CN110790826A - Method for separating and purifying lipopeptide surfactin family compound - Google Patents
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Abstract
The invention discloses a method for separating and purifying lipopeptide surfactin family compounds, belonging to the technical field of fermentation engineering. The invention adopts a method of removing protein by ethanol and removing small molecular impurities by acid precipitation and acid washing to obtain the surfactin family compound with high purity. Wherein the purity of surfactin can reach 94.2%, the recovery rate is 96.3%, the recovery rate of lichenin can reach 95.0%, and the purity can reach 92.6%. The obtained product is milk white powder, has high purity, and does not need further refining. The method for separating and purifying the lipopeptide surfactin family compounds is simple to operate, environment-friendly, low in cost, high in product purity and recovery rate, and capable of being effectively applied to actual industrial production.
Description
Technical Field
The invention relates to a method for separating and purifying lipopeptide surfactin family compounds, belonging to the technical field of fermentation engineering.
Background
Over 30 structurally different bacillus lipopeptides have been discovered in succession since 1949. These lipopeptide compounds have unique and highly similar structures, and are all cyclic heptapeptide compounds synthesized by Non-ribosomal polypeptide pathways (NRPS). Bacillus lipopeptides are divided into four families according to the amino acid sequence in the polypeptide and the cyclization mode: surfactin (surfactin), fengycin (fengycin), iturin (iturin), and kurstatkin, and each family contains a variety of structural analogs. The surfactin family can be further divided into surfactin and lichenin, wherein the surfactin is mainly synthesized by bacillus subtilis and bacillus amyloliquefaciens, and the lichenin is mainly synthesized by bacillus licheniformis. The lipopeptide surfactin family compound has unique biochemical and physicochemical properties: if the surfactant is nontoxic or low-toxic, biodegradable, good in environmental compatibility and resistant to extreme environments (temperature, pH value and salinity), the surfactant is considered to have wide application prospects in the fields of food daily chemicals, environmental engineering, oil extraction industry, cosmetics, medicines and the like, can gradually replace a chemically synthesized surfactant, and is in accordance with the modern industrial production concept of green sustainable development.
Because lipopeptide surfactin family compounds are expensive and industrial production is not realized, the popularization and application of the surfactin family are limited. The main reason for hindering the realization of industrialization is that the downstream separation and purification cost is high, and the current surfactin separation and purification main steps are as follows: (1) centrifugally sterilizing (2) adjusting pH to 2 by hydrochloric acid to precipitate surfactin family; (3) extracting surfactin family compounds in the precipitate by using an organic solvent; (4) further carrying out fine extraction through column chromatography, adsorption resin, ultrafiltration and the like; (5) the organic solvent was removed by rotary evaporation. Wherein, the step (1-3) is a crude extraction process, and the step (4-5) is a fine extraction process. There are many problems in the overall process flow, for example, problems in the crude extraction process include: (1) after acid precipitation, proteins can also be precipitated together with surfactin family compounds, and a lot of small molecular impurities can be brought in, so that the purity after organic solvent extraction is not high, and the literature reports that the purity is generally between 50% and 70%. (2) The surfactin product obtained after the organic solvent extraction is dissolved in the organic solvent, which is not beneficial to the processing and storage of the subsequent products. Problems in the fine lifting process include: the method has the defects of multiple unit operations, complexity, high cost and no contribution to industrial application, and excessively depends on expensive organic solvents, such as methanol, isopropanol, n-butanol, ethyl acetate and the like, which are usually expensive and toxic, so that the cost is increased, the environment is polluted, and the method is not suitable for products in the fields of food, medicine and the like.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for separating and purifying a lipopeptide surfactin family compound. The surfactin family compound is separated and purified from the fermentation liquor, and impurity substances are removed through a series of operations, wherein the impurities mainly comprise some protein and small molecule impurities (mainly salts in a culture medium). The method comprises the steps of firstly removing protein impurities by adopting an organic solvent precipitation method, recovering the organic solvent by rotary evaporation, then removing small molecular impurities while precipitating the surfactin family compound by adopting an acid precipitation mode, and finally removing the residual impurities in the surfactin family compound precipitate by adopting an acid washing mode to obtain a high-purity product. By utilizing the method for separating and purifying surfactin family compounds, the recovery rate of surfactin products can reach 96.3 percent at most, and the purity can reach 94.2 percent; the recovery rate of lichenin can reach 95.0%, and the purity can reach 92.6%.
The first purpose of the invention is to provide a method for separating and purifying lipopeptide surfactin family compounds, which comprises the following operation steps:
(1) centrifuging and sterilizing the fermentation liquor to obtain a supernatant, adding an organic solvent into the supernatant to precipitate and remove protein, removing the protein by suction filtration, and then removing the organic solvent by rotary evaporation;
(2) and (2) adding acid into the fermentation liquor obtained after the organic solvent is removed by rotary evaporation in the step (1), and obtaining a high-purity lipopeptide surfactin family compound product by utilizing acid precipitation and acid washing.
In one embodiment, the organic solvent in step (1) of the process is ethanol.
In one embodiment, in step (1), the fermentation liquid is added with ethanol as an organic solvent, and the mixture is left to stand for precipitation for 2 to 4 hours.
In one embodiment, when the method of step (1) employs an organic solvent to precipitate the protein, the pH of the system is between 6 and 9.
In one embodiment, the protein is removed by suction filtration through a 0.48 μm filter in step (1) of the process.
In one embodiment, the fermentation broth in step (1) of the process: the volume ratio of the organic solvent is 1:1-1: 2.
In one embodiment, the organic solvent is isolated in step (1) of the process by rotary evaporation at 40hpa, 30 ℃.
In one embodiment, the acid in step (2) of the process is H2SO4And HCl.
In one embodiment, in step (2), an acid is added to the fermentation broth after the organic solvent is removed by rotary evaporation until the pH value of the system is 2-3.
In one embodiment, the method step (2) is performed at 1200rpm for 5min when the precipitate is collected by centrifugation.
In one embodiment, the method step (2) acid washes the precipitate 1-5 times.
In one embodiment, the method step (2) acid washes the precipitate twice.
In one embodiment, the method for separating and purifying the lipopeptide surfactin family compound comprises the following steps: centrifuging and sterilizing the fermentation liquor to obtain a supernatant, adding equal volume of ethanol into the supernatant to precipitate and remove protein, removing the protein by suction filtration, and then removing the organic solvent by rotary evaporation; adding acid into the fermentation liquor after the organic solvent is removed by rotary evaporation, adjusting the pH value to 2, and obtaining the high-purity lipopeptide surfactin family compound product by utilizing acid precipitation and acid washing.
The invention has the beneficial effects that:
(1) the method can effectively separate and purify lipopeptide surfactin family compounds, and under the optimal condition, the recovery rate of the surfactin product can reach 96.3 percent, and the purity can reach 94.2 percent; the recovery rate of the lichenin can reach 95.0 percent, and the purity can reach 92.6 percent;
(2) the method adopts the organic solvent to remove protein and then uses the acidic aqueous solution to remove small molecular impurities to separate and purify surfactin family compounds, the purity of the separated product is high, fine extraction is not needed, the operation steps are simple, the cost is low, and the method can be applied to industrial production;
(3) the lipopeptide surfactin family compound precipitate obtained after acid washing can be directly frozen and dried to be prepared into white powder, and the product does not need to be decolorized, thereby being beneficial to the transportation, storage and practical application of the product;
(4) the reagents used in the method for separating and purifying the lipopeptide surfactin family compounds are nontoxic, have small dosage, can be recycled, are economical and environment-friendly, and meet the environment-friendly concept.
Drawings
FIG. 1 Standard Curve for protein concentration in example 2.
FIG. 2 Effect of three organic solvents on protein removal and product recovery in examples 4 and 6.
FIG. 3 Effect of different concentrations of ethanol on protein removal and product recovery in example 5.
FIG. 4 Effect on protein removal and product recovery at different ammonium sulfate concentrations in comparative example 1.
FIG. 5 Effect of the combination of ethanol and ammonium sulfate deproteinization and product recovery in comparative example 2.
FIG. 6 Effect of different pH on ethanol deproteinization and product recovery in example 7.
FIG. 7 influence of acid precipitation in example 8 and acid washing in example 9 on surfactin purity and recovery.
FIG. 8 comparison of appearance of surfactin isolated and purified by the method of the present invention and the method of comparative example 3; fig. 8(a) is a product in example 9, and fig. 8(b) is a product in comparative example 3.
Detailed Description
The present invention will be further illustrated by the following examples and comparative examples.
Example 1 preparation of fermentation broth
The fermentation strain is Bacillus amyloliquefaciens MT45, is separated from China Maotai Daqu, is stored in China general microbiological culture Collection center with the preservation number of CGMCC:12593, and is disclosed in a patent with the publication number of CN106635869A in 5/10.2017.
The LB medium comprises the following components: 5.0g/L of yeast powder and 10.0g/L, NaCl 10.0.0 g/L of tryptone.
Optimizing the components of the culture medium: 65g/L of sucrose, 8g/L of ammonium nitrate, 3.73g/L of peptone, 4.08g/L of potassium dihydrogen phosphate, 10g/L of disodium hydrogen phosphate, 0.096g/L of magnesium sulfate, 7 mu M of calcium chloride, 4 mu M of ferrous sulfate and 4 mu M of EDTA.
Bacillus amyloliquefaciens MT45 was activated in LB medium at 37 ℃ and 200rpm, cultured for 12 hours, inoculated in a 250mL flask containing 50mL of optimized medium at 2% inoculum size, and cultured for 72 hours at 30 ℃ and 200 rpm. Collecting fermentation liquor, centrifuging and sterilizing, and collecting supernatant for separating and extracting surfactin.
Example 2 detection of protein content in fermentation broth
Firstly, a standard curve is established, Bovine Serum Albumin (BSA) is prepared into the concentrations of 0, 0.0625, 0.125, 0.25, 0.5, 0.75 and 1G/L, and the absorbance of different bovine serum albumin solutions is measured at 595nm after the Coomassie brilliant blue G-250 solution is added.
And (3) reacting the fermentation liquor with Coomassie brilliant blue G-250, measuring an absorbance value at 595nm, and establishing a standard curve to calculate the content of the protein in the fermentation liquor. As shown in fig. 1, the set-up criteria are: 2.1295x-0.0275, R20.9949. The initial fermentation broth was found to contain 0.129g/L protein.
Example 3 calculation of recovery and purity of surfactin and protein removal
And (3) recovery rate:
purity:
protein removal rate:
calculating the content of surfactin: the surfactin concentration in the culture medium was quantitatively analyzed by Ultra Performance Liquid Chromatography (UPLC). Using Waters BEH C18Separating with liquid phase gradient chromatography column (100mm × 2.1mm,1.7 μm particle); the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is HPLC grade methanol; elution gradient: 0.1min, 70% B; 0.1-2.0 min, 70% B; 2.0-8.0 min, 70% -100% B; 8.0-10 min, 100% B; 10.1min, 70% B, 10.1-13 min 70% B; the flow rate was 0.3 mL/min. The ultraviolet detection wavelength is 205 nm. Preparing surfactin methanol standard solutions with different concentration gradients, and making a surfactin standard curve according to the peak area and the concentration change. And calculating the surfactin content according to the peak area of the corresponding peak-off time of the sample to be detected by an insertion method. Since various homologs of surfactin exist, the peak areas of different homologs are added to obtain the total surfactin in the system.
Example 4 removal of proteins from fermentation broth Using ethanol as organic solvent
25mL of the fermentation broth prepared in example 1 was taken, added with ethanol as an organic solvent in an equal volume ratio, adjusted to pH 6 with HCl and NaOH, left to stand for precipitation for 2 hours, and then protein was removed by suction filtration through a 0.48 μm filter. And (3) carrying out rotary evaporation on the mixture of the fermentation liquor and the ethanol at 40hpa and 30 ℃ to separate the organic solvent ethanol, and recovering the ethanol obtained by rotary evaporation for secondary utilization. The method for detecting the content of the protein in the example 2 is used for detecting the content of the residual protein in the fermentation liquor after the organic solvent is removed by the rotary evaporation, and the method for calculating the recovery rate and the purity of the surfactin in the fermentation liquor after the organic solvent is removed by the rotary evaporation is used for detecting the recovery rate and the purity of the surfactin in the fermentation liquor after the organic solvent is removed by the rotary evaporation. As can be seen from FIG. 2, the efficiency of protein removal using ethanol was about 84%, the product recovery was greater than 99%, and the product purity was 5.5%.
Example 5 removal of proteins from fermentation broth with ethanol of varying concentrations
Further explore the influence of adding ethanol in different proportions on protein removal efficiency, product purity and recovery rate. Setting the volume ratio of the organic solvent ethanol to the fermentation liquor as follows: 1:0.2, 1:0.5, 1:0.8, 1:1. 1:1.5, 1:2, and the other experimental conditions were the same as in example 4, and the effect of different concentrations of ethanol on protein removal and product recovery was tested. As can be seen from FIG. 3, as the content of ethanol is higher, the protein removal rate and the product purity are improved, and the addition amount of ethanol has no influence on the recovery rate of the product. When the volume ratio of the fermentation liquor to the ethanol is 1:1, the highest protein removal rate is about 84%, the product purity is 5.5%, and the ethanol concentration is further increased, so that the protein removal efficiency and the product purity are not obviously influenced.
Example 6 removal of proteins from fermentation broths Using different organic solvents
The organic solvent ethanol in example 4 was replaced by methanol and pentanediol, other experimental conditions were unchanged, and the effect of methanol and pentanediol on protein removal and product recovery was tested. As can be seen from FIG. 2, the efficiency of protein removal with methanol is about 84% as compared to ethanol, and the efficiency of protein removal with pentanediol is slightly lower, about 81%. The two solvents are added for protein removal, so that the product loss is avoided, the product recovery rate is more than 99%, and the product purity is not obviously different from that of example 4.
Compared with the influences of three organic solvents, namely ethanol, methanol and pentanediol on the protein removal efficiency, the product recovery rate and the purity, the protein removal efficiency of the ethanol and the methanol is about 84%, the protein removal efficiency of the pentanediol is slightly low, the product recovery rate and the purity are not significantly different, but the methanol has certain toxicity, the boiling point of the pentanediol is high, and the subsequent efficiency of the operation of removing the organic solvent by rotary evaporation can be reduced.
Comparative example 1 removal of protein from fermentation broth by salting out method
Removing protein in the fermentation liquor by using a saturated ammonium sulfate solution, taking 25mL of the fermentation liquor prepared in the example 1, adding the saturated ammonium sulfate solutions with different concentrations into the fermentation liquor, setting the volume ratios of the fermentation liquor to the saturated ammonium sulfate solutions to be 1:0.5, 1:0.8, 1:1, 1:1.5 and 1:2 respectively, standing and precipitating for 2 hours, and then filtering and removing the protein by using a filter membrane with the size of 0.48 mu m. Subjecting the mixture of the fermentation broth and ethanol to rotary evaporation at 30 deg.C under 40hpa to separate ethanol, and subjecting the organic solvent to rotary evaporationRecovering the ethanol obtained by steaming for secondary utilization. The method for detecting the content of the protein in the example 2 is used for detecting the content of the residual protein in the fermentation liquor after the organic solvent is removed by the rotary evaporation, and the method for calculating the recovery rate and the purity of the surfactin in the fermentation liquor after the organic solvent is removed by the rotary evaporation is used for detecting the recovery rate and the purity of the surfactin in the fermentation liquor after the organic solvent is removed by the rotary evaporation. The effect of removing protein from the fermentation broth by salting out is shown in FIG. 4, and it was found that the protein removing efficiency decreased with increasing salt solution concentration, at VSample (I):VAmmonium sulfateThe best effect is achieved when the ratio is 1:0.5, and the protein removal efficiency is about 75 percent. However, under the condition, flocculation precipitation also occurs on a large amount of surfactin, so that the recovery rate of the product is greatly reduced. When the concentration of the ammonium sulfate is increased, although the protein removing efficiency is reduced and the recovery rate of surfactin is increased, the purity of the product in the fermentation liquor is reduced to 1 percent due to the introduction of a large amount of ammonium sulfate. When the salting-out method is adopted to remove protein, surfactin and protein are precipitated together, so that the effect of only removing protein in fermentation liquor and retaining the product cannot be achieved.
Comparative example 2 removal of proteins from fermentation broth by combination of ethanol and ammonium sulfate
According to VSample (I)：VEthanolTo further investigate the effect of adding 0%, 5%, 10%, 20%, 30%, 50% by volume of ammonium sulfate on the protein removal efficiency on the basis of the addition of ethanol at a ratio of 1:1, 25mL of the fermentation broth prepared in example 1 was taken, saturated ammonium sulfate solutions of different concentrations were added to the fermentation broth, the volume ratios of the saturated ammonium sulfate solutions were set to 0%, 5%, 10%, 20%, 30%, 50%, and the mixture was allowed to stand for 2 hours to precipitate, and then the protein was removed by suction filtration through a 0.48 μm filter. And (3) carrying out rotary evaporation on the mixture of the fermentation liquor and the ethanol at 40hpa and 30 ℃ to separate the organic solvent ethanol, and recovering the ethanol obtained by rotary evaporation for secondary utilization. The method for detecting the content of the protein in the example 2 is used for detecting the content of the residual protein in the fermentation liquor after the organic solvent is removed by the rotary evaporation, and the method for calculating the recovery rate and the purity of the surfactin in the fermentation liquor after the organic solvent is removed by the rotary evaporation is used for detecting the recovery rate and the purity of the surfactin in the fermentation liquor after the organic solvent is removed by the rotary evaporation. As can be seen from fig. 5, the higher the ammonium sulfate concentration, the protein removal rate was rather significantly decreased,and the purity of the product is also obviously reduced due to the addition of a large amount of salt, but the product recovery rate is not influenced. The addition of ammonium sulfate does not improve the protein removal efficiency, but reduces the product purity. Therefore, the method for removing protein by using ethanol has the best effect without adding ammonium sulfate.
Example 7 removal of proteins from fermentation broths Using ethanol as an organic solvent at different pH values
On the basis of example 4, the effect of different pH on protein removal was further investigated. Adjusting pH to 5, 6, 7, 8, 9 and 10 by HCl and NaOH respectively, testing pH values according to V under different pH values under the same experimental conditions as example 4Sample (I)：VEthanolEffect of ethanol addition on protein removal and product recovery in a 1:1 ratio. As a result, as shown in fig. 6, when the pH was 6 or 7, the protein removal rate was highest, and both peracid and overbase decreased the protein removal rate. Therefore, when ethanol is used for removing protein, the optimal pH value is 6-7.
Example 8 removal of Small molecule impurities by acid precipitation
25mL of the fermentation broth obtained in example 4 after removal of ethanol, a small amount of H was added2SO4And (3) standing the solution for 4-6h until the surfactin precipitates are completely separated out when the pH of the solution is 2. And centrifuging at the rotating speed of 1200rpm for 5min, collecting the precipitate after centrifugation, and detecting the recovery rate and the purity of surfactin in the precipitate by using the method for detecting the recovery rate and the purity of surfactin in example 3. As can be seen from FIG. 7, the purity of surfactin obtained in the precipitation after adjusting the pH to 2 can reach 70%, and the recovery rate of acid precipitation surfactin is 98.8%.
Example 9 removal of residual impurities from fermentation broth in surfactin by acid wash
The precipitate collected after centrifugation in example 8 was taken and 25mL of H pH 2 was used2SO4The aqueous solution is subjected to acid washing for a plurality of times. The recovery rate and purity of surfactin in the precipitate are detected by the method for detecting the recovery rate and purity of surfactin in example 3. As can be seen from fig. 7, the product purity can be significantly improved to 91.1% by the first acid washing, and the product recovery rate is 97.1%. With the increase of the acid washing times, the product purity is gradually increased, and the product recovery rate is slightly reduced. ByAfter the acid washing is carried out for more than 2 times, the purity of the surfactin is not obviously improved, but the loss of the surfactin is further increased, so that after the surfactin is obtained by acid precipitation, H with the pH of 2 is adopted2SO4The acid washing of the aqueous solution is optimal for 2 times, the recovery rate of the product is 96.3 percent, and the purity can reach 94.2 percent.
Comparative example 3 separation and purification by acid precipitation and methanol precipitation
Taking 25mL of the fermentation liquid prepared in the example 1, carrying out centrifugal sterilization on the fermentation liquid, taking a supernatant, adjusting the pH of the supernatant to 2 by using 6mol/L HCl, and standing the supernatant in a refrigerator at 4 ℃ overnight; and centrifuging to collect surfactin sediment, and extracting surfactin by using a pure methanol solvent to obtain a surfactin crude extract. The recovery of surfactin was calculated to be 96% and the purity to be 60% according to the method in example 3.
Example 10: comparison of appearance of surfactin isolated and purified by the method of the present invention and the method of comparative example 3
The product obtained in example 9 by removing protein by ethanol precipitation, acid precipitation and acid water washing to remove small molecular impurities is subjected to vacuum freeze drying to obtain surfactin product, and the appearance of the product is shown in fig. 8 (a). Taking the product obtained in the comparative example 3 through acid precipitation and methanol extraction, removing methanol through rotary evaporation and performing vacuum freeze drying to obtain the surfactin product, wherein the appearance of the surfactin product is shown in figure 8 (b). As can be seen from the figure, the surfactin product separated and purified by the acid precipitation and methanol extraction method is yellow powder and has more impurities, and the surfactin product purified by the method disclosed by the invention is milk white in color and high in product purity.
Example 11 calculation of lichenin recovery and purity, protein removal
And (3) recovery rate:
purity:
protein removal rate:
calculating the content of the lichenin: the concentration of lichenin in the medium was quantified by High Performance Liquid Chromatography (HPLC). By C18Performing liquid phase gradient separation on the chromatographic column; the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is HPLC grade methanol; elution gradient: 0-10 min, 60% -75% B; 10-50 min, 75% -95% B; 50-55 min, 95% -100% B; the flow rate was 0.6 mL/min. The ultraviolet detection wavelength is 210 nm. Preparing methanol standard solutions of different concentration gradients of lichenin, and making a lichenin standard curve according to the peak area and concentration change. And calculating the content of the lichenin according to the peak area of the corresponding peak-off time of the sample to be detected by an insertion method. Since lichenin also exists in various homologues, the peak areas of different homologues are added to obtain the total amount of lichenin in the system.
Example 12: separating and purifying lichenin
The fermentation strain is Bacillus licheniformis MT-6 or MT-15, is separated from high-temperature Daqu, is stored in China general microbiological culture Collection center with the preservation numbers of CGMCC 3962 and CGMCC 3963 respectively. These two strains were disclosed in patent publication No. CN101955980A on 26/1/2011. After culturing the strain in the same manner as in example 1, 25mL of the fermentation broth was taken, to which ethanol was added in an equal volume ratio, and the mixture was allowed to stand for 2 hours to precipitate, and then the protein was removed by suction filtration through a 0.48 μm filter. And (3) carrying out rotary evaporation on the mixture of the fermentation liquor and the ethanol at 40hpa and 30 ℃ to separate the organic solvent ethanol, and recovering the ethanol obtained by rotary evaporation for secondary utilization. Adding a small amount of H into the fermentation liquor obtained after removing the organic solvent ethanol by rotary evaporation2SO4And (5) standing the solution for 4-6h until the lichenin precipitate is completely separated out. Centrifuging at 1200rpm for 5min, collecting precipitate after centrifugation, and adding 25mL of H with pH of 22SO4The precipitate was twice acid washed with water solution and vacuum freeze dried to obtain lichenin product with 95.0% recovery and 92.6% purity as calculated in example 11. Therefore, the optimal process conditions for separating and purifying surfactin are also applicable to lichenin.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for separating and purifying lipopeptide surfactin family compounds is characterized by comprising the following operation steps:
(1) centrifuging and sterilizing the fermentation liquor to obtain a supernatant, adding an organic solvent into the supernatant to precipitate and remove protein, removing the protein by suction filtration, and then removing the organic solvent by rotary evaporation;
(2) and (2) adding acid into the fermentation liquor obtained after the organic solvent is removed by rotary evaporation in the step (1), and obtaining a surfactin product with high purity by utilizing acid precipitation and acid washing.
2. The method for separating and purifying lipopeptide surfactin family compound as claimed in claim 1, wherein the organic solvent in step (1) is ethanol.
3. The method for separating and purifying lipopeptide surfactin family compound as claimed in claim 1, wherein the pH of the system is between 6-9 when organic solvent is used to precipitate the protein in step (1).
4. The method for separating and purifying lipopeptide surfactin family compound according to any one of claims 1 to 3, wherein the fermentation broth in step (1): the volume ratio of the organic solvent is 1:1-1: 2.
5. The method for separating and purifying lipopeptide surfactin family compound as claimed in claim 1, wherein the acid in step (2) is H2SO4And HCl.
6. The method for separating and purifying lipopeptide surfactin family compound as claimed in claim 1 or 5, wherein in the step (2), acid is added to the fermentation broth after removing the organic solvent by rotary evaporation until the pH value of the system is 2-3.
7. The method for separating and purifying lipopeptide surfactin family compound as claimed in any one of claims 1, 5 or 6, wherein step (2) comprises subjecting the precipitate to 1-5 acid washing.
8. The method for separating and purifying lipopeptide surfactin family compound as claimed in any one of claims 1-7, which can obtain surfactin and lichenin.
9. A surfactant comprising the surfactin and lichenin of claim 8.
10. The method for separating and purifying lipopeptide surfactin family compound as described in any one of claims 1 to 7, which is applied to the fields of food daily chemical industry, environmental engineering, oil extraction industry, cosmetics and medicine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112079899A (en) * | 2020-09-30 | 2020-12-15 | 陕西科技大学 | Method for separating antibacterial lipopeptide from bacillus amyloliquefaciens fermentation liquor |
| CN112851754A (en) * | 2021-01-06 | 2021-05-28 | 铜仁职业技术学院 | Method for separating and purifying surfactin by extraction-back extraction |
| CN114276793A (en) * | 2021-12-28 | 2022-04-05 | 大连理工大学 | Environment-friendly natural gas hydrate inhibitor and application thereof |
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| CN112079899A (en) * | 2020-09-30 | 2020-12-15 | 陕西科技大学 | Method for separating antibacterial lipopeptide from bacillus amyloliquefaciens fermentation liquor |
| CN112079899B (en) * | 2020-09-30 | 2022-04-08 | 陕西科技大学 | Method for separating antibacterial lipopeptide from bacillus amyloliquefaciens fermentation liquor |
| CN112851754A (en) * | 2021-01-06 | 2021-05-28 | 铜仁职业技术学院 | Method for separating and purifying surfactin by extraction-back extraction |
| CN112851754B (en) * | 2021-01-06 | 2023-04-25 | 铜仁职业技术学院 | Extraction-back extraction separation and purification method for surfactin |
| CN114276793A (en) * | 2021-12-28 | 2022-04-05 | 大连理工大学 | Environment-friendly natural gas hydrate inhibitor and application thereof |
| CN114276793B (en) * | 2021-12-28 | 2022-09-20 | 大连理工大学 | Environment-friendly natural gas hydrate inhibitor and application thereof |
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