CN104726515A - Method for extracting bacterial exopolysaccharide rich in fucose - Google Patents
Method for extracting bacterial exopolysaccharide rich in fucose Download PDFInfo
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- CN104726515A CN104726515A CN201510122909.9A CN201510122909A CN104726515A CN 104726515 A CN104726515 A CN 104726515A CN 201510122909 A CN201510122909 A CN 201510122909A CN 104726515 A CN104726515 A CN 104726515A
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Abstract
The invention discloses a method for extracting bacterial exopolysaccharide rich in fucose, and belongs to the technical field of bioengineering. The method comprises the following steps: carrying out ventilation culture on escherichia hermannii XPF-1 producing exopolysaccharide to obtain a polysaccharide fermentation liquor rich in fucose; carrying out centrifugal separation, ultra-filtration concentration, alcohol solvent precipitation, low-temperature vacuum drying, and crushing to obtain a bacterial exopolysaccharide product rich in the fucose. On one hand, the solvent dosage of a follow-up process can be reduced, and on the other hand, small molecular substances and water-soluble pigment substances can also be removed; macromolecular substances such as polysaccharides can be precipitated; meanwhile, residual nutrients, inorganic salts, lipid-soluble pigments, metabolism by-products and the like are removed; small molecular substances can be further removed; and target polysaccharides are purified. The polysaccharides are biological substances, and are relatively sensitive to temperature change, and therefore, the risk of molecular structure change of the polysaccharides is reduced by the technology of vacuum drying and crushing at 60 DEG C at most.
Description
Technical field
The present invention relates to a kind of extracting method being rich in the extracellular polysaccharide of bacteria of Fucose, belong to technical field of bioengineering.
Background technology
Biopolymer molecule microorganism being had to provide protection that microbial polysaccharide is bacterium, yeast, the microorganism such as mould and blue-green algae produce in metabolic process.Microbial polysaccharide is with a wide range of applications in a lot of fields, as the biological chemistry amendment in thickening material, suspension agent, emulsifying agent, biological flocculant, microbe oil production, preservation agent, anticancer pharmaceuticals, wrapping material etc.Because microbial polysaccharide adopts fermentation production technology to obtain, with short production cycle, the grain, waste residue, waste liquid etc. of various easy degraded can be utilized under manual control condition to produce.Because microbial polysaccharide potential use is constantly developed, application prospect is more more wide than animals and plants polysaccharide.Current only have the microbial polysaccharide of little kind and producing strains to produce for industry, agricultural and pharmaceutical prod, therefore be necessary screening, be separated new microbial polysaccharide producing strains, microbial polysaccharide product is obtained, to further investigate the chemical structure, physico-chemical property, biological activity etc. of microbial polysaccharide and to explore further by separation and extraction technology.
Microbial polysaccharide has many biological activitys, as physiological functions such as antiviral, immuno-stimulatings.Nearest investigator has found to be rich in the oligosaccharides of Fucose and polysaccharide fraction has the physiologically actives such as anti-oxidant, antitumor and anti-inflammatory, has a good application prospect at makeup and field of medicaments.
The nearest screening and separating of Southern Yangtze University is produced to a strain and is rich in the microorganism strains of Fucose: He Shi dust wish bacterium (
escherichia hermannii) XPF-1(industrial microorganism, 2013,43 (2): 28-32), the monose of the exocellular polysaccharide of its secretion consists of: rhamnosyl, Fucose, seminose, glucose, glucuronic acid, galn and semi-lactosi form, the mol ratio of each component is 2.8:11.4:1.0:36.4:3.0:0.5:14.6, and Fucose accounting reaches 16.4%.By Optimal Medium and fermentation condition, this bacterial strain produces the productive rate being rich in the exocellular polysaccharide of Fucose and reaches 11.4 g/L, has possessed the basic condition of industrial application.
Summary of the invention
The object of this invention is to provide a kind of extracting method being rich in the extracellular polysaccharide of bacteria of Fucose, to obtain the microbial polysaccharide product being rich in Fucose.
Technical scheme of the present invention: a kind of extracting method being rich in the extracellular polysaccharide of bacteria of Fucose, wishes bacterium XPF-1 by a strain He Shi dust and carries out aerlbic culture, obtains the extracellular polysaccharide of bacteria fermented liquid being rich in Fucose; Then centrifugal supernatant liquor, gained supernatant liquor obtains trapped fluid through ultrafiltration and concentration, adds alcohol precipitation, through centrifugal acquisition throw out, then use a small amount of ethanol centrifuge washing 2 ~ 4 times, through 60 DEG C and following low-temperature vacuum drying, pulverize and obtain the extracellular polysaccharide of bacteria raw product being rich in Fucose.
The described extracting method being rich in the extracellular polysaccharide of bacteria of Fucose, concrete steps are:
(1) ferment: wish bacterium XPF-1 for starting strain with He Shi dust,
Seed culture based component is: glucose 10g/L, yeast powder 3g/L, wort extract 3g/L, Tryptones 5g/L, pH 7.0, with pure water preparation, at 121 DEG C of sterilizing 20 min; Agar 20g/L is added in solid medium;
Wish the mono-bacterium colony of bacterium XPF-1 with inoculating needle picking one ring He Shi dust, be seeded in the 500mL triangle shaking flask containing 100mL seed culture medium, be placed in shaking table and cultivate 18 ~ 30h with 250rpm speed oscillation;
Then continue in the fermentation medium to cultivate, fermentation medium components is: maltose 30 g/L, compound nitrogen source 5g/L, K
2hPO
42g/L, MgSO
47H
2o 0.1g/L, initial pH 6.0, prepares with pure water;
Leavening temperature is 30 DEG C, and inoculum size counts 5% by volume, and fermenter volume is 20L, and liquid amount is 14L, and mixing speed is 500 ~ 800rpm, and strength ofdraft is 1.0vvm; Ventilating fermentation 4d obtains the Exopolysaccharide Production From The Fermentation liquid being rich in Fucose;
(2) first time is centrifugal: Exopolysaccharide Production From The Fermentation liquid step (1) gained being rich in Fucose, with 5000 ~ 10000 r/min centrifugation 10 ~ 30 min, obtains supernatant liquor;
(3) ultrafiltration: by gained supernatant liquor through ultrafiltration and concentration, the molecular weight cut-off of ultra-filtration membrane is 10000 ~ 40000 Da, and ultrafiltration pressure is 0.15 ~ 0.3 MPa, obtains trapped fluid;
(4) alcohol precipitation, second time are centrifugal: add alcoholic solvent by step (3) gained trapped fluid, the interpolation volume ratio of alcoholic solvent and trapped fluid is 1.5 ~ 3 ︰ 1, standing precipitating 3h, then centrifugal 10 ~ 20 min under 3000 ~ 8000 r/min, collecting precipitation thing;
(5) wash: in the throw out that step (4) obtains, add a small amount of alcoholic solvent wash, again carry out centrifugal collecting precipitate; In washing process, the interpolation volume ratio of alcoholic solvent is 0.3 ~ 0.8 times of solid substance volume, and centrifuge washing technique is 3000 ~ 8000 r/min, and centrifugation time is 10 ~ 20 min; Centrifugal washing times is 2 ~ 4 times;
(6) aftertreatment: step (5) collected the throw out drying at service temperature is no more than 60 DEG C after gained washing, pulverize, 0.05-0.10MPa vacuum-drying 20-26h, be then crushed to 200-400 order, obtain the extracellular polysaccharide of bacteria that product is rich in Fucose.
Alcoholic solvent described in step (4)-(5) is ethanol and/or the Virahol of arbitrary proportion.
Collect solvent slop in leaching process, can reuse after distillation procedure reclaims.
Described compound nitrogen source is NaNO
380%, yeast powder 20%.
Beneficial effect of the present invention: except containing except desired polysaccharide product in fermented liquid of the present invention, also containing materials such as residual nutritive substance, inorganic salt, metabolic by-prods, thalline and cell debriss, centrifugally operated can remove thalline material and insoluble impurities for the first time, obtain supernatant liquor and carry out ultrafiltration and concentration, the alcoholic solvent consumption of subsequent technique can be reduced on the one hand, small-molecule substance and water colo(u)r material can also be removed on the other hand.Alcoholic solvent precipitating is adopted after obtaining ultra-filter retentate, precipitable go out the macromolecular substance such as polysaccharide, remove residual nutrition, inorganic salt, fat-soluble pigment and metabolic by-prods etc., centrifuge washing afterwards further removes small-molecule substance, purification desired polysaccharide material simultaneously.Because polysaccharide is biological substance, more responsive to temperature variation, therefore adopt low-temperature vacuum drying technique, reduce the risk that polysaccharide molecule structure changes.
Biological material specimens a: strain is produced and is rich in the microorganism strains of Fucose: He Shi dust wish bacterium (
escherichia hermannii) XPF-1, be disclosed in industrial microorganism, 2013,43 (2): 28-32.
Accompanying drawing explanation
Fig. 1 is rich in the extraction schematic flow sheet of the extracellular polysaccharide of bacteria of Fucose.
Embodiment
Embodiment 1: the preparation of being rich in the extracellular polysaccharide of bacteria fermented liquid of Fucose
Seed culture medium (g/L): glucose 10, yeast powder 3, wort extract 3, Tryptones about 5, pH 7.0, with pure water preparation, 121 DEG C of sterilizing 20 min.Solid medium adds agar 20.Wish the mono-bacterium colony of bacterium XPF-1 with inoculating needle picking one ring He Shi dust, be seeded in the 500mL triangle shaking flask containing 100mL seed culture medium, be placed in shaking table and cultivate 24h with 250rpm speed oscillation;
Fermention medium (g/L): maltose 30, compound nitrogen source 5 (wherein NaNO
380%, yeast powder 20%), K
2hPO
42, MgSO
47H
2o 0.1, initial pH 6.0, prepares with pure water.
Fermentation condition: leavening temperature is 30 DEG C, inoculum size 5% (v/v), fermenter volume is 20L, and liquid amount is 14L, and mixing speed is that 500 ~ 800rpm(regulates in real time according to oxygen dissolving value), strength ofdraft is 1.0vvm.Ventilating fermentation 4d obtains the extracellular polysaccharide of bacteria being rich in Fucose, and polysaccharide yield is 11.4 g/L, and fermentation broth viscosity is 1160 mPas.
Embodiment 2:
Gained fermented liquid 14L, removes thalline through centrifugal (10000 r/min, 20min), through ultrafiltration and concentration, (molecular weight cut-off is 10000Da to gained supernatant liquor, working pressure 0.2MPa) obtain trapped fluid 5L, add 15L ethanol, leave standstill precipitating 3h, centrifugal (the 5000r/min of second time, 10min) collecting precipitation thing, again adds a small amount of alcoholic solvent (1.0L), again carries out centrifugal (5000r/min, 10min) collecting precipitation thing, centrifuge washing 2 collecting precipitation things.
Gained throw out is cut fritter, is laid in tray, insert in vacuum drying oven, drying temperature is 55oC, and vacuum tightness is 0.09 MPa, and time of drying is about 24h, collects drying solid powder, adopts pulverizer to pulverize, must be rich in the extracellular polysaccharide of bacteria 120g of Fucose.
Claims (5)
1. be rich in an extracting method for the extracellular polysaccharide of bacteria of Fucose, it is characterized in that: a strain He Shi dust is wished bacterium XPF-1 and carries out aerlbic culture, obtain the extracellular polysaccharide of bacteria fermented liquid being rich in Fucose; Then centrifugal supernatant liquor, gained supernatant liquor obtains trapped fluid through ultrafiltration and concentration, adds alcohol precipitation, through centrifugal acquisition throw out, then use a small amount of ethanol centrifuge washing 2 ~ 4 times, through 60 DEG C and following low-temperature vacuum drying, pulverize and obtain the extracellular polysaccharide of bacteria raw product being rich in Fucose.
2. be rich in the extracting method of the extracellular polysaccharide of bacteria of Fucose according to claim 1, it is characterized in that concrete steps are:
(1) ferment: wish bacterium XPF-1 for starting strain with He Shi dust,
Seed culture based component is: glucose 10g/L, yeast powder 3g/L, wort extract 3g/L, Tryptones 5g/L, pH 7.0, with pure water preparation, at 121 DEG C of sterilizing 20 min; Agar 20g/L is added in solid medium;
Wish the mono-bacterium colony of bacterium XPF-1 with inoculating needle picking one ring He Shi dust, be seeded in the 500mL triangle shaking flask containing 100mL seed culture medium, be placed in shaking table and cultivate 18 ~ 30h with 250rpm speed oscillation;
Then continue in the fermentation medium to cultivate, fermentation medium components is: maltose 30 g/L, compound nitrogen source 5g/L, K
2hPO
42g/L, MgSO
47H
2o 0.1g/L, initial pH 6.0, prepares with pure water;
Leavening temperature is 30 DEG C, and inoculum size counts 5% by volume, and fermenter volume is 20L, and liquid amount is 14L, and mixing speed is 500 ~ 800rpm, and strength ofdraft is 1.0vvm; Ventilating fermentation 4d obtains the Exopolysaccharide Production From The Fermentation liquid being rich in Fucose;
(2) first time is centrifugal: Exopolysaccharide Production From The Fermentation liquid step (1) gained being rich in Fucose, with 5000 ~ 10000 r/min centrifugation 10 ~ 30 min, obtains supernatant liquor;
(3) ultrafiltration: by gained supernatant liquor through ultrafiltration and concentration, the molecular weight cut-off of ultra-filtration membrane is 10000 ~ 40000 Da, and ultrafiltration pressure is 0.15 ~ 0.3 MPa, obtains trapped fluid;
(4) alcohol precipitation, second time are centrifugal: add alcoholic solvent by step (3) gained trapped fluid, the interpolation volume ratio of alcoholic solvent and trapped fluid is 1.5 ~ 3 ︰ 1, standing precipitating 3h, then centrifugal 10 ~ 20 min under 3000 ~ 8000 r/min, collecting precipitation thing;
(5) wash: in the throw out that step (4) obtains, add a small amount of alcoholic solvent wash, again carry out centrifugal collecting precipitate; In washing process, the interpolation volume ratio of alcoholic solvent is 0.3 ~ 0.8 times of solid substance volume, and centrifuge washing technique is 3000 ~ 8000 r/min, and centrifugation time is 10 ~ 20 min; Centrifugal washing times is 2 ~ 4 times;
(6) aftertreatment: step (5) collected the throw out drying at service temperature is no more than 60 DEG C after gained washing, pulverize, 0.05-0.10MPa vacuum-drying 20-26h, then be crushed to 200-400 order, obtain the extracellular polysaccharide of bacteria that product is rich in Fucose.
3. be rich in the extracting method of the extracellular polysaccharide of bacteria of Fucose according to claim 2, it is characterized in that: the alcoholic solvent described in step (4) ~ (5) is ethanol and/or the Virahol of arbitrary proportion.
4. be rich in the extracting method of the extracellular polysaccharide of bacteria of Fucose according to claim 2, it is characterized in that: collect solvent slop in leaching process, can reuse after distillation procedure reclaims.
5. be rich in the extracting method of the extracellular polysaccharide of bacteria of Fucose according to claim 2, it is characterized in that: described compound nitrogen source is NaNO
380%, yeast powder 20%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111234047A (en) * | 2020-03-30 | 2020-06-05 | 西南大学 | Exopolysaccharide rich in fucose and preparation method and application thereof |
| CN111248258A (en) * | 2020-03-30 | 2020-06-09 | 西南大学 | Application of fucose polysaccharide in preparation of preservative and preservative film coating agent thereof |
| CN117646048A (en) * | 2023-12-08 | 2024-03-05 | 重庆智合生物医药有限公司 | Production method for shortening fermentation time of fucoidin |
Citations (1)
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|---|---|---|---|---|
| CN102657674A (en) * | 2012-04-11 | 2012-09-12 | 山东大学 | Application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities |
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2015
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102657674A (en) * | 2012-04-11 | 2012-09-12 | 山东大学 | Application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities |
Non-Patent Citations (1)
| Title |
|---|
| 冯雪萍: "产高粘度微生物多糖的菌种筛选及其单糖组分分析", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111234047A (en) * | 2020-03-30 | 2020-06-05 | 西南大学 | Exopolysaccharide rich in fucose and preparation method and application thereof |
| CN111248258A (en) * | 2020-03-30 | 2020-06-09 | 西南大学 | Application of fucose polysaccharide in preparation of preservative and preservative film coating agent thereof |
| CN111234047B (en) * | 2020-03-30 | 2021-04-23 | 西南大学 | Exopolysaccharide rich in fucose and preparation method and application thereof |
| CN117646048A (en) * | 2023-12-08 | 2024-03-05 | 重庆智合生物医药有限公司 | Production method for shortening fermentation time of fucoidin |
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Application publication date: 20150624 |