CN111662943A - Preparation method of lipopeptide biosurfactant - Google Patents
Preparation method of lipopeptide biosurfactant Download PDFInfo
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- CN111662943A CN111662943A CN202010391728.7A CN202010391728A CN111662943A CN 111662943 A CN111662943 A CN 111662943A CN 202010391728 A CN202010391728 A CN 202010391728A CN 111662943 A CN111662943 A CN 111662943A
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- lipopeptide
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- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Abstract
The invention discloses a preparation method of a lipopeptide biosurfactant, wherein the lipopeptide biosurfactant is a lipopeptide biosurfactant product obtained by fermenting and culturing bacillus subtilis strains, and the preparation method comprises the following steps: (1) constructing a lipopeptide-producing bacillus subtilis strain; (2) primary fermentation culture; (3) secondary fermentation culture; (4) and (5) separating and purifying. The preparation method is simple and has short period. The prepared lipopeptide biosurfactant has good tolerance to temperature and mineralization degree and strong bioactivity.
Description
Technical Field
The invention relates to a preparation process of a biosurfactant, in particular to a preparation method of a lipopeptide biosurfactant.
Background
The surfactant is a substance capable of remarkably changing the surface tension of a liquid or the interfacial tension between two phases, and has good emulsification, foaming, defoaming, solubilization, dispersion and other effects, so that the surfactant is widely applied to various fields of national economy and almost covers all fine chemical engineering fields. As the demand for these products increases, the global surfactant market is in a steady state of development. Synthetic surfactants are typically produced using petroleum substrates and are widely used in food, crop protection, soap and detergent, cleaning, oilfield chemicals, personal care, plastics, pharmaceuticals, rubber, textile, paper, ink and printing industries. At present, the demand and consumption of the synthetic surfactant are relatively stable, market analysis considers that the demand of the part of products is increased mainly because of lower cost and easy preparation, and in addition, a large amount of offshore drilling activities and shale gas exploitation increase, which provide abundant raw materials for the production of the synthetic surfactant and further promote the development of the market.
In consideration of environmental sustainability and increasingly severe global environmental situation, various countries are continuously strengthening the control and management of environmental problems, and strict environmental laws and regulations are issued and incorporated into long-term development plans. The demand for synthetic surfactants will be severely impacted in the coming years and as concerns over the use of synthetic surfactants increase, many environmentally friendly and biodegradable products are emerging on the market. The environmental and health risks that may be posed by synthetic surfactants have caused many governments and industry people to resume their consideration of the biodegradability of surfactants, thereby creating a global hot and commercial opportunity for the development of biosurfactants.
The european union has established a biosurfactant market and is in a global lead in terms of both consumption and throughput, and therefore the european union also sets forth numerous regulatory regimes for assessing and controlling the use of surfactants in different areas, such as REACH (chemical registration, assessment, licensing and restriction) for ensuring that all commercially available detergents, soaps and care products are not harmful to the end user and the environment. According to REACH regulations, surfactant manufacturers need risk assessment of traditional synthetic surfactants and require them to produce environmentally friendly biodegradable surfactants, which all contribute significantly to the development of the biosurfactant market. Besides the promotion effect of the newly established regulation on the biosurfactant market, many other factors promote the biosurfactant market development, for example, the consumer knows the biological products more clearly and the demand is increased gradually; crude oil price fluctuations; economic resurgence and increased consumer expenditures for everyday items.
Over the last three years, the european biosurfactant market has accounted for 50% of the global market. The market currently maintains an average annual growth rate of substantially 6%. In 2015, the global biosurfactant market has a total value of $ 19.5 billion and a sales volume of $ 35.7 million tons, while in 2015, the global biosurfactant market has a total value of $ 340 billion and a sales volume of 1480 million tons. At present, the market of the biosurfactant accounts for less than 6 percent of the market share of the biosurfactant, along with the severe trend of environmental protection, the exhaustion of the traditional fossil energy, the green concept of people is firm, the trend that the biosurfactant replaces the traditional petroleum-based surfactant is obvious, the whole market of the biosurfactant is continuously amplified and continuously increased, and the industry is in the trend of accumulating and waiting for outbreak of thin spray.
However, the biosurfactant market is fraught with opportunities and faces many challenges. The use of biosurfactants is limited, for example, by long production cycle, high cost, complex process, few suppliers, etc. However, the economic and social value that biosurfactants can create in the future can help the market overcome these challenges.
Besides being paid attention to in some special fields such as petroleum industry and environmental engineering, the biosurfactant not only gradually replaces chemically synthesized surfactants in some fields along with the rapid development of biotechnology and related technical means, but also breaks through the application field of the traditional surfactant, the research of the biosurfactant is in the initial stage in China and is also an emerging field in developed countries, the industrialization level of the biosurfactant is low, the technology is immature, and the biosurfactant has great development potential. Microorganisms have shorter generation times than animal and plant growth rates, and thus it is easier to produce biosurfactants by microorganisms. Therefore, the microbial fermentation is used for producing the excellent biosurfactant serving as a surfactant family to replace a chemically synthesized surfactant or serve as an upgrading and updating product of the chemically synthesized surfactant, and the method has great social benefit, economic benefit and ecological benefit.
Disclosure of Invention
The invention mainly aims to overcome the defects of high cost and complex process in the existing biosurfactant preparation process group and provide a preparation method of a lipopeptide biosurfactant. The preparation process of the lipopeptide biosurfactant utilizes a compound mutagenesis technology to breed high-yield strains, screens out high-efficiency strains, establishes a method for rapidly detecting the high-yield strains of the biosurfactant and evaluating the potential of the high-yield strains, and provides conditions for the production and research of the lipopeptide biosurfactant.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme.
The invention provides a preparation method of a lipopeptide biosurfactant, wherein the lipopeptide biosurfactant is a lipopeptide biosurfactant product obtained by fermenting and culturing bacillus subtilis strains, and the preparation method comprises the following steps:
(1) construction of lipopeptide-producing Bacillus subtilis strains: acquiring a gene sequence of the lipopeptide produced by the bacillus subtilis, carrying out PCR amplification, constructing a plasmid containing the gene, and then introducing the plasmid into a host bacillus subtilis cell to complete construction of a lipopeptide-producing bacillus subtilis strain;
(2) primary fermentation culture: inoculating the constructed bacillus subtilis strain into a shake flask for culture, inoculating 1-ring strain into every 500ml of culture medium in the shake flask, and fermenting under a certain condition to obtain a primary shake flask strain;
(3) secondary fermentation culture: transferring the shake flask strain prepared in the step into a fermentation tank filled with a culture medium, wherein the volume ratio of the shake flask strain to the culture medium is 1:20, and performing fermentation culture under certain conditions to obtain a secondary fermentation strain;
(4) separation and purification: and centrifuging the fermentation liquor obtained in the step to remove thalli, adding chloroform/methanol (1-2/1-3, V/V) with the same volume into supernate, extracting, taking the lower layer liquor of the extraction liquid, carrying out reduced pressure evaporation at 50-70 ℃ to obtain a crude product of the biosurfactant, washing chloroform again, and evaporating at 50-70 ℃ to obtain a relatively pure lipopeptide biosurfactant.
In some preferred embodiments of the present invention, the formulation of the shake flask culture medium in the step (2) is: 3-4% of rice bran oil and NH4NO 0.4-1.5%, glucose 20-30%, NaCl 0.05-1.0%, KH2PO40.05～1.0%、K2HPO40.05-1.0%, yeast extract 0.005-0.1%, composite trace element 0.005-0.01%, and balance water, and the pH is 6.0-7.5.
In some preferred embodiments of the invention, the composite trace element is mainly compounded by 1.0g/L of zinc oxide, 1.5g/L of tin oxide, 1.2g/L of manganese sulfate, 1.1g/L of copper chloride, 0.9g/L of cobalt oxide and water.
In some preferred embodiments of the present invention, the fermentation conditions in the step (2) are: the temperature is 40-50 ℃, the pH is 7.0-7.5, the stirring speed is 120r/min, and the fermentation time is 72-96 h.
In some preferred embodiments of the present invention, the fermentation conditions in the step (3) are: the temperature is 40-50 ℃, the pH is 7.0-7.5, the stirring speed is 90r/min, and the fermentation time is 10-12 h.
In some preferred embodiments of the present invention, the medium formulation of the fermenter in the step (3) comprises: carbon source, nitrogen source, inorganic salt and nutrient elements.
In some preferred embodiments of the invention, the carbon source is selected from the group consisting of glucose, lactose, sucrose, starch, maltose, soy molasses, glycerol, sucrose molasses, and mixtures of one or more thereof.
In some preferred embodiments of the invention, the nitrogen source is selected from the group consisting of soybean cake meal, peptone, beef powder, corn steep liquor, yeast powder, ammonium nitrate, ammonium sulfate, urea, and mixtures thereof.
In some preferred embodiments of the invention, the inorganic salts and the nutrient elements are selected from K2HPO4、NaH2PO4、MgSO4、FeSO4、CaCO3、CaCl2KCl and NaCl.
In some preferred embodiments of the present invention, the centrifugation conditions in step (4) are; the rotating speed is 8000-10000 r/min, and the time is 10-20 min.
By the technical scheme, the invention at least has the following advantages: the invention is an improvement of the prior art, and the lipopeptide biosurfactant is a lipopeptide biosurfactant product obtained by fermenting and culturing bacillus subtilis strains. The lipopeptide biosurfactant provided by the invention has the advantages of simple preparation method, mild conditions and short culture period. The obtained biological surfactant of the lipopeptide has high purity, strong activity, good tolerance to temperature and salt and wide application prospect.
In conclusion, the preparation method of the special lipopeptide biosurfactant can effectively improve the yield of products and shorten the culture period. The method has the advantages and practical value, does not have similar design publication or use but is really innovative in the similar method, has great improvement on the method or the function, has great technical progress, produces good and practical effects, and has multiple enhanced effects compared with the prior method, thereby being more suitable for practical use, having industrial wide utilization value and being a novel, improved and practical new design.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given of preferred embodiments of the present invention with reference to the accompanying drawings.
Drawings
FIG. 1 is a CMC value for the critical micelle concentration of a lipopeptide biosurfactant;
FIG. 2 is a graph of the thermal stability of a lipopeptide biosurfactant;
FIG. 3 is a graph showing the effect of mineralization on interfacial tension of aqueous lipopeptide solutions.
Detailed Description
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Obtaining the gene sequence of the lipopeptide produced by the bacillus subtilis from a database, carrying out PCR amplification, constructing a plasmid containing the gene, and then introducing the plasmid into host bacillus subtilis cells to complete the construction of the lipopeptide-producing bacillus subtilis strain. Then culturing by taking the constructed lipopeptide-producing bacillus subtilis as a strain, wherein the culturing process is totally divided into primary fermentation culture and secondary fermentation culture. The primary fermentation culture process comprises the step of inoculating the constructed bacillus subtilis strain into a shake flask for culture, wherein the formula of a shake flask culture medium is as follows: rice bran oil 3%, NH4NO 1.5%, glucose 30%, NaCl 0.05%, KH2PO41.0%、K2HPO40.05 percent, 0.005 percent of yeast extract, 0.01 percent of composite trace elements (formed by compounding 1.0g/L of zinc oxide, 1.5g/L of tin oxide, 1.2g/L of manganese sulfate, 1.1g/L of copper chloride, 0.9g/L of cobalt oxide and water), the balance of water, and the pH value of the mixture is 6.0-7.5. Inoculating 1-ring strain into every 500ml of culture medium in a shake flask, and fermenting for 96h at the temperature of 50 ℃, the pH value of 7.0-7.5 and the stirring speed of 120r/min to obtain a primary shake flask strain. Secondary fermentation culture: transferring the shake flask strain prepared in the above steps into a fermentation tank filled with a culture medium, wherein the culture medium formula of the fermentation tank comprises: glucose 50%, peptone 7%, K2HPO42%、NaH2PO43%、MgSO41.5%、FeSO41.2 percent and the balance of water. The volume ratio of the shake flask strain to the culture medium is 1:20, and the secondary fermentation strain is obtained by fermentation culture for 10 hours at the temperature of 40 ℃, the pH value of 7.0-7.5 and the stirring speed of 90 r/min. Centrifuging the obtained fermentation liquor at 10000r/min for 10min to remove thalli, adding chloroform/methanol (1-2/1-3, V/V) with the same volume into supernate, extracting, taking the lower layer liquid of the extraction liquid, and evaporating under reduced pressure at 60 ℃ to dryness to obtain a crude biosurfactantAnd washing the mixture with chloroform at 60 ℃ again and evaporating the mixture to dryness to obtain the relatively pure lipopeptide biosurfactant.
Example 2
Obtaining the gene sequence of the lipopeptide produced by the bacillus subtilis from a database, carrying out PCR amplification, constructing a plasmid containing the gene, and then introducing the plasmid into host bacillus subtilis cells to complete the construction of the lipopeptide-producing bacillus subtilis strain. Then culturing by taking the constructed lipopeptide-producing bacillus subtilis as a strain, wherein the culturing process is totally divided into primary fermentation culture and secondary fermentation culture. The primary fermentation culture process comprises the step of inoculating the constructed bacillus subtilis strain into a shake flask for culture, wherein the formula of a shake flask culture medium is as follows: rice bran oil 4%, NH4NO 1.0%, glucose 25%, NaCl 0.08%, KH2PO41.0%、K2HPO41.0 percent of yeast extract, 0.1 percent of composite trace elements (formed by compounding 1.0g/L of zinc oxide, 1.5g/L of tin oxide, 1.2g/L of manganese sulfate, 1.1g/L of copper chloride, 0.9g/L of cobalt oxide and water), the balance of water, and the pH value of the mixture is 6.0-7.5. Inoculating 1-ring strain into every 500ml of culture medium in a shake flask, and fermenting for 72 hours at the temperature of 45 ℃, the pH value of 7.0-7.5 and the stirring speed of 120r/min to obtain a primary shake flask strain. Secondary fermentation culture: transferring the shake flask strain prepared in the above steps into a fermentation tank filled with a culture medium, wherein the culture medium formula of the fermentation tank comprises: glucose 50%, peptone 7%, K2HPO42%、NaH2PO43%、MgSO41.5%、FeSO41.2 percent and the balance of water. The volume ratio of the shake flask strain to the culture medium is 1:20, and the secondary fermentation strain is obtained by fermenting and culturing for 12 hours at the temperature of 50 ℃, the pH value of 7.0-7.5 and the stirring speed of 90 r/min. Centrifuging the obtained fermentation liquor at 10000r/min for 20min to remove thalli, adding chloroform/methanol (1-2/1-3, V/V) with the same volume into supernate, extracting, taking the subnatant of the extract, carrying out reduced pressure evaporation at 60 ℃ to obtain a crude product of the biosurfactant, washing the chloroform again, and evaporating to dryness at 60 ℃ to obtain a relatively pure lipopeptide biosurfactant.
Example 3
Obtaining the gene sequence of lipopeptide produced by bacillus subtilis from a database, carrying out PCR amplification, constructing a plasmid containing the gene, and then introducing the plasmid into a host bacillus subtilisAnd (3) completing the construction of the lipopeptide-producing bacillus subtilis strain in bacillus cells. Then culturing by taking the constructed lipopeptide-producing bacillus subtilis as a strain, wherein the culturing process is totally divided into primary fermentation culture and secondary fermentation culture. The primary fermentation culture process comprises the step of inoculating the constructed bacillus subtilis strain into a shake flask for culture, wherein the formula of a shake flask culture medium is as follows: rice bran oil 4%, NH4NO 1.0%, glucose 25%, NaCl 0.08%, KH2PO41.0%、K2HPO41.0 percent of yeast extract, 0.1 percent of composite trace elements (formed by compounding 1.0g/L of zinc oxide, 1.5g/L of tin oxide, 1.2g/L of manganese sulfate, 1.1g/L of copper chloride, 0.9g/L of cobalt oxide and water), the balance of water, and the pH value of the mixture is 6.0-7.5. Inoculating 1-ring strain into every 500ml of culture medium in a shake flask, and fermenting for 84h at the temperature of 40 ℃, the pH value of 7.0-7.5 and the stirring speed of 120r/min to obtain a primary shake flask strain. Secondary fermentation culture: transferring the shake flask strain prepared in the above steps into a fermentation tank filled with a culture medium, wherein the culture medium formula of the fermentation tank comprises: glucose 40%, peptone 12%, K2HPO43%、NaH2PO44%、MgSO41.5%、FeSO41.2 percent and the balance of water. The volume ratio of the shake flask strain to the culture medium is 1:20, and the secondary fermentation strain is obtained by fermenting and culturing for 12 hours at the temperature of 45 ℃, the pH value of 7.0-7.5 and the stirring speed of 90 r/min. Centrifuging the obtained fermentation liquor at 8000r/min for 20min to remove thalli, adding chloroform/methanol (1-2/1-3, V/V) with the same volume into supernate, extracting, taking the subnatant of the extract, carrying out reduced pressure evaporation at 60 ℃ to obtain a crude product of the biosurfactant, washing chloroform again, and evaporating to dryness at 60 ℃ to obtain a relatively pure lipopeptide biosurfactant.
Example 4
Obtaining the gene sequence of the lipopeptide produced by the bacillus subtilis from a database, carrying out PCR amplification, constructing a plasmid containing the gene, and then introducing the plasmid into host bacillus subtilis cells to complete the construction of the lipopeptide-producing bacillus subtilis strain. Then culturing by taking the constructed lipopeptide-producing bacillus subtilis as a strain, wherein the culturing process is totally divided into primary fermentation culture and secondary fermentation culture. The primary fermentation culture process comprises inoculating the Bacillus subtilis strain into shake flask for culture, and shake flask cultureThe formula of the nutrient medium is as follows: rice bran oil 4%, NH4NO 1.0%, glucose 20%, NaCl 1.0%, KH2PO40.05%、K2HPO41.0 percent of yeast extract, 0.1 percent of composite trace elements (formed by compounding 1.0g/L of zinc oxide, 1.5g/L of tin oxide, 1.2g/L of manganese sulfate, 1.1g/L of copper chloride, 0.9g/L of cobalt oxide and water), the balance of water, and the pH value of the mixture is 6.0-7.5. Inoculating 1-ring strain into every 500ml of culture medium in a shake flask, and fermenting for 96h at the temperature of 40 ℃, the pH value of 7.0-7.5 and the stirring speed of 120r/min to obtain a primary shake flask strain. Secondary fermentation culture: transferring the shake flask strain prepared in the above steps into a fermentation tank filled with a culture medium, wherein the culture medium formula of the fermentation tank comprises: glucose 40%, peptone 12%, K2HPO43%、NaH2PO44%、MgSO41.5%、FeSO41.2 percent and the balance of water. The volume ratio of the shake flask strain to the culture medium is 1:20, and the secondary fermentation strain is obtained by fermentation culture for 10 hours at the temperature of 50 ℃, the pH value of 7.0-7.5 and the stirring speed of 90 r/min. Centrifuging the obtained fermentation liquor at 8000r/min for 10min to remove thalli, adding chloroform/methanol (1-2/1-3, V/V) with the same volume into supernate, extracting, taking the subnatant of the extract, carrying out reduced pressure evaporation at 60 ℃ to obtain a crude product of the biosurfactant, washing chloroform again, and evaporating to dryness at 60 ℃ to obtain a relatively pure lipopeptide biosurfactant.
Test example 1 evaluation of Properties of lipopeptide biosurfactant
Test products: lipopeptide biosurfactant prepared in example 1
1. Measurement of lipopeptide surfactant Critical Micelle Concentration (CMC)
Determination of critical micelle concentration: the purified sample obtained in example 1 was diluted with water in equal times, and when the concentration of the solution was less than 0.035mg/mL, the surface tension of water decreased accordingly as the concentration increased, and when the concentration of the sample was more than 0.035mg/mL, the surface tension of water could not be further decreased by increasing the concentration of the sample, indicating that the critical micelle concentration of the sample was about 0.035mg/mL, and the results are shown in FIG. 1.
2. Effect of temperature and mineralization on lipopeptide biosurfactants
(1) Temperature of
Preparation of CMC (Critical micelle concentration, 35 mg. L) with distilled Water pH7.0-1) Aqueous lipopeptide solutions at concentrations useful for determining the effect of temperature on the surface activity of a lipopeptide biosurfactant. The interfacial tension values were measured after cooling to room temperature at different temperatures for different times (1 or 2 h). The result shows that the temperature is 50-120 ℃ and has no influence on the interfacial tension of the lipopeptide after being treated for 1-2 hours, and the interfacial tension of the lipopeptide biosurfactant is still not reduced after being subjected to heat treatment for two hours at 120 ℃, which indicates that the lipopeptide biosurfactant has stronger tolerance to temperature change, and the result is shown in figure 2.
(2) Degree of mineralization
At CMC (critical micelle concentration, 35 mg. L)-1) NaCl was added to the lipopeptide aqueous solution at a concentration of 0.0 wt%, 2.0 wt%, 4.0 wt%, 6.0 wt%, 8.0 wt%, 10.0 wt%, 12.0 wt%, 14.0 wt%, 16.0 wt%, 20.0 wt%, and the interfacial tension values of the lipopeptide solutions at different NaCl concentrations were measured at 45 ℃. The results show that NaCl had no substantial effect on its surface tension until the concentration reached 14.0%. However, when the NaCl concentration is further increased, the surface activity is gradually suppressed and the interfacial tension value is gradually increased. The above results show that the lipopeptide biosurfactant has very high salt tolerance, and salt concentrations up to 14.0 wt% have substantially no effect on the surface tension of the fermentation broth, and the results are shown in FIG. 3.
In conclusion, the lipopeptide biosurfactant of the invention has good tolerance and mineralization degree. The lipopeptide biosurfactant is prepared by fermenting and culturing bacillus subtilis strains. The lipopeptide biosurfactant provided by the invention has the advantages of simple preparation method, mild conditions and short culture period. The obtained biological surfactant of the lipopeptide has high purity, good tolerance to temperature and salt and wide application prospect.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for preparing lipopeptide biosurfactant is characterized in that the lipopeptide biosurfactant is obtained by fermenting and culturing bacillus subtilis strains, and the method comprises the following steps:
(1) construction of lipopeptide-producing Bacillus subtilis strains: acquiring a gene sequence of the lipopeptide produced by the bacillus subtilis, carrying out PCR amplification, constructing a plasmid containing the gene, and then introducing the plasmid into a host bacillus subtilis cell to complete construction of a lipopeptide-producing bacillus subtilis strain;
(2) primary fermentation culture: inoculating the constructed bacillus subtilis strain into a shake flask for culture, inoculating 1-ring strain into every 500ml of culture medium in the shake flask, and fermenting under a certain condition to obtain a primary shake flask strain;
(3) secondary fermentation culture: transferring the shake flask strain prepared in the step into a fermentation tank filled with a culture medium, wherein the volume ratio of the shake flask strain to the culture medium is 1:20, and performing fermentation culture under certain conditions to obtain a secondary fermentation strain;
(4) separation and purification: and centrifuging the fermentation liquor obtained in the step to remove thalli, adding chloroform/methanol (1-2/1-3, V/V) with the same volume into supernate, extracting, taking the lower layer liquor of the extraction liquid, carrying out reduced pressure evaporation at 50-70 ℃ to obtain a crude product of the biosurfactant, washing chloroform again, and evaporating at 50-70 ℃ to obtain a relatively pure lipopeptide biosurfactant.
2. The preparation method according to claim 1, wherein the formulation of the shake flask culture medium in the step (2) is: 3-4% of rice bran oil and NH40.4-1.5% of NO and grape20-30% of sugar, 0.05-1.0% of NaCl and KH2PO40.05～1.0%、K2HPO40.05-1.0%, yeast extract 0.005-0.1%, composite trace element 0.005-0.01%, and balance water, and the pH is 6.0-7.5.
3. The preparation method of claim 2, wherein the composite trace element is mainly compounded by 1.0g/L of zinc oxide, 1.5g/L of tin oxide, 1.2g/L of manganese sulfate, 1.1g/L of copper chloride, 0.9g/L of cobalt oxide and water.
4. The production method according to claim 1, wherein the fermentation conditions in the step (2) are: the temperature is 40-50 ℃, the pH is 7.0-7.5, the stirring speed is 120r/min, and the fermentation time is 72-96 h.
5. The production method according to claim 1, wherein the fermentation conditions in the step (3) are: the temperature is 40-50 ℃, the pH is 7.0-7.5, the stirring speed is 90r/min, and the fermentation time is 10-12 h.
6. The manufacturing method according to claim 1, wherein the culture medium formulation of the fermenter in the step (3) includes: carbon source, nitrogen source, inorganic salt and nutrient elements.
7. The method according to claim 6, wherein the carbon source is selected from the group consisting of glucose, lactose, sucrose, starch, maltose, soy molasses, glycerol, and sucrose molasses.
8. The method according to claim 6, wherein the nitrogen source is selected from the group consisting of soybean cake powder, peptone, beef powder, corn steep liquor, yeast powder, ammonium nitrate, ammonium sulfate and urea.
9. The method according to claim 6, wherein the inorganic salt and the nutrient are selected from K2HPO4、NaH2PO4、MgSO4、FeSO4、CaCO3、CaCl2KCl and NaCl.
10. The production method according to claim 1, wherein the centrifugation conditions in the step (4) are; the rotating speed is 8000-10000 r/min, and the time is 10-20 min.
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