CN101760456A - Preparation method of novel high polymer material of pullulan - Google Patents
Preparation method of novel high polymer material of pullulan Download PDFInfo
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- CN101760456A CN101760456A CN200810162760A CN200810162760A CN101760456A CN 101760456 A CN101760456 A CN 101760456A CN 200810162760 A CN200810162760 A CN 200810162760A CN 200810162760 A CN200810162760 A CN 200810162760A CN 101760456 A CN101760456 A CN 101760456A
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Abstract
The invention discloses a preparation method of novel high polymer material of pullulan. The method takes cane molasses as material and screens the material through microwave-ultraviolet ray compound mutagenesis method to obtain quality bacterial strains capable of high yield of polysaccharide, then through cultivation under optimized fermentation conditions, pullulan can be obtained and the yield is high. The method is simple to operate and saves cost. Therefore, the method has good application prospect in industrial production of pullulan.
Description
Technical field
The invention relates to a kind of preparation method of novel high polymer material of pullulan, belong to macromolecular material or microbial fermentation field.
Background technology
Polysaccharide is a family macromolecule compound, has a wide range of applications at food, chemistry and medicine industry.The traditional natural polysaccharide and the macromolecular compound of chemosynthesis all have its limitation.Microbial polysaccharide has (far below plant and Sargassum polysaccharides) with short production cycle, and output, quality and price are subjected to season, weather effect little, can be applied to industries such as food, medicine, makeup, the advantage that can be degraded by microorganisms safely.Microbial polysaccharide research has in recent years obtained fast development, wherein some suitability for industrialized production.
Pullulan (pullulan, be called for short Pul), have another name called the mould polysaccharide of short stalk, it is the exocellular polysaccharide of the mould generation of falx of sprouting, with α-1, it is main that the 6-glycosidic link constitutes the homotype polysaccharide in conjunction with maltose, be glucose by α-1, the 4-glycosidic link is combined into trisaccharide maltose, two ends are again with α-1, the 6-glycosidic link is with other trisaccharide maltose combination, and macromolecule polysaccharide so repeatedly is formed by connecting.
Research work for pullulan originates in West Germany, and the Englishman also does a lot aspect theoretical.Japan compares the especially research of production technique and product application of system, and obtains many achievements.Pullulan can be by starch hydrolyzates, and sucrose or other carbohydrate direct fermentation are produced.It is soluble in water, and viscosity is lower, is difficult for gelation, and not aging, machine-shaping arbitrarily has no side effect, and is a kind of up-and-coming industrial polysaccharide.Along with the development research and the application (as trehalose etc.) of other biological polyoses, the advantage of pullulan draws attention day by day, is used widely in food, medicine, light industry, chemical industry and oil field.
In the wrapping material field, because products such as film, plastic containers easily cause environmental pollution in the petrochemical industry, therefore seeking degradable high polymer material causes increasing attention.The container made from pullulan dissolves than PVC fast 3～4 times in water, and it is biosynthetic microbiological deterioration material, can be eaten as nutrition by bacterium, does not discharge toxic gas during burning, is real environmentfriendly products.
In foodstuffs industry, pullulan can be used as the wrapping material of fruit, oleaginous food, tealeaves etc., and it also can be used as thickening material etc. and gives food special quality, improves mouthfeel, gives color and prevents mummification etc.
In paper industry, the aqueous solution of pullulan has good film-forming properties, and the no vegetable fibre paper of producing with it has good water-absorbent, is convenient to write and print, and makes the fine papers that binder formulation also can be produced special purpose with it.The husky mould that pullulan is formed as binding agent does not produce gas, dust, noise and vibration when casting
About the production method and the working condition of pullulan, people have done careful research.When carrying out commercial production, generally be substrate with the starch hydrolyzates, its concentration is 10%～15%.Also contain acetone, phosphoric acid salt and basic salt in the substratum.The initial pH of nutrient solution is adjusted to about 6.5, and during the fermentation, it can slowly descend, and particularly in 24h originally, reduces to about 3.5 at last.Bud is short to obstruct the mould maximum growth phase in preceding 75h, and the optimum yield of pullulan obtains in preceding 100h.Nutrient solution need stir and logical oxygen, and temperature remains on about 30 ℃.Select suitable fermentation condition and bacterial strain can obtain the big product of molecular weight.After fermentation finishes, the mould cell of the short stalk of bud is removed from nutrient solution by filtering.With organic solvents in particular is the separable pullulan crude product that obtains of ethanol precipitation, utilizes ultrafiltration and ion exchange resin further to purify.
The pullulan biosynthesizing is extremely complicated, dynamic, as to be subjected to a multifactor impact process.The wherein consumption of the formation of microbial growth, product, substrate, dissolved oxygen amount, pH value etc. all are among the continuous variation.Select best fermentation initial pH value, bacterial classification, inoculum size and bottling amount, can regulate the cell concentration and the ratio of different shape in the fermented liquid, parameters such as residual sugar, pH value and dissolved oxygen amount change in the regulation and control fermenting process, fermentation is carried out to the direction of high yield polysaccharide, improved the output and the transformation efficiency of pullulan greatly.
Produce the used short stalk of the bud trichoderma strain of pullulan fermentation, can from crude substance, separate obtaining.From the short stalk of the bud trichoderma strain that nature obtains, the ability of generally producing pullulan is lower, and employing physics or chemical process mutagenesis can filter out strain excellent, improves the mould fermentation efficiency of the short stalk of bud, reduces the pigment generation.
The present invention is raw material with the cane molasses, and the method by microwave-ultraviolet compounded mutagenesis filters out good high yield sugar bacterial strain, cultivates the pullulan productive rate height that obtains through the fermentation condition of optimizing.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of novel high polymer material of pullulan.
(1) mutagenic treatment
Monospore suspension preparation: on slant medium, add sterilized water, scrape the spore of getting the culture surface, make rough spore suspension with transfering loop.With this suspension vibration 20min, filter suspension, make the monospore suspension of the about 10/ml of spore concentration.
It is under the irradiation dose of lethality rate＞95% that microwave-ultraviolet complex mutation is handled, after handling with microwave irradiation earlier, and dilution coating then, 28 ℃ of-30 ℃ of cultivations, select the bacterial strain that grows fine then and carry out the ultraviolet mutagenesis processing, then dilution coating, 28 ℃ of-30 ℃ of cultivations.
(2) screening of production bacterial strain
The bacterium colony primary dcreening operation: strain culturing that will be after mutagenesis, the bacterial strain that the desalination of picking colony color, paramophia or spore content are abundant, the inclined-plane is preserved.
Shake a bottle primary dcreening operation: the dissociant that the bacterium colony primary dcreening operation is obtained is inoculated in the fermention medium, and shake-flask culture is chosen polysaccharide yield mutant strain high or that pigment content obviously reduces, carries out next step and shakes the multiple sieve of bottle.
Shake the multiple sieve of bottle: will shake the dissociant that filters out at the beginning of the bottle and continue shake-flask culture, and select the highest bacterial strain of polysaccharide yield.
(3) generation of pullulan
With shaking the bacterial strain elder generation process seed culture that the bottle sieve obtains, change fermentation culture then over to.At last with fermented liquid in the centrifugal 15min of 4000r/min, separation of supernatant and bacterial sediment, with the dehydrated alcohol of 2 times of volumes of supernatant liquor adding, vibration shakes up, and 4 ℃ of placements are spent the night, polysaccharide is fully precipitated, the centrifugal 15min of 4000r/min abandons supernatant again, and precipitation is successively with acetone, ether washing, dry to constant weight for 80 ℃, promptly get pullulan.
The method screening high yield sugar bacterial strain of complex mutation of the present invention, effective, the output of sugar height of resulting mutant strain, and fermented liquid is of light color, resulting pullulan is faint yellow, only needs can obtain the pullulan that color is pure, quality is good again through simple follow-up bleaching process again.This method is simple to operate, save cost again, and this method will have good prospects for application in the industrialized production of pullulan.
Claims (6)
1. the preparation method of a novel high polymer material of pullulan is characterized in that may further comprise the steps:
(1) mutagenic treatment
Monospore suspension preparation: on slant medium, add sterilized water, scrape the spore of getting the culture surface, make rough spore suspension with transfering loop.With this suspension vibration 20min, filter suspension, make the monospore suspension of the about 10/ml of spore concentration.
It is under the irradiation dose of lethality rate＞95% that microwave-ultraviolet complex mutation is handled, after handling with microwave irradiation earlier, and dilution coating then, 28 ℃ of-30 ℃ of cultivations, select the bacterial strain that grows fine then and carry out the ultraviolet mutagenesis processing again, then dilution coating, 28 ℃ of-30 ℃ of cultivations.
(2) screening of production bacterial strain
The bacterium colony primary dcreening operation: strain culturing that will be after mutagenesis, the bacterial strain that the desalination of picking colony color, paramophia or spore content are abundant, the inclined-plane is preserved.
Shake a bottle primary dcreening operation: the dissociant that the bacterium colony primary dcreening operation is obtained is inoculated in the fermention medium, and shake-flask culture is chosen polysaccharide yield mutant strain high or that pigment content obviously reduces, carries out next step and shakes the multiple sieve of bottle.
Shake the multiple sieve of bottle: will shake the dissociant that filters out at the beginning of the bottle and continue shake-flask culture, and select the highest bacterial strain of polysaccharide yield.
(3) generation of pullulan
With shaking the bacterial strain elder generation process seed culture that the multiple sieve of bottle obtains, change fermentation culture then over to.At last with fermented liquid in the centrifugal 15min of 4000r/min, separation of supernatant and bacterial sediment, with the dehydrated alcohol of 2 times of volumes of supernatant liquor adding, vibration shakes up, and 4 ℃ of placements are spent the night, polysaccharide is fully precipitated, the centrifugal 15min of 4000r/min abandons supernatant again, and precipitation is successively with acetone, ether washing, dry to constant weight for 80 ℃, promptly get pullulan.
2. according to said mutagenic treatment in the right (1), it is characterized in that used slant medium is potato dextrose agar (PDA): potato 300g/L, glucose 20g/L, agar 20g/L, paraxin 0.1g/L, 121 ℃ of autoclaving 20min.The condition of microwave treatment is frequency 2450Hz, and power is 800W, and the treatment time is 140-160s; The condition that ultraviolet mutagenesis is handled is: with the ultraviolet lamp of 15W, fixed distance is about 30cm, and irradiation time is 60-70s.
3. according to the screening of said production bacterium in the right (2), it is characterized in that the bacterial strain after the mutagenesis is cultivated 3-5d for 28 ℃, place 6-8d for 4 ℃ then.
4. according to the generation of said pullulan in the claim (3), it is characterized in that the seed culture condition is: inoculum size 5%, liquid amount 20%, 28-30 ℃, 180r/min shaking culture 36h; Seed culture medium is: sucrose 50.0g/L, K
2HPO
45.0g/L, (NH4)
2SO
40.6g/L, yeast extract paste 3.0g/L, NaCl 1.0g/L, MgSO
47H
2O 0.2g/L, pH6.5,121 ℃ of sterilization 30min.
5. according to the generation of said pullulan in the claim (3), it is characterized in that the condition of fermentation culture is: inoculum size 15%, liquid amount 30%, 28-30 ℃, 200r/min shaking culture 140-145h; Fermention medium: sucrose 80.0g/L, K
2HPO
46.0g/L, NH
4NO
30.2g/L, yeast extract paste 0.8g/L, NaCl 1.0g/L, CaCO
30.2g/L, pH6,121 ℃ of sterilization 30min.
6. according to said pullulan in the claim (3), it is characterized in that it is a kind of novel macromolecular material, can be widely used in food, medicine, light industry, chemical industry and oil field.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810162760A CN101760456A (en) | 2008-12-11 | 2008-12-11 | Preparation method of novel high polymer material of pullulan |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222233A (en) * | 2016-08-03 | 2016-12-14 | 四川剑南春（集团）有限责任公司 | A kind of daqu fermentation power detection culture medium, the method for its detection daqu fermentation power of preparation method and application |
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
-
2008
- 2008-12-11 CN CN200810162760A patent/CN101760456A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| CN106222233A (en) * | 2016-08-03 | 2016-12-14 | 四川剑南春（集团）有限责任公司 | A kind of daqu fermentation power detection culture medium, the method for its detection daqu fermentation power of preparation method and application |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
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Open date: 20100630 |