CN101173210A - Method for sifting and producing generation agent of dual-rhamnolipid - Google Patents
Method for sifting and producing generation agent of dual-rhamnolipid Download PDFInfo
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- CN101173210A CN101173210A CNA2007101327735A CN200710132773A CN101173210A CN 101173210 A CN101173210 A CN 101173210A CN A2007101327735 A CNA2007101327735 A CN A2007101327735A CN 200710132773 A CN200710132773 A CN 200710132773A CN 101173210 A CN101173210 A CN 101173210A
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Abstract
The invention discloses a method for screening and preparing culture seed for dirhamnolipids, which comprises the following steps: choosing the soil sample polluted by grease chronically as the strain source in strain screening; adopting vegetable oil as the liquid culture medium of single carbon source, and coating with the vegetable oil on the hexadecyl trimethyl ammonium bromide agar plate with stronger selectivity after enrichment; then selecting suspect strain depending on the morphology and growth vigour of the bacterial colony; testing the surfactivity of the strain under primary screening after fermentation culture; analyzing the fermentation broth of the strain with high activity directly with TLC method; then selecting the strain with high dirhamnolipids productivity; extracting the rhamnolipids from the strain fermentation broth under screening with extraction method; obtaining purer dirhamnolipids with column chromatographic separation method. The invention overcomes the disadvantage that the strain is screened only based on the total output of rhamnolipids, and the strain with high dirhamnolipids output is obtained difficultly in the prior screening method, and has the advantages of high accuracy, high rhamnolipids output of the screened strain, more particularly higher dirhamnolipids output, and simple preparation technology.
Description
One, technical field
The present invention relates to a kind of preparation method, the generation bacterium of a kind of pair of rhamnolipid screening specifically and preparation method with bio-surfactant of pharmaceutical use.
Two, background technology
Rhamnolipid is the surfactant that a class is produced by microbial metabolism.Except that the general utility functions with synthetic surfactant, rhamnolipid product itself is nontoxic, and environmental friendliness can be degraded by microorganism 100%, can be widely used in a lot of fields of industry, agricultural and daily life.Discovering in recent years, rhamnolipid has antibiotic activity, especially wherein two rhamnolipids can suppress breast cancer cell growth (Bioresource Technology 2007,98:1149～1153), (U.S.Patent 5,466 to reach tetter (Journal of Dermatological Science2005,40:141～143) such as assisting psoriatic, neurodermatitis and autoimmune disease, 675,1999) treatment.These are found to be two rhamnolipids and lay a good foundation in the application of field of medicaments, also are subjected to people's extensive concern simultaneously.
The rhamnolipid that microbial metabolism produces is a mixture, and the activeconstituents that wherein can be used in field of medicaments is two rhamnolipids, and the content of two rhamnolipids produces very big difference because of the difference of bacterial classification.Therefore, it is crucial obtaining two more bacterial classifications of rhamnolipid output.The use of present oily plate screening method is comparatively general, and bio-surfactant can promote the utilization of microorganism to nonpolar indissoluble substrates such as oils, adopts oil to obtain bio-surfactant easily as single carbon source screening and produces bacterium.(Bioresource Technology 2007,98:1149～1153) (document 1) mentioned above and (Journal of Industrial Microbiology ﹠amp; Biotechnology 1997,19,232～238) provide the method for two rhamnolipids preparations in (document 2).The bacterial classification that the former uses is the self-sizing bacterial classification, and the organic bacterial classification that provides in Shanghai has been provided the latter.They are to be the substratum fermentation of single carbon source with vegetables oil, adopt behind the organic solvent extraction methods such as column chromatography, TLC and high performance liquid chromatography separate two rhamnolipids.
The method of the two rhamnolipids of above-mentioned preparation has following deficiency: it all is as selecting foundation with rhamnolipid output that (1) strain screening still adopts existing bacterial classification, can not guarantee two rhamnolipid output, and the result shows that two rhamnolipid output are very low really.Rhamnolipid output is 17g/L-24g/L in the document 2, wherein two rhamnolipid (main component Rh
2C
10C
10) and single rhamnolipid (main component RhC
10C
10) mass ratio be 1: 4, though the specifying information of output is not provided in the document 1, wherein have two rhamnolipid Rh of anti-breast cancer function
2C
10C
10Only account for extract crude extract 0.1%.(2) adopt oily plate screening the microorganism that can utilize vegetables oil to make single carbon source all can be screened, even select the bacterial classification of the good generation thing tensio-active agent of growing way also to be difficult to guarantee that the thing tensio-active agent that produces is a rhamnolipid.Therefore, this method selectivity is relatively poor, and workload is big.
Three, summary of the invention
The object of the present invention is to provide a kind of selectivity strong, the especially two high bacterial screening methods of rhamnolipid output of rhamnolipid output height.
Another object of the present invention provides a kind of method of utilizing the two rhamnolipids of Production by Microorganism Fermentation preparation.
The technical solution that realizes the object of the invention is: the generation bacterial screening method of a kind of pair of rhamnolipid may further comprise the steps:
A, choose by the collection in worksite pedotheque of grease contamination as the bacterial screening source;
The inoculation vegetables oil is made the enrichment medium of carbon source after b, the sample immersion treatment, gets enrichment culture liquid behind cultivation 24～72h;
C, enrichment culture liquid gradient dilution is coated the cetyl trimethylammonium bromide plate culture medium cultivate 24～96h, kind is protected on single bacterium colony inclined-plane that the picking growing way is good and quantity is many;
The bacterial classification that d, picking inclined-plane are preserved inserts measures surfactivity after fermention medium is cultivated 48～120h, selecting active higher strain fermentating liquid supernatant to carry out TLC analyzes, developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent, the two rhamnolipid content of preliminary judgement, selection Rf value is 0.46 the dense i.e. generation bacterium reservation of pair rhamnolipids of bacterial classification of spot colors, and subzero 26 ℃ of glycerine are preserved.
The preparation method of a kind of pair of rhamnolipid may further comprise the steps:
A, choose by the collection in worksite pedotheque of grease contamination as the bacterial screening source;
The inoculation vegetables oil is made the enrichment medium of carbon source after b, the sample immersion treatment, gets enrichment culture liquid behind cultivation 24～72h;
C, enrichment culture liquid gradient dilution is coated the cetyl trimethylammonium bromide plate culture medium cultivate 24～96h, kind is protected on single bacterium colony inclined-plane that the picking growing way is good and quantity is many;
The bacterial classification that d, picking inclined-plane are preserved inserts measures surfactivity after fermention medium is cultivated 48～120h, selecting active higher strain fermentating liquid supernatant to carry out TLC analyzes, developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent, the two rhamnolipid content of preliminary judgement, selection Rf value is 0.46 the dense i.e. generation bacterium reservation of pair rhamnolipids of bacterial classification of spot colors, and subzero 26 ℃ of glycerine are preserved;
E, will screen gained bacterial classification inoculation LB substratum, cultivate 18～36h after switching go into fermention medium, cultivate 72～120h for 25-38 ℃;
After f, the fermentation ends with fermented liquid bactofugation body;
G, adjustment supernatant liquor pH=1.0～5.0 add the chloroform methanol mixture and make extraction agent, stir, leave standstill separatory, keep the machine phase;
H, remove solvent under reduced pressure, obtain the rhamnolipid crude extract with Rotary Evaporators;
I, crude extract is carried out chromatographic separation in silicagel column, with the eluent wash-out, part is collected;
J, TLC detect separation case, and developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent was that 0.46 elutriant merges with the Rf value, and evaporated under reduced pressure gets required pair of rhamnolipid.
Compared with prior art, the present invention has following remarkable advantage: (1) has selected for use vegetables oil to make the liquid nutrient medium enrichment culture of sole carbon source in bacterial classification primary dcreening operation process, make bio-surfactant produce bacterium and in enrichment process, become dominant microflora, dilution is afterwards coated the cetyl trimethylammonium bromide agar plate and is screened, the Gram-positive bacteria growing is relatively poor in this substratum, intestinal bacteria can not grow, and Pseudomonas aeruginosa well-grown on this substratum of product rhamnolipid, and have following feature: bacterium colony is canescence, flat unformed, spread or slightly spread to periphery, surface wettability, the diffusion of periphery of bacterial colonies substratum has water colo(u)r, so its selectivity is stronger, has reduced workload.(2) again in the sieve process,, increased the TLC analytic process except doing the standard with output.The few glycosyl of the two rhamnolipids of single rhamnolipid, polarity a little less than, adopt chloroform: methyl alcohol: acetate v/v/v=65: 15: 2 when making developping agent, their Rf value is respectively 0.75 and 0.46, therefore can roughly judge two rhamnolipid proportions by the colour developing spot, further improve the screening accuracy.(3) in the bio-surfactant superior strain that screening obtains, rhamnolipid produces bacterium and accounts for 66% of picking bacterial strain total amount, and wherein rhamnolipid is produced in pseudomonas Pseudomonas sp.SC-02 fermentation, and production peak can reach 24g/L.The two rhamnolipid output of target product are 16.3g/L, account for 68% of rhamnolipid total mass, and two rhamnolipids account for rhamnolipid ultimate production 20% in the document 2, and this result is better than document 2. these bacterial strains and produces in two rhamnolipids, have confirmed to have the Rh that suppresses the breast cancer cell splitting function
2C
10C
10Account for 90% of two rhamnolipid total masses, account for 23% of general extractive quality, account for 0.1% of general extractive far above document 1 report.(4) this pair rhamnolipid preparation method technical process is simple, and equipment requirements is low, cooperates in this method each ratio ethyl acetate and methyl alcohol to make eluent, and common silica gel column chromatography method can be isolated purer two rhamnolipids from the fermented liquid extract.
Below in conjunction with accompanying drawing the present invention is described in further detail.
Four, description of drawings
Fig. 1 is the generation bacterium screening of the two rhamnolipids of the present invention and preparation method's schema.
Fig. 2 is the generation bacterium screening of the two rhamnolipids of the present invention and preparation method's generation bacterium screening process figure.
Fig. 3 is the schema of the generation bacterium screening of the two rhamnolipids of the present invention and preparation method's the two rhamnolipids of microbe fermentation method preparation.
Five, embodiment
In conjunction with Fig. 1, the screening of generation bacterium and the preparation method of the two rhamnolipids of the present invention may further comprise the steps:
A, choose by the collection in worksite pedotheque of grease contamination as the bacterial screening source;
The inoculation vegetables oil is made the enrichment medium of carbon source after b, the sample immersion treatment, gets enrichment culture liquid behind cultivation 24～72h;
C, enrichment culture liquid gradient dilution is coated the cetyl trimethylammonium bromide plate culture medium cultivate 24～96h, kind is protected on single bacterium colony inclined-plane that the picking growing way is good and quantity is many;
The bacterial classification that d, picking inclined-plane are preserved inserts measures surfactivity after fermention medium is cultivated 48～120h, selecting active higher strain fermentating liquid supernatant to carry out TLC analyzes, developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent, the two rhamnolipid content of preliminary judgement, selection Rf value is 0.46 the dense i.e. generation bacterium reservation of pair rhamnolipids of bacterial classification of spot colors, and subzero 26 ℃ of glycerine are preserved;
E, will screen gained bacterial classification inoculation LB substratum, cultivate 18～36h after switching go into fermention medium, cultivate 72～120h for 25-38 ℃;
After f, the fermentation ends with fermented liquid bactofugation body;
G, adjustment supernatant liquor pH=1.0～5.0 add the chloroform methanol mixture and make extraction agent, stir, leave standstill separatory, keep the machine phase;
H, remove solvent under reduced pressure, obtain the rhamnolipid crude extract with Rotary Evaporators;
I, crude extract is carried out chromatographic separation in silicagel column, with the eluent wash-out, part is collected;
I, TLC detect separation case, and developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent was that 0.46 elutriant merges with the Rf value, and evaporated under reduced pressure gets required pair of rhamnolipid.
Embodiment 1: in conjunction with Fig. 2, the generation bacterial screening method of the two rhamnolipids of the present invention is promptly gathered pedotheque near for a long time by the kitchen sewage draining exit of grease contamination, takes by weighing the adding of 10g pedotheque stirring and evenly mixing is housed in the beaker of 50mL deionized water.Get 20mL access 80mL enrichment medium after soaking 1h, the 250mL Erlenmeyer flask places 32 ℃ of shaking tables to cultivate rotating speed 230r/min.Take out pregnant solution behind the 36h, gradient dilution, getting extension rate respectively is 10
4, 10
5, 10
6Bacteria suspension coating cetyl trimethylammonium bromide nutrient agar, place incubator, 30 ℃ cultivate 48h (envrionment temperature be lower than this temperature also can, grow up to being convenient to observe form but need to prolong incubation time to bacterium colony), observe colonial morphology, select with form colony number bacterial classification in the majority, especially should select to have the bacterium colony of following characteristics: canescence, flat unformed, spread or slightly spread to periphery, surface wettability, the normal diffusion of periphery of bacterial colonies substratum has water colo(u)r.Transfering loop picking purpose bacterium colony is inoculated in fermention medium, the 250mL Erlenmeyer flask, and liquid amount 80mL, 30 ℃ of shaking tables are cultivated 220r/min.Take out behind the 72h, the centrifugal 15min of 10000r/min, get supernatant liquor and use oil extraction method and surface tension method test surfaces activity respectively, select the oil extraction circle greater than 10cm, and the bacterial classification that surface tension is lower than 30mN/m keeps as the high yield bacterium, supernatant liquor kapillary point sample tlc silica gel plate can be noted waiting for that the moisture of point sample last time volatilizes before each point sample at same position point sample repeatedly for increasing sample concentration.Preparation developping agent (chloroform: methyl alcohol: acetate=65: 15: 2) in chromatography cylinder, silica-gel plate is put into, take out when treating developping agent apart from silica-gel plate top 1cm, volatilize developping agent, spray developer (developer: the 3g phenol and the 5mL vitriol oil are dissolved in the 95mL dehydrated alcohol) hair dryer heating colour developing to silica-gel plate with the glass atomizer.Rhamnolipid produces fermented liquid two pale brown color spot points can occur after launching colour developing, the Rf value is respectively 0.75 and 0.46, and selecting the Rf value is that the dense bacterial classification of 0.46 place's spot colors keeps.Rhamnolipid generation bacterium is 22 strains in the surface-active bacterial classification of 29 strain fermented liquid tools that this method obtains, and therefrom selects two more bacterial strains of rhamnolipid output.The highest bacterial classification of two rhamnolipid output that obtains for this screening of bacterial strain SC-02 wherein, subzero 26 ℃ of glycerine are preserved bacterial classification.
Embodiment 2: the generation bacterial screening method of the two rhamnolipids of the present invention, promptly near the lampblack absorber vapor pipe, gather pedotheque, and take by weighing the adding of 10g pedotheque stirring and evenly mixing is housed in the beaker of 50mL deionized water.Get 10mL access 40mL enrichment medium after soaking 1h, the 150mL Erlenmeyer flask places not to be higher than 40 ℃ of shaking tables cultivations, rotating speed 230r/min.Behind 24-72h, take out pregnant solution, gradient dilution, getting extension rate respectively is 10
3, 10
4, 10
5Bacteria suspension coating cetyl trimethylammonium bromide nutrient agar, place incubator, cultivate 24-96h for 40 ℃, observe colonial morphology, select, especially should select to have the bacterium colony of following characteristics: canescence with form colony number bacterial classification in the majority, flat unformed, spread or slightly spread to periphery, surface wettability, the normal diffusion of periphery of bacterial colonies substratum has water colo(u)r.Transfering loop picking purpose bacterium colony is inoculated in fermention medium, the 150mL Erlenmeyer flask, and liquid amount 40mL, 38 ℃ of shaking tables are cultivated 220r/min.Behind 48-120h, take out, the centrifugal 15min of 10000r/min, get supernatant liquor and use oil extraction method and surface tension method test surfaces activity respectively, select the oil extraction circle greater than 8cm, and the bacterial classification that surface tension is lower than 30mN/m keeps as the high yield bacterium, supernatant liquor kapillary point sample tlc silica gel plate can be noted waiting for that the moisture of point sample last time volatilizes before each point sample at same position point sample repeatedly for increasing sample concentration.Preparation developping agent (chloroform: methyl alcohol: acetate v/v/v=65: 15: 2) in chromatography cylinder, silica-gel plate is put into, take out when treating developping agent apart from silica-gel plate top 1cm, volatilize developping agent, spray developer (developer: the 3g phenol and the 5mL vitriol oil are dissolved in the 95mL dehydrated alcohol) hair dryer heating colour developing to silica-gel plate with the glass atomizer.Rhamnolipid produces fermented liquid two pale brown color spot points can occur after launching colour developing, the Rf value is respectively 0.75 and 0.46, and selecting the Rf value is that the dense bacterial classification of 0.46 place's spot colors keeps.
The active method method of above-mentioned oil extraction is as follows: cut-off directly cleans through washing composition for the culture dish of 16cm, behind twice of the deionized water rinsing, add 60mL deionized water water, add the 0.5mL n-hexadecane on the water surface and form the skim oil film, slowly add at the oil film center 30 μ L through centrifugal fermented liquid supernatant liquid, form a circle around the center oil film is pressed against, the circle diameter approximately is directly proportional with surfactant content.The oil extraction circle is big more, and instruction card surface-active agent output is high more.The surface tension method method is as follows: adopt the surface tension instrument test.Surface tension is more little, illustrates that surfactivity is high more.
Above-mentioned each culture medium preparation method is as follows:
Basis ion substratum (g/L): NaNO
31.0, KH
2PO
42.0, K
2HPO
41.0, MgSO
47H
2O 0.50, and KCl 0.1, CaCl
22H
2O 0.01, FeSO
47H
2O 0.012, yeast extract 0.01, liquid microelement 0.05ml/L.Method for making: it is that 7.2～7.4,121 ℃ of maintenance 20min sterilization postcooling are standby to room temperature that pH is transferred in the dissolving of mentioned component Hybrid Heating, 1mol/LNaOH solution.
Liquid microelement (g/L): H
3BO
30.26, CuSO
45H
2O 0.5, MnSO
4H
2O 0.5, MoNa
2O
42H
2O0.06, ZnSO
47H
2O 0.7.Method for making: successively that above each component is soluble in water in proportion, shake up, standby.
Cetyl trimethylammonium bromide plate culture medium (g/L): extractum carnis 3, peptone 10, NaCl5, cetyl trimethylammonium bromide 0.3, agar 20.Method for making: except that agar, with the dissolving of mentioned component Hybrid Heating, transferring pH is 7.4～7.6, adds agar, after 121 ℃ of 20min sterilizations, makes dull and stereotyped standby.
Enrichment medium: add 25g/L soybean oil (other plant oil also can) in the basic ion substratum.Method for making: basic ion substratum and soybean oil are mixed in proportion after 121 ℃ of 20min sterilizations respectively, and be standby.
Fermention medium: add 40g/L sunflower seed oil, soybean oil or glycerine in the basic ion substratum.Method for making: basic ion substratum and vegetables oil are mixed in proportion after 121 ℃ of 20min sterilizations respectively, and be standby.If select glycerine to make carbon source, method for making is as follows: add glycerine in the basic ion substratum, treat whole dissolvings, and 121 ℃ of 20min sterilizations, standby.
Embodiment 3: in conjunction with Fig. 3, the preparation method of the two rhamnolipids of the present invention is about to the superior strain SC-02 inoculation LB substratum that screening obtains, inoculum size 2%, 32 ℃ of shaking tables are cultivated rotating speed 220r/min, incubation time 18-36h (24h is better), seed liquor inserts glycerine and makes carbon source (also optional sunflower seed oil, soybean oil) fermention medium, liquid amount account for shake bottle long-pending 25%, 25-38 ℃ of (30 ℃ are better) shaking table cultivated, rotating speed 230r/min, incubation time 72-120h.Take out centrifugal treating, 10000r/min, 30min gets supernatant liquor, and it is 2.0 that 6mol/LHCl solution is transferred pH value.Adding doubles the supernatant liquor volume at twice, and chloroform/methanol (v/v=2: 1) mixed solvent, magnetic agitation 20min leaves standstill 6h, and the separating funnel separatory merges organic phase.Remove solvent under reduced pressure under 40 ℃ of the Rotary Evaporators, obtain the rhamnolipid crude extract.Next the rhamnolipid crude extract is carried out separation and purification, adopt silica gel column chromatography method.Carry out recrystallization after using acetone, methyl alcohol or ethyl acetate with the products therefrom dissolving, be further purified.
LB substratum (g/L): tryptone 10, yeast extract 5, NaCl 10.Method for making: it is that 7.2～7.4,121 ℃ of maintenance 20min sterilization postcooling are standby to room temperature that pH is transferred in the dissolving of mentioned component Hybrid Heating, 1mol/LNaOH solution.
A, dress post: (26 * 2.5cm), the ethyl acetate of 1/2 volume of at first packing in post is opened the lower end piston to wet method dress post, slowly adds the column chromatography silica gel that ethyl acetate is soaked then from the top, raps shaft while adding with rubber suction bulb.After the dress post is finished, continue to add the ethyl acetate balance of about 1 column volume.
B, go up sample: the glycolipid crude product is dissolved in few ethyl acetate of trying one's best, when treating that ethyl acetate is apart from the about 1.5cm in silica gel top in the post, slowly and continuously adds sample with pipettor, last sample process is wanted slowly and continuous, and will make sample be tiled in the silica gel top.Close the lower end piston, wait sample slowly to infiltrate silica gel inside, add an absorbent cotton in the upper end, beginning wash-out, 1/s of control flow velocity.
C, wash-out: launch with eluent ethyl acetate to yellow band earlier, use ethyl acetate/methanol (v/v=10: 1) successively, ethyl acetate/methanol (v/v=4: 1), ethyl acetate/methanol (v/v=2: 1), ethyl acetate/methanol (v/v=1: 1) each 100mL wash-out.Every 10mL collects once, changes the ampoule numbering over to and preserves.
D, column chromatography interpretation of result: the surfactivity that adopts the oil extraction method to test each bottle effluent liquid, TLC detects separation case, launch colour developing after, select and only show that a pale brown color spot point and Rf value are 0.46 sample merging, the Rotary Evaporators concentrating under reduced pressure, two rhamnolipids that must be purer.Showing pale brown color spot point and Rf value and be 0.76 sample is single rhamnolipid, and with its merging, evaporated under reduced pressure gets single rhamnolipid.
E, above-mentioned each sample is dissolved in ethyl acetate, adopts other solvents, as methyl alcohol, acetone also can, static, recrystallization, the two rhamnolipids that arrive purelyr.
Adopt the analysis of liquid chromatography/mass spectrometry combined instrument, negative ion detects, SRM scanning, and two rhamnolipids account for 68% of rhamnolipid total mass, Rh
2C
10C
10Account for 90% of two rhamnolipid total masses, account for extract crude extract 23%, also contain a small amount of Rh in two in addition rhamnolipids
2C
8C
10, Rh
2C
10C
8, Rh
2C
10C
12, Rh
2C
12C
10Etc. component.
Claims (6)
1. the generation bacterial screening method of two rhamnolipids may further comprise the steps:
A, choose by the collection in worksite pedotheque of grease contamination as the bacterial screening source;
The inoculation vegetables oil is made the enrichment medium of carbon source after b, the sample immersion treatment, gets enrichment culture liquid behind cultivation 24～72h;
C, enrichment culture liquid gradient dilution is coated the cetyl trimethylammonium bromide plate culture medium cultivate 24～96h, kind is protected on single bacterium colony inclined-plane that the picking growing way is good and quantity is many;
The bacterial classification that d, picking inclined-plane are preserved inserts measures surfactivity after fermention medium is cultivated 48～120h, selecting active higher strain fermentating liquid supernatant to carry out TLC analyzes, developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent, the two rhamnolipid content of preliminary judgement, selection Rf value is 0.46 the dense i.e. generation bacterium reservation of pair rhamnolipids of bacterial classification of spot colors, and subzero 26 ℃ of glycerine are preserved.
2. the preparation method of two rhamnolipids may further comprise the steps:
A, choose by the collection in worksite pedotheque of grease contamination as the bacterial screening source;
The inoculation vegetables oil is made the enrichment medium of carbon source after b, the sample immersion treatment, gets enrichment culture liquid behind cultivation 24～72h;
C, enrichment culture liquid gradient dilution is coated the cetyl trimethylammonium bromide plate culture medium cultivate 24～96h, kind is protected on single bacterium colony inclined-plane that the picking growing way is good and quantity is many;
The bacterial classification that d, picking inclined-plane are preserved inserts measures surfactivity after fermention medium is cultivated 48～120h, selecting active higher strain fermentating liquid supernatant to carry out TLC analyzes, developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent, the two rhamnolipid content of preliminary judgement, selection Rf value is 0.46 the dense i.e. generation bacterium reservation of pair rhamnolipids of bacterial classification of spot colors, and subzero 26 ℃ of glycerine are preserved;
E, will screen gained bacterial classification inoculation LB substratum, cultivate 18～36h after switching go into fermention medium, cultivate 72～120h for 25-38 ℃;
After f, the fermentation ends with fermented liquid bactofugation body;
G, adjustment supernatant liquor pH=1.0～5.0 add the chloroform methanol mixture and make extraction agent, stir, leave standstill separatory, keep the machine phase;
H, remove solvent under reduced pressure, obtain the rhamnolipid crude extract with Rotary Evaporators;
I, crude extract is carried out chromatographic separation in silicagel column, with the eluent wash-out, part is collected;
J, TLC detect separation case, and developping agent is a chloroform: methyl alcohol: acetate v/v/v=65: 15: 2, phenol sulfuric acid chromogenic reagent was that 0.46 elutriant merges with the Rf value, and evaporated under reduced pressure gets required pair of rhamnolipid.
3. the screening of generation bacterium and the preparation method of pair rhamnolipid according to claim 1 and 2, it is characterized in that: the enrichment culture process environment temperature of step b is 25-40 ℃.
4. the screening of generation bacterium and the preparation method of pair rhamnolipid according to claim 1 and 2, it is characterized in that: the culture temperature of step c is 25-40 ℃.
5. the preparation method of pair rhamnolipid according to claim 2 is characterized in that: the eluent of step I is methyl alcohol, chloroform, methylene dichloride, ethyl acetate or their mixture.
6. the screening of generation bacterium and the preparation method of pair rhamnolipid according to claim 2 is characterized in that: carry out recrystallization after using acetone, methyl alcohol or ethyl acetate with the products therefrom dissolving, be further purified.
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| CN102796781A (en) * | 2012-08-08 | 2012-11-28 | 中国海洋石油总公司 | Method for producing rhamnolipid by virtue of fermentation and separation of pseudomonas aeruginosa |
| JP2014111595A (en) * | 2012-11-26 | 2014-06-19 | Evonik Industries Ag | Process for isolation of rhamnolipids |
| US9884883B2 (en) | 2015-01-12 | 2018-02-06 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| CN110882263A (en) * | 2019-12-20 | 2020-03-17 | 青岛市肿瘤医院 | Application of Monostroma nitidum oligosaccharose and Monostroma nitidum oligosaccharose derivative in resisting breast cancer |
| US10829507B2 (en) | 2017-02-06 | 2020-11-10 | Stepan Company | Decolorization of concentrated rhamnolipid composition |
| CN112553267A (en) * | 2020-12-11 | 2021-03-26 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of synbiotics for regulating and controlling glycolipid metabolic activity |
| CN112626148A (en) * | 2020-12-11 | 2021-04-09 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of synbiotics |
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2007
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| CN102796781A (en) * | 2012-08-08 | 2012-11-28 | 中国海洋石油总公司 | Method for producing rhamnolipid by virtue of fermentation and separation of pseudomonas aeruginosa |
| JP2014111595A (en) * | 2012-11-26 | 2014-06-19 | Evonik Industries Ag | Process for isolation of rhamnolipids |
| US9884883B2 (en) | 2015-01-12 | 2018-02-06 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| US10829507B2 (en) | 2017-02-06 | 2020-11-10 | Stepan Company | Decolorization of concentrated rhamnolipid composition |
| CN110882263A (en) * | 2019-12-20 | 2020-03-17 | 青岛市肿瘤医院 | Application of Monostroma nitidum oligosaccharose and Monostroma nitidum oligosaccharose derivative in resisting breast cancer |
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| CN112553267A (en) * | 2020-12-11 | 2021-03-26 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of synbiotics for regulating and controlling glycolipid metabolic activity |
| CN112626148A (en) * | 2020-12-11 | 2021-04-09 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of synbiotics |
| CN112553267B (en) * | 2020-12-11 | 2022-06-14 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of synbiotics for regulating and controlling glycolipid metabolic activity |
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