CN103059107A - Method for purifying sodium surfactin - Google Patents
Method for purifying sodium surfactin Download PDFInfo
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- CN103059107A CN103059107A CN2013100160684A CN201310016068A CN103059107A CN 103059107 A CN103059107 A CN 103059107A CN 2013100160684 A CN2013100160684 A CN 2013100160684A CN 201310016068 A CN201310016068 A CN 201310016068A CN 103059107 A CN103059107 A CN 103059107A
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Abstract
The invention relates to the field of microbial fermentation and discloses a method for purifying sodium surfactin. The method comprises the following steps of: taking a sodium surfactin fermentation liquid to centrifugally collect supernatant and collecting a trapped liquid after membrane filtration; adjusting the pH value of the trapped liquid to 5.0-6.6 and centrifugally collecting supernatant; and adding an acid solution to the solution obtained in the step 2 until the pH value is 2.0-5.0, collecting the precipitate, slowly adding an alkaline solution to carry out neutralization reaction, thus obtaining the finished product of sodium surfactin after drying the reaction liquid, wherein the purity of the finished product of sodium surfactin reaches 85-88%. The method has beneficial effects that in order to further improve the purity of the finished product of sodium surfactin, the finished product of sodium surfactin is dissolved by utilizing a dissolving agent and the upper organic phase concentrated crystal is collect, thus obtaining the purified product of sodium surfactin is obtained; the purity of the purified product of sodium surfactin reaches 90-93%; further, the supernatant obtained through centrifugation in the step 2 is chromatographically purified by utilizing ion exchange resins and the purity reaches 93-98%; and the sodium surfactin can be widely applied in the field of food as a food additive.
Description
Technical field
The present invention relates to the microbial fermentation field, relate to specifically a kind of purification process of sodium bacillus subtilis lipopeptide.
Background technology
Tensio-active agent (Surfactants) refers to significantly to reduce solvent surface tension and liquid-liquid interface tension force, and has the material of a fixed structure, lypohydrophilic character and special absorption property, comprises chemical surfactant and bio-surfactant.Chemical surfactant mostly is to come take oil as the raw material chemosynthesis, usually can bring serious problem of environmental pollution in production and use procedure.Bio-surfactant (Biosurfactants) is to have surface-active biomacromolecule material by the class that Institute of Micro-biology produces, except having the reduction surface tension, outside the same functions such as stable emulsion and increase foam, also has the not available characteristic of general synthetic surfactant, as has a structure diversity, lower toxicity, higher biodegradability, good biocompatibility, high whipability, to extreme temperature, pH value and salt concn have higher selectivity and specificity, the characteristics such as can be obtained by cheap renewable raw materials production, therefore in environmental protection, oil production, medicine, the fields such as food-processing have larger applied research and are worth.
Sodium bacillus subtilis lipopeptide is the sodium salt of surfaction, and surfaction is translated into Surfactin, is a kind of by the synthetic cyclic peptide of subtilis, uses mainly with sodium-salt form.Since nineteen sixty-eight was found first by people such as Arima, Surfactin was one of bio-surfactant that surfactivity is the strongest, research is more always, is widely used in foodstuffs industry, environment-industry as good biocompatible surfaces promoting agent.Except having stronger surfactivity, Surfactin can also increase the biodegradable of hydrophobic hydro carbons, in the biological restoration that is subjected to hydrocarbon contamination soil and ocean, play a significant role, it can also be combined with the part heavy metal and remove heavy metal in polluted soil and the settling, and has certain anti-microbial activity etc.Therefore have larger applied research is worth in fields such as environmental protection, oil production, high-end makeup, medicine, food-processings.
The Surfactin production cost is higher, 3 ~ 10 times of chemical-biological tensio-active agent, wherein the processing costs in the extraction of product and downstream accounts for the overwhelming majority of productive expense, especially the singularity owing to product and thalline causes fermentation broth viscosity large when pre-treatment sodium bacillus subtilis lipopeptide fermented liquid, and bacterium liquid separation costs is high.Therefore, product separation and the method for purification of exploitation cheap and simple are very necessary.
2000, Randhir etc. proposed to separate Surfactin by foam recovery.Huei-Li Chen in 2007 etc. utilize Acid precipitation and PVDF resin absorption that Surfactin is carried out purification operations.Tribute state letter waited the centrifugal removal thalline of human supercentrifuge in 2007, and the supernatant liquor acidifying is carried out solvent extraction after getting the Surfactin crude extract.Huei-Li Chen in 2008 etc. utilize again ammonium sulfate that Surfactin is carried out mixed salt out, collect Surfactin by ultrafiltration and nanofiltration again.Mohd Hafez Mohd Isa in 2008 etc. utilize ultrafiltration membrance filter to collect Surfactin.But above technique is the laboratory means, and then cost is higher if carry out suitability for industrialized production.Therefore the surfaction sodium purifying method of developing a kind of cheap and simple that is suitable for suitability for industrialized production is significant.
Summary of the invention
In view of this, the present invention seeks to be the laboratory means for prior art, the defective that production cost is high provides a kind of cheap and simple, is suitable for the purification process of the sodium bacillus subtilis lipopeptide of suitability for industrialized production.
For realizing purpose of the present invention, the present invention adopts following technical scheme:
A kind of purification process of sodium bacillus subtilis lipopeptide comprises:
Step 1: get the sodium bacillus subtilis lipopeptide fermented liquid, centrifugal collection supernatant liquor is collected trapped fluid behind membrane filtration;
Step 2: trapped fluid pH is transferred to 5.0-6.6, centrifugal collection supernatant liquor;
Step 3: add acid solution to pH value 2.0-5.0 to step 2 gained solution, collecting precipitation slowly adds alkaline solution and carries out neutralization reaction after the washing, obtain Bacitracin sodium finished product after the reaction solution drying.
Further, purification process step 1 of the present invention also is included in centrifugally frontly carries out pretreated step to the sodium bacillus subtilis lipopeptide fermented liquid, and described pre-treatment is that with sodium bacillus subtilis lipopeptide fermented liquid dilution 1-10 doubly adjust pH transfers to 6.0-8.5.
As preferably, the described centrifugal separating factor of step 1 is 5000-17000g.
As preferably, the film of the described membrane filtration of step 1 is ceramic membrane.
As preferably, the described centrifugal separating factor of step 2 is 5000-17000g.
As preferably, the described acid solution of step 3 is one or more the mixture in formic acid, acetic acid, propionic acid, butyric acid, hydrochloric acid, lactic acid, oxysuccinic acid, tartrate, oxalic acid, citric acid, fumaric acid, the Sorbic Acid.
As preferably, the described alkaline solution of step 3 is one or more the mixture in sodium hydroxide, potassium hydroxide, the sodium bicarbonate.
As preferably, also comprise the solvent extraction step, described solvent is extracted as gets sodium bacillus subtilis lipopeptide finished product adding solvating agent, at 10-50 ℃ of stirring and leaching 1-5 hour, leaves standstill 1-3 hour, collects organic phase, condensing crystal.
As preferably, described solvating agent is a kind of in normal hexane, acetone, ethanol, ethyl acetate, methyl alcohol, ether, chloroform, the methylene dichloride.
As preferably, by g/mL, the mass volume ratio of described Bacitracin sodium finished product and solvating agent is 1:2-1:50.
As preferably, also comprise the centrifugal gained supernatant liquor of step 2 spent ion exchange resin chromatography purification, collect the step of chromatographic solution.
As preferably, described resinbed is analysed purifying and is regulated pH to 6.0-10.0 for getting the centrifugal gained supernatant liquor of step 2, and then spent ion exchange resin chromatography absorption is carried out gradient elution with the different pH value aqueous solution, the elutriant of collection pH5.5-6.5.
The purification process of sodium bacillus subtilis lipopeptide of the present invention at first utilizes centrifugal removal thalline, then utilize molecule to be easy to the characteristic of polymerization, process by the film of certain pore size and to remove small molecular weight impurity and pigment, film trapped fluid pH is transferred to 2.0-6.6, utilize fractionation precipitation polar impurity and centrifugal, remove the impurity small molecular weight impurities such as protein that are mixed with in the surfaction, afterwards with the centrifuged supernatant acidifying, adopt isoelectric point precipitation that surfaction is settled down, in the adding alkaline solution and behind the salify, drying can obtain the sodium bacillus subtilis lipopeptide finished product.Make sodium bacillus subtilis lipopeptide finished product purity behind the high performance liquid chromatography detection purifying and reach 85%-88%.In order further to improve Bacitracin sodium finished product purity, the purification process of sodium bacillus subtilis lipopeptide of the present invention comprises that also solvent extracts sodium bacillus subtilis lipopeptide finished product step.Utilize solvating agent dissolving sodium bacillus subtilis lipopeptide finished product, collect the upper organic phase condensing crystal, obtain the Bacitracin sodium pure product, purity reaches 90-93%.Further, also comprise and utilize resinbed to analyse the step of purification step 2 centrifugal gained supernatant liquors, make the surfaction sodium pure product purity that makes reach 93-98%.The purification process of sodium bacillus subtilis lipopeptide of the present invention makes surfaction sodium pure product purity 85%-98%, can be used as foodstuff additive and is widely used in field of food.
Embodiment
The embodiment of the invention discloses a kind of purification process of sodium bacillus subtilis lipopeptide.Those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, and the related personnel obviously can change or suitably change and combination method as herein described within not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
A kind of purification process of sodium bacillus subtilis lipopeptide comprises:
Step 1: get the sodium bacillus subtilis lipopeptide fermented liquid, centrifugal collection supernatant liquor is collected trapped fluid behind membrane filtration;
Step 2: trapped fluid pH is transferred to 5.0-6.6, centrifugal collection supernatant liquor;
Step 3: add acid solution to pH value 2.0-5.0 to step 2 gained solution, collecting precipitation slowly adds alkaline solution and carries out neutralization reaction after the washing, obtain Bacitracin sodium finished product after the reaction solution drying.
Purification process of the present invention at first utilizes centrifugal removal thalline, then utilize molecule to be easy to the characteristic of polymerization, process by the film of certain pore size and to remove small molecular weight impurity and pigment, film trapped fluid pH is transferred to 2.0-6.6, utilize fractionation precipitation polar impurity and centrifugal, remove the impurity small molecular weight impurities such as protein that are mixed with in the surfaction, afterwards with the centrifuged supernatant acidifying, adopt isoelectric point precipitation that surfaction is settled down, in the adding alkaline solution and behind the salify, drying can obtain the sodium bacillus subtilis lipopeptide finished product.
Surfaction adopts the method for fermentation to prepare more at present, because fermented liquid complicated component, there are a large amount of thalline, and thalline self-dissolving meeting causes impurity to increase, therefore purification process of the present invention at first utilizes the thalline in the centrifugal removal fermented liquid, for the subsequent handlings such as purifying create favorable conditions.
The iso-electric point of surfaction is 2-5, in fermented liquid pH value during near the surfaction iso-electric point, thereby the part surfaction can mix with thalline by Precipitation, when centrifugal removal thalline, the surfaction of separating out and thalline mix by centrifugal and remove, cause the loss of surfaction, reduce the yield of surfaction.Therefore for fear of surfaction meeting Precipitation, guarantee the yield of surfaction, purification process step 1 of the present invention also is included in centrifugally frontly carries out pretreated step to the surfaction fermented liquid, described pre-treatment is that with surfaction fermented liquid dilution 1-10 doubly adjust pH transfers to 6.5-8.5.
Centrifugal is exactly the powerful centrifugal force that utilizes the centrifuge rotor high speed rotating to produce, and the settling velocity of particle in the accelerating liquid is opened the separating substances of different settling ratios and buoyant density in the sample.The important indicator of weighing the whizzer separation performance is separating factor, and it represents separated material suffered centrifugal force and the ratio of its gravity in rotary drum, and separating factor is larger, and separation is also rapider usually, and separating effect is better.In order to reach the effect of better removal thalline, the centrifugal separating factor of the described sodium bacillus subtilis lipopeptide fermented liquid of purification process step 1 of the present invention is 5000g-17000g.
Whizzer of a great variety, as preferably, whizzer of the present invention is a kind of in horizontal centrifuge, link-suspended basket centrifuge, pocket type whizzer, tubular-bowl centrifuge, the disk centrifugal separator.
Purification process of the present invention utilizes molecule to be easy to the characteristic of polymerization behind centrifugal removal thalline, by the film of certain pore size centrifugal gained supernatant liquor is carried out filtration treatment.
Membrane filtration is a kind of screening process relevant with the membrane pore size size, take the pressure difference of film both sides as motivating force, take film as filtration medium, under certain pressure, when stoste flow through the film surface, the many tiny micropore that gathers in the film surface only allowed water and small-molecule substance by becoming through liquid, and volume then is trapped within the liquid feeding side of film in the stoste greater than the material in film surface micropore footpath, become trapped fluid, thereby realize the purpose to the separation and purification of stoste.
The selection of membrane pore size has material impact to the separation and purification effect of membrane filtration.In order to reach the small molecular weight impurity removed in the surfaction and the purpose of pigment, the film of the described membrane filtration of purification process step 1 of the present invention is ceramic membrane.
Further, the aperture of ceramic composite membrane of the present invention is preferably 10nm-800nm.20nm-100nm more preferably
The performance of ceramic membrane performance and temperature just have direct relation, and therefore, for the effect of membrane filtration, the film temperature of the described membrane filtration of purification process step 1 of the present invention is preferably 10-50 ℃.
The fermented liquid complicated component of fermentation preparation surfaction except having a large amount of thalline, also contains the protein that do not utilized fully by microorganism etc.In order further to improve Bacitracin sodium finished product purity, purification process step 2 of the present invention is carried out centrifugal removal of impurities with the collection trapped fluid of membrane filtration, to remove the impurity such as protein that are mixed with in the surfaction.Wherein, described centrifugal removal of impurities is transferred pH to 5.0-6.6, centrifugal collection supernatant liquor for getting membrane filtration gained trapped fluid.Utilize the target product surfaction different from impurity iso-electric points such as protein, the principle of natural subsidence can occur in the impurity such as protein when pH value of solution reaches its iso-electric point, film trapped fluid pH is transferred to 5.0-6.6, the impurity such as target product surfaction and protein are separated, centrifugal except impurity such as deproteinizes.
Wherein, described film trapped fluid pH is preferably 4.0-6.5.
In order to reach the effect of the impurity such as better removal protein, alleviate the pressure of follow-up refining step, centrifugal separating factor is preferably 5000g-17000g described in the described centrifugal removal step of purification process step 2 of the present invention.
Purification process step 3 of the present invention is carried out acid precipitation with step 2 gained solution.Utilize isoelectric point precipitation, regulate centrifuged supernatant pH value to the surfaction iso-electric point with acid, surfaction is settled down.Wherein, described acid precipitation adds acid solution to pH value 2.0-5.0, collecting precipitation for getting centrifugal removal of impurities gained supernatant liquor.Preferably, add acid solution to pH value 3.0-4.0.
Wherein, the described acid solution of step 3 is one or more the mixture in formic acid, acetic acid, propionic acid, butyric acid, hydrochloric acid, lactic acid, oxysuccinic acid, tartrate, oxalic acid, citric acid, fumaric acid, the Sorbic Acid.
Further, described acid precipitation also comprises the water washing and precipitating step, better to remove the impurity that is mixed with in the surfaction that settles down.
Purification process step 3 of the present invention is got precipitation adding alkaline solution and is carried out neutralization reaction, makes Bacitracin sodium, and drying can obtain Bacitracin sodium finished product.Wherein said neutralization reaction refers to add the alkaline solution reaction to neutral, namely reacts to pH6.5-8.5.
Wherein, as preferably, the temperature of described neutralization reaction is 20-55 ℃.
As preferably, described alkaline solution is one or more the mixture in sodium hydroxide, potassium hydroxide, the sodium bicarbonate.
Dry general reference is removed the various operations of moisture or other hygroscopic waters from wet stock.Tan by the sun to remove moisture under the sunlight as in daily life moist material being placed, industrially remove moisture in air, industrial gasses or the organic liquid with silica gel, lime, the vitriol oil etc.In Chemical Manufacture, drying is often referred to the wet solid materials of the heating such as warm air, stack gas and infrared rays, and make the vaporization of wherein contained moisture or solvent and remove, be a kind of unit operation that belongs to the caloic transmittance process.Dry purpose is to make material be convenient to store, transport and use, or satisfies the further needs of processing.According to the method for supplying of heat, multiple dry type is arranged: 1. convection drying: warm air or stack gas are directly contacted with wet stock, rely on transmission of heat by convection to the material heat supply, steam is then taken away by air-flow.Convection drying is most widely used aborning, and it comprises air stream drying, spraying drying, fluidized drying, revolving drum drying and carriage-type drying etc.; 2. conductive drying: wet stock directly contacts with heating-wall, and heat is passed to wet stock by thermal conduction by wall, and steam is discharged by air extractor.It comprises roller drying, lyophilize, vacuum rake type drying etc.; 3. radiant drying: heat projects the wet stock surface in the radiative transfer mode, is converted into heat energy after being absorbed, and steam is discharged by air extractor, such as ultra red ray drying; 4. dielectric heating drying: wet stock is placed in the high-frequency electric field, rely on the electric energy heating and make the moisture vaporization, comprise radio-frequency drying, microwave drying.
Wherein, as preferably, the described drying of purification process step 3 of the present invention is spraying drying.
The present invention adopts high performance liquid chromatography to detect the Bacitracin sodium finished product that purification process of the present invention prepares, purity 85-88%.
Further, in order to improve the purity of Bacitracin sodium finished product, purification process of the present invention also comprises the solvent extraction step.Utilize solvating agent dissolving sodium bacillus subtilis lipopeptide finished product, collect the upper organic phase condensing crystal, obtain the Bacitracin sodium pure product.
In some specific embodiments, solvent of the present invention is extracted as gets sodium bacillus subtilis lipopeptide finished product adding solvating agent, at 10-50 ℃ of stirring and leaching 1-5 hour, leaves standstill 1-3 hour, collects organic phase, condensing crystal.
Wherein, as preferably, described solvating agent is a kind of in normal hexane, acetone, ethanol, ethyl acetate, methyl alcohol, ether, chloroform, the methylene dichloride.
As preferably, by g/mL, the mass volume ratio of described Bacitracin sodium finished product and solvating agent is 1:2-1:50.Be that described solvating agent add-on is that every 1g sodium bacillus subtilis lipopeptide finished product adds solvating agent 2-50mL.
In some specific embodiments, solvent of the present invention extracts the sodium bacillus subtilis lipopeptide finished product and has added solubilizing agent, to promote the dissolving of sodium bacillus subtilis lipopeptide finished product.
Wherein, as preferably, described solubilizing agent is one or more in poly yamanashi esters, polyoxyethylene fatty acid ester class, squalane, Tweens, spans, sulfated castor oil, sodium lauryl sulphate, the sodium laurylsulfonate.
As preferably, the consumption of described solubilizing agent accounts for the 1v/v%-5v/v% of abstraction reaction liquid, and namely the volume fraction of solubilizing agent is 1%-5% described in the described abstraction reaction liquid.
The present invention adopts high performance liquid chromatography to detect Bacitracin sodium finished product and obtain Bacitracin sodium pure product, purity 90-93% after solvent extracts.
In some specific embodiments, the purification process of sodium bacillus subtilis lipopeptide of the present invention also comprises the centrifugal gained supernatant liquor of step 2 spent ion exchange resin chromatography purification, collects the step of chromatographic solution.
Chromatography (Chromatography) is the abbreviation of " chromatographic analysis ", is the difference of utilizing each component physical properties, the method that multicomponent mixture is separated.Chromatography is divided into adsorption chromatography, partition chromatography, ion exchange chromatography, gel permeation chromatography, affinity chromatography etc.Wherein ion-exchange resin purification is the method that the permutoid reaction that occurs between the ion that utilizes in ion exchange resin and the solution is separated.
As preferably, resinbed of the present invention is analysed purifying and is regulated pH to 6.0-10.0 for getting the centrifugal gained supernatant liquor of step 2, and then spent ion exchange resin chromatography absorption is carried out gradient elution with the different pH value aqueous solution, the elutriant of collection pH5.5-6.5.
Ion exchange resin of the present invention is a kind of in the neutral polymeric adsorbent of Sephadex LH-20 resin, charged ion exchange resin AG1-X4, XAD-7, strongly acidic styrene type cation exchange resin, the polyvinylidene difluoride (PVDF) PVDF resin.
The present invention adopt high performance liquid chromatography detect through centrifugal removal thalline, membrane filtration processings, centrifugal removal of impurities, resinbed analyse purifying, acid precipitation, in and salify, acquisition Bacitracin sodium pure product after extracting finally by solvent, purity 93-98%.
In order further to understand the present invention, below in conjunction with embodiment the purification process of a kind of sodium bacillus subtilis lipopeptide provided by the invention is elaborated.
Wherein the method for high-performance liquid chromatogram determination sodium bacillus subtilis lipopeptide finished product purity is as follows:
Chromatographic condition: chromatographic column: 5 μ m C18 posts, 200 * 4.6mm; Moving phase: 80% acetonitrile+20%3.8mM trifluoroacetic acid (TFA), through the organic membrane filtration of 0.22 μ m, ultrasonic degas 20min; Flow velocity: 1.0mL/min; Column temperature: 27 ℃; Sample size: 20 μ L; Ultraviolet detection wavelength: 205nm.
Sample preparation: accurately take by weighing sodium bacillus subtilis lipopeptide standard substance 10mg, be mixed with the reference liquid mother liquor of 2mg/mL with 5mL moving phase, drawing the 1mL mother liquor is the reference liquid of 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL with two times of gradient method dilutions, saves backup in refrigerator freezing.
Accurately take by weighing 50.0mg (being accurate to 0.0001g) and make the sodium bacillus subtilis lipopeptide testing sample to the 50mL volumetric flask, add moving phase and be mixed with the 1mg/mL sample solution, supersound extraction 20 minutes left standstill 30 minutes, behind the organic filtering with microporous membrane of 0.22 μ m, collect filtrate and treat Liquid Detection.
Measure: according to concentration sample introduction from low to high, detected result is processed through Shimadzu LC Solution workstation and is drawn typical curve, processes in detail operation and sees " high performance liquid chromatography working specification " with the sodium bacillus subtilis lipopeptide reference liquid.Under above-mentioned chromatographic condition, qualitative according to retention time, the external standard peak area quantification.Be the peak area of sodium bacillus subtilis lipopeptide according to the peak area sum between standard substance high-efficient liquid phase chromatogram 6-30 minute.
The sodium bacillus subtilis lipopeptide response value should be in the typical curve linearity range in the testing sample, again sample introduction analysis after exceeding the concentration linearity range and then should diluting.
The result calculates: analytical results is processed through Shimadzu LC Solution workstation and is drawn testing sample purity, and " high performance liquid chromatography working specification " seen in the operation of workstation integrated.
Embodiment 1: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get the surfaction fermented liquid, the centrifugal 30min of 5000g collects centrifugal gained supernatant liquor, is 30 ℃ ultrafiltration membrance filter through 50nm, film temperature, collection membrane filters trapped fluid, transfer pH to 5.0-5.5, the centrifugal 10min of 10000g collects supernatant liquor, add hydrochloric acid to pH value 3.0-4.0, collecting precipitation adds sodium hydroxide solution at 20 ℃ and carries out neutralization reaction, namely gets the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.The sodium bacillus subtilis lipopeptide finished product purity that high effective liquid chromatography for measuring makes is 85%.
Embodiment 2: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get the surfaction fermented liquid, the centrifugal 10min of 12000g collects centrifugal gained supernatant liquor, is 20 ℃ ultrafiltration membrance filter through 50nm, film temperature, collection membrane filters trapped fluid, transfer pH to 5.0-6.0, the centrifugal 10min of 10000g collects supernatant liquor, add formic acid to pH value 3.0-5.0, collecting precipitation adds potassium hydroxide solution at 25 ℃ and carries out neutralization reaction, namely gets the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.The sodium bacillus subtilis lipopeptide finished product purity that high effective liquid chromatography for measuring makes is 86%.
Embodiment 3: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get 10 times of surfaction fermented liquid dilutions, adjust pH transfers to 7.0-7.5, the centrifugal 5min of 17000g, collect centrifugal gained supernatant liquor, be 30 ℃ ultrafiltration membrance filter through 50nm, film temperature, collection membrane filters trapped fluid, transfers pH to 5.0-5.5, the centrifugal 10min of 10000g, collect supernatant liquor, add hydrochloric acid to pH value 2.0-3.0, collecting precipitation, add sodium hydroxide solution at 20 ℃ and carry out neutralization reaction, namely get the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.The sodium bacillus subtilis lipopeptide finished product purity that high effective liquid chromatography for measuring makes is 88%.
Embodiment 4: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get 5 times of surfaction fermented liquid dilutions, adjust pH transfers to 6.5-7.5, the centrifugal 10min of 12000g, collect centrifugal gained supernatant liquor, be 20 ℃ ultrafiltration membrance filter through 20nm, film temperature, collection membrane filters trapped fluid, transfers pH to 5.0-6.0, the centrifugal 10min of 10000g, collect supernatant liquor, add acetic acid to pH value 3.0-5.0, collecting precipitation, add sodium hydrogen carbonate solution at 25 ℃ and carry out neutralization reaction, namely get the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.The sodium bacillus subtilis lipopeptide finished product purity that high effective liquid chromatography for measuring makes is 87%.
Embodiment 5: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get 4 times of surfaction fermented liquid dilutions, adjust pH transfers to 7.0-8.5, the centrifugal 15min of 12000g, collect centrifugal gained supernatant liquor, be 30 ℃ ultrafiltration membrance filter through 50nm, film temperature, collection membrane filters trapped fluid, transfers pH to 4.0-6.5, the centrifugal 10min of 10000g, collect supernatant liquor, add formic acid to pH value 3.0-5.0, collecting precipitation, add potassium hydroxide solution at 50 ℃ and carry out neutralization reaction, namely get the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.Get the sodium bacillus subtilis lipopeptide finished product, the amount that adds 10mL by every 1g sodium bacillus subtilis lipopeptide finished product adds acetone, 10 ℃ of stirring and leaching 5 hours, leaves standstill 1 hour, collects organic phase, and condensing crystal namely gets the surfaction sodium pure product.The surfaction sodium pure product purity that high effective liquid chromatography for measuring makes is 90%.
Embodiment 6: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get 6 times of surfaction fermented liquid dilutions, adjust pH transfers to 6.5-7.0, the centrifugal 20min of 8000g, collect centrifugal gained supernatant liquor, be 50 ℃ ultrafiltration membrance filter through 50nm, film temperature, collection membrane filters trapped fluid, transfers pH to 5.0-5.5, the centrifugal 10min of 10000g, collect supernatant liquor, add formic acid to pH value 2.0-3.0, collecting precipitation, add sodium hydroxide solution at 20 ℃ and carry out neutralization reaction, namely get the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.Get the sodium bacillus subtilis lipopeptide finished product, the amount that adds 2mL by every 1g sodium bacillus subtilis lipopeptide finished product adds ethyl acetate, adds the 1v/v% sodium lauryl sulphate, 50 ℃ of stirring and leaching 1 hour, left standstill 3 hours, collect organic phase, condensing crystal namely gets the surfaction sodium pure product.The surfaction sodium pure product purity that high effective liquid chromatography for measuring makes is 93%.
Embodiment 7: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get 5 times of surfaction fermented liquid dilutions, adjust pH transfers to 6.5-7.5, the centrifugal 10min of 12000g, collect centrifugal gained supernatant liquor, through 20nm, the film temperature is 20 ℃ ultrafiltration membrance filter, and collection membrane filters trapped fluid, transfers pH to 5.0-6.0, the centrifugal 10min of 10000g, collect supernatant liquor, regulate pH to 6.0, then with the neutral polymeric adsorbent chromatography absorption of XAD-7, carry out gradient elution with the different pH value aqueous solution, collect the elutriant of pH5.5-6.5, add citric acid to pH value 3.0-5.0, collecting precipitation, add potassium hydroxide solution at 25 ℃ and carry out neutralization reaction, namely get the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.Get the sodium bacillus subtilis lipopeptide finished product, the amount that adds 50mL by every 1g sodium bacillus subtilis lipopeptide finished product adds ethanol, 20 ℃ of stirring and leaching 2 hours, leaves standstill 2 hours, collects organic phase, and condensing crystal namely gets the surfaction sodium pure product.The surfaction sodium pure product purity that high effective liquid chromatography for measuring makes is 95%.
Embodiment 8: the purifying of sodium bacillus subtilis lipopeptide of the present invention
Get 10 times of surfaction fermented liquid dilutions, adjust pH transfers to 7.0-8.0, the centrifugal 30min of 5000g, collect centrifugal gained supernatant liquor, through 50nm, the film temperature is 10 ℃ ultrafiltration membrance filter, and collection membrane filters trapped fluid, transfers pH to 5.0-5.5, the centrifugal 10min of 10000g, collect supernatant liquor, regulate pH to 10.0, then with the absorption of Sephadex LH-20 resin chromatography, carry out gradient elution with the different pH value aqueous solution, collect the elutriant of pH5.5-6.5, add hydrochloric acid to pH value 2.0-3.0, collecting precipitation, add potassium hydroxide solution at 25 ℃ and carry out neutralization reaction, namely get the sodium bacillus subtilis lipopeptide finished product after reaction solution is spray-dried.Get the sodium bacillus subtilis lipopeptide finished product, the amount that adds 30mL by every 1g sodium bacillus subtilis lipopeptide finished product adds normal hexane, adds the 5v/v% sodium laurylsulfonate, 30 ℃ of stirring and leaching 4 hours, left standstill 1 hour, collect organic phase, condensing crystal namely gets the surfaction sodium pure product.The surfaction sodium pure product purity that high effective liquid chromatography for measuring makes is 98%.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (12)
1. the purification process of a sodium bacillus subtilis lipopeptide is characterized in that, comprising:
Step 1: get the sodium bacillus subtilis lipopeptide fermented liquid, centrifugal collection supernatant liquor is collected trapped fluid behind membrane filtration;
Step 2: trapped fluid pH is transferred to 5.0-6.6, centrifugal collection supernatant liquor;
Step 3: add acid solution to pH value 2.0-5.0 to step 2 gained solution, collecting precipitation slowly adds alkaline solution and carries out neutralization reaction, obtains Bacitracin sodium finished product after the reaction solution drying.
2. described purification process according to claim 1 is characterized in that, step 1 also is included in centrifugally frontly carries out pretreated step to the sodium bacillus subtilis lipopeptide fermented liquid, and described pre-treatment is that with sodium bacillus subtilis lipopeptide fermented liquid dilution 1-10 doubly adjust pH transfers to 6.0-8.5.
3. described purification process according to claim 1 and 2 is characterized in that the described centrifugal separating factor of step 1 is 5000-17000g.
4. described purification process according to claim 1 and 2 is characterized in that the film of the described membrane filtration of step 1 is ceramic membrane.
5. described purification process according to claim 1 and 2 is characterized in that the described centrifugal separating factor of step 2 is 5000-17000g.
6. described purification process according to claim 1 and 2, it is characterized in that the described acid solution of step 3 is one or more the mixture in formic acid, acetic acid, propionic acid, butyric acid, hydrochloric acid, lactic acid, oxysuccinic acid, tartrate, oxalic acid, citric acid, fumaric acid, the Sorbic Acid.
7. described purification process according to claim 1 and 2 is characterized in that, the described alkaline solution of step 3 is one or more the mixture in sodium hydroxide, potassium hydroxide, the sodium bicarbonate.
8. the described purification process of any one is characterized in that according to claim 1-7, also comprises the solvent extraction step, described solvent is extracted as gets sodium bacillus subtilis lipopeptide finished product adding solvating agent, at 10-50 ℃ of stirring and leaching 1-5 hour, leaves standstill 1-3 hour, collect organic phase, condensing crystal.
9. described purification process according to claim 8 is characterized in that, described solvating agent is a kind of in normal hexane, acetone, ethanol, ethyl acetate, methyl alcohol, ether, chloroform, the methylene dichloride.
10. described purification process according to claim 8 is characterized in that by g/mL, the mass volume ratio of described Bacitracin sodium finished product and solvating agent is 1:2-1:50.
11. the described purification process of any one is characterized in that according to claim 1-10, also comprises the centrifugal gained supernatant liquor of step 2 spent ion exchange resin chromatography purification, collects the step of chromatographic solution.
12. described purification process according to claim 11, it is characterized in that, described resinbed is analysed purifying and is regulated pH to 6.0-10.0 for getting the centrifugal gained supernatant liquor of step 2, then spent ion exchange resin chromatography absorption, carry out gradient elution with the different pH value aqueous solution, collect the elutriant of pH5.5-6.5.
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