CN100366727C - Prussian blue producing strain and prussian blue producing process with the strain - Google Patents
Prussian blue producing strain and prussian blue producing process with the strain Download PDFInfo
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- CN100366727C CN100366727C CNB2004100657630A CN200410065763A CN100366727C CN 100366727 C CN100366727 C CN 100366727C CN B2004100657630 A CNB2004100657630 A CN B2004100657630A CN 200410065763 A CN200410065763 A CN 200410065763A CN 100366727 C CN100366727 C CN 100366727C
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Abstract
The present invention discloses a strain for producing pullulan and a method for producing the pullulan by using the strain, which relates to the production of extracellular water-soluble polysaccharide by using glucose metabolism. The present invention belongs to the technical field of biological engineering. The strain provided by the present invention is classified and named aureobasidium pullulans G-58, and the preservation number of the strain is CGMCC No. 1234. The strain is used for producing the pullulan by processes of the fermentation culture and fermentation liquid post treatment of heat treatment, decolorization, sedimentation, desalination, drying, etc. The pullulan of an obtained product has the advantages of light color, high polysaccharide yield, excellent viscoelasticity, emulsifying property, plasticity, and no toxicity or harm, and is widely applied to industries of food, medicines, cosmetics, etc. The pigment content of the fermentation liquid of a fermentation process is low, and a post treatment process can be greatly simplified. The post treatment process of the fermentation liquid has the advantages of reasonable design, simple operation, low cost, high yield, good decolorization and desalination effect and good water solubility. The quality of the obtained product meets requirements. The present invention provides a possibility for the industrial large-scale production of the pullulan with low price.
Description
Technical field
A kind of bacterial strain of producing Propiram is produced the method for Propiram with utilizing this bacterial strain, relates to and utilizes carbohydrate metabolism production extracellular water-soluble polysaccharide, belongs to technical field of bioengineering.
Background technology
The Propiram definite designation is the extracellular water-soluble polysaccharide that Aureobasidium pullulans (Aureobasidium pullulans) utilizes carbohydrate metabolism to produce in culturing process for the short mould polysaccharide of stalk (pullulan) is called pullulan or Propiram again.Its molecule be by trisaccharide maltose with α-1, the straight-chain polysaccharide that the 6-glycosidic link is formed by connecting, 1,4 key and 1,6 key ratio are 2: 1 in the molecule, the polymerization degree is 100～5000, molecular weight is (4～200) * 104.Because this unique mode of connection, the mould polysaccharide of short stalk has splendid visco-elasticity, emulsifying property, plasticity-, and is nontoxic, is widely used in industries such as food, medicine, makeup.
The mould polysaccharide membrane of short stalk has glass pattern gloss and transparency and intensity, and oil resistant, heat-resisting envelope are edible; Its special nature is lower than the ventilation property of other polymeric membranes, and gases such as oxygen, nitrogen, carbonic acid gas and fragrance almost can not see through.The mould polysaccharide membrane of short stalk is widely used in fruit, vegetables, egg, particularly oleaginous food as food fresh keeping membrane, as ham, instant noodles, meat etc.The mould polysaccharide soln neutrality of short stalk, it is little that viscosity is influenced by temperature, pH value, and good salt tolerant and acid resistance are arranged, and therefore can be used for the thickening of high salt food and food flavouring, and as soy sauce, shrimp paste, thickening effectiveness obviously is better than other polysaccharide such as Viscogum BE, carrageenin.The mould polysaccharide of short stalk digests very slowly under the effect of animal digestion organ endoenzyme, utilizes its low digestibility, can make low calorific food and beverage.Find again that recently the mould polysaccharide of short stalk has the effect that makes bifidus bacillus propagation, treatment constipation and suppress tumour.
Have only Japanese Lin Yuan company that produced in small quantities is arranged at present, cost an arm and a leg.The mass-produced first cause of the short mould polysaccharide of stalk of restriction is during the fermentation, Aureobasidium pullulans can be secreted and similar black of melanochrome (melanin pigmentation) or blackish green material, this material sticks on the mould polysaccharide of short stalk securely, be difficult to remove with general method, can remove with the Sevag fractionating process, but to remove melanochrome fully, must repeat repeatedly, and about 15% polysaccharide is lost in each fractionation meeting, final yield is very low, so must screening hang down the pigment bacterial strain, to overcome the influence of melanochrome to polysaccharide product.
Summary of the invention
The purpose of this invention is to provide a strain Aureobasidium pullulans variant CGMCC No.1234 and utilize this bacterial strain to produce the method for Propiram, comprising zymotechnique and fermented liquid aftertreatment technology.Propiram is the polyose colloid with good filming and fragrance parcel performance, is the preferred materials that refreshing tablets is produced.
Technical scheme of the present invention is described Aureobasidium pullulans variant, its classification called after Aureobasidium pullulans (Aureobasidium pullulans) G-58, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.1234.The method of utilizing this bacterial strain to produce Propiram is:
A) zymotechnique
Bacterial classification is CGMCC No.1234;
Seed culture medium is in g/L: sucrose 50, (NH
4)
2SO
40.8, K
2HPO
44, MgSO
40.2, NaCl0.2, yeast extract paste 1.5, pH6; 121 ℃, the 30min sterilization.
Fermention medium is in g/L: sucrose 50-100, (NH
4)
2SO
40.4-0.8, K
2HPO
44-8, MgSO
40.4, NaCl0.2, MnCl
20.005, yeast extract paste 0.3-0.5, pH6.0-7.0; 121 ℃, the 30min sterilization.
The seed culture condition: get 28 ℃ of slant strains one rings of cultivating 4 days, be inoculated in seed culture medium, liquid amount is for adorning 100mL-120mL in the 500mL triangular flask, and 28 ℃, 200r/min shake-flask culture were seed liquor in 36 hours.
10 liters of fermentor cultivation conditions: 10 liters of fermentor tanks, 6 liters of liquid amounts, inoculum size 2%; 28 ℃ of temperature, tank pressure 0.5kg/cm
2, stirring velocity 200～600r/min, air flow 1.5～6L/min, fermentation time 96 hours.
1.5m
3Fermentor cultivation condition: 1.5m
3Fermentor tank, 900 liters of liquid amounts, inoculum size 2%; 28 ℃ of temperature, tank pressure 0.5kg/cm
2, stirring velocity 200～600r/min, air flow 1.5～6L/min, fermentation time 96 hours.
Best fermention medium is counted with g/L: sucrose 50, (NH
4)
2SO
40.6, K
2HPO
46, MgSO
40.4 NaCl 0.2, MnCl
20.005, yeast extract paste 0.4, pH6.5; 121 ℃, the 30min sterilization;
Best 10 liters or 1.5m
3Fermentor cultivation condition: stirring velocity 400r/min, air flow 3L/min, fermentation time 96 hours.
B) fermented liquid aftertreatment technology
Fermented liquid I, centrifugal I, thermal treatment, centrifugal II, decolouring, centrifugal III, industrial spirit precipitation, centrifugal IV, desalination, filtration II after filtration makes liquid Propiram product; Or the continuation drying makes solid Propiram product.
Concrete is operating as:
Filter I:8 layer gauze suction filtration,
Centrifugal I:3000r/min, 30min,
Thermal treatment: the supernatant liquor after the centrifugal I is used for thermal treatment, uses NaHCO
3With the HCl adjust pH, 70 ℃, 10min, pH7.0,
Centrifugal II:4000r/min, 30min,
Decolouring: activated carbon decolorizing, activated carbon dosage are 0.5%, 15 ℃, pH7.0,
Centrifugal III:4000r/min, 30min,
The industrial spirit precipitation: the supernatant liquor of centrifugal III adds 2 times of volume industrial spirit, stirring, 4 ℃ of placement 12h,
Centrifugal IV:2000r/min, 15min,
Desalination: the precipitation of centrifugal IV is washed 3 times with 65% alcohol,
Filter II:8 layer gauze suction filtration,
It is water-soluble to filter the precipitation that II obtains, and is mixed with solid content and is 20% the aqueous solution,
Evaporation: above-mentioned solution evaporation to solid content 60%-70%, is liquid Propiram product; Or spraying drying, make solid Propiram product.
Analytical procedure:
Fermented liquid color and absorbance measurement: adopt the pigment in the solution after 721 type spectrophotometric determinations extract polysaccharide.Distilled water is blank, and length scanning, wavelength transfer to maximum transmission rate (654nm), surveys with this wavelength that each fermented liquid 4000r/min, 30min are centrifugal to go supernatant liquor absorbancy behind the thalline again.
Biomass is measured: quantitatively measure fermented liquid, and centrifugal 30min under the 4000r/min, inclining supernatant liquor and is used for sugar determination.Centrifugal 2 times of distilled water wash of sedimentary thalline are dried to constant weight under 100 ℃, measure biomass with weighting method.
Crude polysaccharides is measured: centrifuged supernatant adds 2 times of dehydrated alcohols, stirs, and places liquid for 4 ℃, and the mould polysaccharide of short stalk is fully precipitated.Precipitated liquid must be lacked the mould polysaccharide precipitation of stalk with the centrifugal 30min of 4500r/min, and precipitation is washed with anhydrous propanone, anhydrous diethyl ether for 3 times more successively with the washing of 95% alcohol, and 70 ℃ are dried to constant weight, promptly get to lack the mould polysaccharide crude of stalk, weigh, and calculate output.
Beneficial effect of the present invention: the inventive method provides a kind of Aureobasidium pullulans variant CGMCC No.1234 of production Propiram of mutagenic and breeding voluntarily, ferment and fermented liquid aftertreatment products obtained therefrom Propiram lighter color with this variant, be pale yellow or oyster white, the polysaccharide yield height, product has splendid visco-elasticity, emulsifying property, plasticity-, nontoxic, is widely used in industries such as food, medicine, makeup.Described zymotechnique fermented liquid pigment content is low, can simplify aftertreatment technology greatly, the fermented liquid aftertreatment technology is reasonable in design, simple to operate, cost is low, yield is high, the decolouring desalting effect is good, good water solubility, quality product reaches requirement, for the lower Propiram of large-scale industrialization production prices provides possibility.
The biological material specimens preservation
A kind of bacterial strain of producing Propiram, this bacterial strain is Aureobasidium pullulans (Aureobasidium pullulans) G-58, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCCNo.1234, and preservation date is on October 19th, 2004.
Embodiment
Embodiment 1
10 liters of fermentor cultivation
Bacterial classification is CGMCC No.1234;
Seed culture medium is in g/L: sucrose 50, (NH
4)
2SO
40.8, K
2HPO
44, MgSO
40.2, NaCl0.2, yeast extract paste 1.5; PH6; 121 ℃, the 30min sterilization;
Fermention medium is counted with g/L: sucrose 50, (NH
4)
2SO
40.6, K
2HPO
46, MgSO
40.4 NaCl 0.2, MnCl
20.005, yeast extract paste 0.4, pH6.5; 121 ℃, the 30min sterilization;
The seed culture condition: get 28 ℃ of slant strains one rings of cultivating 4 days, be inoculated in seed culture medium, liquid amount is for adorning 100mL-120mL in the 500mL triangular flask, and 28 ℃, 200r/min shake-flask culture obtained the 120mL seed liquor in 36 hours;
10 liters of fermentor cultivation conditions: 10 liters of fermentor tanks, 6 liters of liquid amounts, inoculum size just in time is the 120mL seed liquor by 2%; 28 ℃ of temperature, tank pressure 0.5kg/cm
2, stirring velocity 400r/min, air flow 3L/min, fermentation time 96 hours.Polysaccharide yield reaches 24.1g/L, and sugared transformation efficiency reaches 48.2%.
Embodiment 2
1.5m
3Fermentor cultivation
Seed culture medium, seed culture condition, fermention medium are with embodiment 1.
1.5m
3Fermentor cultivation condition: 1.5m
3Fermentor tank, 900 liters of liquid amounts, inoculum size is 18 liters of seed liquor by 2%, 28 ℃ of temperature, tank pressure 0.5kg/cm
2, stirring velocity 400r/min, air flow 3L/min, fermentation time 96 hours, polysaccharide yield reaches 25.6g/L, and sugared transformation efficiency reaches 51.2%.
Embodiment 3
Carry out aftertreatment with embodiment 1 or embodiment 2 gained fermented liquids, post-treatment condition is: fermented liquid I, centrifugal I, thermal treatment, centrifugal II, decolouring, centrifugal III, industrial spirit precipitation, centrifugal IV, desalination, filtration II after filtration makes liquid Propiram product; Or the continuation drying makes solid Propiram product.
Concrete is operating as:
Filter I:8 layer gauze suction filtration,
Centrifugal I:3000r/min, 30min,
Thermal treatment: the supernatant liquor after the centrifugal I is used for thermal treatment, uses NaHCO
3With the HCl adjust pH, 70 ℃, 10min, pH7.0, before the thermal treatment, absorbancy is 0.295, after the thermal treatment, absorbancy is 0.300; Before the thermal treatment, polysaccharide content is 31.5g/L, and after the thermal treatment, polysaccharide content is 29.8g/L, and polysaccharide yield is 94.5%.
Centrifugal II:4000r/min, 30min,
Decolouring: activated carbon decolorizing, activated carbon dosage are 0.5%, 15 ℃, pH7.0, and before the decolouring, absorbancy is 0.300, and after the decolouring, absorbancy is 0.265; Before the decolouring, polysaccharide content is 29.3g/L, and after the decolouring, polysaccharide content is 28.7g/L, and polysaccharide yield is 98%.
Centrifugal III:4000r/min, 30min,
The industrial spirit precipitation: the supernatant liquor of centrifugal III adds 2 times of volume industrial spirit, stirring, 4 ℃ of placement 12h,
Centrifugal IV:2000r/min, 15min,
Desalination: the precipitation of centrifugal IV is washed 3 times with 65% alcohol,
Filter II:8 layer gauze suction filtration,
It is water-soluble to filter the precipitation that II obtains, and is mixed with solid content and is 20% the aqueous solution,
Evaporation: above-mentioned solution evaporation to solid content 60%-70%, is liquid Propiram product; Or spraying drying, make solid Propiram product.
Claims (4)
1. bacterial strain of producing Propiram, its classification called after Aureobasidium pullulans (Aureobasidiumpullulans) G-58 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.1234.
2. method of utilizing the described bacterial strain of claim 1 to produce Propiram is characterized in that:
A) zymotechnique
Bacterial classification is CGMCC No.1234;
Seed culture medium is in g/L: sucrose 50, (NH
4)
2SO
40.8, K
2HPO
44, MgSO
40.2, NaCl0.2, yeast extract paste 1.5, pH6; 121 ℃, the 30min sterilization;
Fermention medium is in g/L: sucrose 50, (NH
4)
2SO
40.4-0.8, K
2HPO
44-8, MgSO
40.4 NaCl 0.2, MnCl
20.005, yeast extract paste 0.3-0.5, pH6.0-7.0; 121 ℃, the 30min sterilization; The seed culture condition: get 28 ℃ of slant strains one rings of cultivating 4 days, be inoculated in seed culture medium, liquid amount is for adorning 100mL-120mL in the 500mL triangular flask, and 28 ℃, 200r/min shake-flask culture were seed liquor in 36 hours;
10 liters of fermentor cultivation conditions: 10 liters of fermentor tanks, 6 liters of liquid amounts, inoculum size 2%; 28 ℃ of temperature, tank pressure 0.5kg/cm
2, stirring velocity 200～600r/min, air flow 1.5～6L/min, fermentation time 96 hours;
1.5m
3Fermentor cultivation condition: 1.5m
3Fermentor tank, 900 liters of liquid amounts, inoculum size 2%; 28 ℃ of temperature, tank pressure 0.5kg/cm
2, stirring velocity 200～600r/min, air flow 1.5～6L/min, fermentation time 96 hours;
B) fermented liquid aftertreatment technology
Fermented liquid I, centrifugal I, thermal treatment, centrifugal II, decolouring, centrifugal III, industrial spirit precipitation, centrifugal IV, desalination, filtration II after filtration makes liquid Propiram product; Or the continuation drying makes solid Propiram product.
3. method according to claim 2, zymotechnique wherein is:
Fermention medium is in g/L: sucrose 50, (NH
4)
2SO
40.6, K
2HPO
46, MgSO
40.4, NaCl0.2, MnCl
20.005, yeast extract paste 0.4, pH6.5; 121 ℃, the 30min sterilization;
10 liters or 1.Sm
3Fermentor cultivation condition: stirring velocity 400r/min, air flow 3L/min, fermentation time 96 hours.
4. method according to claim 2, fermented liquid aftertreatment technology wherein is:
Filter I:8 layer gauze suction filtration,
Centrifugal I:3000r/min, 30min,
Thermal treatment: the supernatant liquor after the centrifugal I is used for thermal treatment, uses NaHCO
3With the HCl adjust pH, 70 ℃, 10min, pH7.0,
Centrifugal II:4000r/min, 30min,
Decolouring: activated carbon decolorizing, activated carbon dosage are 0.5%, 15 ℃, pH7.0,
Centrifugal III:4000r/min, 30min,
The industrial spirit precipitation: the supernatant liquor of centrifugal III adds 2 times of volume industrial spirit, stirring, 4 ℃ of placement 12h,
Centrifugal IV:2000r/min, 15min,
Desalination: the precipitation of centrifugal IV is washed 3 times with 65% alcohol,
Filter II:8 layer gauze suction filtration,
It is water-soluble to filter the precipitation that II obtains, and is mixed with solid content and is 20% the aqueous solution,
Evaporation: above-mentioned solution evaporation to solid content 60%-70%, is liquid Propiram product;
Or spraying drying, make solid Propiram product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100657630A CN100366727C (en) | 2004-11-16 | 2004-11-16 | Prussian blue producing strain and prussian blue producing process with the strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100657630A CN100366727C (en) | 2004-11-16 | 2004-11-16 | Prussian blue producing strain and prussian blue producing process with the strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1609188A CN1609188A (en) | 2005-04-27 |
| CN100366727C true CN100366727C (en) | 2008-02-06 |
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Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100465191C (en) * | 2006-11-03 | 2009-03-04 | 天津市工业微生物研究所 | Drying method of bearing blue polysaccharide |
| CN101215592B (en) * | 2008-01-15 | 2011-08-17 | 天津市工业微生物研究所 | Fermentation method for producing pullulan polysaccharide |
| CN101974543B (en) * | 2010-08-18 | 2012-05-23 | 江南大学 | Extracellular pullulanase encoding gene derived from bacilli and application thereof |
| CN101988036B (en) * | 2010-11-01 | 2013-04-03 | 吉林农业大学 | Aureobasidium pullulans strain as well as preparation method and application thereof in producing pigment-free pullulan |
| ES2556985T3 (en) | 2011-01-11 | 2016-01-21 | Capsugel Belgium Nv | New hard capsules comprising pululane |
| IL275345B2 (en) * | 2011-12-02 | 2023-04-01 | Prairie Aqua Tech | Microbial-based process for highquality protein concentrate |
| CN102492752B (en) * | 2011-12-16 | 2013-09-11 | 天津北洋百川生物技术有限公司 | Method for co-producing pullulan polysaccharide and melanin by Aureobasidium pullulan |
| CN103233049B (en) * | 2013-05-10 | 2014-12-10 | 天津科技大学 | Method for fermentation production of xylitol using aureobasidium pullulans mutant strain |
| CN103320332A (en) * | 2013-07-18 | 2013-09-25 | 新疆农业科学院微生物应用研究所 | Radiation-resistant fungus and its application in melanin separation and extraction |
| CN104195091B (en) * | 2014-09-12 | 2018-10-16 | 甘肃省农业科学院生物技术研究所 | The preparation method of a kind of solid biologic microbial inoculum and with the solid biologic microbial inoculum of its preparation |
| CN105420127B (en) * | 2016-01-18 | 2020-07-03 | 陕西省微生物研究所 | High-yield strain of high-molecular-weight pullulan and method for producing high-molecular-weight pullulan by using high-yield strain |
| CA3059527A1 (en) | 2017-04-14 | 2018-10-18 | Capsugel Belgium Nv | Pullulan capsules |
| BR112019021396A2 (en) | 2017-04-14 | 2020-04-28 | Capsugel Belgium Nv | pullulan manufacturing process |
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| EP0538049A1 (en) * | 1991-10-16 | 1993-04-21 | SHIN-ETSU BIO, Inc. | High molecular weight pullulan and method for its production |
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