CN100534999C - Process of extracting rhanolipid as biosurfactant - Google Patents
Process of extracting rhanolipid as biosurfactant Download PDFInfo
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- CN100534999C CN100534999C CNB2006101368800A CN200610136880A CN100534999C CN 100534999 C CN100534999 C CN 100534999C CN B2006101368800 A CNB2006101368800 A CN B2006101368800A CN 200610136880 A CN200610136880 A CN 200610136880A CN 100534999 C CN100534999 C CN 100534999C
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Abstract
The present invention is process of extracting rhamnolipid as biosurfactant. The process includes the following steps: centrifuging rhamnolipid fermenting liquid at 4 deg.c in 8000 rpm to eliminate thallus, adding ammonium sulfate in 60-120 mg/L, stilling in a fridge at 4 deg.c for 10 hr, and centrifuging to eliminate protein precipitate; diluting with icy salt solution, eliminating protein precipitate to obtain supernatant, stilling in a fridge at 4 deg.c for residual grease to demulsify and agglomerate in the surface of the supernatant, skimming the grease; regulating the solution pH to below 2, stilling in a fridge at 4 deg.c for 10 hr; centrifuging at 4 deg.c and 8000 rpm for 30 min and freeze drying the collected yellowish precipitate to obtain rhamnolipid product. The technological process of the present invention features low production cost and environment friendship.
Description
Technical field
The present invention relates to extracting method by the bio-surfactant of microorganisms.
Background technology
Rhamnolipid is by the surface-active amphipathic compound of having of microorganisms; it is compared with the tensio-active agent of chemosynthesis; except that having identical characteristics such as reduction surface tension, stable emulsion and foaming; its surfactivity and emulsifying capacity are stronger; also having characteristics such as not available nontoxic, the readily biodegradable of general synthetic surfactant, is a kind of green product that helps environment protection.The molecular structure type of rhamnolipid is various, has many special functional groups, and specificity is strong, can be used for some special dimensions.It has the potential using value in industrial aspect such as oil recovery, environment remediation, medicine, makeup, washing composition and food.
Yet, the very low and fermented liquid complicated component of the output of rhamnolipid in microbial fermentation solution, its separation, extraction and concentration technology not only difficulty are bigger, also become the major portion of rhamnolipid production cost, and its expense accounts for 60%～70% of total production cost.Thereby to select suitable product extracting method be an important step that guarantees the production technique success.Domestic extraction process has generally comprised processes such as centrifugation, chloroform-methanol extraction, rotary evaporation and silica gel column chromatography at present.For large-scale production, need a large amount of chloroforms make solvent, be irrational economically, and chloroform is a kind of highly toxic organic chloride, human body and environment are had harm.Expensive and the high toxicity of producing has determined large-scale production of rhamnolipid and application to be restricted.
It is the productive rate and concrete use etc. of how to improve rhamnolipid that domestic research to rhanolipid as biosurfactant at present, patented technology relate to more.Bio-surfactant as microorganisms can strengthen the oils extraction, certain unit has developed a kind of rhamnolipid biological surface activator, with other surfactant compound, be used for tertiary oil recovery, average recovery ratio improves 20% (number of patent application 99107581.1) than water.For by the sea of oil pollution or soil, can accelerate the degraded of oils; Also be useful on the soil of counterweight metallic pollution and phreatic reparation etc.Someone has studied solubilising and the desorption of rhamnolipid to polycyclic aromatic hydrocarbons in the soil, the somebody has studied the fermentative production of rhamnolipid, be intended to improve the content of rhamnolipid, be applied to better in the consumer garbage compost processing (number of patent application 03118042.6).Yet because rhamnolipid is present in the fermented liquid of microorganism, and content is very low, only is 25g/L as the maximum production of the disclosed rhamnolipid of CN1275429A; The content of rhamnolipid is 30g/L～40g/L in the disclosed fermented liquid of another investigator, but complicated component in the fermented liquid, the technology of the rhamnolipid that separation and Extraction is purer is comparatively complicated, there is the 70-90% of being about all will be used on middle and lower reaches extraction and the separating technology in its production cost, thereby the large-scale application of rhamnolipid just is restricted.Research extraction rhamnolipid simply and effectively environment protection method has caused many scholars' interest.
Summary of the invention
The objective of the invention is, defective at the prior art existence, propose a kind of extraction process of rhanolipid as biosurfactant, it can effectively reduce the production cost of rhamnolipid, significantly reduce in the production process the harm of environment, and it is dropped in the practical application better.
Technical scheme of the present invention is that the processing step that described rhanolipid as biosurfactant extracts is:
(1) getting the fermented liquid that rhamnolipid content is not less than 28g/L, is under 3.5 ℃ of-4.5 ℃ of conditions with this fermented liquid in temperature, is after the centrifugal 18-22min of 7800rpm-8200rpm removes thalline with the rotating speed, gets supernatant A;
(2) after the amount of pressing 60mg/L-120mg/L in supernatant A added ammonium sulfate, the refrigerator of putting into 3.5 ℃-4.5 ℃ left standstill 11h-13h, centrifugal then removal protein throw out, supernatant liquor B;
(3) supernatant liquor B is suitably diluted with 0 ℃-4 ℃ ice salt solution, put into 3.5 ℃ of-4.5 ℃ of refrigerators and leave standstill, extremely remaining grease breakdown of emulsion condenses behind the supernatant liquor surface, and it is scraped off gently, gets supernatant C;
(4) the pH value with supernatant C transfers to below 2.0, and the refrigerator of putting into 3.5 ℃-4.5 ℃ leaves standstill 11h-13h, then:
(5) with gained liquid behind centrifugal 25min-35min under 3.5 ℃-4.5 ℃ and the 7800rpm-8200rpm condition, light yellow precipitate, collect this throw out;
(6) light yellow precipitate of collecting is carried out lyophilize, promptly obtain the rhamnolipid product.
Below the present invention made further specify.
Above-mentioned steps (1) to (3) is the pre-treatment to the raw material rhamnolipid fermentation liquor, and through after the pre-treatment of these three steps, the bacterium in the fermented liquid, Residual oil and protein impurity have all obtained removal.
Lyophilize in the above-mentioned steps (6) can be adopted following operating process:
1. product (light yellow precipitate) is put into wide mouthful beaker, in cryogenic refrigerator (30 ℃) freezing 30 minutes;
2. the temperature with the freezing well of freeze drier transfers to-45 ℃;
3. will freeze good product and be placed on the pallet of freeze drier, and cover plexiglass tent and check its resistance to air loss;
4. start vacuum pump and vacuumize, the exsiccant time is 24～36 hours.
As the raw materials used rhamnolipid fermentation liquor of extraction process of the present invention, can adopt prior art products, or adopt the prior art preparation.For example, described rhamnolipid fermentation liquor can be obtained by following approach:
1, the activation of bacterial classification and preservation: buy Pseudomonas aeruginosa (Pseudomonas aeruginosa) CCTCCAB93066 from China typical culture collection center (CCTCC), bacterial classification activated back switching is deposited on the beef extract-peptone slant medium.The composition of substratum is shown in Table 1.Slant culture to the vigorous back of thalli growth (24～36h), the test tube mouth is wrapped with the kraft paper plug, 4 ℃ refrigerator preservation is put on the inclined-plane, preservation term is 1～3 month, goes down to posterity once in general 2 months.Separate obtaining the pure culture bacterium when going down to posterity earlier by plate streaking, it is seeded on the slant medium that goes down to posterity preserves again.
The go down to posterity composition of slant medium of table 1.
| Extractum carnis | Peptone | NaCl | Agar | The pH value |
| 2.0g/L | 5.0g/L | 5.0g/L | 20.0g/L | 7.0 |
2. the production of rhamnolipid fermentation liquor: press the preparation of culture medium prescription shown in the tabulation 2 seed culture fluid, get 80mL and in the 500mL Erlenmeyer flask, sterilize, in Erlenmeyer flask, cultivated 24 hours from the inclined-plane inoculation P.A-AB93066 (Pseudomonas aeruginosa) that preserves.Culture condition: 37 ℃ of temperature, pH value 6.5,200 rev/mins of rotating speeds.
Table 2 seed culture medium
| Extractum carnis | Peptone | NaCl |
| 3.0g/L | 5.0g/L | 5.0g/L |
Fermentative medium formula is: rough soya-bean oil 100～120g/L, NaNO
37.5～8.5g/L, KH
2PO
40.5～1.5g/L, Na
2HPO
4.12H
2O 0.5～1.5g/L, FeSO
4.7H
2O 0.1g/L, MgSO
4.7H
2O0.1g/L, CaCl
2.2H
2O 0.1g/L, pH7.0.Be total to 1.8L by above recipe configuration fermentation culture (not comprising soya-bean oil), be respectively charged in the triangular flask of 8 1000mL, because of oils sterilization separately, so soya-bean oil (must add 15～20% water) is divided in 8 Boiling tubes in addition, triangular flask and test tube seal with gauze earlier, wrap one deck kraft paper again, and tighten with cotton thread; With 2 in newspaper bag 10mL transfer pipet; Above-mentioned triangular flask, Boiling tube and transfer pipet are sterilized in Autoclave, and temperature is controlled between 120-125 ℃, and sterilization time is 30 minutes; From the above-mentioned 500mL triangular flask that seed culture fluid is housed, draw 7mL seed culture medium (cultivating 24h) respectively with transfer pipet, insert in the fermentation culture of 8 bacterium of having gone out.Be seeded on the Bechtop and finish, culture condition: 37 ℃, pH value 7.0, and with 250 rev/mins of shaking culture, incubation time is 96 hours, promptly obtains described rhamnolipid fermentation liquor, wherein rhamnolipid content is not less than 28g/L.
Rhamnolipid product by above-mentioned explained hereafter by the analysis revealed of liquid-matter coupling to sample, contains the rhamnolipid of two kinds of structures, and molecular weight is respectively 650 and 504, is respectively two rhamnolipids and single rhamnolipid.
The mensuration of rhamnolipid product: will carry out high performance liquid chromatography-mass spectrometry through cryodesiccated rhamnolipid product and identify.High performance liquid chromatography plays the effect of sample separation, for mass spectroscopy is prepared.The evaluation of sample is carried out in Hunan University's chemical-biological sensing and metrology National Key Laboratory.Concrete instrument and operational condition are as follows:
The instrument title: the model that U.S. Thermo Finngan company produces is the LC/MS LC-MS instrument chromatographic condition of LCO Advantage: chromatographic column Inertsil ODS-3 post (150mm * 2.1mm * 5 μ m).Sample size: 20 μ L, moving phase is acetonitrile: 1% aqueous acetic acid.In the different wash-out periods, the volume ratio difference of moving phase.Acetonitrile in 0～15min: the volume ratio of 1% acetic acid is 50: 50, and the volume ratio of moving phase is 100: 0 in 16～23min, and the volume ratio of moving phase is again 50: 50 in 24～30min.Flow velocity is 0.3mL/min.Column temperature: 25 ℃, capillary temperature is 300 ℃.
The mass spectrum condition: electron spray(ES) (ESI) source, negative ion (NEG) detects.Scanning of the mass spectrum total mass number scope: 300～1000m/z.
Scanning result is seen Fig. 1-Fig. 3.
Each peak to total mass spectrum is analyzed one by one, and the m/z that obtains two peaks of time=13.54min and 16.32min meets the molecular weight rule of rhamnolipid, as Fig. 2, shown in Figure 3.In Fig. 2 and Fig. 3, the m/z of measured matter is respectively 650 and 503.9, and corresponding molecular weight is respectively 650 and 504, is respectively the molecular weight of two rhamnolipids and single rhamnolipid.The molecular structural formula of two rhamnolipids and single rhamnolipid as shown in Figure 4 and Figure 5.
By the abundance of the total mass spectrum of Fig. 1 as seen, the main component of sample is two rhamnolipids and single rhamnolipid, and purity is very high, and wherein two rhamnolipids account for 60%.Rhamnolipid after extracting is carried out weighing, and output is 28.3g/L, and its surface tension of water-soluble back is 29.48mN/m, and micelle-forming concentration CMC is 25mg/L.
Know-why of the present invention is, after Pseudomonas aeruginosa (Pseudomonas aeruginose) is cultivated by fermentation, produced a large amount of rhamnolipids, but composition is very complicated in the fermented liquid, except that the sandlwood glycolipid, also contain thalline, remaining medium component and proteinate and protein emulsifier, neutral fat, polysaccharide etc.Because it is acid that rhamnolipid is in the aqueous solution, have only when the pH of solution value and to dissolve fully greater than 5 the time, so can extract rhamnolipid with acid precipitation method.But the soya-bean oil of protein matter in the fermented liquid and remnants can produce the serious disturbance effect to the precipitation of rhamnolipid.Reason has two, and the first, when extracting rhamnolipid, need the pH of solution is transferred to below 2.0 with acid precipitation method, produce precipitation thereby the protein matter in this moment fermented liquid reaches iso-electric point, and rhamnolipid can not be separated effectively; The second, the surfactivity of rhamnolipid stably is scattered in the solution the remaining soya-bean oil in the fermented liquid, has formed stable emulsus system with rhamnolipid, thereby hinders the precipitation of rhamnolipid.This experiment is earlier by the centrifugal thalline of removing; Utilize ammonium sulfate to produce sedimentary principle, thereby in supernatant liquor, add an amount of centrifugal again protein matter of removing of ammonium sulfate with protein matter; Add icy salt solution (NaCl solution) remaining soya-bean oil breakdown of emulsion is condensed in the supernatant liquor surface, scrape off Residual oil gently.Behind this three steps preprocessing process, the bacterium in the fermented liquid, Residual oil and protein impurity have all obtained removal.Again the pH value of supernatant liquor is transferred to and leave standstill 12 hours below 2, centrifugal with 8000r/min then, collecting precipitation; The light yellow precipitate of collecting is carried out just obtaining the rhamnolipid product after moisture content is removed in lyophilize.
As known from the above, the present invention is a kind of extraction process of rhanolipid as biosurfactant, be Acid precipitation-freezing extraction method. compare with prior art, the positively effect that it had is, the ultimate capacity of gained rhamnolipid is up to more than the 28g/L, production cost is low more than one times with methods such as centrifugation, chloroform-methanol extraction, rotary evaporation and silica gel column chromatographies, and production process of the present invention does not adopt any objectionable impurities, human body and environment there is not harm, it is a green extraction process cheaply, and operating procedure is simple, is worth promoting.
Description of drawings
Fig. 1 is total mass spectrum of rhamnolipid sample;
Fig. 2 is the mass spectrum of two rhamnolipids;
Fig. 3 is the mass spectrum of single rhamnolipid;
Fig. 4 is the structural formula of single rhamnolipid;
Fig. 5 is the structural formula of two rhamnolipids.
Embodiment
1. the preparation of rhamnolipid fermentation liquor:
1) Pseudomonas aeruginosa is seeded to the seed culture medium from slant medium, carries out shake-flask culture, dress seed culture fluid 80mL in the Erlenmeyer flask of 500mL.Culture condition: 37 ℃ of temperature, 24 hours time, 200 rev/mins of rotating speeds.
2) 250mL that packs in each 1L fermentor tank is the fermention medium of carbon source with soya-bean oil, inoculation Pseudomonas aeruginosa seed culture fluid 7mL, culture condition: 37 ℃ is 7.0 with HCl or NaOH control pH value, and with 250 rev/mins of shaking culture, incubation time is 96 hours.Obtain the about 230mL of fermentation culture of each fermentor tank, the fermented liquid in all fermentor tanks is collected in together.
Annotate: the prescription of go down to posterity slant medium and seed culture medium see Table 1 and table 2 shown in, fermentative medium formula is: rough soya-bean oil 120g/L, NaNO
38.0g/L, KH
2PO
41.0g/L, Na
2HPO
4.12H
2O 1.0g/L, FeSO
4.7H
2O 0.1g/L, MgSO
4.7H
2O 0.1g/L, CaCl
2.2H
2O 0.1g/L, pH7.0.
2. the pre-treatment of rhamnolipid fermentation liquor:
Take out to cultivate the fermented liquid after 96 hours, fermented liquid is 8000 rev/mins at rotating speed, removed thalline when temperature is 4 ℃ in centrifugal 20 minutes, get supernatant liquor after centrifugal.The refrigerator of putting into 4 ℃ behind the ammonium sulfate of supernatant liquor adding 80mg/L left standstill centrifugal again removal protein throw out 12 hours.Removed the sedimentary supernatant liquor of protein (concentration is 0.2 times that the volume of 10% icy salt solution is got fermentating liquid volume) with an amount of icy salt solution (NaCl solution) dilution, putting into 4 ℃ of refrigerators left standstill 8 hours, treat that remaining soya-bean oil breakdown of emulsion condenses in the supernatant liquor surface, scrapes off Residual oil gently.Behind this three steps preprocessing process, the bacterium in the fermented liquid, Residual oil and protein impurity have all obtained removal.
3. the extraction of rhamnolipid:
To transfer to below 2.0 through the pH value of pretreated supernatant liquor, the refrigerator of putting into 4 ℃ left standstill 12 hours; Again with fermented liquid centrifugal 30 minutes and collecting precipitation under the condition of 4 ℃ and 8000r/min; The light yellow precipitate of collecting is carried out lyophilize, obtain the rhamnolipid product.
Cryodesiccated specific operation process is as follows:
1. product is put into wide mouthful beaker, in cryogenic refrigerator (30 ℃) freezing 30 minutes;
2. the temperature with the freezing well of freeze drier transfers to-45 ℃;
3. will freeze good product and be placed on the pallet of freeze drier, and cover plexiglass tent and check its resistance to air loss;
4. start vacuum pump and vacuumize, the exsiccant time is 24～36 hours.
Claims (1)
1. the extraction process of a rhanolipid as biosurfactant is characterized in that, its processing step is:
(1) getting the fermented liquid that rhamnolipid content is not less than 28g/L, is under 3.5 ℃ of-4.5 ℃ of conditions with this fermented liquid in temperature, is after the centrifugal 18-22min of 7800rpm-8200rpm removes thalline with the rotating speed, gets supernatant A;
(2) after the amount of pressing 60mg/L-120mg/L in supernatant A added ammonium sulfate, the refrigerator of putting into 3.5 ℃-4.5 ℃ left standstill 11h-13h, centrifugal then removal protein throw out, supernatant liquor B;
(3) supernatant liquor B is suitably diluted with 0 ℃-4 ℃ ice salt solution, put into 3.5 ℃ of-4.5 ℃ of refrigerators and leave standstill, extremely remaining grease breakdown of emulsion condenses behind the supernatant liquor surface, and it is scraped off gently, gets supernatant C;
(4) the pH value with supernatant C transfers to below 2.0, and the refrigerator of putting into 3.5 ℃-4.5 ℃ leaves standstill 11h-13h, then:
(5) with gained liquid behind centrifugal 25min-35min under 3.5 ℃-4.5 ℃ and the 7800rpm-8200rpm condition, light yellow precipitate, collect this throw out;
(6) light yellow precipitate of collecting is carried out lyophilize, promptly obtain the rhamnolipid product.
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| CN102766172A (en) * | 2011-09-19 | 2012-11-07 | 大庆沃太斯化工有限公司 | Industrial production method of rhamnolipid biosurfactant dry powder |
| DE102013205756A1 (en) * | 2013-04-02 | 2014-10-02 | Evonik Industries Ag | Mixture composition containing rhamnolipids |
| KR102557083B1 (en) | 2015-01-12 | 2023-07-18 | 스테판 컴파니 | Production of rhamnolipid compositions |
| CN110248952B (en) | 2017-02-06 | 2023-11-14 | 斯泰潘公司 | Decolorization of concentrated rhamnolipid compositions |
| CN108191930B (en) * | 2018-01-22 | 2021-02-09 | 中国科学院沈阳应用生态研究所 | Method for extracting rhamnolipid product from fermentation liquor |
| CN109180746A (en) * | 2018-09-14 | 2019-01-11 | 南京工业大学 | A method of isolating and purifying rhamnolipid |
| CN114456880B (en) * | 2020-11-09 | 2023-12-19 | 万华化学(四川)有限公司 | Rhamnolipid kitchen heavy oil stain cleaning agent and preparation method thereof |
| CN114044644B (en) * | 2021-12-17 | 2023-01-24 | 临沂海螺新材料科技有限公司 | Preparation method of ecological concrete water reducing agent |
| CN114522243B (en) * | 2022-01-24 | 2024-04-30 | 江南大学 | Preparation method and antioxidation application of rhamnolipid/fullerene composite material |
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Non-Patent Citations (2)
| Title |
|---|
| Production of rhamnolipids by Pseudomonas aeruginosa. Gloria soberón-Chávez, et al.Appl. Microbiol Biotechnol.,Vol.68 . 2005 |
| Production of rhamnolipids by Pseudomonas aeruginosa. Gloria soberón-Chávez, et al.Appl. Microbiol Biotechnol.,Vol.68. 2005 * |
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