CN109180746A - A method of isolating and purifying rhamnolipid - Google Patents
A method of isolating and purifying rhamnolipid Download PDFInfo
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- CN109180746A CN109180746A CN201811071939.1A CN201811071939A CN109180746A CN 109180746 A CN109180746 A CN 109180746A CN 201811071939 A CN201811071939 A CN 201811071939A CN 109180746 A CN109180746 A CN 109180746A
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- Prior art keywords
- rhamnolipid
- liquid
- isolating
- supernatant
- purifying
- Prior art date
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- FCBUKWWQSZQDDI-UHFFFAOYSA-N rhamnolipid Chemical compound CCCCCCCC(CC(O)=O)OC(=O)CC(CCCCCCC)OC1OC(C)C(O)C(O)C1OC1C(O)C(O)C(O)C(C)O1 FCBUKWWQSZQDDI-UHFFFAOYSA-N 0.000 title claims abstract description 159
- 238000000034 method Methods 0.000 title claims abstract description 54
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 78
- 239000007788 liquid Substances 0.000 claims abstract description 63
- 239000006228 supernatant Substances 0.000 claims abstract description 45
- 239000003208 petroleum Substances 0.000 claims abstract description 39
- 230000001376 precipitating effect Effects 0.000 claims abstract description 28
- 239000002253 acid Substances 0.000 claims abstract description 14
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 5
- 239000011574 phosphorus Substances 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims description 48
- 230000004151 fermentation Effects 0.000 claims description 44
- 238000003756 stirring Methods 0.000 claims description 30
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 14
- 238000000926 separation method Methods 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 12
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 10
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 10
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 6
- 238000002835 absorbance Methods 0.000 claims description 6
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 6
- 238000003760 magnetic stirring Methods 0.000 claims description 6
- 230000006920 protein precipitation Effects 0.000 claims description 6
- 229910021653 sulphate ion Inorganic materials 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 abstract description 20
- 238000011084 recovery Methods 0.000 abstract description 15
- 238000012869 ethanol precipitation Methods 0.000 abstract description 6
- 238000010923 batch production Methods 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract 1
- 231100000252 nontoxic Toxicity 0.000 abstract 1
- 230000003000 nontoxic effect Effects 0.000 abstract 1
- 238000001035 drying Methods 0.000 description 8
- 238000012545 processing Methods 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 239000001117 sulphuric acid Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000003946 protein process Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 3
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000003876 biosurfactant Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
Abstract
The present invention relates to a kind of methods for isolating and purifying rhamnolipid, thallus is removed using the method for centrifugation, collect supernatant, then by the supernatant of collection, 10% ethanol precipitation albumen of fermentating liquid volume is added, using the albumen of the method removal precipitating of centrifugation, collects supernatant, phosphorus acid for adjusting pH, which is added, is precipitated rhamnolipid, generates precipitating；Later by rhamnolipid precipitating is collected by centrifugation.It is finally beaten into petroleum ether and spreads rhamnolipid precipitating, rhamnolipid is allowed uniformly to be scattered in petroleum ether, dissolve the impurity in rhamnolipid, the rhamnolipid that precipitating is purified is collected by centrifugation.Use the final rhamnolipid rate of recovery of method of the invention for 91.82%, rhamnolipid purity is 85%.Compared with prior art, the characteristics of method of the invention has easy to operate, and the rate of recovery is high, non-toxic and safe, is a kind of simple process, is suitble to the simplicity of industrialized batch production, economical, efficient method.
Description
Technical field
The present invention relates to the isolation and purification methods of a kind of isolation and purification method, especially rhamnolipid.
Background technique
Rhamnolipid is to study a kind of more biosurfactant at present, has emulsification, solubilising and reduces interface
The functions such as power, and low toxicity, easily biological-degradable, have well in fields such as petrochemical industry, biological medicine, food hygiene and environmental protections
Application prospect.However the separation costs of rhamnolipid restrict its use cost, because the downstream separation cost of rhamnolipid is logical
Often account for the 60% of its production cost.At present about the separation many places of rhamnolipid in laboratory stage, few formation are industrialized
Scale.
At present to the research of rhamnolipid focus mostly in improve yield on and its application effect on, in terms of extraction process
It studies less.Existing technique mainly includes extraction, and active carbon adsorption, these methods are all different degrees of, and there is some
Defect.Zhao etc. is extracted using chloroform methanol system, and chloroform and methanol are that have very supervirulent chemicals, with putting for production
Greatly, security risk also gradually increases, and is not used to cosmetics and food using the processed rhamnolipid of the reagents such as chloroform
Industry, product quality cannot ensure.Ren Fangxu etc. does not select rhamnolipid using activated carbon adsorption rhamnolipid active carbon
Selecting property, so that separation process low efficiency, and active carbon is on the high side, and it is excessively high to will cause separation costs.
Summary of the invention
This method purpose, which is that, to be overcome the problems of the above-mentioned prior art and provides a kind of easy, economical, efficient
Rhamnolipid isolation and purification method.
The purpose of the present invention can be achieved through the following technical solutions:
A method of isolating and purifying rhamnolipid, which comprises the steps of:
(1) rhamnolipid fermentation liquor is placed in centrifugal enrichment thallus in centrifuge tube, collects sterile fermented liquid supernatant liquid；
(2) dehydrated alcohol is added in the fermented liquid supernatant liquid that step (1) is collected；
(3) magnetic stir bar is added in the fermented liquid supernatant liquid that dehydrated alcohol is added in step (2), be placed on magnetic stirring apparatus
Stirring；
(4) solution after stir process in (3) is placed in centrifugal enrichment protein precipitation in centrifuge tube, collects the supernatant for removing removing protein
Liquid removes the rhamnolipid fermentation liquor of removing protein；
(5) phosphorus acid for adjusting pH is added in the rhamnolipid fermentation liquor for removing removing protein collected in (4) is precipitated rhamnolipid, produces
Raw precipitating, stands a period of time；
(6) fermentation liquid for generating precipitating in (5) is placed in centrifugal enrichment rhamnolipid in centrifuge tube to precipitate, gives up supernatant, it will
The precipitating of generation is dried, and rhamnolipid precipitated products are obtained；
(7) rhamnolipid precipitated products obtained in (6) are uniformly beaten and is spread in petroleum ether, dissolved miscellaneous in rhamnolipid
Matter；
(8) petroleum ether in (7) and rhamnolipid precipitated products mixture are placed in centrifuge tube and are centrifuged, make to be separated by solid-liquid separation, collected
Rhamnolipid solid, the rhamnolipid product purified.
In the step (1), rhamnolipid fermentation liquor is centrifuged method of the invention under the conditions of 10000 rpm, 4 DEG C
20min。
The centrifuge tube is 50 mL conical centrifuge tubes.
In the step (2), the additive amount of dehydrated alcohol is the 10% of rhamnolipid fermentation liquor volume.
In the step (3), the condition of stirring is that 250rpm stirs 9h.
In the step (4), step (6) and step (8), by rhamnolipid fermentation liquor under the conditions of 6000 rpm, 4 DEG C from
10 min of the heart.
Phosphoric acid used in the step (5) is to analyze pure rank phosphoric acid；Add phosphorus acid for adjusting pH to 2, is stood at 4 DEG C
18h。
In the step (7), the mass volume ratio of rhamnolipid precipitated products and petroleum ether is 1/2 ~ 1/8(w/v).
The present invention detects rhamnolipid concentration using anthrone-sulphuric acid method, and step (1) fermented liquid supernatant liquid, step (4) is taken to go
The petroleum ether after supernatant and step (8) separation of solid and liquid after the sour precipitating of the rhamnolipid fermentation liquor of removing protein, step (6)
Quantitative anthrone sulphate reagent is added in liquid, and boiling water bath reacts 10min, and control ultraviolet wavelength is 620nm, is tested and is sent out using microplate reader
Zymotic fluid absorbance substitutes into and calculates rhamnolipid concentration in the standard curve made of standard rhamnose, calculates rhamnolipid loss
Rate.
Compared with prior art, present invention combination ethanol precipitation albumen, phosphoric acid precipitates rhamnolipid, petroleum ether purify mouse
Lee's glycolipid, can obtain the higher rhamnolipid of purity very well, and repeatability is good.The method overcome danger existing in the prior art
It is dangerous big, the defects of rate of recovery is low, and product quality is poor, it is a kind of simple process, is suitble to simplicity, the warp of industrialization batch production
Ji, efficient rhamnolipid isolation and purification method.
Detailed description of the invention
Fig. 1 is the analysis result figure of embodiment 1.
Specific embodiment
The present invention is described in detail with specific example with reference to the accompanying drawing.
Embodiment 1
A method of rhamnolipid being isolated and purified, this method mainly comprises the steps of:
(1) 520mL rhamnolipid fermentation liquor is uniformly sub-packed in 12 50mL conical centrifuge tubes, is centrifuged using low-temperature and high-speed
Machine 10000rpm, 4 DEG C of centrifugation 20min, careful collection supernatant.
(2) supernatant collected in step (1) is poured into graduated cylinder, it is 500mL that correct amount volume, which measures residual volume, is added
Enter the total 50mL of 10% dehydrated alcohol.
(3) fermentation liquid that dehydrated alcohol is added in (2) is poured into 1000mL beaker, magnetic stir bar is added, addition
Magnetic stir bar is tetrafluoro magnetic stir bar A type, and beaker is put on magnetic stirring apparatus and stirs 9h, and setting revolving speed is 150rpm.
(4) fermentation liquid that processing terminate in (3) is uniformly sub-packed in the conical centrifuge tube of 12 50mL, uses low temperature height
Fast centrifuge 6000rpm, 4 DEG C of centrifugation 10min are enriched with protein precipitation, and careful collection removes the supernatant of removing protein, and pours into graduated cylinder
Middle amount volume.Measuring residual volume is 525mL, obtains the rhamnolipid fermentation liquor of removing protein.
(5) rhamnolipid fermentation liquor being collected into (4) is slowly added to phosphoric acid, it is stirring while adding, pH to 2 is adjusted, 4
DEG C place 18h.
(6) fermentation liquid that processing is completed in (5) is used into low-temperature and high-speed centrifuge 6000rpm, 4 DEG C of 10 min of centrifugation, it is quasi-
Really measuring supernatant volume is 505 mL, gives up supernatant, the precipitating of generation is put into 105 DEG C of baking ovens, and drying weighing there are
The thick rhamnolipid of 26.15g, measuring purity is 65.45 %.
(7) the thick rhamnolipid in (6) is added in 200mL petroleum ether, breaks up rhamnolipid using rapid mixer
Solid, until rhamnolipid is dispersed in petroleum ether.
(8) by (7) rhamnolipid and petroleum ether use low-temperature and high-speed centrifuge 6000rpm, 4 DEG C of 10 min of centrifugation,
It is separated by solid-liquid separation, collects rhamnolipid precipitating, be put into 105 DEG C of baking ovens, drying weighing there are 18.5g rhamnolipid, survey
Obtaining purity is 85%.Petroleum ether liquid is collected, measurement volume is 200mL.
Changed using anthrone-sulphuric acid method detection rhamnolipid concentration.Take the fermentation of quantitative deproteinized front and back, acid precipitating front and back
Quantitative anthrone sulphate reagent is added in petroleum ether liquid after liquid and purification, and boiling water bath reacts 10min, and control ultraviolet wavelength is
620nm calculates rhamnolipid in the standard curve that substitution makes of standard rhamnose using microplate reader test broth absorbance
Concentration judges rhamnolipid loss late.The rhamnolipid concentration for measuring supernatant in step (1) is 33.76g/L, measures step
(4) deproteinized post-fermentation liquid rhamnolipid concentration is 31.82g/L in, and the rhamnolipid rate of recovery of ethanol precipitation protein Process is
98.99%, the rhamnolipid concentration for measuring supernatant in step (6) is 1.04g/L, and the rhamnolipid of acid precipitating rhamnolipid returns
Yield is 96.89%, and measuring in step (8) rhamnolipid concentration in petroleum ether liquid is 4.25g/L, petroleum ether method
The rhamnolipid rate of recovery is 91.82%, and to sum up, this method can obtain the very high rhamnose of purity under the premise of higher recovery
Rouge.
Embodiment 2
A method of rhamnolipid being isolated and purified, this method mainly comprises the steps of:
(1) 520mL rhamnolipid fermentation liquor is uniformly sub-packed in 12 50mL conical centrifuge tubes, is centrifuged using low-temperature and high-speed
Machine 10000rpm, 4 DEG C of centrifugation 20min, careful collection supernatant.
(2) supernatant collected in step (1) is poured into graduated cylinder, it is 500mL that correct amount volume, which measures residual volume, is added
Enter the total 50mL of 10% dehydrated alcohol.
(3) fermentation liquid that dehydrated alcohol is added in (2) is poured into 1000mL beaker, magnetic stir bar is added, addition
Magnetic stir bar is tetrafluoro magnetic stir bar A type, and beaker is put on magnetic stirring apparatus and stirs 9h, and setting revolving speed is 150rpm.
(4) fermentation liquid that processing terminate in (3) is uniformly sub-packed in the conical centrifuge tube of 12 50mL, uses low temperature height
Fast centrifuge 6000rpm, 4 DEG C of centrifugation 10min are enriched with protein precipitation, and careful collection removes the supernatant of removing protein, and pours into graduated cylinder
Middle amount volume.Measuring residual volume is 525mL, obtains the rhamnolipid fermentation liquor of removing protein.
(5) rhamnolipid fermentation liquor being collected into (4) is slowly added to phosphoric acid, it is stirring while adding, pH to 2 is adjusted, 4
DEG C place 18h.
(6) fermentation liquid that processing is completed in (5) is used into low-temperature and high-speed centrifuge 6000rpm, 4 DEG C of 10 min of centrifugation, it is quasi-
Really measuring supernatant volume is 505 mL, gives up supernatant, the precipitating of generation is put into 105 DEG C of baking ovens, and drying weighing there are
The thick rhamnolipid of 25.87g, measuring purity is 65.24 %.
(7) the thick rhamnolipid in (6) is added in 150mL petroleum ether, breaks up rhamnolipid using rapid mixer
Solid, until rhamnolipid is dispersed in petroleum ether.
(8) by (7) rhamnolipid and petroleum ether use low-temperature and high-speed centrifuge 6000rpm, 4 DEG C of 10 min of centrifugation,
It being separated by solid-liquid separation, collects rhamnolipid precipitating, be put into 105 DEG C of baking ovens, drying weighing there are 18.35g rhamnolipid,
Measuring purity is 84.29%.Petroleum ether liquid is collected, measurement volume is 150mL.
Changed using anthrone-sulphuric acid method detection rhamnolipid concentration.Take the fermentation of quantitative deproteinized front and back, acid precipitating front and back
Quantitative anthrone sulphate reagent is added in petroleum ether liquid after liquid and purification, and boiling water bath reacts 10min, and control ultraviolet wavelength is
620nm calculates rhamnolipid in the standard curve that substitution makes of standard rhamnose using microplate reader test broth absorbance
Concentration judges rhamnolipid loss late.The rhamnolipid concentration for measuring supernatant in step (1) is 33.76g/L, measures step
(4) deproteinized post-fermentation liquid rhamnolipid concentration is 31.82g/L in, and the rhamnolipid rate of recovery of ethanol precipitation protein Process is
98.99%, the rhamnolipid concentration for measuring supernatant in step (6) is 1.21g/L, and the rhamnolipid of acid precipitating rhamnolipid returns
Yield is 96.43%, and measuring in step (8) rhamnolipid concentration in petroleum ether liquid is 5.6g/L, the mouse of petroleum ether method
Lee's glycolipid rate of recovery is 91.40%, and to sum up, this method can obtain the very high rhamnolipid of purity under the premise of higher recovery.
Embodiment 3
A method of rhamnolipid being isolated and purified, this method mainly comprises the steps of:
(1) 520mL rhamnolipid fermentation liquor is uniformly sub-packed in 12 50mL conical centrifuge tubes, is centrifuged using low-temperature and high-speed
Machine 10000rpm, 4 DEG C of centrifugation 20min, careful collection supernatant.
(2) supernatant collected in step (1) is poured into graduated cylinder, it is 500mL that correct amount volume, which measures residual volume, is added
Enter the total 50mL of 10% dehydrated alcohol.
(3) fermentation liquid that dehydrated alcohol is added in (2) is poured into 1000mL beaker, magnetic stir bar is added, addition
Magnetic stir bar is tetrafluoro magnetic stir bar A type, and beaker is put on magnetic stirring apparatus and stirs 9h, and setting revolving speed is 150rpm.
(4) fermentation liquid that processing terminate in (3) is uniformly sub-packed in the conical centrifuge tube of 12 50mL, uses low temperature height
Fast centrifuge 6000rpm, 4 DEG C of centrifugation 10min are enriched with protein precipitation, and careful collection removes the supernatant of removing protein, and pours into graduated cylinder
Middle amount volume.Measuring residual volume is 525mL, obtains the rhamnolipid fermentation liquor of removing protein.
(5) rhamnolipid fermentation liquor being collected into (4) is slowly added to phosphoric acid, it is stirring while adding, pH to 2 is adjusted, 4
DEG C place 18h.
(6) fermentation liquid that processing is completed in (5) is used into low-temperature and high-speed centrifuge 6000rpm, 4 DEG C of 10 min of centrifugation, it is quasi-
Really measuring supernatant volume is 505 mL, gives up supernatant, the precipitating of generation is put into 105 DEG C of baking ovens, and drying weighing there are
The thick rhamnolipid of 25.46g, measuring purity is 64.86 %.
(7) the thick rhamnolipid in (6) is added in 100mL petroleum ether, breaks up rhamnolipid using rapid mixer
Solid, until rhamnolipid is dispersed in petroleum ether.
(8) by (7) rhamnolipid and petroleum ether use 6000 rpm of low-temperature and high-speed centrifuge, 4 DEG C of 10 min of centrifugation,
It being separated by solid-liquid separation, collects rhamnolipid precipitating, be put into 105 DEG C of baking ovens, drying weighing there are 18.25g rhamnolipid,
Measuring purity is 85.4%.Petroleum ether liquid is collected, measurement volume is 100mL.
Changed using anthrone-sulphuric acid method detection rhamnolipid concentration.Take the fermentation of quantitative deproteinized front and back, acid precipitating front and back
Quantitative anthrone sulphate reagent is added in petroleum ether liquid after liquid and purification, and boiling water bath reacts 10min, and control ultraviolet wavelength is
620nm calculates rhamnolipid in the standard curve that substitution makes of standard rhamnose using microplate reader test broth absorbance
Concentration judges rhamnolipid loss late.The rhamnolipid concentration for measuring supernatant in step (1) is 33.76g/L, measures step
(4) deproteinized post-fermentation liquid rhamnolipid concentration is 31.82g/L in, and the rhamnolipid rate of recovery of ethanol precipitation protein Process is
98.99%, the rhamnolipid concentration for measuring supernatant in step (6) is 1.39g/L, and the rhamnolipid of acid precipitating rhamnolipid returns
Yield is 95.88%, and measuring in step (8) rhamnolipid concentration in petroleum ether liquid is 8.55g/L, petroleum ether method
The rhamnolipid rate of recovery is 90.82%, and to sum up, this method can obtain the very high rhamnose of purity under the premise of higher recovery
Rouge.
Embodiment 4
A method of rhamnolipid being isolated and purified, this method mainly comprises the steps of:
(1) 520mL rhamnolipid fermentation liquor is uniformly sub-packed in 12 50mL conical centrifuge tubes, is centrifuged using low-temperature and high-speed
Machine 10000rpm, 4 DEG C of centrifugation 20min, careful collection supernatant.
(2) supernatant collected in step (1) is poured into graduated cylinder, it is 500mL that correct amount volume, which measures residual volume, is added
Enter the total 50mL of 10% dehydrated alcohol.
(3) fermentation liquid that dehydrated alcohol is added in (2) is poured into 1000mL beaker, magnetic stir bar is added, addition
Magnetic stir bar is tetrafluoro magnetic stir bar A type, and beaker is put on magnetic stirring apparatus and stirs 9h, and setting revolving speed is 150rpm.
(4) fermentation liquid that processing terminate in (3) is uniformly sub-packed in the conical centrifuge tube of 12 50mL, uses low temperature height
Fast centrifuge 6000rpm, 4 DEG C of centrifugation 10min are enriched with protein precipitation, and careful collection removes the supernatant of removing protein, and pours into graduated cylinder
Middle amount volume.Measuring residual volume is 525mL, obtains the rhamnolipid fermentation liquor of removing protein.
(5) rhamnolipid fermentation liquor being collected into (4) is slowly added to phosphoric acid, it is stirring while adding, pH to 2 is adjusted, 4
DEG C place 18h.
(6) fermentation liquid that processing is completed in (5) is used into low-temperature and high-speed centrifuge 6000rpm, 4 DEG C of 10 min of centrifugation, it is quasi-
Really measuring supernatant volume is 505 mL, gives up supernatant, the precipitating of generation is put into 105 DEG C of baking ovens, and drying weighing there are
The thick rhamnolipid of 25.44g, measuring purity is 64.65 %.
(7) the thick rhamnolipid in (6) is added in 50mL petroleum ether, it is solid to break up rhamnolipid using rapid mixer
Body, until rhamnolipid is dispersed in petroleum ether.
(8) by (7) rhamnolipid and petroleum ether use 6000 rpm of low-temperature and high-speed centrifuge, 4 DEG C of 10 min of centrifugation,
It is separated by solid-liquid separation, collects rhamnolipid precipitating, be put into 105 DEG C of baking ovens, drying weighing there are 18.5g rhamnolipid, survey
Obtaining purity is 85.3%.Petroleum ether liquid is collected, measurement volume is 50mL.
Changed using anthrone-sulphuric acid method detection rhamnolipid concentration.Take the fermentation of quantitative deproteinized front and back, acid precipitating front and back
Quantitative anthrone sulphate reagent is added in petroleum ether liquid after liquid and purification, and boiling water bath reacts 10min, and control ultraviolet wavelength is
620nm calculates rhamnolipid in the standard curve that substitution makes of standard rhamnose using microplate reader test broth absorbance
Concentration judges rhamnolipid loss late.The rhamnolipid concentration for measuring supernatant in step (1) is 33.76g/L, measures step
(4) deproteinized post-fermentation liquid rhamnolipid concentration is 31.82g/L in, and the rhamnolipid rate of recovery of ethanol precipitation protein Process is
98.99%, the rhamnolipid concentration for measuring supernatant in step (6) is 1.59g/L, and the rhamnolipid of acid precipitating rhamnolipid returns
Yield is 95.25%, and measuring in step (8) rhamnolipid concentration in petroleum ether liquid is 17.3g/L, petroleum ether method
The rhamnolipid rate of recovery is 90.23%, and to sum up, this method can obtain the very high rhamnose of purity under the premise of higher recovery
Rouge.
Claims (10)
1. a kind of method for isolating and purifying rhamnolipid, which comprises the steps of:
(1) rhamnolipid fermentation liquor is placed in centrifugal enrichment thallus in centrifuge tube, collects sterile fermented liquid supernatant liquid；
(2) dehydrated alcohol is added in the fermented liquid supernatant liquid that step (1) is collected；
(3) magnetic stir bar is added in the fermented liquid supernatant liquid that dehydrated alcohol is added in step (2), be placed on magnetic stirring apparatus
Stirring；
(4) solution after stir process in (3) is placed in centrifugal enrichment protein precipitation in centrifuge tube, collects the supernatant for removing removing protein
Liquid removes the rhamnolipid fermentation liquor of removing protein；
(5) phosphorus acid for adjusting pH is added in the rhamnolipid fermentation liquor for removing removing protein collected in (4) is precipitated rhamnolipid, produces
Raw precipitating, stands a period of time；
(6) fermentation liquid for generating precipitating in (5) is placed in centrifugal enrichment rhamnolipid in centrifuge tube to precipitate, gives up supernatant, it will
The precipitating of generation is dried, and rhamnolipid precipitated products are obtained；
(7) rhamnolipid precipitated products obtained in (6) are uniformly beaten and is spread in petroleum ether, dissolved miscellaneous in rhamnolipid
Matter；
(8) petroleum ether in (7) and rhamnolipid precipitated products mixture are placed in centrifuge tube and are centrifuged, make to be separated by solid-liquid separation, collected
Rhamnolipid solid, the rhamnolipid product purified.
2. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that the step (1)
In, rhamnolipid fermentation liquor is centrifuged 20min under the conditions of 10000 rpm, 4 DEG C.
3. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that the centrifuge tube is point
Bottom centrifuge tube.
4. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that in the step (2),
The additive amount of dehydrated alcohol is the 10% of rhamnolipid fermentation liquor volume.
5. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that in the step (3),
The condition of stirring is that 250rpm stirs 9h.
6. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that the step (4),
In step (6) and step (8), rhamnolipid fermentation liquor is centrifuged 10 min under the conditions of 6000 rpm, 4 DEG C.
7. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that in the step (5)
Phosphoric acid used is to analyze pure rank phosphoric acid；Add phosphorus acid for adjusting pH to 2.
8. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that in the step (5),
18h is stood at 4 DEG C.
9. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that in the step (7),
The mass volume ratio of rhamnolipid precipitated products and petroleum ether is 1/2 ~ 1/8(w/v).
10. a kind of method for isolating and purifying rhamnolipid according to claim 1, which is characterized in that further include: take step
(1) supernatant and step after fermented liquid supernatant liquid, step (4) go the rhamnolipid fermentation liquor of removing protein, step (6) acid to precipitate
(8) quantitative anthrone sulphate reagent is added in the petroleum ether liquid after being separated by solid-liquid separation, and boiling water bath reacts 10min, controls ultraviolet wavelength
Rhamnose is calculated in the standard curve that substitution makes of standard rhamnose using microplate reader test broth absorbance for 620nm
Lipid concentration calculates rhamnolipid loss late.
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Cited By (2)
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| CN112225763A (en) * | 2020-11-16 | 2021-01-15 | 西安润嬴生物科技有限公司 | Separation and purification method of rhamnolipid |
| CN114249779A (en) * | 2021-10-27 | 2022-03-29 | 南京理工大学 | Method for separating rhamnolipid |
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