津北洋百川生物技术有限公司 | Method for increasing pulullan output by controling temperature at two stage and feeding tween 60 |
| CN105624233A (en) * | 2015-12-09 | 2016-06-01 | 天津北洋百川生物技术有限公司 | Method for increasing yield of pullulan |
| CN108118003A (en) * | 2016-11-30 | 2018-06-05 | 财团法人食品工业发展研究所 | Black yeast, culture medium and method for producing β -glucan, and black yeast culture and composition |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| 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 |
| CN111971395A (en) * | 2018-04-04 | 2020-11-20 | 科莱恩国际有限公司 | Fermentation method of ascomycetes |
| CN111971395B (en) * | 2018-04-04 | 2022-03-25 | 科莱恩国际有限公司 | Fermentation method of ascomycetes |
| CN108795777A (en) * | 2018-05-31 | 2018-11-13 | 浙江理工大学 | Aureobasidium pullulans RM 1603 and its application |
| 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 |
| 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 |
| CN113652456A (en) * | 2021-08-02 | 2021-11-16 | 浙江科技学院 | Method for producing pullulan polysaccharide by cell recycling and application |
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