CN101407831A - Method for preparing rhamnolipid from macroporous resin - Google Patents
Method for preparing rhamnolipid from macroporous resin Download PDFInfo
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- CN101407831A CN101407831A CNA2008101672091A CN200810167209A CN101407831A CN 101407831 A CN101407831 A CN 101407831A CN A2008101672091 A CNA2008101672091 A CN A2008101672091A CN 200810167209 A CN200810167209 A CN 200810167209A CN 101407831 A CN101407831 A CN 101407831A
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- rhamnolipid
- ethanol
- macroporous resin
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Abstract
The invention discloses a method for preparing rhamnolipid through macroporous resin, which includes the following steps: ethanol is used as a material and fermenting is carried out for preparing the rhamnolipid; the rhamnolipid is absorbed by the macroporous resin; the ethanol is used for desorption; and then the rhamnolipid is separated, recycled and purified; the waste liquid of ethanol during the regeneration process of the themacroporous resin is recycled and used as the material for fermenting the rhamnolipid. The yield of the rhamnolipid can achieve 15 to 30g/L and the purity is above 80 percent. Not only is the production cost of the rhamnolipid effectively reduced by recycling the waste liquid of ethanol as the material for fermenting, but also the processing cost of the waste liquid of ethanol is reduced; and simultaneously the environment problem brought by discharging the waste liquid which is purified by other organic solvents is avoided. The method has the obvious advantages of broad material sources, low cost, simple production and purifying technique, high yield purity, economy, environment protection, and the like.
Description
Technical field:
The invention belongs to biological technical field, the preparation method that relates to a kind of rhanolipid as biosurfactant, relate to particularly with ethanol be carbon source ferment, with macroporous resin adsorption and carry out the preparation method of the rhamnolipid of the separation purification method of wash-out and ethanol recycling with ethanol.
Background technology:
Rhamnolipid is the secondary metabolite that pseudomonas produces, it is a kind of important bio-surfactant, compare with chemical surfactant, not only have superior surfactivity, interfacial activity, emulsification and solubilising, also have advantages such as nontoxic, easy degraded, have broad application prospects in fields such as food-processing, medical production, daily cosmetics, soil remediation and oil productions, market potential is huge.Yet the production cost of rhamnolipid is higher, purifying process complexity etc. greatly limited the production and the widespread use of rhamnolipid.
The production cost height of rhamnolipid mainly is that high and separation purifying technique complexity two aspect reasons cause owing to the raw materials for production cost.The traditional technology of rhamnolipid is a raw material production with soybean oil, Semen Maydis oil, rapeseed oil and rice bran wet goods vegetables oil mainly, though can obtain higher output, but raw materials cost is very high, and along with being becoming tight grain and oil price increase and supply day, plant oil can not satisfy the demand of rhamnolipid large-scale industrial production to raw material.In addition, rhamnolipid is an amphiphilic species, has good lyotropy.When being fermenting raw materials production rhamnolipid with the plant oil, rhamnolipid combines with the remaining grease of fermentation, form the little emulsion droplet of homodisperse, increased the difficulty of rhamnolipid separation and purification, the rate of recovery is low, and need just can obtain the higher rhamnolipid of purity through purifying repeatedly, generally need to use methyl alcohol etc. to extract, produce the waste liquid of a large amount of contaminate environment.Thereby the rhamnolipid large-scale production need be sought more economically raw material and extraction process succinctly effective, economic environmental protection reliably.
Macroporous resin is the hydrophobic resin of a class chemosynthesis, has that physical and chemical stability height, physical strength are good, wash-out and characteristics such as easy of regenerating.Compare with traditional technology, that macroporous adsorption resin technology has is with short production cycle, required equipment simple, the advantage such as reliable and stable of quality product, has been widely used in the separation and purification of glucosides class effective ingredient in the herbal medicine.Yet being used for the separation and purification of rhamnolipid, macroporous resin still do not have report at present both at home and abroad.
Summary of the invention:
The object of the present invention is to provide that a kind of production technique is simple, with low cost, product purity is high, the rhamnolipid preparation method of economic environmental protection.
In order to achieve the above object, the technical solution used in the present invention is a kind of method of preparing rhamnolipid from macroporous resin, may further comprise the steps:
1, the preparation method of rhamnolipid:
Fermented bacterium is through seed selection, and being suitable for ethanol is that carbon source through fermentation is produced rhamnolipid.
1-1, actication of culture: inoculation is in activation medium, 30 ℃, 150rpm shake-flask culture 18h.
1-2, fermentation condition: 1～3% (v/v) inoculum size, 30～35 ℃, pH 6.0～7.5, and blowing air amount 0.5～1.0vvm fermented 5～7 days, and rhamnolipid concentration reaches 15～30g/L.
1-3, ethanol feeding method: the ethanol initial concentration is 1～2% (v/v), adds 1% (v/v) in per 24 hours, and the total ethanol charging capacity is 3～9% (v/v).
Fermention medium: except that ethanol, also comprise 0.5% yeast extract powder, 0.5% glucose, 0.08%MgSO
4, 0.5% (v/v) trace metal salts solution, consist of (g/L): H
3BO
30.232, ZnSO
47H
2O 0.174, (NH
4)
2SO
4FeSO
46H
2O 0.116, CoSO
47H
2O 0.096, (NH
4)
2Mo
2O
74H
2O 0.022, CuSO
45H
2O 0.008, MnSO
44H
2O0.008.
Strain activation and culture base: 2% (v/v) glycerine, 2% peptone, 1%K
2SO
4, 0.14%MgCl
26H
2O.
2, macroporous resin adsorption is separated rhamnolipid: fermented liquid is transferred pH to 6.0～7.0 after removing thalline by centrifugal or filtration, crosses the macroporous resin column bed with the flow velocity of 1～3BV/h.After impurity was removed in washing, ethanol elution was collected elutriant, and ethanol is removed in underpressure distillation, promptly obtained purity greater than 80% rhamnolipid crude extract.
3, the ethanol waste liquid reclaims, and is used for the fermentation of rhamnolipid: after wash-out finished, washing macroporous resin column bed was collected elutant.The elutant that alcohol concn is high reclaims ethanol through underpressure distillation, can be used for the rhamnolipid wash-out once more; The elutant that alcohol concn is low is behind the mensuration alcohol concn, as the rhamnolipid fermentation raw material.
Rhamnolipid preparation method of the present invention is a raw material with the ethanol waste liquid that produces in the macroporous resin adsorption sepn process.
Compared with prior art, the present invention has following remarkable advantage: (1) raw material cheapness and wide material sources, compare with vegetables oil, ethanol is cheap, and along with the bio-ethanol continuous advancement in technology, the alcoholic acid industrial scale will constantly enlarge, and price will constantly reduce, and ethanol can become the reliable raw material of rhamnolipid large-scale industrial production; (2) purifying process is simple, and replacing vegetables oil with ethanol is the raw material production rhamnolipid, can not produce the grease remnants that are difficult to remove, and ethanol itself is more volatile, can obtain rhamnolipid thereby only separate by macroporous resin adsorption; (3) product purity height, steady quality, rhamnolipid and macroporous resin have very high affinity and selectivity, and ethanol has high desorption rate to rhamnolipid, can effectively guarantee the purity and the quality of product; (4) economic environmental protection, do not use virose solvent in the whole production technology, the ethanol waste liquid that produces in the recovery purge process has not only reduced organic waste water discharging and processing cost as the raw materials for production of rhamnolipid, and realize that resources circulation utilizes again, reduced the production cost of rhamnolipid.
Description of drawings:
8 days fermentation diagrams of Fig. 1 .4% (v/v) ethanol
7 days fermentation diagrams of Fig. 2 .7% (v/v) ethanol
Fig. 3. the rhamnolipid desorption curve
Fig. 4. in the macroporous resin regenerative process, the variation of alcohol concn in the elutant
Fig. 5. ethanol recycling schema
Fig. 6. the rhamnolipid rate ratio of different ethanol raw materials is
Embodiment:
The invention will be further described below with reference to accompanying drawing and specific examples.
Embodiment 1: the seed selection of ethanol tolerance type superior strain
Alcohol concn is higher than 3% (v/v) and suppresses the Pseudomonas aeruginosa growth, for obtaining higher output yield, needs screening ethanol tolerance type superior strain.
Starting strain is inoculated in contains in 2% (v/v) alcoholic acid substratum shake-flask culture after 48 hours, be transferred to and contain shaking in the bottle of 4% (v/v) alcoholic acid substratum and cultivate after 48 hours, be forwarded to again to contain in 2% (v/v) alcoholic acid substratum and cultivated 48 hours, so alternately cultivate for several times.
Produce the rhamnolipid bacterial strain by containing the screening of 4% (v/v) ethanol blue gel plate isolation, with screening inoculation in containing the shaking in the bottle of 4% (v/v) ethanol substratum, after continuous passage cultivated for 5 generations, produce the rhamnolipid bacterial strain with containing the screening of 4% (v/v) ethanol blue gel plate isolation again, each seed selection bacterial strain of shake-flask culture 5 days, after measuring the rhamnolipid output of each bacterial strain respectively, selecting wherein, superior strain is the production bacterial strain.
Ethanol tolerance type superior strain Pseudomonas aeruginosa ER0603 can be in containing 4% (v/v) alcoholic acid substratum normal growth, cultivating rhamnolipid content through 5 days is 6.54g/L.
Substratum composition: 2% or 4% (v/v) ethanol, 0.5% yeast extract powder, 0.5% glucose and 0.08%MgSO
4, 2% (v/v) pH, 6.8 phosphoric acid buffers.
The dull and stereotyped composition of blue gel: except that above-mentioned composition, also contain 0.02%CTAB (hexadecyl trimethyl ammonium bromide) and 25 μ g/ml methylene blues.
Shake-flask culture condition: 30 ℃, 150rpm.
The plate screening culture condition: 30 ℃, 72h.
Starting strain: Pseudomonas aeruginosa CICC 10204
The fermentation in 8 days of embodiment 2:4% (v/v) ethanol
1, bacterial classification: ethanol tolerance type superior strain Pseudomonas aeruginosa ER0603
2, actication of culture:
1) activation medium: 2% (v/v) glycerine, 2% peptone, 1%K
2SO
4, 0.14%MgCl
26H
2O.
2) activation condition: 30 ℃, 150rpm cultivates 12～18h.
3, fermentative production rhamnolipid
1) fermention medium: 0.5% yeast extract powder, 0.5% glucose, 0.08%MgSO
4, 0.5% (v/v) trace metal salts solution, consist of (g/L): H
3BO
30.232, ZnSO
47H
2O 0.174, (NH
4)
2SO
4FeSO
46H
2O 0.116, CoSO
47H
2O 0.096, (NH
4)
2Mo
2O
74H
2O 0.022, CuSO
45H
2O 0.008, MnSO
44H
2O 0.008.
2) fermentation condition: fermentor tank 60% fermented liquid of packing into, the bacterial classification inoculation amount is 1.5%, ventilating ratio is 1.0vvm, mixing speed 200r/min, 30～35 ℃ of temperature, pH 6.5～7.0, ferment 8 days.
3) ethanol feeding method: ethanol initial concentration 2% (v/v), added 1% (v/v) every 24 hours, total charging capacity is 4% (v/v).
4) rhamnolipid output: fermenting process rhamnolipid content, as shown in Figure 1.After the fermentation ends, fermented liquid is removed thalline through the centrifugal 20min of 5000rpm, and measuring rhamnolipid content is 17.4g/L.
The fermentation in 7 days of embodiment 3:7% (v/v) ethanol
1, bacterial classification: with embodiment 2
2, actication of culture: with embodiment 2
3, fermentative production rhamnolipid
1) fermention medium: with embodiment 2
2) fermentation condition: with embodiment 2
3) ethanol feeding method: ethanol initial concentration 2% (v/v), added 1% (v/v) every 24 hours, total charging capacity is 7% (v/v).
4) rhamnolipid output: fermenting process rhamnolipid content, as shown in Figure 2.After the fermentation ends, fermented liquid is removed thalline through the centrifugal 20min of 5000rpm, and measuring rhamnolipid content is 26.6g/L.
Embodiment 4: macroporous resin adsorption is separated rhamnolipid
The preparation of rhamnolipid: with embodiment 3
Fermented liquid is removed thalline through the centrifugal 20min of 5000rpm, transfers pH to 6.5, crosses the macroporous resin column bed with the flow velocity of 2BV/h.The water of 3BV cleans the post bed, removes impurity.95% (v/v) ethanol is collected elution peak, as shown in Figure 3 with 1BV/h flow velocity wash-out macroporous resin.50 ℃ of evaporated under reduced pressure, the gained solid substance is the product rhamnolipid.Through the HPLC quantitative assay, the content of rhamnolipid is 82.2%.
Embodiment 5: macroporous resin washes out the recycling of ethanol waste liquid
Rhamnolipid preparation method: with embodiment 3
Rhamnolipid adsorption separating method: with embodiment 4
After the rhamnolipid wash-out finishes, water cleans the macroporous resin column bed with the flow velocity of 2～3BV/h, form alcohol concn elutant (as shown in Figure 4) from high to low, it can be divided into two portions: the 1BV elutant of first part for collecting earlier, alcohol concn is higher than 50% (v/v); Second section is for collecting 2～3BV elutant subsequently, and alcohol concn is about 10～15% (v/v).First part's concentration is higher than the ethanol waste liquid of 50% (v/v), reclaims through 50 ℃ of underpressure distillation, obtains reclaiming alcohol concn greater than 90% (v/v), as the desorption agent.Second section is measured alcohol concn with potassium dichromate oxidation or additive method, and alcohol concn is 13.4% (v/v) in the elutant, as fermentation raw material.
Ethanol recycling flow process, as shown in Figure 5.
Embodiment 6: different ethanol raw materials are produced the comparison of rhamnolipid output
1, bacterial classification: with embodiment 3
2, actication of culture: with embodiment 3
3, fermentative production rhamnolipid
1) fermention medium: with embodiment 3
2) fermentation condition: with embodiment 3, fermentation time is 7 days.
3) ethanol feeding method: with embodiment 3
4, feed ethanol: be raw material with dehydrated alcohol, 75% (v/v) ethanol and ethanol waste liquid respectively, wherein the ethanol waste liquid is the ethanol waste liquid that produces in the macroporous resin regenerative process, demarcates through potassium dichromate process, and ethanol content is 13.4% (v/v).The output of rhamnolipid is respectively 29.2g/l, 29.1g/l, 29.3g/l, as shown in Figure 6.
Claims (5)
1, a kind of method of preparing rhamnolipid from macroporous resin is characterized in that may further comprise the steps:
1-1, be carbon source with ethanol, the fermentative preparation rhamnolipid:
A) actication of culture: inoculation is in activation medium, 30 ℃, 150rpm shake-flask culture 18h.
B) fermentation condition: 1～3% (v/v) inoculum size, 30～35 ℃, pH 6.0～7.5, and blowing air amount 0.5～1.0vvm fermented 5～7 days, and rhamnolipid concentration reaches 15～30g/L.
C) ethanol feeding method: the ethanol initial concentration is 1～2% (v/v), adds 1% (v/v) in per 24 hours, and the total ethanol charging capacity is 3～9% (v/v).
1-2, macroporous resin adsorption are separated rhamnolipid: after fermented liquid is removed thalline by centrifugal or filtration, transfer pH to 6.0～7.0, cross the macroporous resin column bed with the flow velocity of 1～3BV/h, ethanol is desorption agent wash-out, collection contains the component of rhamnolipid, ethanol is removed in underpressure distillation, obtains purity greater than 80% rhamnolipid crude extract.
1-3, ethanol waste liquid reclaim, and are used for the rhamnolipid fermentation: after wash-out finished, washing macroporous resin column bed was collected elutant.The elutant that alcohol concn is high reclaims ethanol through underpressure distillation, can be used for the rhamnolipid wash-out once more; The elutant that alcohol concn is low is behind the mensuration alcohol concn, as the rhamnolipid fermentation raw material.
2, the method for preparing rhamnolipid from macroporous resin according to claim 1 is characterized in that: use the ethanol waste liquid that washes out from macroporous resin to produce rhamnolipid as fermenting raw materials.
3, the method for preparing rhamnolipid from macroporous resin according to claim 1 is characterized in that:
Except that ethanol, also comprise 0.5% yeast extract powder, 0.5% glucose, 0.08%MgSO in 3-1, the fermention medium
4, 0.5% (v/v) trace metal salts solution, consist of (g/L): H
3BO
30.232, ZnSO
47H
2O 0.174, (NH
4)
2SO
4FeSO
46H
2O 0.116, CoSO
47H
2O 0.096, (NH
4)
2Mo
2O
74H
2O 0.022, CuSO
45H
2O 0.008, MnSO
44H
2O0.008.
3-2, strain activation and culture base composition are 2% (v/v) glycerine, 2% peptone, 1%K
2SO
4, 0.14%MgCl
26H
2O.
4, the method for preparing rhamnolipid from macroporous resin according to claim 1 is characterized in that: fermented bacterium is through seed selection, and being suitable for ethanol is that carbon source through fermentation is produced rhamnolipid.
5, the method for preparing rhamnolipid from macroporous resin according to claim 1 is characterized in that: the desorption agent is 60～100% (v/v) ethanol.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014111595A (en) * | 2012-11-26 | 2014-06-19 | Evonik Industries Ag | Process for isolation of rhamnolipids |
| CN104450825A (en) * | 2014-12-26 | 2015-03-25 | 芝王（天津）生物科技有限公司 | Double-phase fermentation preparation condition optimizing method for rhamnolipid |
| WO2016115048A1 (en) | 2015-01-12 | 2016-07-21 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| WO2016139127A1 (en) * | 2015-03-02 | 2016-09-09 | Unilever Plc | A method of separating rhamnolipids from a fermentation broth |
| US9884883B2 (en) | 2015-01-12 | 2018-02-06 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| US10487294B2 (en) | 2015-03-02 | 2019-11-26 | Conopco, Inc. | Compositions with reduced dye-transfer properties |
| CN111423252A (en) * | 2020-05-29 | 2020-07-17 | 万华化学集团股份有限公司 | Comprehensive treatment method of rhamnolipid fermentation liquor |
| US10829507B2 (en) | 2017-02-06 | 2020-11-10 | Stepan Company | Decolorization of concentrated rhamnolipid composition |
-
2008
- 2008-10-15 CN CNA2008101672091A patent/CN101407831A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014111595A (en) * | 2012-11-26 | 2014-06-19 | Evonik Industries Ag | Process for isolation of rhamnolipids |
| CN104450825A (en) * | 2014-12-26 | 2015-03-25 | 芝王（天津）生物科技有限公司 | Double-phase fermentation preparation condition optimizing method for rhamnolipid |
| WO2016115048A1 (en) | 2015-01-12 | 2016-07-21 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| US9884883B2 (en) | 2015-01-12 | 2018-02-06 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| WO2016139127A1 (en) * | 2015-03-02 | 2016-09-09 | Unilever Plc | A method of separating rhamnolipids from a fermentation broth |
| CN107405537A (en) * | 2015-03-02 | 2017-11-28 | 荷兰联合利华有限公司 | From the method for separation of fermentative broth rhamnolipid |
| US10259837B2 (en) | 2015-03-02 | 2019-04-16 | Conopco, Inc. | Method of separating rhamnolipids from a fermentation broth |
| US10487294B2 (en) | 2015-03-02 | 2019-11-26 | Conopco, Inc. | Compositions with reduced dye-transfer properties |
| CN107405537B (en) * | 2015-03-02 | 2020-01-03 | 荷兰联合利华有限公司 | Method for separating rhamnolipid from fermentation liquor |
| US10829507B2 (en) | 2017-02-06 | 2020-11-10 | Stepan Company | Decolorization of concentrated rhamnolipid composition |
| CN111423252A (en) * | 2020-05-29 | 2020-07-17 | 万华化学集团股份有限公司 | Comprehensive treatment method of rhamnolipid fermentation liquor |
| CN111423252B (en) * | 2020-05-29 | 2022-04-19 | 万华化学(四川)有限公司 | Comprehensive treatment method of rhamnolipid fermentation liquor |
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Open date: 20090415 |