Fermentation medium for efficiently producing lipopeptide by bacillus subtilis, and application and method of fermentation medium

Abstract

The invention provides a fermentation medium for efficiently producing lipopeptide by bacillus subtilis as well as application and a method of the fermentation medium. Each liter of the fermentation medium comprises 20-60g of a carbon source; 5-10 g of a nitrogen source; 1-5g of corn steep liquor dry powder; 1 to 5g of monopotassium phosphate; 1 to 5g of disodium hydrogen phosphate; 0.1 to 0.5 g of magnesium sulfate; 5 to 10 mg of calcium chloride; 5 to 10 mg of manganese sulfate; 5 to 10 mg of ferrous sulfate; and vegetable oil with a volume fraction of 1-5%. The carbon source is selected from at least one of glucose, maltodextrin and brown sugar; the nitrogen source is selected from at least one of diammonium hydrogen phosphate, sodium nitrate and urea; the vegetable oil is selected from at least one of soybean oil and corn oil. Bacillus subtilis with the volume fraction of 1-5% is inoculated into a fermentation culture medium for fermentation and fed-batch culture, and the yield of lipopeptide reaches 48.58 g / L. The method improves the lipopeptide production capability of the bacillus subtilis while eliminating foam, and is suitable for large-scale production and application.

Description

Technical Field

[0001] The present application relates to the technical field of fermentation engineering, and in particular to a fermentation medium for efficiently producing lipopeptides by Bacillus subtilis, and its use and method. Background Art

[0002] Surfactants are lipopeptide-type biosurfactants mainly produced by the secondary metabolism of Bacillus. They are composed of cyclic or linear peptide chains connected to lipid residues. Compared with chemical surfactants, lipopeptides have the advantages of low toxicity, easy biodegradation, and good environmental compatibility. Due to their high foaming biosurfactant properties, they are used in various industrial fields, such as detergents, food emulsifiers, bioremediation and enhanced oil recovery.

[0003] In recent years, the global demand for surfactant has increased rapidly year by year. However, its industrial production is limited by problems such as low fermentation yield and difficulty in controlling foam during the fermentation process. Summary of the invention

[0004] In view of this, the purpose of the present application is to provide a fermentation medium for efficiently producing lipopeptides by Bacillus subtilis, and its use and method.

[0005] Based on the above purpose, the present application provides a fermentation medium for efficiently producing lipopeptides by Bacillus subtilis, comprising: vegetable oil and a basic medium;

[0006] The basic culture medium comprises the following substances: 20-60 g / L carbon source; 5-10 g / L nitrogen source; 1-5 g / L corn steep liquor powder; 1-5 g / L potassium dihydrogen phosphate; 1-5 g / L disodium hydrogen phosphate; 0.1-0.5 g / L magnesium sulfate; 5-10 mg / L calcium chloride; 5-10 mg / L manganese sulfate; 5-10 mg / L ferrous sulfate and 1-5% vegetable oil by volume; the carbon source is selected from at least one of glucose, maltodextrin and brown sugar; the nitrogen source is selected from at least one of diammonium hydrogen phosphate, sodium nitrate and urea; the vegetable oil is selected from at least one of soybean oil and corn oil;

[0007] Wherein, the inoculation amount of the Bacillus subtilis in the fermentation medium is 1-5% by volume.

[0008] In some embodiments, the vegetable oil is selected from soybean oil, and the volume fraction of the soybean oil is 4.8-5.2%.

[0009] In some embodiments, the carbon source is selected from glucose, and the concentration of glucose is 40-50 g / L.

[0010] In some embodiments, the nitrogen source is selected from urea, and the concentration of the urea is 4.8-5.2 g / L.

[0011] In some embodiments, the concentration of the corn steep liquor dry powder is 3-5 g / L.

[0012] In some embodiments, the concentration of potassium dihydrogen phosphate is 2-4 g / L.

[0013] The present application also provides an application of the fermentation medium for efficiently producing lipopeptides by Bacillus subtilis as described in any of the preceding items to increase the lipopeptide content in the fermentation product of Bacillus subtilis.

[0014] The present application also provides a method for efficiently producing lipopeptides by Bacillus subtilis, comprising:

[0015] Inoculating Bacillus subtilis into a liquid seed culture medium and performing seed culture to obtain a fermentation strain;

[0016] Inoculate the fermentation bacteria into the fermentation medium and carry out fermentation culture for 18-24 hours; wherein the inoculation amount of the fermentation bacteria is 1-5% by volume; and the fermentation medium is the fermentation medium as described in any of the preceding items;

[0017] Add feed medium and additional vegetable oil and continue culturing for 30-50 hours.

[0018] In some embodiments, the vegetable oil is soybean oil, and the amount of soybean oil added is 0.5%-2.5% by volume; the feed medium includes: 300-500g / L of carbon source; 5-25g / L of corn syrup powder; 25-50g / L of nitrogen source; and the concentration of the feed medium is 10%-20% by volume.

[0019] In some embodiments, the feeding medium is added in an intermittent feeding manner; the soybean oil is added in a continuous flow feeding manner;

[0020] The culture temperature of the seed culture is the same as that of the fermentation culture, both of which are 30-40°C.

[0021] As can be seen from the above, the fermentation medium for efficiently producing lipopeptides by Bacillus subtilis provided in the present application controls the foam by adding at least one of soybean oil and corn oil during the lipopeptide fermentation process, and is combined with a basic culture medium including: 20-60 g / L of carbon source; 5-10 g / L of nitrogen source; 1-5 g / L of corn slurry powder; 1-5 g / L of potassium dihydrogen phosphate; 1-5 g / L of disodium hydrogen phosphate; 0.1-0.5 g / L of magnesium sulfate; 5-10 mg / L of calcium chloride; 5-10 mg / L of manganese sulfate; and 5-10 mg / L of ferrous sulfate. It can reduce the fermentation cost, improve the ability of Bacillus subtilis to produce lipopeptides while eliminating foam, and thereby increase the fermentation yield of lipopeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 To investigate the effects of different types of vegetable oils on lipopeptide production;

[0024] Figure 2 The effect of soybean oil concentration on lipopeptide production;

[0025] Figure 3 The foaming phenomenon during the fermentation process with different soybean oil addition amounts;

[0026] Figure 4 The effect of carbon source on lipopeptide production;

[0027] Figure 5 The effect of nitrogen source on lipopeptide production;

[0028] Figure 6 The effect of corn steep liquor dry powder concentration on lipopeptide production;

[0029] Figure 7 is the effect of glucose concentration on lipopeptide production;

[0030] Figure 8 This is the effect of potassium dihydrogen phosphate concentration on lipopeptide production. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] Lipopeptides are biosurfactants with excellent surface activity. During the fermentation production process, accompanied by ventilation and stirring, they will inevitably cause serious foam problems. The large accumulation of these foams will lead to a decrease in dissolved oxygen, which is not conducive to the growth of Bacillus subtilis.

[0034] At present, several foam prevention methods are commonly used in the fermentation process. Mechanical or physical methods usually apply shear stress to destroy the foam, but the operation cost is high and it is harmful to cells. The use of defoamers can usually effectively avoid the generation of foam. Currently, one type of defoamers commonly used is chemical synthetics such as polyethers and polysilicone oils. Although the defoaming effect is good, it has certain toxicity to bacterial growth. Another type of defoamer is natural oils such as soybean oil, rice bran oil and animal fats, which can be used as defoamers in the fermentation process.

[0035] Therefore, in the related art, there are problems that foam is easily generated during the process of efficiently producing lipopeptides by Bacillus subtilis and the lipopeptide content is low.

[0036] Based on this, the embodiments of the present application provide a fermentation medium and method for improving the ability of Bacillus subtilis to produce lipopeptides. By optimizing the components of the fermentation medium and adding natural oil soybean oil, soybean oil can be used as a carbon source for the fermentation medium to produce additional effects on the metabolism of the microorganism Bacillus subtilis, and can also play a role in eliminating foam. Through this dual effect, the ability of Bacillus subtilis to ferment and produce lipopeptides can be improved, the yield of lipopeptides can be increased, and the problems of low content and easy foaming in the efficient production of lipopeptides by Bacillus subtilis can be solved to a certain extent.

[0037] The present application embodiment provides a fermentation medium for efficiently producing lipopeptides by Bacillus subtilis, comprising: vegetable oil and a basal medium;

[0038] The basic culture medium comprises substances with the following concentrations: 20-60 g / L of carbon source; 5-10 g / L of nitrogen source; 1-5 g / L of corn steep liquor powder; 1-5 g / L of potassium dihydrogen phosphate; 1-5 g / L of disodium hydrogen phosphate; 0.1-0.5 g / L of magnesium sulfate; 5-10 mg / L of calcium chloride; 5-10 mg / L of manganese sulfate; 5-10 mg / L of ferrous sulfate and 1-5% by volume of vegetable oil; the carbon source is selected from at least one of glucose, maltodextrin and brown sugar; the nitrogen source is selected from at least one of diammonium hydrogen phosphate, sodium nitrate and urea; the vegetable oil is selected from at least one of soybean oil and corn oil; and the inoculation amount of the Bacillus subtilis in the fermentation medium is 1-5% by volume.

[0039] The fermentation medium for efficiently producing lipopeptides by Bacillus subtilis of the embodiment of the present application controls the foam by adding at least one of soybean oil and corn oil during the lipopeptide fermentation process, and is combined with a basic culture medium including: 20-60 g / L of carbon source; 5-10 g / L of nitrogen source; 1-5 g / L of corn steep liquor dry powder; 1-5 g / L of potassium dihydrogen phosphate; 1-5 g / L of disodium hydrogen phosphate; 0.1-0.5 g / L of magnesium sulfate; 5-10 mg / L of calcium chloride; 5-10 mg / L of manganese sulfate; and 5-10 mg / L of ferrous sulfate, which can reduce the fermentation cost, improve the ability of Bacillus subtilis to produce lipopeptides while eliminating foam, and thereby increase the fermentation yield of lipopeptides.

[0040] In some embodiments, the vegetable oil can be selected from soybean oil, and the volume fraction of the soybean oil is 4.8-5.2%. The use of soybean oil has a lower cost and can make the fermentation product of Bacillus subtilis have a higher lipopeptide yield.

[0041] In some embodiments, the carbon source can be selected from glucose, and the concentration of the glucose is 40-50 g / L. Glucose has the advantages of single ingredient and low price, and can make the fermentation product of Bacillus subtilis have a good lipopeptide yield.

[0042] In some embodiments, the nitrogen source can be selected from urea, and the concentration of the urea is 4.8-5.2 g / L. Urea has the advantages of being able to quickly provide a nitrogen source, being low in cost, being non-hazardous, and being easily available, and can enable the fermentation product of Bacillus subtilis to have a higher lipopeptide yield.

[0043] In some of the embodiments, the concentration of the corn steep liquor dry powder can be 3-5 g / L, which can enable a higher lipopeptide yield in the fermentation product of Bacillus subtilis.

[0044] In some of the embodiments, the concentration of potassium dihydrogen phosphate can be 2-4 g / L, which can enable a higher lipopeptide yield in the fermentation product of Bacillus subtilis.

[0045] Based on the same inventive concept, corresponding to the fermentation medium for efficiently producing lipopeptides by Bacillus subtilis in any of the above embodiments, the present application also provides an application of a fermentation medium for efficiently producing lipopeptides by Bacillus subtilis in increasing the lipopeptide content in the fermentation product of Bacillus subtilis.

[0046] Based on the same inventive concept, corresponding to the fermentation medium for efficiently producing lipopeptides by Bacillus subtilis in any of the above embodiments, the present application also provides a method for efficiently producing lipopeptides by Bacillus subtilis.

[0047] The method for efficiently producing lipopeptides by Bacillus subtilis provided in the embodiments of the present application may include:

[0048] Step S100, inoculating Bacillus subtilis into a liquid seed culture medium to perform seed culture to obtain a fermentation strain;

[0049] Step S200, inoculating the fermentation bacteria into a fermentation medium, and fermenting and culturing for 18-24 hours; wherein the inoculation amount of the fermentation bacteria is 1-5% by volume; and the fermentation medium is the fermentation medium for efficiently producing lipopeptides by Bacillus subtilis as described in any of the above embodiments;

[0050] Step S300, adding feed medium and vegetable oil, and continuing culturing for 30-50 hours.

[0051] In some embodiments, in step S100, the liquid seed culture medium may include substances with the following concentrations: sodium chloride 10 g / L, tryptone 10 g / L, and yeast powder 5 g / L.

[0052] The seed culture conditions may be a culture temperature of 30-40°C and a shaker speed of 180-220 r / min. The seed culture may be cultured until the OD value is 5-6.

[0053] In some embodiments, in step S200, the culture conditions may be a culture temperature of 30-40°C, a shaker speed of 180-220 r / min, a stirring speed of 500 r / min in the fermenter, and a culture time of 18-24 h. The initial pH of the fermentation medium may be 6-7, which may be adjusted by adding sodium hydroxide. That is, the culture temperature of the seed culture may be the same as the culture temperature of the fermentation culture, both of which are 30-40°C.

[0054] In some embodiments, the vegetable oil may be soybean oil, and the added amount of the soybean oil may be 0.5%-2.5% by volume.

[0055] In some embodiments, the feed medium may include: 300-500 g / L of carbon source; 5-25 g / L of corn steep liquor powder; 25-50 g / L of nitrogen source; and the concentration of the feed medium is 10%-20% by volume. That is, the added volume of the feed medium is 10%-20% of the added volume of the fermentation medium.

[0056] In some embodiments, the feeding medium is added in an intermittent feeding manner. The soybean oil is added in a continuous flow feeding manner. By adding the feeding medium in an intermittent feeding manner, the fermentation yield of lipopeptides can be increased and a good foam control effect can be achieved.

[0057] The method for efficiently producing lipopeptides by Bacillus subtilis of the embodiment of the present application can improve the ability of Bacillus subtilis to produce lipopeptides by adding soybean oil, optimizing the fermentation medium and the feeding process, thereby increasing the fermentation yield of lipopeptides. At the same time, a large amount of foaming during the fermentation process can be controlled to stabilize the fermentation process, and efficient foam-free production of lipopeptide surfactants is achieved, with a lipopeptide yield of up to 48.58 g / L, which is significantly higher than the lipopeptide yield achieved by the current preparation method. The production cost of lipopeptides can be reduced, and it can be applied to industrial large-scale production.

[0058] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0059] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0060] 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-UV).

[0061] Among them, the specific method of determining the content of lipopeptides by high performance liquid chromatography (HPLC-UV) includes: (1) Chromatographic conditions: chromatographic column is Shimadzu C18 0DS-3 (4.6mmx250mm, 5μm), flow rate is 1.0mL / min, ultraviolet detection wavelength is 205nm, column temperature is room temperature. Mobile phase: chromatographic acetonitrile-0.1% TFA containing 0.1% TFA; injection volume: 20μL; instrument: Shimadzu L-033 chromatograph; gradient setting: 0-7min, A (10%-90%), B (90%-10%); 7-24min, A (90%), B (10%); 24-29min, A (90%-10%), B (10%-90%); 29-36min, A (10%), B (90%).

[0062] (2) Determination method: After the fermentation of Bacillus subtilis in the fermentation medium is completed, the fermentation liquid is collected, ethanol is added for extraction, and after alcohol extraction, it is filtered through a filter membrane and the content is determined by the above-mentioned detection method.

[0063] Among them, in Examples 1 to 10, Bacillus subtilis was used as the starting strain, and the composition of the culture medium was optimized through a large number of experiments to achieve efficient production of lipopeptides.

[0064] Example 1 Screening of vegetable oil

[0065] Experimental methods:

[0066] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, and inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain a fermentation strain.

[0067] 2) Fermentation culture: The fermentation strain is inoculated into the fermentation medium at a volume fraction of 4% for fermentation culture. The initial fermentation medium composition is: 60 g / L carbon source, 5 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 5 g / L nitrogen source, 1 g / L disodium hydrogen phosphate, 0.15 g / L magnesium sulfate, 10 mg / L calcium chloride, 10 mg / L manganese sulfate, and 10 mg / L ferrous sulfate. The initial medium (i.e., initial fermentation medium) is kept unchanged, and different types of vegetable oils are added to the initial medium: corn oil, soybean oil, olive oil, rapeseed oil, and peanut oil for culture. The culture temperature is 37° C., the shaking speed is 220 r / min, and the fermentation is carried out for 24 hours. The fermentation shake bottle is supplemented with a feed medium with a volume fraction of 12% and a soybean oil with a volume fraction of 2%, and the culture is continued at 37° C. and 220 r / min for 48 hours (i.e., the entire fermentation culture duration can be 48 hours). The lipopeptide surfactant is obtained in the fermentation broth, and the lipopeptide content in the fermentation broth is determined after the fermentation is completed.

[0068] 3) The lipopeptide content was determined by high performance liquid chromatography-UV.

[0069] Experimental results: Figure 1 shown.

[0070] Result analysis: Figure 1 The lipopeptide yields of different vegetable oils are as follows: the lipopeptide yield of soybean oil is 23.38 g / L, the lipopeptide yield of corn oil is 21.87 g / L, and the yields of other oils are all lower. The lipopeptide yield of soybean oil is the highest, and compared with corn oil, soybean oil is cheaper and has lower cost. Therefore, soybean oil is determined to be the best vegetable oil.

[0071] Example 2 Screening of soybean oil addition amount

[0072] Experimental methods:

[0073] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, and inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain a fermentation strain.

[0074] 2) Fermentation culture: The fermentation strains were inoculated into the fermentation medium at a volume fraction of 1%-5% for fermentation culture, based on the initial fermentation medium, the initial fermentation medium components were: 60g / L carbon source, 5g / L corn steep liquor powder, 2g / L potassium dihydrogen phosphate, 5g / L nitrogen source, 1g / L disodium hydrogen phosphate, 0.15g / L magnesium sulfate, 10mg / L calcium chloride, 10mg / L manganese sulfate, 10mg / L ferrous sulfate. The other components except soybean oil in the initial medium were kept unchanged, and soybean oil with a volume fraction of 1%, 2%, 3%, 4%, and 5% was added for comparison, the culture temperature was 37°C, the shaking speed was 220r / min, the fermentation was carried out for 24h, the volume fraction of 12% feed medium and 2% soybean oil was supplemented in the fermentation shake bottle, and the culture was continued at 37°C and 220r / min for 48h (that is, the entire fermentation culture duration can be 48h), and the lipopeptide surfactant was obtained in the fermentation broth, and the lipopeptide content in the fermentation broth was determined after the fermentation was completed.

[0075] 3) Determination of lipopeptide content by high performance liquid chromatography (HPLC-UV);

[0076] 4) At this time, the number of live Bacillus subtilis in the fermentation broth is determined by the plate count method: the fermentation broth is diluted in 10-fold increments, 100 μL of each dilution is spread on a plate, and cultured for 24 hours. The number of colonies formed in each plate is recorded, and the total number of bacterial colonies contained in each milliliter of the original sample is calculated based on the dilution factor and the sampling volume.

[0077] Experimental results: Figure 2 and Figure 3 shown.

[0078] Result analysis: Figure 2 The lipopeptide yields corresponding to different additions of soybean oil showed that after 48 hours of culture, when the soybean oil addition was 5%, the lipopeptide yield was the highest, reaching 32.38 g / L. At the same time, the effect of 5% soybean oil addition on the number of viable bacteria was detected. After 48 hours of fermentation with oil, the number of viable bacteria was 5×10 8 cfu / mL. Figure 3 It can be seen that when the soybean oil addition amount is 4-5%, there is less foam in the fermentation shake flask. It can be seen that while improving the ability of Bacillus subtilis to produce lipopeptides, it can also solve the foaming problem in the fermentation process.

[0079] Example 3 Screening of carbon sources

[0080] Experimental methods:

[0081] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain fermentation strains;

[0082] 2) Fermentation culture: 4% of the fermentation strain is inoculated into the fermentation medium for fermentation culture, and 5% of soybean oil is added to the initial fermentation medium. The initial fermentation medium contains: 60 g / L carbon source, 5 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 5 g / L nitrogen source, 1 g / L disodium hydrogen phosphate, 0.15 g / L magnesium sulfate, 10 mg / L calcium chloride, 10 mg / L manganese sulfate, and 10 mg / L ferrous sulfate. Keep the other components of the culture medium unchanged except the carbon source, use brown sugar, maltodextrin and glucose as carbon sources respectively, take 60g / L of carbon source as the standard, adjust the addition amount according to the carbon content to make the final carbon content consistent, and conduct shake flask fermentation experiments. The culture temperature is 37°C, the shaker speed is 220r / min, and the fermentation is carried out for 24h. The fermentation shake flask is supplemented with 12% by volume of feed medium and 2% by volume of soybean oil, and the culture is continued at 37°C and 220r / min for 48h (that is, the entire fermentation culture duration can be 48h). Obtain lipopeptide surfactant in the fermentation broth, and determine the lipopeptide content in the fermentation broth after the fermentation is completed.

[0083] Experimental results: Figure 4 shown.

[0084] Result analysis: Figure 4 The lipopeptide yields corresponding to different carbon sources are shown. It can be seen that the lipopeptide fermentation yields of glucose and brown sugar are basically the same, but compared with brown sugar, glucose has a single ingredient and a low price, so glucose is determined to be the best carbon source.

[0085] Example 4 Screening of nitrogen sources

[0086] Experimental methods:

[0087] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain fermentation strains;

[0088] 2) Fermentation culture: 4% of the fermentation strain was inoculated into the fermentation medium for fermentation culture, and 5% soybean oil was added based on the initial fermentation medium. The components of the initial fermentation medium are: 60g / L carbon source, 5g / L corn steep liquor powder, 2g / L potassium dihydrogen phosphate, 5g / L nitrogen source, 1g / L disodium hydrogen phosphate, 0.15g / L magnesium sulfate, 10mg / L calcium chloride, 10mg / L manganese sulfate, and 10mg / L ferrous sulfate. Glucose was used as the carbon source, and the other components of the medium except the nitrogen source were kept unchanged. Diamine hydrogen phosphate, sodium nitrate, and urea were used as nitrogen sources, respectively, and 5g / L nitrogen source was used as the standard. The addition amount was adjusted according to the nitrogen content to make the final nitrogen content consistent, and the shake flask fermentation was carried out. The culture temperature is 37°C, the shaking speed is 220 r / min, the fermentation is carried out for 24 hours, 12% feed medium and 2% soybean oil are supplemented in the fermentation shake flask, and the culture is continued at 37°C and 220 r / min for 48 hours (that is, the entire fermentation culture time can be 48 hours), and the lipopeptide surfactant is obtained in the fermentation broth. After the fermentation is completed, the lipopeptide content in the fermentation broth is determined.

[0089] Experimental results: Figure 5 shown.

[0090] Result analysis: Figure 5 From the lipopeptide yields corresponding to the different nitrogen sources shown, it can be seen that using urea as a nitrogen source to replace sodium nitrate (hazardous chemical) has the advantages of being easy to obtain, quickly providing nitrogen sources and being low in cost. Therefore, urea is selected as the best nitrogen source.

[0091] Example 5 Screening of corn steep liquor powder concentration

[0092] Experimental methods:

[0093] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain fermentation strains;

[0094] 2) Fermentation culture: 4% of the fermentation strain is inoculated into the fermentation medium for fermentation culture. The initial fermentation medium composition is: 60 g / L carbon source, 5 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 5 g / L nitrogen source, 1 g / L disodium hydrogen phosphate, 0.15 g / L magnesium sulfate, 10 mg / L calcium chloride, 10 mg / L manganese sulfate, and 10 mg / L ferrous sulfate. Based on the initial fermentation medium, 5% soybean oil was added, glucose was used as the carbon source, and urea was used as the nitrogen source. The other components in the medium except the concentration of corn slurry powder were kept unchanged, and corn slurry powder concentrations of 1g / L, 2g / L, 3g / L, 4g / L, and 5g / L were selected for comparison. The culture temperature was 37°C, the shaking speed was 220r / min, and the fermentation was carried out for 24h. 12% feed medium and 2% soybean oil were supplemented in the fermentation shake flask, and the culture was continued at 37°C and 220r / min for 48h (that is, the entire fermentation culture duration can be 48h), and lipopeptide surfactants were obtained in the fermentation broth. After the fermentation was completed, the lipopeptide content in the fermentation broth was determined.

[0095] Experimental results: Figure 6 shown.

[0096] Result analysis: Figure 6 It can be seen from the lipopeptide yields corresponding to different corn syrup dry powder concentrations that when the corn syrup dry powder concentration is 3g / L, the lipopeptide yield is the highest at 34.47g / L. Therefore, it is determined that the corn syrup dry powder concentration is 3g / L as the optimal concentration.

[0097] Example 6 Glucose concentration screening

[0098] Experimental methods:

[0099] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain fermentation strains;

[0100] 2) Fermentation culture: 4% of the fermentation strain is inoculated into the fermentation medium for fermentation culture. The initial fermentation medium composition is: 60 g / L carbon source, 5 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 5 g / L nitrogen source, 1 g / L disodium hydrogen phosphate, 0.15 g / L magnesium sulfate, 10 mg / L calcium chloride, 10 mg / L manganese sulfate, and 10 mg / L ferrous sulfate. Based on the initial fermentation medium, 5% soybean oil was added, glucose was used as the carbon source, urea was used as the nitrogen source, and other components in the medium except the glucose concentration were kept unchanged. Glucose concentrations of 20g / L, 30g / L, 40g / L, 50g / L, and 60g / L were selected for comparison. The culture temperature was 37°C, the shaking speed was 220r / min, and the fermentation was carried out for 24h. 12% feed medium and 2% soybean oil were supplemented in the fermentation shake flask, and the culture was continued at 37°C and 220r / min for 48h (that is, the entire fermentation culture duration can be 48h), and a lipopeptide surfactant was obtained in the fermentation broth. After the fermentation was completed, the lipopeptide content in the fermentation broth was determined.

[0101] Experimental results: Figure 7 shown.

[0102] Result analysis: Figure 7 From the lipopeptide production corresponding to different glucose concentrations, it can be seen that the optimal glucose concentration is 40g / L, and the corresponding lipopeptide production is 38.51g / L.

[0103] Example 7 Potassium dihydrogen phosphate concentration screening

[0104] Experimental methods:

[0105] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, and inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain a fermentation strain.

[0106] 2) Fermentation culture: 4% of the fermentation strain is inoculated into the fermentation medium for fermentation culture. The initial fermentation medium composition is: 60 g / L carbon source, 5 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 5 g / L nitrogen source, 1 g / L disodium hydrogen phosphate, 0.15 g / L magnesium sulfate, 10 mg / L calcium chloride, 10 mg / L manganese sulfate, and 10 mg / L ferrous sulfate. Based on the initial fermentation medium, 5% soybean oil was added, glucose was used as the carbon source, urea was used as the nitrogen source, and the other components in the medium except the concentration of potassium dihydrogen phosphate were kept unchanged. Potassium dihydrogen phosphate with concentrations of 1g / L, 2g / L, 3g / L, 4g / L, and 5g / L were selected for comparison. The culture temperature was 37°C, the shaking speed was 220r / min, and the fermentation was carried out for 24h. 12% feed medium and 2% soybean oil were supplemented in the fermentation shake flask, and the culture was continued at 37°C and 220r / min for 48h. Lipopeptide surfactant was obtained in the fermentation broth, and the lipopeptide content in the fermentation broth was determined after the fermentation was completed.

[0107] Experimental results: Figure 8 shown.

[0108] Result analysis: Figure 8 From the lipopeptide yields corresponding to different concentrations of potassium dihydrogen phosphate, it can be seen that the optimal potassium dihydrogen phosphate concentration is 3g / L, and the corresponding lipopeptide yield is 40.85g / L.

[0109] Example 8 Co-screening of three factors: glucose concentration, corn steep liquor powder concentration, and potassium dihydrogen phosphate concentration

[0110] Experimental method: The initial fermentation medium composition is: carbon source 60g / L, corn steep liquor powder 5g / L, potassium dihydrogen phosphate 2g / L, nitrogen source 5g / L, disodium hydrogen phosphate 1g / L, magnesium sulfate 0.15g / L, calcium chloride 10mg / L, manganese sulfate 10mg / L, ferrous sulfate 10mg / L. At the same time, 5% soybean oil is added.

[0111] Through the above-mentioned single factor experiment, combined with the fermentation characteristics of Bacillus subtilis, three factors that significantly affect the yield of lipopeptides were selected: glucose concentration, corn syrup dry powder concentration, and potassium dihydrogen phosphate concentration. A three-factor three-level orthogonal experiment was designed to further optimize the fermentation medium and finally determine the ratio and concentration of the medium. Among them, the orthogonal experimental factor levels are shown in Table 1. The specific experimental design is shown in Table 2.

[0112] Experimental results: The specific experimental design and range analysis results are shown in Table 2.

[0113] Table 1 Factor level table

[0114]

[0115] Table 2 Orthogonal experimental results of main factors

[0116]

[0117] Result analysis: As shown in Table 2, the lipopeptide production in experimental group 6 was the highest, reaching 42.16 g / L.

[0118] From the range analysis in Table 2, it can be seen that under the experimental factor levels set in this experiment, the influence level of each factor on the production of lipopeptides by Bacillus subtilis fermentation is: corn syrup powder concentration > glucose concentration > potassium dihydrogen phosphate concentration. From the mean analysis, it can be seen that the best combination of culture medium is A2B3C1, that is, glucose concentration 40g / L, corn syrup powder 4g / L, potassium dihydrogen phosphate 2g / L.

[0119] Example 9 uses the optimized conditions of shake flask fermentation as the initial fermentation conditions of the fermenter, and achieves efficient production of lipopeptides by adjusting the fermentation feeding process.

[0120] Experimental methods:

[0121] 1) Seed culture: prepare 500 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast powder, sterilize at 121° C. for 20 min, inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain fermentation strains;

[0122] 2) Fermentation tank culture: Prepare 16L fermentation liquid in a 30L fermentation tank. The fermentation medium composition is: glucose 40g / L, corn steep liquor powder 4g / L, potassium dihydrogen phosphate 2g / L, soybean oil 5%, urea 5g / L, disodium hydrogen phosphate 1g / L, magnesium sulfate 0.15g / L, calcium chloride 10mg / L, manganese sulfate 10mg / L, ferrous sulfate 10mg / L. After loading, sterilize at 115℃ for 15min, cool to 37℃, inoculate the seed liquid into the fermentation tank at 4%, and then incubate at 37℃, stirring speed 500r / min, tank pressure 0.05MPa, and continuously introduce air flow 0.5m 3 / h, ferment to 24h, feed and soybean oil are added by continuous flow addition, and culture is continued to 48h (that is, the entire fermentation culture duration can be 48h). Lipopeptide surfactant is obtained in the fermentation broth.

[0123] The feed medium contained 500 g / L glucose, 30 g / L corn steep liquor powder, and 30 g / L urea. The feed volume was 3 L and the feed acceleration was 10 g / L·h.

[0124] The supplement amount of soybean oil is 2%, and the flow acceleration is 2 g / L·h.

[0125] Experimental results: After the fermentation was completed, the lipopeptide content in the fermentation broth was determined to be 44.71 g / L.

[0126] Example 10 used the optimized conditions of shake flask fermentation as the initial fermentation conditions of the fermenter, and achieved efficient production of lipopeptides by adjusting the fermentation feeding process.

[0127] Experimental methods:

[0128] 1) Seed culture: prepare 500 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast powder, sterilize at 121° C. for 20 min, inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain fermentation strains;

[0129] 2) Fermentation tank culture: Prepare 16L fermentation liquid in a 30L fermentation tank. The fermentation medium composition is: glucose 40g / L, corn steep liquor powder 4g / L, potassium dihydrogen phosphate 2g / L, soybean oil 5%, urea 5g / L, disodium hydrogen phosphate 1g / L, magnesium sulfate 0.15g / L, calcium chloride 10mg / L, manganese sulfate 10mg / L, ferrous sulfate 10mg / L. After loading, sterilize at 115℃ for 15min, cool to 37℃, inoculate the seed liquid into the fermentation tank at 4%, and then incubate at 37℃, stirring speed 500r / min, tank pressure 0.05MPa, and continuously introduce air flow 0.5m 3 / h, ferment to 24h, add feed medium and soybean oil by intermittent feeding, continue to culture to 48h, and obtain lipopeptide surfactant in the fermentation broth.

[0130] The feed medium contained 500 g / L glucose, 30 g / L corn steep liquor powder, and 30 g / L urea, with a volume of 3L.

[0131] The intermittent feeding method of the feed medium is: 1.5L is added each time at 12h and 24h of fermentation.

[0132] The supplement amount of soybean oil is 2%, and the flow acceleration is 2 g / L·h.

[0133] Experimental results: After the fermentation was completed, the lipopeptide content in the fermentation broth was determined to be 48.58 g / L.

[0134] Comparative Example 1: No soybean oil added

[0135] Test method:

[0136] 1) Seed culture: prepare 50 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast powder, sterilize at 121° C. for 20 min, and inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain a fermentation strain.

[0137] 2) Fermentation culture: The fermentation strain is inoculated into the fermentation medium at 4% for fermentation culture. The initial fermentation medium composition is: 60 g / L carbon source, 5 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 5 g / L nitrogen source, 1 g / L disodium hydrogen phosphate, 0.15 g / L magnesium sulfate, 10 mg / L calcium chloride, 10 mg / L manganese sulfate, and 10 mg / L ferrous sulfate. The culture temperature is 37°C, the shaker speed is 220 r / min, the fermentation is carried out for 24 hours, 12% feed medium is added to the fermentation shake bottle, and the culture is continued at 37°C and 220 r / min for 48 hours (that is, the entire fermentation culture duration can be 48 hours), and the lipopeptide surfactant is obtained in the fermentation broth. After the fermentation is completed, the lipopeptide content in the fermentation broth is determined by high performance liquid chromatography, and the number of viable Bacillus subtilis in the fermentation broth is determined by plate count method. The culture was then extended to 72 h, and the lipopeptide content in the fermentation broth was determined by high performance liquid chromatography, and the number of viable Bacillus subtilis in the fermentation broth was determined by the plate count method.

[0138] Experimental results: The lipopeptide production in 48 hours was 10.9 g / L, and the number of viable bacteria in the fermentation broth was 1.2×10 6 cfu / mL, and the culture time was extended to 72h, at which time the number of viable bacteria in the fermentation broth was determined to be 5.3×10 8 cfu / mL, the lipopeptide yield was 12.7 g / L, and there was a lot of foam in the shake flask.

[0139] Comparative Example 2: No soybean oil added

[0140] Experimental methods:

[0141] 1) Seed culture: prepare 500 mL of liquid seed culture solution with the following formula: 10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast powder, sterilize at 121° C. for 20 min, inoculate Bacillus subtilis into the liquid seed culture medium for culture to obtain fermentation strains;

[0142] 2) Fermentation tank culture: Prepare 16L fermentation liquid in a 30L fermentation tank. The fermentation medium composition is: 40g / L glucose, 4g / L corn steep liquor powder, 2g / L potassium dihydrogen phosphate, 5g / L urea, 1g / L disodium hydrogen phosphate, 0.15g / L magnesium sulfate, 10mg / L calcium chloride, 10mg / L manganese sulfate, 10mg / L ferrous sulfate. After loading, sterilize at 115℃ for 15min, and inoculate the seed liquid into the fermentation tank at 4% when cooled to 37℃. Then, incubate at 37℃, stirring speed 500r / min, tank pressure 0.05MPa, and continuously introduce air flow 0.5m 3 / h, ferment to 24h, feed by continuous flow addition, continue to culture to 48h (that is, the entire fermentation culture duration can be 48h), and obtain lipopeptide surfactant in the fermentation broth.

[0143] The feed medium contained 500 g / L glucose, 30 g / L corn steep liquor powder, and 30 g / L urea. The feed volume was 3 L and the feed acceleration was 10 g / L·h.

[0144] Experimental results: During the fermentation process, large-scale foaming occurred and the foam could not be controlled, resulting in material leakage. The final fermentation lipopeptide production was 12.47g / L, and the remaining fermentation liquid volume was less than 10L.

[0145] Result analysis: Comparing Example 2 with Comparative Example 1, it can be seen that when the soybean oil addition amount in Example 2 is 5%, the number of viable bacteria reaches the level of 72 hours in Comparative Example 1 when soybean oil is not added, compared with the culture medium in Comparative Example 1, when soybean oil is added at a rate of 5%. Under the same number of viable Bacillus subtilis, the lipopeptide yield in the fermentation product when soybean oil is added at a rate of 5% is increased by 154.96% compared with the fermentation product without soybean oil addition. Therefore, the ability of Bacillus subtilis to produce lipopeptides is improved after adding soybean oil, and at the same time, Figure 3 It can be seen that there is less foam in the fermentation shake bottle after adding soybean oil, which shows that adding soybean oil also solves the foaming problem in the fermentation process.

[0146] Comparing Example 9 with Comparative Example 2, it can be seen that when the soybean oil addition amount of the fermentation medium in Example 9 is 5%, the lipopeptide yield in the fermentation product of Bacillus subtilis is significantly higher than that without adding soybean oil, and the foam can be well controlled. Therefore, after adding soybean oil, the ability of Bacillus subtilis to produce lipopeptides is improved, and the foaming problem in the fermentation process is solved at the same time.

[0147] 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.

[0148] 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.

[0149] 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 efficiently producing lipopeptides by Bacillus subtilis, characterized in that: include: Vegetable oil and basal medium; The basic culture medium comprises the following substances in the following concentrations: 20-60 g / L carbon source; 5-10 g / L nitrogen source; 1-5 g / L corn steep liquor powder; 1-5 g / L potassium dihydrogen phosphate; 1-5 g / L disodium hydrogen phosphate; 0.1-0.5 g / L magnesium sulfate; 5-10 mg / L calcium chloride; 5-10 mg / L manganese sulfate and 5-10 mg / L ferrous sulfate; the carbon source is selected from at least one of glucose, maltodextrin and brown sugar; the nitrogen source is selected from at least one of diammonium hydrogen phosphate, sodium nitrate and urea; the vegetable oil is selected from at least one of soybean oil and corn oil; the volume fraction of the vegetable oil is 1-5%; Wherein, the inoculation amount of the Bacillus subtilis in the fermentation medium is 1-5% by volume.

2. The fermentation medium for efficiently producing lipopeptides by Bacillus subtilis according to claim 1, characterized in that: The vegetable oil is selected from soybean oil, and the volume fraction of the soybean oil is 4.8-5.2%.

3. The fermentation medium for efficiently producing lipopeptides by Bacillus subtilis according to claim 1, characterized in that: The carbon source is selected from glucose, and the concentration of the glucose is 40-50 g / L.

4. The fermentation medium for efficiently producing lipopeptides by Bacillus subtilis according to claim 1, characterized in that: The nitrogen source is selected from urea, and the concentration of the urea is 4.8-5.2 g / L.

5. The fermentation medium for efficiently producing lipopeptides by Bacillus subtilis according to claim 1, characterized in that: The concentration of the corn steep liquor dry powder is 3-5 g / L.

6. The fermentation medium for efficiently producing lipopeptides by Bacillus subtilis according to claim 1, characterized in that: The concentration of the potassium dihydrogen phosphate is 2-4 g / L.

7. Use of the fermentation medium for efficiently producing lipopeptides by Bacillus subtilis according to any one of claims 1 to 6 in increasing the lipopeptide content in a fermentation product of Bacillus subtilis.

8. A method for efficiently producing lipopeptides by Bacillus subtilis, characterized in that: include: Inoculating Bacillus subtilis into a liquid seed culture medium, and performing seed culture to obtain a fermentation strain; Inoculate the fermentation bacteria into the fermentation medium and carry out fermentation culture for 18-24 hours; wherein the inoculation amount of the fermentation bacteria is 1-5% by volume; and the fermentation medium is the fermentation medium according to any one of claims 1-6; Add feed medium and additional vegetable oil and continue culturing for 30-50 hours.

9. The method according to claim 8, characterized in that The vegetable oil is soybean oil, and the added amount of the soybean oil is 0.5%-2.5% by volume; the feed culture medium comprises: 300-500g / L of carbon source; 5-25g / L of corn steep liquor dry powder; 25-50g / L of nitrogen source; and the concentration of the feed culture medium is 10%-20% by volume.

10. The method according to claim 9, characterized in that The feeding medium is added in an intermittent feeding manner; the soybean oil is added in a continuous flow feeding manner; The culture temperature of the seed culture is the same as that of the fermentation culture, both of which are 30-40°C.

Images