CN103740785A - Method for producing pullulan through high-density fermentation - Google Patents
Method for producing pullulan through high-density fermentation Download PDFInfo
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- CN103740785A CN103740785A CN201310754978.2A CN201310754978A CN103740785A CN 103740785 A CN103740785 A CN 103740785A CN 201310754978 A CN201310754978 A CN 201310754978A CN 103740785 A CN103740785 A CN 103740785A
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Abstract
The invention discloses a method for producing pullulan through high-density fermentation, belongs to the field of microbial fermentation, and aims to overcome the defects of a conventional optimal regulation method, further improve the yield of the pullulan, improve the efficiency of the pullulan fermentation process, and reduce generation of pigments in the fermentation process. According to the method, the pH and the dissolved oxygen are regulated in two stages, so that the high-density fermentation is realized, the yield of the pullulan is improved by over 50 percent, the generation of the pigments is reduced to a certain extent, troubles are reduced for subsequent extraction, and the cost of the pullulan is further reduced.
Description
Technical field
The invention belongs to microorganism fermentation field, be specifically related to a kind of method that high density fermentation is produced pulullan polysaccharide, a kind of method that particularly two stage regulation and control pH and dissolved oxygen are produced pulullan polysaccharide.
Background technology
Microbial polysaccharide mainly refers to the polysaccharide that most of bacterium, a small amount of fungi and algae produce.The advantage and with short production cycle such as safe owing to having, side effect is little, physicochemical property is unique, be not subject to the restriction of the conditions such as season, region, disease and pest and it is received much concern at food and non-food product industry, especially at field of medicaments, there is huge application potential.Common microbial polysaccharide has: xanthan gum, gelling gum, heat setting glue, pulullan polysaccharide etc.
BIOSYNTHESIS OF MONOSE AND major part belongs to high viscosity fermentation, because microbial polysaccharide belongs to secondary metabolite more, in microorganism, in stationary phase, starting a large amount of generations, is all that BIOSYNTHESIS OF MONOSE AND is divided into two stages so research at present improves the method for microbial polysaccharide output: the first stage is by optimal conditions, thalline to be produced in a large number substantially; Subordinate phase is by change condition, polysaccharide to be produced in a large number after thalline enters stationary phase.
High density fermentation adopts two-stage process, can reduce the equipment scale in yeast culture stage on the one hand, save large fermentation tank watt consumption and energy expenditure during the fermentation, can improve on the other hand the synthetic production intensity of cell cultures and polysaccharide, make production process reach energy-saving and cost-reducing object.
In prior art, be all to improve pulullan polysaccharide output by induction mutation of bacterium, medium optimization mostly, but because pulullan polysaccharide fermentation belongs to high viscosity fermentation, in the fermentation later stage, along with the increase of viscosity, thalline reduces greatly to the utilising efficiency of substrate, and pigment formation is serious, subsequent extracted process has been brought to great inconvenience, and the method for optimizing by tradition regulates and controls, to the increase of pulullan polysaccharide output, be not often clearly, and fermentation efficiency can not obtain fine raising.
Summary of the invention
In order to solve the deficiency in traditional optimization regulating method, further increase pulullan polysaccharide output, improve the efficiency in pulullan polysaccharide fermenting process, reduce the generation of pigment in fermenting process, the present invention is by two stage regulation and control pH and dissolved oxygens, thereby realize high density fermentation, greatly improve the production intensity of pulullan polysaccharide.
Technical solution of the present invention is as follows:
High density fermentation is produced the method for pulullan polysaccharide, and by two stage regulation and control pH and dissolved oxygens, the first stage makes thalline raised growth, and thalline obtains a large amount of accumulation; Subordinate phase changes pH and dissolved oxygen, and pulullan polysaccharide is produced in a large number.Specifically comprise the steps:
(1) bacterial strain is connected to seed culture medium and carries out shaking flask activation, rotating speed is 150-250rpm, and temperature is 28 ℃, and incubation time is 30-35 hour.
(2) seed culture fluid after activation is with 2-5%(v/v) amount be connected in new seed shaking flask and carry out enlarged culturing, rotating speed is 150-250rpm, temperature is 28 ℃, incubation time is 16-20 hour.
(3) by step (2) gained seed liquor with 2-5%(v/v) amount be connected in 5L fermentor tank and carry out fermentation culture, according to thalli growth situation, be OD
620value regulates and controls pH and dissolved oxygen: as thalline OD
620< 0.7(seed dry weight is less than 12g/L) time, when dissolved oxygen is down to 60% left and right, dissolved oxygen is connected with stirring, dissolved oxygen is maintained between 50%-60%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH that it is stabilized between 2.8-3.2 simultaneously thalline is produced in a large number; As thalline OD
620>=0.7(seed dry weight is not less than 12g/L) time, dissolved oxygen is set between 25%-30%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH that it is stabilized between 4.8-5.2 simultaneously and make thalline synthesize in a large number pulullan polysaccharide; 28 ℃ of temperature, fermentation time 72-96 hour.
(4) every 8 hours sampling records, measure indices: pH, dissolved oxygen, dry cell weight, pulullan polysaccharide output.
Described bacterial strain is specially Aureobasidium pullulans (Aureobasidium pullulans) BCSWGHPL101, on December 25th, 2012, be preserved in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC No.7055, Classification And Nomenclature: Aureobasidium pullulans Aureobasidium pullulans.
Described dry cell weight is measured: accurately get the centrifugal 20min of 30mL fermented liquid 5000rpm, remove supernatant, centrifugal 2-3 time of thalline distilled water wash, then carries out suction filtration, and 80 ℃ of oven for drying are weighed, and this quality is multiplied by 1000 divided by 30 being thick dry weight, with g/L, remembers.
Described pulullan polysaccharide raw sugar is measured: accurately gets the centrifugal 20min of 30mL fermented liquid 15000rpm and removes thalline, and twice dehydrated alcohol alcohol precipitation for supernatant liquor, gained precipitation is dried and is weighed, and this quality is multiplied by 1000 divided by 30 being thick output, with g/L, remembers.
Beneficial effect
The invention provides a kind of method of producing pulullan polysaccharide, simple to operate, not only solved problem low to substrate utilization ratio in Propiram fermentation process with high viscosity, and increased the output of pulullan polysaccharide, improved the production intensity of pulullan polysaccharide, and reduced to a certain extent the generation of pigment, for subsequent extracted has reduced trouble, further reduced the cost of pulullan polysaccharide.
Embodiment
Embodiment 1
(1) CGMCC No.7055 bacterial strain is connected to seed culture medium and carries out shaking flask activation, rotating speed is 150rpm, and temperature is 28 ℃, and incubation time is 35 hours.
(2) seed culture fluid after activation is with 2-5%(v/v) amount be connected in new seed shaking flask and carry out enlarged culturing, rotating speed is 150rpm, temperature is 28 ℃, incubation time is 20 hours.
(3) by gained seed liquor with 2-5%(v/v) amount be connected in 5L fermentor tank and carry out fermentation culture, according to thalli growth situation, be OD
620value regulates and controls pH and dissolved oxygen: as thalline OD
620< 0.7(seed dry weight is less than 12g/L) time, when dissolved oxygen is down to 60% left and right, dissolved oxygen connect with stirring, make dissolved oxygen maintain 50%, to utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L to regulate and control pH simultaneously and make it thalline be produced in a large number between being stabilized in 2.8; As thalline OD
620>=0.7(seed dry weight is not less than 12g/L) time, dissolved oxygen is set as to 25%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH to make it make thalline synthetic pulullan polysaccharide in a large number between being stabilized in 4.8 simultaneously; 28 ℃ of temperature, rotating speed 300rpm, fermentation time 72 hours.
Seed culture medium consists of: sucrose 100g/L, yeast soak powder 3g/L, sodium-chlor 2.5g/L, ammonium sulfate 1g/L, MgSO47H2O0.4g/L, K2HPO41g/L, FeSO47H2O0.05g/L, and all the other are water, pH7.0.
Fermentation culture based component: sucrose 150g/L, urea 2g/L, sodium-chlor 2.5g/L, MgSO47H2O0.4g/L, K2HPO42g/L, FeSO47H2O0.05g/L, all the other are water, initial pH6.0.
After having fermented, the thick dry weight of thalline is 16.53g/L, and Crude polysaccharides output is: 78.8g/L.Compare output with the controlled trial that regulates and controls (pH nature, ventilating ratio 1:1, rotating speed 300rpm) and improved 57.6%; After centrifugal, supernatant OD value under 654nm is 0.132, and regulates and controls to compare (OD value is 0.25), and pigment has reduced by 47.2%.
Note: saturated sodium sulfate dissolved oxygen is zero; Rotating speed 500rpm, when ventilation is 7L/min, dissolved oxygen is 100%.
Embodiment 2
(1) CGMCC.NO.7055 bacterial strain is connected to seed culture medium and carries out shaking flask activation, rotating speed is 200rpm, and temperature is 28 ℃, and incubation time is 32 hours.
(2) seed culture fluid after activation is with 2-5%(v/v) amount be connected in new seed shaking flask and carry out enlarged culturing, rotating speed is 200rpm, temperature is 28 ℃, incubation time is 18 hours.
(3) by gained seed liquor with 2-5%(v/v) amount be connected in 5L fermentor tank and carry out fermentation culture, according to thalli growth situation, be OD
620value regulates and controls pH and dissolved oxygen: as thalline OD
620< 0.7(seed dry weight is less than 12g/L) time, when dissolved oxygen is down to 60% left and right, dissolved oxygen is connected with stirring, make dissolved oxygen maintain 55%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH to make it be stabilized in 3.0 simultaneously thalline is produced in a large number; As thalline OD
620>=0.7(seed dry weight is not less than 12g/L) time, dissolved oxygen is set as to 28%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH to make it make thalline synthetic pulullan polysaccharide in a large number between being stabilized in 5.0 simultaneously; 28 ℃ of temperature, rotating speed 300rpm, fermentation time 88 hours.
Seed culture medium consists of: sucrose 100g/L, yeast soak powder 3g/L, sodium-chlor 2.5g/L, ammonium sulfate 1g/L, MgSO47H2O0.4g/L, K2HPO41g/L, FeSO47H2O0.05g/L, and all the other are water, pH7.0.
Fermentation culture based component: sucrose 150g/L, urea 2g/L, sodium-chlor 2.5g/L, MgSO47H2O0.4g/L, K2HPO42g/L, FeSO47H2O0.05g/L, all the other are water, initial pH6.0.
After having fermented, the thick dry weight of thalline is 16.63g/L, and Crude polysaccharides output is: 80.67g/L.Compare output with the controlled trial that regulates and controls (pH nature, ventilating ratio 1:1, rotating speed 300rpm) and improved 61.34%; After centrifugal, supernatant OD value under 654nm is 0.112, and regulates and controls to compare (OD value is 0.25), and pigment has reduced by 55.2%.
Embodiment 3
(1) CGMCC.NO.7055 bacterial strain is connected to seed culture medium and carries out shaking flask activation, rotating speed is 250rpm, and temperature is 28 ℃, and incubation time is 30 hours.
(2) seed culture fluid after activation is with 2-5%(v/v) amount be connected in new seed shaking flask and carry out enlarged culturing, rotating speed is 250rpm, temperature is 28 ℃, incubation time is 16 hours.
(3) by gained seed liquor with 2-5%(v/v) amount be connected in 5L fermentor tank and carry out fermentation culture, according to thalli growth situation, be OD
620value regulates and controls pH and dissolved oxygen: as thalline OD
620< 0.7(seed dry weight is less than 12g/L) time, when dissolved oxygen is down to 60% left and right, dissolved oxygen is connected with stirring, dissolved oxygen is set as to 60%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH to make it be stabilized in 3.2 simultaneously thalline is produced in a large number; As thalline OD
620>=0.7(seed dry weight is not less than 12g/L) time, dissolved oxygen is maintained between 30%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH to make it make thalline synthetic pulullan polysaccharide in a large number between being stabilized in 5.2 simultaneously; 28 ℃ of temperature, rotating speed 300rpm, fermentation time 96 hours.
Seed culture medium consists of: sucrose 100g/L, yeast soak powder 3g/L, sodium-chlor 2.5g/L, ammonium sulfate 1g/L, MgSO47H2O0.4g/L, K2HPO41g/L, FeSO47H2O0.05g/L, and all the other are water, pH7.0.
Fermentation culture based component: sucrose 150g/L, urea 2g/L, sodium-chlor 2.5g/L, MgSO47H2O0.4g/L, K2HPO42g/L, FeSO47H2O0.05g/L, all the other are water, initial pH6.0.
After having fermented, the thick dry weight of thalline is 18.70g/L, and Crude polysaccharides output is: 76.2g/L.Compare output with the controlled trial that regulates and controls (pH nature, ventilating ratio 1:1, rotating speed 300rpm) and improved 52.4%; After centrifugal, supernatant OD value under 654nm is 0.129, and regulates and controls to compare (OD value is 0.25), and pigment has reduced by 48.4%.
Claims (2)
1. high density fermentation is produced a method for pulullan polysaccharide, and concrete steps are as follows:
(1) bacterial strain is connected to seed culture medium and carries out shaking flask activation, rotating speed is 150-250rpm, and temperature is 28 ℃, and incubation time is 30-35 hour;
(2) seed culture fluid after activation is with 2-5%(v/v) amount be connected in new seed shaking flask and carry out enlarged culturing, rotating speed is 150-250rpm, temperature is 28 ℃, incubation time is 16-20 hour;
(3) by step (2) gained seed liquor with 2-5%(v/v) amount be connected in 5L fermentor tank and carry out fermentation culture, according to thalli growth situation, be OD
620value regulates and controls pH and dissolved oxygen: as thalline OD
620< 0.7(seed dry weight is less than 12g/L) time, when dissolved oxygen is down to 60% left and right, dissolved oxygen is connected with stirring, dissolved oxygen is maintained between 50%-60%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH that it is stabilized between 2.8-3.2 simultaneously thalline is produced in a large number; As thalline OD
620>=0.7(seed dry weight is not less than 12g/L) time, dissolved oxygen is set between 25%-30%, utilize the hydrochloric acid of 3mol/L and the sodium hydroxide of 3mol/L regulation and control pH that it is stabilized between 4.8-5.2 simultaneously and make thalline synthesize in a large number pulullan polysaccharide; 28 ℃ of temperature, fermentation time 72-96 hour.
2. the method that a kind of high density fermentation as claimed in claim 1 is produced pulullan polysaccharide, is characterized in that: described bacterial classification is Aureobasidium pullulans (Aureobasidium pullulans) BCSWGHPL101, deposit number CGMCC No.7055.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107641634A (en) * | 2017-11-02 | 2018-01-30 | 北京艾普希隆生物科技有限公司 | A kind of production technology of Aureobasidium pullulans fermenting and producing pulullan polysaccharide |
| CN108795777A (en) * | 2018-05-31 | 2018-11-13 | 浙江理工大学 | Aureobasidium pullulans RM 1603 and its 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107641634A (en) * | 2017-11-02 | 2018-01-30 | 北京艾普希隆生物科技有限公司 | A kind of production technology of Aureobasidium pullulans fermenting and producing pulullan polysaccharide |
| CN108795777A (en) * | 2018-05-31 | 2018-11-13 | 浙江理工大学 | Aureobasidium pullulans RM 1603 and its application |
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Application publication date: 20140423 |