CN101100687A - Method for preparing beta-polymalic acid and simultaneously coproducing byproduct prussian blue - Google Patents
Method for preparing beta-polymalic acid and simultaneously coproducing byproduct prussian blue Download PDFInfo
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- CN101100687A CN101100687A CNA2007100583965A CN200710058396A CN101100687A CN 101100687 A CN101100687 A CN 101100687A CN A2007100583965 A CNA2007100583965 A CN A2007100583965A CN 200710058396 A CN200710058396 A CN 200710058396A CN 101100687 A CN101100687 A CN 101100687A
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- propiram
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Abstract
Beta-polyoxysuccinic acid and Prussian blue as by-product are prepared by: inoculating activated fungus in fermenting medium for 8-14 days at 24-30 deg.C and 120rpm, separating solid from liquid to remove mycelia, adding alcohol with equal volume to separate Prussian blue deposit and melanochrome, concentrating supernatant and centrifuging with double-volume alcohol to separate Prussian blue, re-concentrating, adding four volume alcohol to deposit beta-polyoxysuccinic acid, and dissolving into water, removing residual melanochrome and micro-molecular materials, and frozen drying to obtain beta-polyoxysuccinic acid, dissolving separated Prussian blue in mixed alcohol-butanone to remove melanochrome, and dialyzing and frozen drying to obtain Prussian blue. It can remove melanochrome effectively to quality purity and reduce pollution by discharged Prussian blue and melanochrome.
Description
Technical field
Technical scheme of the present invention belongs to biological process synthesising biological degraded macromolecular material and grinds the field.
Background technology
The study on the synthesis of Biodegradable polyester starts from the sixties in 20th century.1969, may there be the polymkeric substance that contains the oxysuccinic acid structural unit in these bacterium of supposition such as the microbiologist Shimada of research penicillium cyclopium (Pencillium cyclopium) and Matsushima, this polymkeric substance can suppress the acid protease activity of penicillium cyclopium, confirms that finally this polymkeric substance is polymalic acid (PMA).1979, Vert etc. synthesized water-soluble fatty adoption ester---polymalic acid first, and it is only monomer with the oxysuccinic acid.1989, Fisher etc. isolated PMA from slime mould (Physarum Polycephalum) cell, and found that it is the inhibitor of archaeal dna polymerase α.Up to now, be that the foreign scholar of representative is to synthetic, the performance of PMA with should be used as more deep research with Vert, Cammas, Kajiyama etc.; Domestic up to just having the scholar that the synthetic of PMA cooked fundamental research with performance in recent years.
Polymalic acid (PMA) is a kind of aliphatic polyester base polymer, is the new bio polymer of completely biodegradable.PMA can be degraded to oligopolymer earlier by certain micro-organisms, then forms the oxysuccinic acid monomer, and eventual degradation is CO
2And H
2O, its degraded product is nontoxic, can not produce secondary pollution to environment, is a kind of ideal Biodegradable material.PMA mainly contains α, β, three kinds of structures of γ, and being present in biology intravital mainly is the β type, and it has good water-solubility, biodegradable, biocompatibility and Bioabsorbable, and it has easy metabolic and easily modification property.Therefore, β-PMA can be used as pharmaceutical carrier and microencapsulation material, biomedical material, as operating sutures and bandage etc., also can be used as makeup, packaging material for food etc.
Propiram is a kind of exocellular polysaccharide by product that Aureobasidium pullulans produces, and is soluble in water, is insoluble to ethanol.It is a kind of have fabulous film forming, one-tenth fibre, choke, bonding, nontoxicity biopolymer, has been widely used in fields such as biological medicine and food.
The preparation of PMA mainly contains two kinds of chemical synthesis and biological fermentation process.At the beginning of the research, its synthetic method mainly concentrates on chemical synthesis, is divided into two kinds of direct polymerization method and ring-opening polymerization methods again.That the direct polymerization method obtains mainly is γ type PMA, and relative molecular mass is lower, and actual application value is less, and catalyzer is poisonous, and obtain product difficult the separation.The ring-opening polymerization method is divided into lactide and lactone open loop method again, and that the lactide open loop obtains is α type PMA, and lactone open loop method can obtain β type PMA, but the step of this method formation lactone is more, uneconomical or productive rate is too low.Biological fermentation process can obtain β type PMA, and molecular weight is relatively large, and required raw material is simple and easy to, and the reaction conditions gentleness has bigger development prospect.But, for a long time, microbial method prepares the international and domestic laboratory level that all is in of research of polymalic acid, and have that PMA yields poorly, problem that separation and purification difficulty and melanochrome are difficult to remove, and, owing to ignored the separation and purification of by product Propiram, the manufacturing cost of polymalic acid can be in any more, cause environmental problem again.In fact, making up polymalic acid production genetic engineering bacterium is to solve product separation and purification problem the best way.Yet under the situation that high yield polymalic acid construction of genetic engineering does not also have to realize, the removal of by product Propiram is one of the problem that need pay much attention to of polymalic acid production always.
Summary of the invention
Technical problem to be solved by this invention is: a kind of preparation method who obtains the Beta-polymalic acid of by product Propiram simultaneously is provided, realize two product fermentations of Beta-polymalic acid and Propiram, effectively remove melanochrome, reduce because the discarded environmental pollution that causes of by product Propiram.
The present invention solves this technical problem the technical scheme that is adopted:
The first step, culture medium preparation
1. activation medium: i.e. potato sucrose substratum (PSA): potato 200g/L, sucrose 20g/L, agar 20g/L, pH5.6,121 ℃ of high pressure steam sterilizations 30 minutes;
2. the preparation of fermention medium: glucose 120g/L, NaNO
32g/L, KH
2PO
40.1g/L, MgSO
47H
2O 0.2g/L, KCl 0.5g/L, CaCO
330g/L, 4.0,121 ℃ of high pressure steam sterilizations of pH 15 minutes, CaCO
3Independent dry sterilization, 180 ℃ 2 hours;
Second step, actication of culture
With freeze-drying lactobacillus---(Aureobasidium pullulans As3.837) adopts PSA to carry out activation culture 40-90 hour at 22-26 ℃ to Aureobasidium pullulans;
The 3rd step, seed culture
The single bacterium of the activation that picking second step makes on solid plate is inoculated in the L pipe that the fermention medium that the first step makes is housed, at 24-30 ℃, and under the 120r/min condition shaking culture 40-100 hour;
The 4th step, inoculation fermentation
The inoculum that the 3rd step was made is inoculated in the fermention medium with 0.8-2%, and at 24-30 ℃, the shaking culture fermentation is 8-14 days under the 120r/min condition;
The 5th step, separation and purification
1. the initial gross separation of Beta-polymalic acid and Propiram: after the 4th step fermentation ends, centrifugal, solid-liquid separation is removed thalline; Fermented liquid is regulated pH to 5.0, add equal-volume ethanol, the black flocks is has adsorbed melanic Propiram, and faint yellow clear liquor is molten Beta-polymalic acid liquid; Filtering separation; Add the long-pending ethanol of diploid after molten Beta-polymalic acid liquid concentrated, a small amount of flocks of appearance is a Propiram, filters and incorporates in the Propiram of primary deposition; The inspissation of the revolving secondary supernatant liquor that contracts obtains the comparatively Beta-polymalic acid concentrated solution of purifying;
2. the purifying of Beta-polymalic acid: top Beta-polymalic acid solution after 1. concentrating is added 4 times of volume ethanol, place 4 ℃ of refrigerator overnight, the heavy Beta-polymalic acid precipitation of adularescent is separated out; Be dissolved in once more in the water of 50-100 times of weight, melanochrome and some small-molecule substances in the Beta-polymalic acid are removed in dialysis or ultrafiltration 2 days, and lyophilize gets Beta-polymalic acid white powder product;
3. the purifying of Propiram: preparation ethanol/mixed solvent of 6: 4 of butanone ratio, with the top Propiram that 1. obtains and 5-15 times of this solvent, stirred 2 days, Propiram is fully separated with melanochrome; The Propiram crude product is soluble in water, remove residual melanochrome with the dialysis of conventional dialysis method; With the lyophilize of purified Propiram solution, get the little yellow Propiram solid of by product.
The present invention compared with prior art, beneficial effect is: the first, use this preparation method to ferment and can obtain the two products of Beta-polymalic acid and Propiram with Aureobasidium pullulans; The second, can effectively remove melanochrome, improve product purity; The 3rd, owing to the separation and purification of by product Propiram, both can reduce the production cost of Beta-polymalic acid, can avoid again because Propiram with the melanic discarded environmental problem that causes, reduces environmental pollution.
Embodiment
Embodiment 1
Prepare Beta-polymalic acid and Propiram as follows:
The first step, culture medium preparation
1. activation medium: i.e. potato sucrose substratum (PSA): potato 200g/L, sucrose 20g/L, agar 20g/L, pH5.6,121 ℃ of high pressure steam sterilizations 30 minutes;
2. the preparation of fermention medium: glucose 120g/L, NaNO
32g/L, KH
2PO
40.1g/L, MgSO
47H
2O 0.2g/L, KCl 0.5g/L, CaCO
330g/L, pH4.0,121 ℃ of high pressure steam sterilizations 15 minutes, CaCO
3Independent dry sterilization, 180 ℃ 2 hours;
Second step, actication of culture
Adopt the PSA substratum of the first step configuration to carry out activation culture 48 hours the Aureobasidium pullulans (Aureobasidium pullulans, As 3.837) that is stored in-70 ℃ the glycerine pipe at 24 ℃.
The 3rd step, seed culture
Single bacterium that picking second step makes on solid plate is inoculated in the L pipe that the fermention medium that the 5mL the first step makes is housed, and at 24 ℃, shaking culture is 90 hours under the 120rpm condition.
The 4th step, inoculation fermentation
The seed culture fluid that inoculum size with 0.8% made for the 3rd step is inoculated in the fermention medium, and at 28 ℃, the shaking culture fermentation is 14 days under the 120rpm condition.
The 5th step, separation and purification
1. the initial gross separation of Beta-polymalic acid and Propiram: after the 4th step fermentation ends, centrifugal solid-liquid separates, and removes thalline; Fermented liquid is regulated pH to 5.0, add equal-volume ethanol, the black flocks is has adsorbed melanic Propiram, and faint yellow clear liquor is Beta-polymalic acid solution; Filtering separation; To add the long-pending ethanol of diploid after the Beta-polymalic acid solution concentration, a small amount of flocks of appearance is a Propiram, filters and incorporates in the Propiram of primary deposition; The inspissation of the revolving secondary supernatant liquor that contracts obtains the comparatively Beta-polymalic acid concentrated solution of purifying;
2. the purifying of Beta-polymalic acid: top Beta-polymalic acid solution after 1. concentrating is added 4 times of volume ethanol, place 4 ℃ of refrigerator overnight, adularescent Beta-polymalic acid precipitation is separated out; Be dissolved in once more in the water of 50 times of weight, dialysed two days, remove melanochrome and some small-molecule substances in the Beta-polymalic acid, lyophilize gets Beta-polymalic acid white powder product;
3. the purifying of Propiram: preparation ethanol/mixed solvent of 6: 4 of butanone ratio, with the top Propiram that 1. obtains and 8 times of these solvent, stirred 2 days, Propiram is fully separated with melanochrome; The Propiram crude product is soluble in water, remove residual melanochrome with the dialysis tubing dialysis.With the lyophilize of purified Propiram solution, get the fine and close membranaceous little yellow Propiram solid of by product.
Embodiment 2
Prepare Beta-polymalic acid and Propiram as follows:
The first step, culture medium preparation
1. activation medium: i.e. potato sucrose substratum (PSA): potato 200g/L, sucrose 20g/L, agar 20g/L, pH5.6,121 ℃ of high pressure steam sterilizations 30 minutes;
2. the preparation of fermention medium: glucose 120g/L, NaNO
32g/L, KH
2PO
40.1g/L, MgSO
47H
2O 0.2g/L, KCl 0.5g/L, CaCO
330g/L pH4.0,121 ℃ of high pressure steam sterilizations 15 minutes, CaCO
3Independent dry sterilization, 180 ℃ 2 hours;
Second step, actication of culture
The PSA substratum that adopts the first step to make the Aureobasidium pullulans (Aureobasidium pullulans, As 3.837) that is stored in-70 ℃ the glycerine pipe carried out activation culture 48 hours at 24 ℃.
The 3rd step, seed culture
Single bacterium that picking second step makes on solid plate is inoculated in the L pipe that the fermention medium that the 5mL the first step makes is housed, and at 26 ℃, shaking culture is 72 hours under the 120rpm condition.
The 4th step, inoculation fermentation
Inoculum size with 1% is inoculated in seed culture fluid in the fermention medium, and at 28 ℃, the shaking culture fermentation is 9 days under the 120rpm condition.
The 5th step, separation and purification
1. the initial gross separation of Beta-polymalic acid and Propiram: centrifugal 10 minutes of the fermented liquid 13000rpm that the 4th step obtained removes thalline.Fermented liquid is regulated pH5.0, add isopyknic dehydrated alcohol, observed the cotton-shaped floating matter of black and generated, this is Propiram, centrifugal telling.Part melanochrome is attached on the Propiram, is together told.Faint yellow Beta-polymalic acid clear liquor adds 2 times of volume dehydrated alcohols, the centrifugal Propiram that separates once more after concentrating.
2. the purifying of Beta-polymalic acid: after the supernatant liquor rotary evaporation behind the primary sedimentation concentrated, add the dehydrated alcohol of about 4 times of volumes, place 4 ℃ of refrigerator overnight, carry out secondary sedimentation, promptly have Beta-polymalic acid to settle out.Find that sedimentary Beta-polymalic acid is present in colloidal state in the mixed solution of second alcohol and water, promptly have Beta-polymalic acid to precipitate to wherein adding 2% (w/w) NaCl.With the Beta-polymalic acid of gained precipitation be dissolved in once more 100 water doubly in, remove residual melanochrome with the dialysis tubing dialysis.Beta-polymalic acid solution lyophilize with after the dialysis promptly gets white Beta-polymalic acid product.
3. the purifying of Propiram: top 1. twice isolated Propiram and melanic mixture are placed ethanol/mixed solvent of 6: 4 of butanone ratio, 80rpm stirred 2 days on magnetic stirring apparatus, filtration obtains the Propiram crude product, discards to contain melanic mixed solution.The Propiram crude product is soluble in water, remove residual melanochrome with the dialysis tubing dialysis.Propiram solution in the lyophilize dialysis tubing obtains the faint yellow Propiram of by product.
Embodiment 3
Prepare Beta-polymalic acid and Propiram as follows:
The first step, culture medium preparation
1. activation medium: i.e. potato sucrose substratum (PSA): potato 200g/L, sucrose 20g/L, agar 20g/L, pH5.6,121 ℃, high pressure steam sterilization 30 minutes;
2. the preparation of fermention medium: glucose 120g/L, NaNO
32g/L, KH
2PO
40.1g/L, MgSO
47H
2O 0.2g/L, KCl 0.5g/L, CaCO
330g/L, 4.0,121 ℃ of high pressure steam sterilizations of pH 15 minutes, CaCO
3Independent dry sterilization, 180 ℃ 2 hours;
Second step, actication of culture
The PSA substratum that adopts the first step to make the Aureobasidium pullulans (Aureobasidium pullulans, As 3.837) that is stored in-70 ℃ the glycerine pipe carried out activation culture 90 hours at 22 ℃.
The 3rd step, seed culture
Single bacterium that picking second step makes on solid plate is inoculated in the L pipe that the fermention medium that the 5mL the first step makes is housed, and at 28 ℃, shaking culture is 60 hours under the 120rpm condition.
The 4th step, inoculation fermentation
The seed culture fluid that inoculum size with 2% made for the 3rd step is inoculated in the fermention medium, and at 26 ℃, the shaking culture fermentation is 8 days under the 120rpm condition.
The 5th step, separation and purification
1. the initial gross separation of Beta-polymalic acid and Propiram: after the 4th step fermentation ends, centrifugal solid-liquid separates, and removes thalline; Fermented liquid is regulated pH to 5.0, add equal-volume ethanol, the black flocks is has adsorbed melanic Propiram, and faint yellow clear liquor is Beta-polymalic acid solution; Filtering separation; To add the long-pending ethanol of diploid after the Beta-polymalic acid solution concentration, a small amount of flocks of appearance is a Propiram, filters and incorporates in the Propiram of primary deposition; The inspissation of the revolving secondary supernatant liquor that contracts obtains the comparatively Beta-polymalic acid concentrated solution of purifying;
2. the purifying of Beta-polymalic acid: top Beta-polymalic acid solution after 1. concentrating is added 4 times of volume ethanol, place 4 ℃ of refrigerator overnight, adularescent Beta-polymalic acid precipitation is separated out; Be dissolved in once more in the water of 50 times of weight, adopt the tubular fibre separator, carry out ultrafiltration with the conventional ultrafiltration method, it is 5000D that the carrying of film stayed molecular weight, remove melanochrome and some small-molecule substances in the Beta-polymalic acid, lyophilize gets Beta-polymalic acid white powder product;
3. the purifying of Propiram: earlier the top Propiram that 1. obtains is dissolved in the distilled water of 5 times of weight, 80 ℃ of water-baths heated 2 hours, removed Pullulanase; Preparation ethanol/mixed solvent of 6: 4 of butanone ratio with above-mentioned Propiram and 8 times of these solvent, stirred 2 days, and Propiram is fully separated with melanochrome; The Propiram crude product is soluble in water, remove residual melanochrome with the dialysis tubing dialysis.With the lyophilize of purified Propiram solution, get the fine and close membranaceous little yellow Propiram solid of by product.
Claims (5)
1. obtain the preparation method of the Beta-polymalic acid of by product Propiram simultaneously, may further comprise the steps:
The first step, culture medium preparation
1. activation medium: i.e. potato sucrose substratum (PSA): potato 200g/L, sucrose 20g/L, agar 20g/L, pH5.6,121 ℃ of high pressure steam sterilizations 30 minutes;
2. the preparation of fermention medium: glucose 120g/L, NaNO
32g/L, KH
2PO
40.1g/L, MgSO
47H
2O 0.2g/L, KCl 0.5g/L, CaCO
330g/L, pH4.0,121 ℃ of high pressure steam sterilizations 15 minutes, CaCO
3Independent dry sterilization, 180 ℃ 2 hours;
Second step, actication of culture
With freeze-drying lactobacillus---(Aureobasidium pullulan As3.837) adopts PSA to carry out activation culture 40-90 hour at 22-26 ℃ to Aureobasidium pullulans;
The 3rd step, seed culture
The single bacterium of the activation that picking second step makes on solid plate is inoculated in the L pipe that the fermention medium that the first step makes is housed, at 24-30 ℃, and under the 120r/min condition shaking culture 40-100 hour;
The 4th step, inoculation fermentation
The inoculum that the 3rd step was made is inoculated in the fermention medium with 0.8-2%, and at 24-30 ℃, the shaking culture fermentation is 8-14 days under the 120r/min condition;
The 5th step, separation and purification
1. the initial gross separation of Beta-polymalic acid and Propiram: after the 4th step fermentation ends, centrifugal, solid-liquid separation is removed thalline; Fermented liquid is regulated pH to 5.0, add equal-volume ethanol, the black flocks is has adsorbed melanic Propiram, and faint yellow clear liquor is molten Beta-polymalic acid liquid; Filtering separation; Add the long-pending ethanol of diploid after molten Beta-polymalic acid liquid concentrated, a small amount of flocks of appearance is a Propiram, filters and incorporates in the Propiram of primary deposition; The inspissation of the revolving secondary supernatant liquor that contracts obtains the comparatively Beta-polymalic acid concentrated solution of purifying;
2. the purifying of Beta-polymalic acid: top Beta-polymalic acid solution after 1. concentrating is added 4 times of volume ethanol, place 4 ℃ of refrigerator overnight, the heavy Beta-polymalic acid precipitation of adularescent is separated out; Be dissolved in once more in the water of 50-100 times of weight, melanochrome and some small-molecule substances in the Beta-polymalic acid are removed in dialysis or ultrafiltration 2 days, and lyophilize gets PMA white powder product;
3. the purifying of Propiram: preparation ethanol/mixed solvent of 6: 4 of butanone ratio, with the top Propiram that 1. obtains and 5-15 times of this solvent, stirred 2 days, Propiram is fully separated with melanochrome; The Propiram crude product is soluble in water, remove residual melanochrome with the dialysis of conventional dialysis method; With the lyophilize of purified Propiram solution, get the little yellow Propiram solid of by product.
2. the method for preparing Beta-polymalic acid and Propiram according to claim 1 is characterized in that, the actication of culture temperature is 24 ℃, and the activation culture time is 48-60 hour.
3. the method for preparing Beta-polymalic acid and Propiram according to claim 1 is characterized in that the seed culture temperature is 28 ℃, and incubation time is 60 hours.
4. the method for preparing Beta-polymalic acid and Propiram according to claim 1 is characterized in that, in the Beta-polymalic acid purification step, also adds 2% (w/w) NaCl solid when adding 4 times of volume ethanol, so that PMA fully precipitates.
5. the method for preparing Beta-polymalic acid and Propiram according to claim 1, it is characterized in that, in the purification step of Propiram, earlier Propiram is dissolved in the distilled water of 5-15 times of weight, 80 ℃ of water-baths heated 2 hours, remove Pullulanase, after the cooling, add ethanol/butanone mixed solvent again and remove melanochrome.
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CN102492740A (en) * | 2011-12-16 | 2012-06-13 | 天津北洋百川生物技术有限公司 | Method for producing poly(malic acid) and pullulan together by using Aureobasidium pullulans |
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CN101487034B (en) * | 2009-02-23 | 2013-06-05 | 中国科学院过程工程研究所 | Preparation of beta-poly malic acid and salt thereof |
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CN101487034B (en) * | 2009-02-23 | 2013-06-05 | 中国科学院过程工程研究所 | Preparation of beta-poly malic acid and salt thereof |
CN101979499B (en) * | 2009-10-29 | 2012-11-14 | 天津北洋百川生物技术有限公司 | Mutant aureobasidium pullulans TKPM00006 for massively producing beta-poly(malic acid) and culture method thereof |
US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
CN102220248A (en) * | 2011-05-20 | 2011-10-19 | 浙江大学 | Bacterial strain for producing PMLA [Poly (Beta-L-malic acid)] and method for producing PMLA by fermentation of bacterial strain |
CN102492740A (en) * | 2011-12-16 | 2012-06-13 | 天津北洋百川生物技术有限公司 | Method for producing poly(malic acid) and pullulan together by using Aureobasidium pullulans |
CN102492740B (en) * | 2011-12-16 | 2013-08-07 | 天津北洋百川生物技术有限公司 | Method for producing poly(malic acid) and pullulan together by using Aureobasidium pullulans |
CN103045662A (en) * | 2012-12-31 | 2013-04-17 | 天津北洋百川生物技术有限公司 | Fermentation medium for improving output and purity of beta-polymalic acid produced by zymotechnics |
CN103103225A (en) * | 2012-12-31 | 2013-05-15 | 天津北洋百川生物技术有限公司 | Method for preparing beta-polymalic acid of high purity |
US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
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