Fermentation medium for increasing surfactin content, application thereof and fermentation method

Abstract

The present application provides a fermentation medium for increasing the content of surfactin, its application and fermentation method. The fermentation medium is used for fermentation of Bacillus subtilis; the fermentation medium includes the following substances in weight percentage: 4-7% carbon source; 0.65-2.25% nitrogen source; 0.13-0.6% phosphate; 0.015-0.05% magnesium sulfate; 0.0007-0.02% calcium chloride; 0.0005-0.001% manganese sulfate; 0.0006-0.06% iron sulfate; 0.5-1% amino acid; 0.5-3% soybean fiber, and the balance is water; wherein the pH of the fermentation medium is 6.5-7; the inoculation amount of Bacillus subtilis liquid in the fermentation medium is 3-5% by volume fraction; wherein the carbon source is selected from at least one of molasses, glucose, brown sugar, maltodextrin and starch; the nitrogen source includes a combination of at least one of corn slurry powder, yeast extract and yeast powder and urea.

Description

Technical Field

[0001] The present application relates to the technical field of fermentation engineering, and in particular to a fermentation medium for increasing the content of surfactin, its application and fermentation method. Background Art

[0002] Biosurfactants are hydrophobic and hydrophilic metabolites produced by bacteria, yeast, fungi and other microorganisms from various substrates (sugars, oils, alkanes, etc.). They can reduce surface and interfacial tension, produce micelles, and enhance the solubility of hydrocarbons in water or water in hydrocarbons. Biosurfactants have received widespread attention in recent years due to their low toxicity, surface activity and environmental friendliness.

[0003] Surfactin is one of the most effective lipopeptide biosurfactants produced by various strains of Bacillus subtilis. Its main structure is a cyclic lipopeptide composed of 12-16 carbon chain length fatty acids and 7 amino acids connected by lactone bonds. In addition to its high surface activity, low toxicity, high biodegradability and biocompatibility, surfactin also has antibacterial, antiviral, antitumor properties and inhibits the formation of fibrin clots. These excellent properties make it have broad application prospects in the fields of petroleum industry, environmental control, cosmetics, agriculture, medicine, etc.

[0004] In the related art, the production of surfactin is low. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide a fermentation medium for increasing the content of surfactin, its application and fermentation method.

[0006] Based on the above purpose, the present application provides a fermentation medium for increasing the content of surfactin, the fermentation medium is used for fermentation of Bacillus subtilis; the fermentation medium comprises the following substances in weight percentage: 4-7% carbon source; 0.65-2.25% nitrogen source; 0.13-0.6% phosphate; 0.015-0.05% magnesium sulfate; 0.0007-0.02% calcium chloride; 0.0005-0.001% manganese sulfate; 0.0006-0.06% iron sulfate; 0.5-1% amino acid; 0.5-3% soybean fiber, and the balance is water; wherein the pH of the fermentation medium is 6.5-7; the inoculation amount of the Bacillus subtilis liquid in the fermentation medium is 3-5% by volume fraction;

[0007] The carbon source is selected from at least one of molasses, glucose, brown sugar, maltodextrin and starch; the nitrogen source includes a combination of at least one of corn syrup powder, yeast extract and yeast powder and urea.

[0008] In some embodiments, the soybean fiber is added in an amount of 1-3%.

[0009] In some embodiments, the nitrogen source is selected from a combination of yeast extract and urea; the mass percentage of the yeast extract is 0.45-0.55%; the mass percentage of the urea is 0.45-0.55%.

[0010] In some embodiments, the carbon source is selected from brown sugar, and the mass percentage of the brown sugar is 4-7%.

[0011] In some embodiments, the phosphate is selected from a combination of potassium dihydrogen phosphate and disodium hydrogen phosphate; and the amino acid is selected from leucine.

[0012] The present application also provides an application of the fermentation medium with increased surfactin content as described in any of the preceding items to increase the surfactin content in a fermentation product of Bacillus subtilis.

[0013] The present application also provides a fermentation method for increasing the content of surfactin, comprising:

[0014] Inoculating Bacillus subtilis into a liquid seed culture medium, and performing seed culture for 16-18 hours to obtain a fermentation strain;

[0015] Inoculate the fermentation bacteria into the fermentation medium and carry out fermentation culture for 22-26 hours; wherein the inoculation amount of the fermentation bacteria is 3-5% by volume; and the fermentation medium is the fermentation medium described in any of the above embodiments;

[0016] Feed medium was added and fed-batch culture was carried out for 46-50 h.

[0017] In some embodiments, the feed medium includes brown sugar, urea and leucine; the feed medium is added in an amount such that the ratio of the added volume of the feed medium to the volume of the fermentation medium is 10-30%.

[0018] In some embodiments, the feed medium comprises the following substances in percentage by weight: 20-35% brown sugar, 1-2.5% urea and 4-5% leucine.

[0019] In some embodiments, the culture temperature in the seed culture, the fermentation culture and the fed-batch culture is 30-40°C.

[0020] As can be seen from the above, the fermentation medium for increasing the content of surfactin provided by the present application is used for fermentation of Bacillus subtilis; the fermentation medium is provided to include the following substances in weight percentage: carbon source 4-7%; nitrogen source 0.65-2.25%; phosphate 0.13-0.6%; magnesium sulfate 0.015-0.05%; calcium chloride 0.0007-0.02%; manganese sulfate 0.0005-0.001%; ​​iron sulfate 0.0006-0.06%; amino acid 0.5-1%; soybean fiber 0.5- 4%, and the balance is water; wherein the pH of the fermentation medium is 6.5-7; the inoculation amount of the Bacillus subtilis culture in the fermentation medium is 3-5% by volume; wherein the carbon source is selected from at least one of molasses, glucose, brown sugar, maltodextrin and starch; and the nitrogen source comprises a combination of at least one of corn slurry powder, yeast extract and yeast powder and urea, which can significantly increase the yield of surfactant produced by Bacillus subtilis fermentation while inhibiting foam, thereby facilitating industrial large-scale production and having great industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 Schematic diagram of the process of the fermentation method for increasing the content of surfactin according to an embodiment of the present application.

[0023] Figure 2 The results are the significant differences between Example 1, Example 2 and Example 4.

[0024] Figure 3 It is the foam suppression effect diagram in Example 2 and Example 3. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0026] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should be understood by people with ordinary skills in the field to which the present application belongs. The "first", "second" and similar words used in the embodiments of the present application do not represent any order, quantity or importance, but are only used to distinguish different components. "Including" or "comprising" and similar words mean that the elements or objects appearing in front of the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

[0027] Although surfactin has important characteristics that are widely used in industry, its high production cost and low yield in actual production limit its application. Therefore, improving the yield of surfactin and reducing production costs remain the fundamental solutions for the commercial application of surfactin. In the related art, surfactin is produced by fermentation of Bacillus subtilis. Since it requires ventilation and stirring, it is easy to produce foam, so there are problems such as easy foaming and low surfactin yield. Optimization of culture medium formula is one of the effective means to increase the yield of surfactin.

[0028] Based on this, the present invention provides a fermentation medium and a fermentation method for increasing the content of surfactant, and by adding soybean fiber, the yield of surfactant produced by Bacillus subtilis fermentation can be significantly increased while suppressing foam, thereby facilitating industrial large-scale production and having great industrial application prospects. It can solve the current problems of low yield and high cost of surfactant to a certain extent.

[0029] The embodiment of the present application provides a fermentation medium for increasing the content of surfactin, the fermentation medium is used for fermentation of Bacillus subtilis; the fermentation medium comprises the following substances in weight percentage: 4-7% carbon source; 0.65-2.25% nitrogen source; 0.13-0.6% phosphate; 0.015-0.05% magnesium sulfate; 0.0007-0.02% calcium chloride; 0.0005-0.001% manganese sulfate; 0.0006-0.06% iron sulfate; 0.5-1% amino acid; 0.5-4% soybean fiber, and the balance is water; wherein the pH of the fermentation medium is 6.5-7; the inoculation amount of the Bacillus subtilis liquid in the fermentation medium is 3-5% by volume fraction;

[0030] The carbon source is selected from at least one of molasses, glucose, brown sugar, maltodextrin and starch; the nitrogen source includes a combination of at least one of corn syrup powder, yeast extract and yeast powder and urea.

[0031] In some embodiments, the carbon source can be selected from brown sugar, and the mass percentage of the brown sugar can be 4-7%. Compared with molasses, glucose, maltodextrin and starch, this mass percentage of brown sugar can better increase the content of surfactin obtained by fermentation of Bacillus subtilis.

[0032] In some embodiments, the nitrogen source is selected from a combination of yeast extract and urea; the mass percentage of the yeast extract is 0.45-0.55%; the mass percentage of the urea is 0.45-0.55%. Compared with corn syrup powder and yeast powder, the yeast extract can better increase the content of surfactant obtained by fermentation of Bacillus subtilis.

[0033] In some embodiments, the phosphate may be selected from a combination of potassium dihydrogen phosphate and disodium hydrogen phosphate. The mass percentage of potassium dihydrogen phosphate may be 0.03%, and the mass percentage of disodium hydrogen phosphate may be 0.1%.

[0034] In some embodiments, the amino acid can be selected from leucine. As a component of surfactin, leucine can provide an essential component for the growth of surfactin, thereby better increasing the content of surfactin obtained by fermentation of Bacillus subtilis.

[0035] In some embodiments, the soybean fiber can be added in an amount of 1-3%, which can better increase the content of surfactant obtained by fermentation of Bacillus subtilis. The addition of soybean fiber can increase the viscosity of the fermentation medium, making the fermentation medium more viscous, thereby inhibiting the foam generated during the fermentation process to a certain extent, and at the same time promoting the growth of Bacillus subtilis cells and the production of the product surfactant.

[0036] Based on the same inventive concept, the present application also provides an application of the aforementioned fermentation medium with increased surfactin content to increase the surfactin content in a fermentation product of Bacillus subtilis. The Bacillus subtilis may be the Bacillus subtilis with a deposit number of CGMCC No.24947.

[0037] Based on the same inventive concept, the present application also provides a fermentation method for increasing the content of surfactin, such as Figure 1 As shown, this may include:

[0038] Step S100, inoculating Bacillus subtilis into a liquid seed culture medium, and performing seed culture for 16-18 hours to obtain a fermentation strain;

[0039] Step S200, inoculating the fermentation bacteria into the fermentation medium, and fermenting and culturing for 22-26 hours; wherein the inoculation amount of the fermentation bacteria is 3-5% by volume; and the fermentation medium is the fermentation medium as described in any of the previous embodiments;

[0040] Step S300, adding feed medium and performing fed-batch culture for 46-50 hours.

[0041] In some embodiments, the feed medium includes brown sugar, urea and leucine; the feed medium is added in an amount such that the ratio of the added volume of the feed medium to the volume of the fermentation medium is 9-11%.

[0042] In some embodiments, the feed medium includes the following substances in weight percentage: 20-35% brown sugar, 1-2.5% urea and 4-5% leucine. Generally, since the fermentation medium itself is already relatively viscous, the viscous state can be maintained without adding soy fiber in the subsequent feed medium, and it can also have a certain ability to inhibit foaming and promote the growth of Bacillus subtilis cells and the production of product surfactin.

[0043] In some embodiments, the dissolved oxygen content in the seed culture, the fermentation culture and the fed-batch culture can be controlled to be DO>20%, and the culture temperature can be 30-40° C. Under the shaking table culture condition, the shaking table speed can be 180-220 r / min. Under the fermenter culture condition, the speed can be 300-500 rpm.

[0044] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0045] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0046] The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the following examples, Bacillus subtilis was used as the starting strain, and the Bacillus subtilis may be the Bacillus subtilis with a deposit number of CGMCC No. 24947. In the following examples, the lipopeptide content was determined by high performance liquid chromatography (HPLC).

[0047] Specifically, HPLC quantitative assays may include:

[0048] (1) Chromatographic conditions: chromatographic column Shimadzu C18 ODS-3 (4.6 mm × 250 mm, 5 μm), flow rate 1.0 mL / min, detection wavelength 205 nm, column temperature room temperature. Mobile phase: 0.1% TFA-acetonitrile-0.1% TFA; Instrument: Shimadzu L-033 chromatograph; Gradient setting: 0-7 min, A (10%-90%), B (90%-10%); 7-24 min, A (90%), B (10%); 24-29 min, A (90%-10%), B (10%-90%); 29-36 min, A (10%), B (90%).

[0049] (2) Determination method: After the fermentation of Bacillus subtilis in the fermentation medium is completed, the fermentation liquid is collected, extracted with ethanol, filtered through a filter membrane after alcohol extraction, and the content is determined by the above-mentioned detection method.

[0050] Example 1: Effect of nitrogen source on surfactin production

[0051] Experimental method: A single colony of Bacillus subtilis was inoculated into LB liquid culture medium and cultured at 37°C, 200rpm for 16-18h, inoculated into the following fermentation medium with different nitrogen sources at an inoculum rate of 4v / v%, fermented at 37°C, 200rpm for 24h, and a feed medium with a volume of 10% of the initial culture medium volume was added, and cultured at 37°C, 200rpm until the end of the fermentation for 48h.

[0052] Basic components (i.e. fermentation medium components): 6.5% maltodextrin, 0.5% urea, 0.03% potassium dihydrogen phosphate, 0.1% disodium hydrogen phosphate, 0.015% magnesium sulfate, 0.0007% calcium chloride, 0.0006% manganese sulfate, 0.0006% iron sulfate, 0.5% leucine, pH 7. Add 0.5% corn steep liquor powder or yeast extract or yeast powder or other nitrogen source to the fermentation medium of the above composition.

[0053] The feed medium formula was: 32.5% maltodextrin, 2.5% urea, 5% leucine, and natural pH. The content of surfactin in the fermentation broth obtained after 48 hours of fermentation was determined by HPLC.

[0054] Experimental results: as shown in Table 1 below.

[0055] Table 1: Effects of different nitrogen sources on surfactin production

[0056]

[0057]

[0058] Results analysis: As shown in Table 1, adding different types of nitrogen sources to the fermentation medium has different effects on the surfactin content in the fermentation product of Bacillus subtilis. Among them, the surfactin content in the fermentation product of Bacillus subtilis obtained by adding yeast extract as a nitrogen source is the highest.

[0059] Example 2: Effect of Carbon Source on Surfactin Yield

[0060] Experimental method: A single colony of Bacillus subtilis was inoculated into LB liquid culture medium and cultured at 37°C, 200 rpm for 16-18 h. It was then inoculated into the following fermentation medium with different nitrogen sources at a rate of 4 v / v%, and fermented at 37°C, 200 rpm for 24 h. A feed medium with a volume of 10% of the initial culture medium volume was added, and the culture was continued at 37°C, 200 rpm until the end of the fermentation at 48 h.

[0061] Basic components (i.e. fermentation medium components): 0.5% yeast extract, 0.5% urea, 0.03% potassium dihydrogen phosphate, 0.1% disodium hydrogen phosphate, 0.015% magnesium sulfate, 0.0007% calcium chloride, 0.0006% manganese sulfate, 0.0006% iron sulfate, 0.5% leucine, pH 7. Add 7% of one of the carbon sources such as maltodextrin, glucose, brown sugar, molasses, starch, etc. to the above composition.

[0062] Feed medium formula: 2.5% urea, 5% leucine, natural pH. Add 35% of molasses or glucose or brown sugar or maltodextrin or starch and other carbon sources to the above composition.

[0063] The content of surfactin in the fermentation broth obtained after 48 h of fermentation was determined by HPLC.

[0064] Experimental results: See Table 2 and Figure 3 .in, Figure 3 The control group is a diagram of the foam state when the carbon source is brown sugar.

[0065] Table 2: Effects of different carbon sources on surfactin production

[0066]

[0067] Results analysis: As shown in Table 2, adding different types of carbon sources to the fermentation medium has different effects on the surfactin content in the fermentation product of Bacillus subtilis. Among them, the surfactin content in the fermentation product of Bacillus subtilis obtained by adding brown sugar as a carbon source is the highest.

[0068] Example 3: Effect of legume substances on surfactin production

[0069] Experimental method: A single colony of Bacillus subtilis was inoculated into LB liquid culture medium and cultured at 37°C, 200rpm for 16-18h, inoculated into the following fermentation medium with different nitrogen sources at an inoculum rate of 4v / v%, fermented at 37°C, 200rpm for 24h, and a feed medium with a volume of 10% of the initial culture medium volume was added, and cultured at 37°C, 200rpm until the end of the fermentation for 48h.

[0070] Basic components (i.e., components of fermentation medium): 7% brown sugar, 0.5% yeast extract, 0.5% urea, 0.03% potassium dihydrogen phosphate, 0.1% disodium hydrogen phosphate, 0.015% magnesium sulfate, 0.0007% calcium chloride, 0.0006% manganese sulfate, 0.0006% iron sulfate, 0.5% leucine, pH 7. 3% of one of the bean substances such as soybean meal, soybean powder, soybean fiber, etc. is added to the above composition.

[0071] Feed medium formula: brown sugar 35%, urea 2.5%, leucine 5%, natural pH.

[0072] The content of surfactin in the fermentation broth obtained after 48 h of fermentation was determined by HPLC.

[0073] Experimental results: See Table 3 and Figure 3 .

[0074] Table 3: Effects of different types of soybean substances on surfactin production

[0075]

[0076] Result analysis: As shown in Table 3, adding different types of soybean substances to the fermentation medium can inhibit foam to a certain extent, but the effects on the surfactin content in the fermentation product of Bacillus subtilis are different. Among them, after adding soybean powder and soybean fiber, compared with the control in Example 2 where the carbon source is brown sugar, only a small amount of foam can be inhibited. After adding soybean meal, compared with the control in Example 2 where the carbon source is brown sugar, a large amount of foam can be inhibited. Soybean meal is the residue left by soybeans during the oil extraction process, mainly containing protein, fiber, minerals and other components. Soybean powder is a powder directly processed from soybeans as raw materials, mainly containing protein, fat, carbohydrates, minerals and dietary fiber and other substances. Soybean fiber mainly refers to the general term for macromolecular sugars that cannot be digested by digestive enzymes, including cellulose, pectin, xylan, mannose and other substances. Therefore, adding soybean fiber as a soybean substance has a better effect of inhibiting foam, and the surfactin content in the obtained fermentation product of Bacillus subtilis is the highest.

[0077] Example 4: Effect of soybean fiber concentration on surfactin production

[0078] Experimental method: A single colony of Bacillus subtilis was inoculated into LB liquid culture medium and cultured at 37°C, 200rpm for 16-18h, inoculated into the following fermentation medium with different nitrogen sources at an inoculum rate of 4v / v%, fermented at 37°C, 200rpm for 24h, and a feed medium with a volume of 10% of the initial culture medium volume was added, and cultured at 37°C, 200rpm until the end of the fermentation for 48h.

[0079] Basic components (i.e., fermentation medium components): 7% brown sugar, 0.5% yeast extract, 0.5% urea, 0.03% potassium dihydrogen phosphate, 0.1% disodium hydrogen phosphate, 0.015% magnesium sulfate, 0.0007% calcium chloride, 0.0006% manganese sulfate, 0.0006% iron sulfate, 0.5% leucine, pH 7. Soybean fiber was added to the above composition at a concentration of 0.5%, 1%, or 3%.

[0080] Feed medium formula: brown sugar 35%, urea 2.5%, leucine 5%, natural pH.

[0081] The content of surfactin in the fermentation broth obtained after 48 h of fermentation was determined by HPLC.

[0082] Experimental results: See Table 4 and Figure 2 .

[0083] Table 4: Effects of different concentrations of soybean fiber on the surface active ingredients

[0084]

[0085] Results analysis: As shown in Table 4, adding different concentrations of soybean fiber to the fermentation medium has different effects on the surfactin content in the fermentation product of Bacillus subtilis. Among them, the surfactin content in the fermentation product of Bacillus subtilis obtained by adding 3% soybean fiber is the highest.

[0086] By comparing Example 4 with Example 1 and Example 2, it can be seen that after adding soybean fiber at a concentration of 3%, the surfactin content in the fermentation product of Bacillus subtilis increased by nearly 50% compared with the yeast extract type nitrogen source in Example 1 without adding soybean fiber. Compared with the brown sugar type carbon source in Example 2 without adding soybean fiber, the surfactin content in the fermentation product of Bacillus subtilis increased by nearly 30%.

[0087] Figure 2 is a comparison chart of the yields under the best conditions in Example 1, Example 2, and Example 4, Figure 2It can be seen that P≤0.001 between Example 1 and Example 2, Example 4 and Example 1, and Example 4 and Example 2, indicating that there are extremely significant statistical differences, indicating that adding soybean fiber can increase the content of surfactant while inhibiting foam.

[0088] Example 5: Production of surfactin in a 30L fermenter

[0089] Test method: a single colony of Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 16-18 hours, and then inoculated into the following fermentation medium at a rate of 4 v / v% for fermentation. After 16 hours of fermentation, feed medium was added and culture was continued until the end of the fermentation at 48 hours. Fermentation conditions: temperature 37°C, ventilation: 20m 2 / h, rotation speed: 300-500rpm, keep DO>20%, use NaOH to adjust and maintain pH above 6.0.

[0090] Basic components (i.e., components of the fermentation medium): brown sugar 7%, yeast extract 0.5%, urea 0.5%, potassium dihydrogen phosphate 0.03%, disodium hydrogen phosphate 0.1%, magnesium sulfate 0.015%, calcium chloride 0.0007%, manganese sulfate 0.0006%, iron sulfate 0.0006%, leucine 0.5%, pH 7. Soybean fiber 3%.

[0091] Feed medium formula: brown sugar 35%, urea 2.5%, leucine 5%, natural pH. The added volume of feed medium accounts for 10% of the volume of fermentation medium.

[0092] The fermentation broth obtained after 48 h of fermentation was determined by HPLC.

[0093] Experimental results: The measured surfactin content was 22.04 g / L.

[0094] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0095] While the disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications and variations of these embodiments will be apparent to those skilled in the art in light of the foregoing description.

[0096] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A fermentation medium for increasing the content of surfactin, characterized in that: The fermentation medium is used for fermentation of Bacillus subtilis; the fermentation medium comprises the following substances in weight percentage: 4-7% carbon source; 0.65-2.25% nitrogen source; 0.13-0.6% phosphate; 0.015-0.05% magnesium sulfate; 0.0007-0.02% calcium chloride; 0.0005-0.001% manganese sulfate; 0.0006-0.06% iron sulfate; 0.5-1% amino acid; 0.5-3% soybean fiber, and the balance is water; wherein the pH of the fermentation medium is 6.5-7; the inoculation amount of the Bacillus subtilis liquid in the fermentation medium is 3-5% by volume fraction; The carbon source is selected from at least one of molasses, glucose, brown sugar, maltodextrin and starch; the nitrogen source includes a combination of at least one of corn syrup powder, yeast extract and yeast powder and urea.

2. The fermentation medium for increasing the surfactin content according to claim 1, characterized in that: The added amount of the soybean fiber is 1-3%.

3. The fermentation medium for increasing the surfactin content according to claim 2, characterized in that: The nitrogen source is selected from a combination of yeast extract and urea; the mass percentage of the yeast extract is 0.45-0.55%; the mass percentage of the urea is 0.45-0.55%.

4. The fermentation medium for increasing the surfactin content according to claim 1, characterized in that: The carbon source is selected from brown sugar, and the mass percentage of the brown sugar is 4-7%.

5. The fermentation medium for increasing the surfactin content according to claim 1, characterized in that: The phosphate is selected from a combination of potassium dihydrogen phosphate and disodium hydrogen phosphate; and the amino acid is selected from leucine.

6. Use of the fermentation medium with increased surfactin content as claimed in any one of claims 1 to 5 in increasing the surfactin content in a fermentation product of Bacillus subtilis.

7. A fermentation method for increasing the content of surfactin, characterized in that: include: Inoculating Bacillus subtilis into a liquid seed culture medium, and performing seed culture for 16-18 hours to obtain a fermentation strain; Inoculate the fermentation strain into the fermentation medium and carry out fermentation culture for 22-26 hours; wherein the inoculation amount of the fermentation strain is 3-5% by volume; the fermentation medium is the fermentation medium according to any one of claims 1-6; Feed medium was added and fed-batch culture was carried out for 46-50 h.

8. The fermentation method for increasing the surfactin content according to claim 7, characterized in that: The feed medium comprises brown sugar, urea and leucine; the addition amount of the feed medium is such that the ratio of the addition volume of the feed medium to the volume of the fermentation medium is 9-11%.

9. The fermentation method for increasing the surfactin content according to claim 8, characterized in that: The feed medium comprises the following substances in percentage by weight: 20-35% brown sugar, 1-2.5% urea and 4-5% leucine.

10. The fermentation method for increasing the surfactin content according to claim 7, characterized in that: The culture temperature in the seed culture, the fermentation culture and the fed-batch culture is 30-40°C.

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