CN1313498C - Process for producing pullulan - Google Patents
Process for producing pullulan Download PDFInfo
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- CN1313498C CN1313498C CNB200510008439XA CN200510008439A CN1313498C CN 1313498 C CN1313498 C CN 1313498C CN B200510008439X A CNB200510008439X A CN B200510008439XA CN 200510008439 A CN200510008439 A CN 200510008439A CN 1313498 C CN1313498 C CN 1313498C
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Abstract
The present invention relates to a method for producing pullulan. The present invention comprises the following steps that (1) aureobasidium pullulan as original strains is induced by ultraviolet rays and nitrosoguanidine (NTG), and subsequently, the induced strains which have less melanin secretion and high conversion rate are selected from a large number of induced strains are selected in the process of selecting and breeding the strains; (2) a seed culture medium and a fermentation culture medium are prepared; (3) the strains obtained in step (1) are cultured at 30 to 33 DEG C, and then, the strains are inoculated in the seed culture medium which is sterilized and cooled for the cultivation so as to obtain seed liquid in the process of culturing seeds; (4) the sterilized and cooled fermentation culture medium and the seed liquid obtained in step (3) are added in a fermentation tank for fermentation to obtain fermentation liquid in the process of fermentation, wherein the inoculated amount of the seed liquid is from 1.5 to 5%; (5) the pullulan is extracted from the fermentation liquid so as to obtain the finished pullulan. The polysaccharide conversion rate reaches 60 to 70 g/L in the present invention, and the polysaccharide yield of the pullulan reaches 60 to 70%. The obtained pullulan is white or yellow.
Description
Technical field
The present invention relates to a kind of method of producing Propiram.
Background technology
In chemical industry, foodstuffs industry and medicine industry, do not produce the nuisanceless plastics of high temperature or toxic gas after thirsting for for a long time obtaining to be decomposed, burning by soil microorganisms, temperature head almost there is not the edible film that rerum natura changes, is difficult for seeing through oxygen, the high-strength structureal adhesives of acid and alkali-resistance and useful to the environment body, and can be in order to producing the natural matter of valuable industrial goods, what adapt with these requirements is the polysaccharide Propiram.Propiram is a kind of novel polymeric biomaterial, has fabulous film forming, becomes characteristics such as fiber, choke, bonding, easy processing, nontoxicity, has been widely used in fields such as food, medicine, light industry, chemical industry and oil.
Present most of Propiram produces bacterium and produces dark green and even black pigment simultaneously, even these pigments adopt any special measures of various extraction decolourings all can't dispel in the extraction process of back, the Propiram of production is that black or pigment are very dark; In addition, part bacterial strain yield is low, production cost is high, can't suitability for industrialized production.
Summary of the invention
The present invention is directed to the existing Propiram of producing the method generation of Propiram is the very dark and general not high problem of yield of black or pigment, a kind of method of producing Propiram is proposed, by cultivating the hypopigmented production bacterial strain of high yield, preparing suitable medium and select suitable production technique, realize the suitability for industrialized production of light Propiram.
The method of production Propiram provided by the present invention may further comprise the steps:
(1) seed selection of bacterial classification: with the Aureobasidium pullulans is that starting strain carries out ultraviolet ray, nitrosoguanidine (NTG) mutagenesis, the screening secretion mutagenic strain that melanochrome is less and transformation efficiency is higher from a large amount of mutagenic strains; Specifically may further comprise the steps: the preparation of a, spore suspension; B, ultraviolet mutagenesis; C, nitrosoguanidine are handled; D, screening; E, preservation;
(2) culture medium preparation: each component of seed culture medium and weight percent thereof are ammoniacal liquor 0.018～0.022%, K
2HPO
40.2～0.45%, MgSO
40.015～0.023%, NaCl0.09～0.11%, yeast extract paste 0.13～0.15%, (the DE value is meant the quite weight percent of glucose of sugar contained in the solution for the DE value for all the other, be suitable dextrose equivalent value) 48～55 Semen Maydis powder liquefier, glucose solution or maltose solution, pH value is 6.0～7.0; Each component of fermention medium and weight percent thereof are ammoniacal liquor 0.018～0.045%, K
2HPO
40.2～0.55%, MgSO
40.015～0.045%, NaCl0.09～0.12%, yeast extract paste 0.12～0.16%, all the other are 6.0～7.0 for Semen Maydis powder liquefier, glucose solution or the maltose solution of DE value 48～55, pH value;
(3) seed culture: the bacterial classification that step (1) is obtained is in 30～33 ℃, tank pressure 0.5kg/cm
2Under cultivate, stir and also to feed sterile air, stirring velocity 400～600r/min, air flow 1.5～3L/min cultivated 36～42 hours, was inoculated in the refrigerative seed culture medium of having sterilized then and cultivated, and obtained seed liquor;
(4) fermentation: the seed liquor that cooled fermention medium and the step of will sterilizing (3) obtains together adds in the fermentor tank, the weight ratio of seed liquor and fermention medium is 1.5～5%, stir and feed sterile air, under 29～33 ℃ of temperature, ferment, obtain fermented liquid, stirring velocity 400～600r/min, air flow 3～6L/min, fermentation time are 72～86 hours;
(5) extract: from fermented liquid Propiram is extracted, can adopt traditional isolation technique, with the fermented liquid dilution, high speed centrifugation is removed thalline again such as earlier, decolours then, concentrates, dry, pulverizing, obtains the finished product Propiram.
The seed selection of described bacterial strain may further comprise the steps: the preparation of (1) spore suspension: with starting strain on the wort inclined-plane 30 ℃ cultivated 3～4 days, cultivating the back washes spore with physiological saline, receive in the more rich substratum of nutrition, vibration 4～5h makes spore activation rudiment; Centrifugation, with the flushing of pH 6.0tris damping fluid once, wash out from centrifuge tube with same damping fluid again, forward in the little triangular flask, break up with the granulated glass sphere vibration, filter by one deck rice paper or absorbent cotton then, make monospore suspension, behind the blood counting chamber counting, spore concentration is adjusted to 10
7/ ml; (2) ultraviolet mutagenesis: get during mutagenic treatment in the culture dish that the 5ml spore suspension is put in diameter 6cm, put into aseptic stirring iron core, liquid layer thickness is 2mm; The preheating 20～30 minutes of turning on light earlier before the processing, stable back starts the induction stirring ware, and it is even to make bacteria suspension accept uviolizing, breakdown culture dish lid covers the ware lid immediately after 10 minutes, take out processing tank, coating culture dish flat board is dull and stereotyped with the black cloth parcel under ruddiness, carries out shaking culture; (3) nitrosoguanidine is handled: with above-mentioned ultraviolet mutagenesis bacterial strain spore suspension and concentration is that the nitroso guanidine solution of 2mg/ml mixed by 1: 4, act on 2～3h down at 26～28 ℃, handle the back centrifugation, and wash centrifugal more than 10 times repeatedly with physiological saline, dilute 104 times after returning to original volume with physiological saline at last, get 0.1ml and be coated with dull and stereotyped the cultivation; (4) screening: carry out flat board after nitrosoguanidine is handled and cultivate, separation screening promptly gets desired bacterial strain repeatedly; (5) preservation: isolated inoculation after growth fully on the slant medium, is placed 4 ℃ of left and right sides refrigerator preservations, and the preservation ambient moisture was transplanted once below 50～70% in per 4～6 months.
The collocation method of described tris damping fluid is: get Tutofusin tris (being called for short tris) 6.1 grams, add oxysuccinic acid 5.8 grams, be dissolved in the 980ml water, regulate pH to 6.0 with 1mol/lNaOH, be settled to 1000ml again.
The preparation method of described Semen Maydis powder liquefier is, Semen Maydis powder added water and amylase liquefies, and glucide wherein is dissolved in the liquefier, isolates the solids component in the liquefier, promptly obtains the Semen Maydis powder liquefier.
It is 7.0 that the pH value of described fermention medium is preferably.
Described seed culture can be at 30～33 ℃ of tank pressure 0.5kg/cm
2Under carry out, stir and also to feed sterile air, stirring velocity 400～600r/min, air flow 1.5～3L/min cultivated 36～42 hours.
Described fermentation can be carried out under 29～33 ℃ of temperature, stirs also to feed sterile air, and stirring velocity 400～600r/min, air flow 3～6L/min, fermentation time are 72～86 hours.
The present invention is that starting strain carries out ultraviolet ray, nitrosoguanidine (NTG) mutagenesis and the low pigment that filters out and the higher mutagenic strain of transformation efficiency with the Aureobasidium pullulans, on the fermention medium that contains W-Gum, glucose or maltose, carry out the fermentative production Propiram, the polysaccharide transformation efficiency reaches 60～70g/L, the pulullan polysaccharide yield can reach 60～70%, and the Propiram that obtains is white or faint yellow.
Embodiment
Embodiment 1
The method of this production Propiram may further comprise the steps:
(1) seed selection of bacterial classification: with the Aureobasidium pullulans is that starting strain carries out ultraviolet ray, nitrosoguanidine (NTG) mutagenesis, screening secretion mutagenic strain that melanochrome is less and transformation efficiency is higher and preservation (concrete step is as indicated above) from a large amount of mutagenic strains;
(2) culture medium preparation: Semen Maydis powder is added water and amylase liquefies, glucide wherein is dissolved in the liquefier, isolate the solids component in the liquefier, the Semen Maydis powder liquefier that obtains is used to prepare seed culture medium and fermention medium;
Contain ammoniacal liquor 0.21 gram in every kilogram of seed culture medium, K
2HPO
44.0 gram, MgSO
40.19 gram, the NaCl1.05 gram, yeast extract paste 1.49 grams, all the other are 6.0 for the Semen Maydis powder liquefier of DE value 50, pH value, adopt H
2SO
4, Ca (OH)
2As acidity regulator;
Contain ammoniacal liquor 0.41 gram in every kilogram of fermention medium, K
2HPO
45.05 gram, MgSO
40.39 gram, the NaCl1.09 gram, yeast extract paste 1.45 grams, all the other are 7.0 for the Semen Maydis powder liquefier of DE value 50, pH value, adopt H
2SO
4, Ca (OH)
2As acidity regulator;
(3) seed culture: the bacterial classification that step (1) obtains was cultivated 72 hours down in 30 ℃, be inoculated in the refrigerative seed culture medium of having sterilized then, at 30 ℃ of tank pressure 0.5kg/cm
2Down, stir and feed sterile air, stirring velocity 600r/min, air flow 1.5L/min cultivated 36 hours, obtained seed liquor;
(4) fermentation: the seed liquor that cooled fermention medium and the step of will sterilizing (3) obtains together adds in 50 tons of fermentor tanks, 40 tons of fermentation can liquid measures, inoculum size is 2%, 30 ℃ of temperature, stir and feed sterile air, stirring velocity 400r/min, air flow 3L/min, fermentation time is 86 hours, obtains fermented liquid;
(5) extract: from fermented liquid Propiram is extracted, can adopt traditional isolation technique, with the fermented liquid dilution, high speed centrifugation is removed thalline more earlier, decolours then, concentrates, dry, pulverizing, obtains the finished product Propiram.
Embodiment 2
The method of this production Propiram may further comprise the steps:
(1) seed selection of bacterial classification: with the Aureobasidium pullulans is that starting strain carries out ultraviolet ray, nitrosoguanidine (NTG) mutagenesis, screening secretion mutagenic strain that melanochrome is less and transformation efficiency is higher and preservation (concrete step is as indicated above) from a large amount of mutagenic strains;
(2) culture medium preparation: glucose is made solution, be used to prepare seed culture medium and fermention medium;
Contain ammoniacal liquor 0.20 gram in every kilogram of seed culture medium, K
2HPO
44.1 gram, MgSO
40.21 gram, the NaCl1.01 gram, yeast extract paste 1.49 grams, all the other are the glucose solution of DE value 52, pH value is 6.0, adopts H
2SO
4, Ca (OH)
2As acidity regulator;
Contain ammoniacal liquor 0.41 gram in every kilogram of fermention medium, K
2HPO
44.05 gram, MgSO
40.41 gram, the NaCl1 gram, yeast extract paste 1.41 grams, all the other are the glucose solution of DE value 52, pH value is 7.0, adopts H
2SO
4, Ca (OH)
2As acidity regulator;
(3) seed culture: the bacterial classification that step (1) obtains was cultivated 72 hours down in 31 ℃, be inoculated in the refrigerative seed culture medium of having sterilized then, at 31 ℃ of tank pressure 0.5kg/cm
2Down, stir and feed sterile air, stirring velocity 500r/min, air flow 2.5L/min cultivated 38 hours, obtained seed liquor;
(4) fermentation: the seed liquor that cooled fermention medium and the step of will sterilizing (3) obtains together adds in 50 tons of fermentor tanks, 40 tons of fermentation can liquid measures, inoculum size is 3%, 30 ℃ of temperature, stir and feed sterile air, stirring velocity 400r/min, air flow 3L/min, fermentation time is 80 hours, obtains fermented liquid;
(5) extract: from fermented liquid Propiram is extracted, can adopt traditional isolation technique, with the fermented liquid dilution, high speed centrifugation is removed thalline more earlier, decolours then, concentrates, dry, pulverizing, obtains the finished product Propiram.
Embodiment 3
The method of this production Propiram may further comprise the steps:
(1) seed selection of bacterial classification: with the Aureobasidium pullulans is that starting strain carries out ultraviolet ray, nitrosoguanidine (NTG) mutagenesis, screening secretion mutagenic strain that melanochrome is less and transformation efficiency is higher and preservation (concrete step is as indicated above) from a large amount of mutagenic strains;
(2) culture medium preparation: Semen Maydis powder is added water and amylase liquefies, glucide wherein is dissolved in the liquefier, isolate the solids component in the liquefier, the Semen Maydis powder liquefier that obtains is used to prepare seed culture medium and fermention medium;
Contain ammoniacal liquor 0.19 gram in every kilogram of seed culture medium, K
2HPO
42.5 gram, MgSO
40.16 gram, the NaCl0.95 gram, yeast extract paste 1.36 grams, all the other are 6.3 for the Semen Maydis powder liquefier of DE value 49, pH value, adopt H
2SO
4, Ca (OH)
2As acidity regulator;
Contain ammoniacal liquor 0.25 gram in every kilogram of fermention medium, K
2HPO
43.20 gram, MgSO
40.22 gram, the NaCl0.95 gram, yeast extract paste 1.25 grams, all the other are 6.8 for the Semen Maydis powder liquefier of DE value 49, pH value, adopt H
2SO
4, Ca (OH)
2As acidity regulator;
(3) seed culture: the bacterial classification that step (1) obtains was cultivated 75 hours down in 30 ℃, be inoculated in the refrigerative seed culture medium of having sterilized then, at 30 ℃ of tank pressure 0.5kg/cm
2Down, stir and feed sterile air, stirring velocity 450r/min, air flow 2L/min cultivated 40 hours, obtained seed liquor;
(4) fermentation: the seed liquor that cooled fermention medium and the step of will sterilizing (3) obtains together adds in 50 tons of fermentor tanks, 40 tons of fermentation can liquid measures, inoculum size is 4%, 30 ℃ of temperature, stir and feed sterile air, stirring velocity 550r/min, air flow 5L/min, fermentation time is 76 hours, obtains fermented liquid;
(5) extract: from fermented liquid Propiram is extracted, can adopt traditional isolation technique, with the fermented liquid dilution, high speed centrifugation is removed thalline more earlier, decolours then, concentrates, dry, pulverizing, obtains the finished product Propiram.
Claims (5)
1. method of producing Propiram is characterized in that may further comprise the steps:
(1) seed selection of bacterial classification: with the Aureobasidium pullulans is that starting strain carries out ultraviolet ray, nitrosoguanidine mutagenesis, the screening secretion mutagenic strain that melanochrome is less and transformation efficiency is higher from a large amount of mutagenic strains; Specifically may further comprise the steps: the preparation of a, spore suspension; B, ultraviolet mutagenesis; C, nitrosoguanidine are handled; D, screening; E, preservation;
(2) culture medium preparation: each component of seed culture medium and weight percent thereof are ammoniacal liquor 0.018～0.022%, K
2HPO
40.2～0.45%, MgSO
40.015～0.023%, NaCl 0.09～0.11%, yeast extract paste 0.13～0.15%, and all the other are Semen Maydis powder liquefier, glucose solution or the maltose solution of dextrose equivalent value 48～55, pH value is 6.0～7.0; Each component of fermention medium and weight percent thereof are ammoniacal liquor 0.018～0.045%, K
2HPO
40.2～0.55%, MgSO
40.015～0.045%, NaCl 0.09～0.12%, yeast extract paste 0.12～0.16%, and all the other are Semen Maydis powder liquefier, glucose solution or the maltose solution of dextrose equivalent value 48～55, pH value is 6.0～7.0:
(3) seed culture: the bacterial classification that step (1) is obtained is in 30～33 ℃, tank pressure 0.5kg/cm
2Under cultivate, stir and also to feed sterile air, stirring velocity 400～600r/min, air flow 1.5～3L/min cultivated 36～42 hours, was inoculated in the refrigerative seed culture medium of having sterilized then and cultivated, and obtained seed liquor;
(4) fermentation: the seed liquor that cooled fermention medium and the step of will sterilizing (3) obtains together adds in the fermentor tank, the weight ratio of seed liquor and fermention medium is 1.5～5%, stir and feed sterile air, under 29～33 ℃ of temperature, ferment, obtain fermented liquid, stirring velocity 400～600r/min, air flow 3～6L/min, fermentation time are 72～86 hours;
(5) extract: from fermented liquid, Propiram is extracted.
2. the method for production Propiram according to claim 1, it is characterized in that: the seed selection of described bacterial strain may further comprise the steps: the preparation of (1) spore suspension: with starting strain on the wort inclined-plane 30 ℃ cultivated 3～4 days, cultivating the back washes spore with physiological saline, receive in the more rich substratum of nutrition, vibration 4～5h makes spore activation rudiment; Centrifugation, with the flushing of pH 6.0tris damping fluid once, wash out from centrifuge tube with same damping fluid again, forward in the little triangular flask, break up with the granulated glass sphere vibration, filter by one deck rice paper or absorbent cotton then, make monospore suspension, behind the blood counting chamber counting, spore concentration is adjusted to 10
7/ ml; (2) ultraviolet mutagenesis: get during mutagenic treatment in the culture dish that the 5ml spore suspension is put in diameter 6cm, put into aseptic stirring iron core, liquid layer thickness is 2mm; The preheating 20～30 minutes of turning on light earlier before the processing, stable back starts the induction stirring ware, and it is even to make bacteria suspension accept uviolizing, breakdown culture dish lid covers the ware lid immediately after 10 minutes, take out processing tank, coating culture dish flat board is dull and stereotyped with the black cloth parcel under ruddiness, carries out shaking culture; (3) nitrosoguanidine is handled: with above-mentioned ultraviolet mutagenesis bacterial strain spore suspension and concentration is that the nitroso guanidine solution of 2mg/ml mixes by 1:4, act on 2～3h down at 26～28 ℃, handle the back centrifugation, and wash centrifugally more than 10 times repeatedly with physiological saline, return to physiological saline at last and dilute 10 after original volume
4Doubly, get 0.1ml and be coated with dull and stereotyped the cultivation; (4) screening: carry out flat board after nitrosoguanidine is handled and cultivate, separation screening promptly gets desired bacterial strain repeatedly; (5) preservation: isolated inoculation after growth fully on the slant medium, is placed 4 ℃ of left and right sides refrigerator preservations, and the preservation ambient moisture was transplanted once below 50～70% in per 4～6 months.
3. the method for production Propiram according to claim 2, it is characterized in that: the collocation method of described tris damping fluid is: get Tutofusin tris 6.1 grams, add oxysuccinic acid 5.8 grams, be dissolved in the 980ml water, regulate pH to 6.0 with 1mol/l NaOH, be settled to 1000ml again.
4. the method for production Propiram according to claim 1, it is characterized in that: the preparation method of described Semen Maydis powder liquefier is, Semen Maydis powder is added water and amylase liquefies, glucide wherein is dissolved in the liquefier, isolate the solids component in the liquefier, promptly obtain the Semen Maydis powder liquefier.
5. the method for production Propiram according to claim 1 is characterized in that: the pH value of described fermention medium is 7.0.
Priority Applications (1)
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|---|---|---|---|
| CNB200510008439XA CN1313498C (en) | 2005-02-21 | 2005-02-21 | Process for producing pullulan |
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|---|---|---|---|
| CNB200510008439XA CN1313498C (en) | 2005-02-21 | 2005-02-21 | Process for producing pullulan |
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|---|---|
| CN1654482A CN1654482A (en) | 2005-08-17 |
| CN1313498C true CN1313498C (en) | 2007-05-02 |
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Cited By (2)
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| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
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| 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 |
| 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 |
| CN102634556A (en) * | 2012-03-29 | 2012-08-15 | 陈洁 | Method for utilizing potato starch waste water to ferment pullulan |
| CN103088085B (en) * | 2012-12-31 | 2014-08-27 | 天津北洋百川生物技术有限公司 | Method for fermenting pulullan polysaccharide by preparing culture medium from starch wastewater and malt sprouts |
| CN103243135A (en) * | 2012-12-31 | 2013-08-14 | 天津北洋百川生物技术有限公司 | Novel culture medium for producing pulullan and method for fermenting and producing pulullan |
| CN103060204B (en) * | 2012-12-31 | 2014-02-26 | 天津北洋百川生物技术有限公司 | Mutant strain for mass production of pulullan and cultural method thereof |
| CN103626885A (en) * | 2013-11-14 | 2014-03-12 | 江南大学 | Clean production method of Pulullan |
| CN105695347A (en) * | 2016-04-28 | 2016-06-22 | 廊坊梅花生物技术开发有限公司 | Strain producing pullulan, application thereof and pullulan production method |
| CA3059529A1 (en) | 2017-04-14 | 2018-10-18 | Capsugel Belgium Nv | Process for making pullulan |
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| EP0812919A1 (en) * | 1991-10-16 | 1997-12-17 | SHIN-ETSU BIO, Inc. | High molecular weight pullulan and method for its production |
| CN1216780A (en) * | 1997-11-05 | 1999-05-19 | 中国科学院微生物研究所 | Tech. and process for extracting pullulan from fermentation liquor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
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| CN1654482A (en) | 2005-08-17 |
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| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 515011 M3 in 11 districts of Jinyuan Industrial City, Chaoshan Road, Shantou City, Guangdong Province Patentee after: GUANGDONG FUWEI FRUITS & NUTS MANUFACTURING CO.,LTD. Address before: 515011 M3 in 11 districts of Jinyuan Industrial City, Chaoshan Road, Shantou City, Guangdong Province Patentee before: Shantou Fuwei Fruits & Nuts Manufacturing Co.,Ltd. |
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| CP01 | Change in the name or title of a patent holder | ||
| CP03 | Change of name, title or address |
Address after: 515000 M3 and D buildings of 13-07 and 13-08 blocks of Jinyuan Industrial City, Chaoshan Road, Shantou City, Guangdong Province Patentee after: GUANGDONG FUWEI HEALTH TECHNOLOGY Co.,Ltd. Address before: 515011 M3 in 11 districts of Jinyuan Industrial City, Chaoshan Road, Shantou City, Guangdong Province Patentee before: GUANGDONG FUWEI FRUITS & NUTS MANUFACTURING CO.,LTD. |
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| CP03 | Change of name, title or address |