CN100366727C - A strain for producing pullulan and a method for producing pullulan using the strain - Google Patents
A strain for producing pullulan and a method for producing pullulan using 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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- pullulan
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- 229920001218 Pullulan Polymers 0.000 title abstract description 9
- 239000004373 Pullulan Substances 0.000 title abstract description 9
- 235000019423 pullulan Nutrition 0.000 title abstract description 9
- 238000004519 manufacturing process Methods 0.000 title abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 230000001580 bacterial effect Effects 0.000 claims abstract description 16
- 241000223678 Aureobasidium pullulans Species 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 238000001556 precipitation Methods 0.000 claims abstract description 13
- 238000010612 desalination reaction Methods 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 5
- ZBAFFZBKCMWUHM-UHFFFAOYSA-N propiram Chemical compound C=1C=CC=NC=1N(C(=O)CC)C(C)CN1CCCCC1 ZBAFFZBKCMWUHM-UHFFFAOYSA-N 0.000 claims description 23
- 229950003779 propiram Drugs 0.000 claims description 23
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 11
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000007669 thermal treatment Methods 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 3
- 238000012807 shake-flask culturing Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims 2
- 230000008020 evaporation Effects 0.000 claims 2
- 238000000967 suction filtration Methods 0.000 claims 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- 238000005507 spraying Methods 0.000 claims 1
- 150000004676 glycans Chemical class 0.000 abstract description 28
- 229920001282 polysaccharide Polymers 0.000 abstract description 28
- 239000005017 polysaccharide Substances 0.000 abstract description 28
- 235000013305 food Nutrition 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000049 pigment Substances 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 238000004042 decolorization Methods 0.000 abstract 2
- 239000002537 cosmetic Substances 0.000 abstract 1
- 238000004945 emulsification Methods 0.000 abstract 1
- 238000010438 heat treatment Methods 0.000 abstract 1
- 230000004060 metabolic process Effects 0.000 abstract 1
- 239000012528 membrane Substances 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- HVGQWHMSVYODLJ-GFCCVEGCSA-N melanochrome Natural products CC1(C)Oc2cc3OC(=CC(=O)c3c(O)c2C[C@H]1O)CO HVGQWHMSVYODLJ-GFCCVEGCSA-N 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 230000023852 carbohydrate metabolic process Effects 0.000 description 2
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- -1 Viscogum BE Chemical class 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000008446 instant noodles Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种生产普鲁兰的菌株与利用该菌株生产普鲁兰的方法，涉及利用糖代谢生产细胞外水溶性多糖，属于生物工程技术领域。本发明提供的菌株，其分类命名为出芽短梗霉(Aureobasidium pullulans)G-58，保藏编号为CGMCC No.1234；用该菌株经过发酵培养和发酵液后处理：热处理、脱色、沉淀、脱盐、干燥等工艺生产普鲁兰。所得产品普鲁兰色泽浅，多糖得率高，具有极佳的粘弹性、乳化性、可塑性、无毒无害，广泛应用于食品、医药、化妆品等行业。所述发酵工艺发酵液色素含量低，可大大简化后处理工艺，发酵液后处理工艺设计合理、操作简单、成本低、得率高、脱色脱盐效果好，水溶性好，产品质量达到要求，为工业化大规模生产价格较低的普鲁兰提供了可能。The invention discloses a strain for producing pullulan and a method for producing pullulan by using the strain, involving the production of extracellular water-soluble polysaccharide by utilizing sugar metabolism, and belongs to the technical field of bioengineering. The bacterial strain provided by the present invention is classified as Aureobasidium pullulans (Aureobasidium pullulans) G-58, and the preservation number is CGMCC No.1234; use this bacterial strain to undergo fermentation and post-treatment of fermented liquid: heat treatment, decolorization, precipitation, desalination, Drying and other processes are used to produce pullulan. The obtained product pullulan has light color, high polysaccharide yield, excellent viscoelasticity, emulsification, plasticity, non-toxic and harmless, and is widely used in food, medicine, cosmetics and other industries. The fermentation process has low pigment content in the fermentation liquid, which can greatly simplify the post-treatment process. The post-treatment process of the fermentation liquid has reasonable design, simple operation, low cost, high yield, good decolorization and desalination effect, good water solubility, and the product quality meets the requirements. Pullulan, which is lower in price for industrial mass production, offers the possibility.
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.
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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 | A gene encoding extracellular pullulanase derived from bacillus and its application |
| 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 |
| WO2012095746A2 (en) | 2011-01-11 | 2012-07-19 | Capsugel Belgium Nv | New hard capsules |
| KR102312454B1 (en) * | 2011-12-02 | 2021-10-14 | 프레리 아쿠아 테크 | Microbial-based process for high-quality 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 |
| AU2018253392B2 (en) | 2017-04-14 | 2023-11-02 | Capsugel Belgium Nv | Process for making pullulan |
| JP2020516653A (en) | 2017-04-14 | 2020-06-11 | カプスゲル・ベルギウム・ナムローゼ・フェンノートシャップＣａｐｓｕｇｅｌ Ｂｅｌｇｉｕｍ ＮＶ | Pullulan capsule |
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