Method for producing surfactin by promoting fermentation of bacillus subtilis through magnetoelectric coupling

Abstract

The invention discloses a method for promoting fermentation of bacillus subtilis to produce surfactin by utilizing magnetoelectric coupling. The method comprises the following steps: adding magnesium ions into a fermentation system of bacillus subtilis, and carrying out continuous static magnetic field treatment and electrostatic field treatment on the fermentation system to obtain surfactin. According to the method disclosed by the invention, the content of the surfactin in the fermentation liquor is effectively increased through the synergistic effect of magnetoelectric coupling treatment and magnesium ions, a clean, effective and non-transgenic method is provided for increasing the yield of the surfactin, and the economic value of the application of the surfactin in food chemical industry, biological medicine or agriculture is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentation engineering, and particularly relates to a method for promoting the fermentation production of surfactin by Bacillus subtilis using magnetoelectric coupling. Background Art

[0002] Surfactin is a lipopeptide biosurfactant produced by Bacillus subtilis. Its structure includes a hydrophilic peptide ring composed of 7 amino acids and a hydrophobic β-hydroxy fatty acid chain composed of 13 - 17 carbon atoms, and it is the biosurfactant with the strongest surface activity currently discovered. Surfactin has broad application prospects in the fields of oil exploitation, medicine and pesticides, daily chemical products, food and chemical industry, etc. due to its excellent emulsifying performance, broad-spectrum antibacterial activity, low toxicity and high biodegradability. And compared with chemically synthesized surfactants, surfactin is more in line with the concept of green health and sustainable development. However, the low yield and high production cost seriously restrict the industrial production and application of surfactin. Currently, many studies have improved the yield of surfactin by optimizing the medium composition and fermentation conditions, using cost-effective agricultural wastes as raw materials and adopting genetic engineering techniques, etc. The high input costs or relatively high operation difficulties required by these methods limit their applications, and little attention has been paid to assisting surfactin production through physical field technology in the existing research progress. Summary of the Invention

[0003] The main object of the present invention is to provide a method for promoting the fermentation production of surfactin by Bacillus subtilis using magnetoelectric coupling to overcome the deficiencies of the prior art.

[0004] To achieve the foregoing invention object, the technical solutions adopted by the present invention include:

[0005] One aspect of the present invention provides a method for promoting the fermentation production of surfactin by Bacillus subtilis using magnetoelectric coupling, which includes: adding a magnesium ion-containing compound to the fermentation system of Bacillus subtilis, and subjecting the Bacillus subtilis to continuous static magnetic field and electrostatic field treatments to obtain surfactin.

[0006] Another aspect of the present invention provides a method for culturing Bacillus subtilis, which includes:

[0007] Activating and culturing the strain of Bacillus subtilis to obtain a seed solution;

[0008] Adding the seed solution to a fermentation medium to form a fermentation system, adding magnesium ions, and subjecting the fermentation system to continuous static magnetic field treatment and electrostatic field treatment to obtain surfactin.

[0009] Another aspect of the present invention also provides a culture device for Bacillus subtilis, which is used for the culture method of Bacillus subtilis. And the culture device includes:

[0010] A sample chamber, at least used for accommodating a container loaded with a fermentation system;

[0011] A magnetic field generating unit, at least used for forming a static magnetic field in the sample chamber;

[0012] An electric field generating unit, at least used for forming an electrostatic field in the sample chamber;

[0013] A shaker unit, at least used for carrying the container and rotating it; the shaker unit is placed in the sample chamber;

[0014] A temperature regulating unit, at least used for regulating the temperature in the sample chamber;

[0015] A control unit, connected to the magnetic field generating unit, the electric field generating unit, the shaker unit, and the temperature regulating unit, and at least used for regulating the working states and working parameters of the magnetic field generating unit, the electric field generating unit, the shaker unit, and the temperature regulating unit.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] (1) The synergistic effect of magnetoelectric coupling treatment and magnesium ions in the culture medium is beneficial to promoting the growth and metabolic process of Bacillus subtilis, and the magnetoelectric coupling treatment is beneficial to promoting Bacillus subtilis to absorb magnesium ions in the culture medium, promoting the synthesis of surfactin, and improving the economic value for the application of surfactin in food chemical industry, biomedicine and agriculture.

[0018] (2) After adding a magnesium ion-containing compound to the fermentation medium and performing magnetoelectric coupling treatment in the present invention, the content of surfactin in the fermentation broth is effectively increased, laying a foundation for the commercial application of surfactin.

[0019] (3) The magnetoelectric coupling treatment selected in the present invention, as an emerging physical field-assisted processing technology, has the characteristics of strong penetration ability, no secondary pollution, convenient operation, relatively low operation cost, etc., providing technical feasibility for industrial processing and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is a three-dimensional model diagram of the culture device of Bacillus subtilis in a typical embodiment of the present invention;

[0022] Figure 2 It is a comparison diagram of the intracellular and extracellular magnesium ion concentrations of Bacillus subtilis of the present invention with and without magnetoelectric coupling treatment.

[0023] Explanation of reference numerals:

[0024] 1. Sample chamber; 2. Magnetic field generating unit; 3. Electric field generating unit; 4. Shaker unit; 5. Temperature regulating unit; 6. Control unit. Detailed implementation manners

[0025] As an environmentally friendly, economically feasible, convenient and sustainable method, electromagnetic field technology shows great potential in promoting the growth and metabolism of microorganisms such as bacteria, fungi and microalgae through various biological effects to produce nutrients, enzymes and various high-value products. The effects of electromagnetic fields on microorganisms depend on several factors, including exposure time, electromagnetic intensity and fermentation broth composition. Existing studies have found that electromagnetic fields may affect the biochemical processes of microorganisms through pathways such as mediating transmembrane transport of nutrients, electron transfer in photosynthesis and respiration, enzyme activity and gene expression. In addition, the effectiveness of optimizing the Mg 2+ concentration as a feasible strategy to increase the surfactin production of Bacillus subtilis has been studied. As a cofactor, magnesium ions enhance the activity of phosphoadenylyl transferase (Sfp) by forming complexes with enzymes, thus promoting the synthesis of surfactin; at the same time, the magnetic isotope effect of magnesium ions is considered to be a medium for magnetic field regulation of enzyme-catalyzed reactions. Therefore, it is necessary to further study the synergistic effect of magnesium ions on the fermentation of Bacillus subtilis in a magnetoelectric coupling environment; the effects of electric and magnetic fields on various microorganisms and their metabolism are also worthy of further study; and it has great value to develop a miniaturized magnetoelectric coupling device suitable for shaker oscillation and apply it to the study of microorganisms and their metabolism.

[0026] In view of the problems existing in the above-mentioned prior art, through extensive and in-depth research by the inventors of the present invention, a method for promoting the fermentation of Bacillus subtilis to produce surfactin by magnetoelectric coupling is provided. The method mainly designs a culture device for Bacillus subtilis to generate a constant and adjustable static magnetic field and electrostatic field, and simultaneously cooperate with magnesium ions in the culture medium. During the cultivation of Bacillus subtilis, a static magnetic field and an electrostatic field are applied to assist its liquid fermentation, thereby promoting its growth and metabolism, solving the problem of low fermentation yield of surfactin and the technical problems that the existing methods for improving the fermentation of Bacillus to produce surfactin generally have high implementation costs and complex operations, in order to improve the ability of Bacillus subtilis to ferment and produce surfactin on the premise of environmental friendliness and economic feasibility.

[0027] The technical solution, its implementation process, principle, etc. will be further explained as follows.

[0028] As an aspect of the technical solution of the present invention, a method for promoting the fermentation of Bacillus subtilis to produce surfactin by magnetoelectric coupling includes: adding magnesium ions to the fermentation system of Bacillus subtilis, and performing continuous static magnetic field treatment and electrostatic field treatment on the fermentation system to obtain surfactin.

[0029] In some embodiments, the concentration of magnesium ions in the fermentation system is 0.5 - 1.5 mmol / L.

[0030] In some preferred embodiments, the concentration of magnesium ions in the fermentation system is 1.3 mmol / L.

[0031] In some embodiments, the magnesium ions are derived from magnesium ion-containing compounds such as at least any one of anhydrous magnesium sulfate or magnesium chloride, but are not limited thereto.

[0032] In some embodiments, the intensity of the static magnetic field treatment is 10 - 20 mT, and the time of the static magnetic field treatment is 72 h.

[0033] In some preferred embodiments, the intensity of the static magnetic field treatment is 16 mT, and the time of the static magnetic field treatment is 72 h.

[0034] In some embodiments, the intensity of the electrostatic field treatment is 4 - 8 V / cm, and the time of the electrostatic field treatment is 72 h.

[0035] In some preferred embodiments, the intensity of the electrostatic field treatment is 6 V / cm, and the time of the electrostatic field treatment is 72 h.

[0036] The method provided by the present invention effectively increases the content of surfactin in the fermentation broth after adding a certain concentration of magnesium ions to the culture medium and treating it with an electromagnetic field, reaching a maximum of 4.195 g / L, laying a foundation for the commercial application of surfactin.

[0037] As another aspect of the present invention, a method for culturing Bacillus subtilis provided by it includes:

[0038] Activating and culturing the strain of Bacillus subtilis to obtain a seed solution;

[0039] Adding the said seed solution to a fermentation medium to form a fermentation system, adding magnesium ions, and subjecting the fermentation system to continuous static magnetic field treatment and electrostatic field treatment to obtain surfactin.

[0040] In some embodiments, the method for culturing Bacillus subtilis specifically includes: thawing the preserved Bacillus subtilis ATCC 21223 strain and inoculating it onto a solid agar medium for incubation, then inoculating a single colony into a seed medium, and then placing it in a shaker unit for activation and culture to obtain a seed solution.

[0041] In some preferred embodiments, the solid agar medium includes LB solid medium.

[0042] In some more preferred embodiments, the components of the solid agar medium include 10 - 12 g / L peptone, 5 - 7 g / L yeast powder, 10 - 12 g / L sodium chloride, and 0.2 - 0.4 g / L agar powder.

[0043] In some preferred embodiments, the seed medium includes liquid LB medium.

[0044] In some more preferred embodiments, the components of the seed medium include 10 - 12 g / L peptone, 5 - 7 g / L yeast powder, and 10 - 12 g / L sodium chloride.

[0045] In some embodiments, the method for culturing Bacillus subtilis specifically further includes: adding the said seed solution to a fermentation medium, placing it in a culture device for fermentation culture, and performing continuous static magnetic field treatment and electrostatic field treatment to obtain surfactin.

[0046] In some preferred embodiments, the fermentation medium includes a basal salt medium.

[0047] In some more preferred embodiments, the components of the fermentation medium include 40 g / L glucose, 20 g / L tryptone, 40 mmol / L disodium hydrogen phosphate, 30 mmol / L potassium dihydrogen phosphate, 0.007 mmol / L calcium chloride, 0.5 - 1.5 mmol / L magnesium ion-containing compound, 0.004 mmol / L ethylenediaminetetraacetic acid, 0.2 mmol / L ferrous sulfate heptahydrate, and 0.01 mmol / L manganese sulfate monohydrate.

[0048] In a typical embodiment, the culturing method of the Bacillus subtilis specifically comprises the following steps:

[0049] (1) Strain activation

[0050] The Bacillus subtilis ATCC 21223 strain stored at -80°C was slowly thawed and inoculated onto a solid agar medium. After incubating at 37°C for 24 h, a single colony was picked with an inoculation loop and inoculated into a seed medium. Then, the shake flask was placed in a shaker incubator at 30°C and activated for 12 h at a rotation speed of 180 r / min.

[0051] (2) Scale-up culture

[0052] The seed liquid was added to the fermentation medium at an inoculation amount of 3%, and then the shake flask was placed in a shaker incubator at 30°C and cultured for 72 h at a rotation speed of 180 r / min, and a continuous static magnetic field and an electrostatic field were applied for treatment to ferment and prepare surfactin.

[0053] In some embodiments, the content of surfactin in the fermentation system is 0.5 - 4.5 g / L.

[0054] In some embodiments, the magnesium ion concentration in the fermentation system is 0.5 - 1.5 mmol / L.

[0055] In some preferred embodiments, the magnesium ion concentration in the fermentation system is 1.3 mmol / L.

[0056] In some embodiments, the magnesium ion is derived from a magnesium ion-containing compound, and the magnesium ion-containing compound includes at least any one of anhydrous magnesium sulfate or magnesium chloride, but is not limited thereto.

[0057] In some embodiments, the intensity of the static magnetic field treatment is 10 - 20 mT, and the time of the static magnetic field treatment is 72 h.

[0058] In some preferred embodiments, the intensity of the static magnetic field treatment is 16 mT, and the time of the static magnetic field treatment is 72 h.

[0059] In some embodiments, the intensity of the electrostatic field treatment is 4-8 V / cm, and the time of the electrostatic field treatment is 72 h.

[0060] In some preferred embodiments, the intensity of the electrostatic field treatment is 6 V / cm, and the time of the electrostatic field treatment is 72 h.

[0061] Another aspect of the present invention further provides a culture device for Bacillus subtilis, which is used for the culture method of Bacillus subtilis, and the culture device includes:

[0062] A sample chamber, at least for accommodating a container loaded with a fermentation system;

[0063] A magnetic field generating unit, at least for forming a static magnetic field in the sample chamber;

[0064] An electric field generating unit, at least for forming an electrostatic field in the sample chamber;

[0065] A shaker unit, at least for carrying the container and rotating it; the shaker unit is placed in the sample chamber;

[0066] A temperature regulating unit, at least for regulating the temperature in the sample chamber;

[0067] A control unit, which is connected to the magnetic field generating unit, the electric field generating unit, the shaker unit, and the temperature regulating unit, and at least for regulating the working states and working parameters of the magnetic field generating unit, the electric field generating unit, the shaker unit, and the temperature regulating unit.

[0068] Please refer to Figure 1 , the culture device for Bacillus subtilis (which can also be called "magnetoelectric coupling oscillation constant temperature incubator") includes: a sample chamber 1, a magnetic field generating unit 2, an electric field generating unit 3, a shaker unit 4, a temperature regulating unit 5, and a control unit 6. Among them, the sample chamber 1 is used for accommodating a shaking flask; the magnetic field generating unit 2 is used for forming a constant and adjustable static magnetic field in the sample chamber; the electric field generating unit 3 is used for forming a constant and adjustable electrostatic field in the sample chamber; the shaker unit 4 is used for carrying the shaking flask and has a controllable rotation speed; the shaker unit 4 is placed in the sample chamber 1; the temperature regulating unit 5 is used for regulating the temperature in the sample chamber; the control unit 6 is connected to the magnetic field generating unit 2, the electric field generating unit 3, the shaker unit 4, and the temperature regulating unit 5, and is used for regulating the working states and working parameters of the magnetic field generating unit 2, the electric field generating unit 3, the shaker unit 4, and the temperature regulating unit 5.

[0069] During the cultivation of Bacillus subtilis, the magnetic field generating unit 2 applies a static magnetic field, and the electric field generating unit 3 applies an electrostatic field to assist Bacillus subtilis in its life activities.

[0070] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Those skilled in the art who make modifications or equivalent replacements on the basis of understanding the technical solutions of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, shall be covered by the protection scope of the present invention.

[0071] For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For those reagents or instruments without indicating the manufacturer, they can all be obtained through commercial purchase. The remaining raw materials not mentioned and the commercial purchase selections of the instruments are all conventional selections and do not involve the core technical means of the present invention.

[0072] The strain information involved in the following embodiments is as follows:

[0073] The Bacillus subtilis (ATCC 21332) used in this experiment is from the China Center for Industrial Culture Collection.

[0074] The culture media involved in the following embodiments are as follows:

[0075] Solid agar medium (LB solid medium): 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 0.2 g / L agar powder.

[0076] Seed medium (liquid LB medium): 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0077] Fermentation medium (MSM medium): 40 g / L glucose, 20 g / L tryptone, 40 mM disodium hydrogen phosphate, 30 mM potassium dihydrogen phosphate, 0.007 mM calcium chloride, 0.5 - 1.5 mM anhydrous magnesium sulfate or magnesium sulfate, 0.004 mM ethylenediaminetetraacetic acid, 0.2 mM ferrous sulfate heptahydrate, and 0.01 mM manganese sulfate monohydrate.

[0078] The detection methods involved in the following embodiments are as follows:

[0079] Determination of cell biomass (mg): After culturing for 72 h, collect the culture medium and measure the absorbance OD at 600 nm 600 to represent the biomass.

[0080] Detection of surfactin content (mg / L): The fermentation medium was centrifuged at 8000 r / min for 15 min, and after separating the supernatant, the content of surfactin was quantitatively analyzed using ultra-high performance liquid chromatography (UPLC). The chromatographic column was a BEH C18 column (150×2.1 mm, 1.7 μm), the injection volume was 20 μL, the column temperature was 28 °C, the system running flow rate was 0.8 mL / min, the mobile phase was 90% (v / v) methanol and 10% (v / v) water (containing 0.05% trifluoroacetic acid), and the absorbance was monitored at 205 nm.

[0081] Measurement of the residual sugar content (mg / L) in the fermentation medium: The dinitrosalicylic acid (DNS) method was used to determine the residual sugar content in the supernatant of the fermentation broth. The supernatant was obtained by removing the bacterial cell precipitate after centrifuging the fermentation broth at 8000 r / min for 15 min. A standard curve of the residual sugar content was established by measuring the absorbance at 540 nm, and the fitting equation was y = 1.531x + 0.013, R 2 = 0.9997 (where x is the absorbance at 540 nm and y [mg / L] is the residual sugar content).

[0082] Example 1

[0083] A method for promoting the fermentation production of surfactin by Bacillus subtilis using magnetoelectric coupling, the specific steps are as follows:

[0084] (1) After thawing the frozen Bacillus subtilis ATCC 21332, it was inoculated on LB solid medium. After incubating at 37 °C for 24 h, a single colony was picked with an inoculation loop and transferred to a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium. It was cultured in a shaker incubator at 30 °C for 14 h, and the rotation speed was 180 r / min.

[0085] (2) Take 3 mL of the activated seed culture solution (OD 600 = 1.0) and inoculate it into 100 mL of fermentation media with magnesium ion concentrations of 0.5 mmol / L, 0.7 mmol / L, 0.9 mmol / L, 1.1 mmol / L, 1.3 mmol / L, and 1.5 mmol / L respectively, and incubate in a constant temperature shaking incubator at 30 °C and 180 r / min for 72 h.

[0086] After culturing for 72 h, the medium was collected and the absorbance OD 600 was measured at 600 nm to represent the biomass. The medium was centrifuged at 8000 r / min for 15 min, and the supernatant was separated to measure the residual sugar content and surfactin content respectively. The results are shown in Table 1.

[0087] Table 1 Effects of different magnesium ion concentrations on the growth and metabolism of Bacillus subtilis

[0088]

[0089] Table 1 shows that the increase in magnesium ion concentration within the experimental range promotes the biomass and surfactin accumulation of Bacillus subtilis and the absorption of nutrients. Among them, when the magnesium ion concentration reaches 1.3 mmol / L, the biomass and surfactin yield of Bacillus subtilis both reach relatively the highest levels, and the residual sugar content in the culture medium is the lowest. This indicates that when the magnesium ion concentration is 1.3 mmol / L, Bacillus subtilis has stronger growth and metabolic capabilities and the highest utilization efficiency of the substrate. Higher concentrations of magnesium ions may affect the acid-base balance of the culture medium and are instead unfavorable for the growth and metabolism of the bacteria.

[0090] Example 2

[0091] The specific implementation method is the same as that of Example 1, with the difference being that:

[0092] The concentration of magnesium sulfate in the fermentation medium is 1.3 mmol / L. After inoculating the bacteria, they are cultured in an incubator, and static magnetic field intensities of 10 mT, 12 mT, 14 mT, 16 mT, 18 mT, and 20 mT are applied respectively. A control group is set up, and the control group is not subjected to magnetic field treatment, while other culture conditions are the same as those of the above experimental groups.

[0093] After culturing for 72 h, the culture medium is collected and the absorbance OD is measured at 600 nm 600 to represent the biomass. The culture medium is centrifuged at 8000 r / min for 15 min to separate the bacterial cell precipitate and collect the supernatant. After drying the bacterial cell precipitate, the biomass is measured, and the residual sugar content and surfactin content are measured in the supernatant respectively. The results are shown in Table 2.

[0094] Table 2 Effects of different static magnetic field intensities on the growth and metabolism of Bacillus subtilis

[0095]

[0096]

[0097] It can be seen from Table 2 that applying static magnetic fields with different intensities to the fermentation system has different degrees of promoting effects on the growth and metabolism of Bacillus subtilis. Among them, the promoting effect is the most obvious when a static magnetic field of 16 mT is applied. This indicates that the magnetic field effect has a "window effect", that is, the magnetic field only has a specific impact at a specific intensity. The magnetic field may affect the activity of related enzymes through the magnesium isotope effect of some active centers containing magnesium metal ions, and then change the rate of anabolic reactions. In addition, the magnetic field promotes its growth, substrate consumption, and the synthesis of surfactin by affecting the transfer and transmission of electrons or ions in the anabolic reactions of the bacteria.

[0098] Example 3

[0099] The specific implementation method is the same as that of Example 1, with the difference that:

[0100] The concentration of magnesium sulfate in the fermentation medium is 1.3 mmol / L. After inoculating the bacteria, a static magnetic field of 16 mT is applied and electrostatic fields of 4 V / cm, 5 V / cm, 6 V / cm, 7 V / cm, and 8 V / cm are respectively applied, and the culture is carried out in an incubator. A control group is set without electric field treatment, and other culture conditions are the same as those of the above experimental group.

[0101] After culturing for 72 h, the bacterial liquid is collected and the absorbance OD is measured at 600 nm 600 to represent the biomass. The medium is centrifuged at 8000 r / min for 15 min, and the supernatant is separated to measure the residual sugar content and the surfactin content respectively. The results are shown in Table 3.

[0102] Table 3 Effects of different electrostatic field intensities on the growth and metabolism of Bacillus subtilis

[0103]

[0104] It can be seen from Table 3 that compared with the control group, different electrostatic field intensity treatments all have a promoting effect on the growth of Bacillus subtilis and the utilization of nutrients. Among them, applying 6 V / cm to the fermentation system can achieve the best promotion effect to the greatest extent. While a higher electric field may inhibit carbohydrate metabolism and its corresponding enzyme activities and have a penetrating effect on the microbial cell membrane, thus offsetting its promoting effect on the growth and metabolism of the bacteria.

[0105] Comparative Example 1

[0106] Bacillus subtilis was fermented and cultured under the conditions of not applying magnetic field or electric field treatment and the medium without magnesium ions, single magnesium ion treatment, single magnetic field treatment, single electric field treatment, and simultaneous magnetic field and electric field treatment while the medium without magnesium ions. A comparative experiment was carried out under the same other culture conditions, and the results are shown in Table 4.

[0107] Table 4 Comparative examples with magnetic field, electric field treatment, and magnesium ions as key parameters

[0108]

[0109] It can be seen from Table 4 that applying an appropriate static magnetic field and electrostatic field during the fermentation and culture of Bacillus subtilis and adding a certain concentration of magnesium ions to the medium can produce a better growth and metabolism promoting effect than a single treatment method, indicating that under certain conditions, magnetoelectric coupling can produce a synergistic effect with magnesium ions to promote the synthesis of surfactin.

[0110] The above experimental results illustrate that in different embodiments, different magnesium ion concentrations in the culture medium, as well as different magnetic and electric field strengths, will affect the accumulation of Bacillus subtilis biomass, the substrate consumption rate, and the synthesis of surfactin. As can be seen from Tables 1 to 3, the optimal magnesium ion concentration in the culture medium is 1.3 mmol / L; the optimal magnetic field treatment strength is 16 mT; the optimal electric field treatment strength is 6 V / cm. Especially after the culture medium containing 1.3 mmol / L magnesium ions is treated with a static magnetic field of 16 mT and an electrostatic field of 6 V / cm, the cell biomass and substrate consumption ratio are increased by 59.13% and 40.77% compared with the control group (static magnetic field 0 mT, electrostatic field 0 V / cm, magnesium ion 0 mmol / L). In particular, the surfactin yield reaches 4.195 g / L, which is 3.906 g / L higher than that of the control group, improving the economic value for the application of surfactin in food chemistry, biomedicine, and agriculture.

[0111] In addition, the magnesium ion concentrations inside and outside the cells were measured by inductively coupled plasma mass spectrometry (ICP-MS), and the specific method refers to GB 5009.268-2016. Figure 2 The results show that compared with the single magnesium ion treatment group in the control group (static magnetic field 0 mT, electrostatic field 0 V / cm, magnesium ion 1.3 mmol / L) and the magnetoelectric coupling synergistic 1.3 mmol / L group in Example 3 (static magnetic field 16 mT, electrostatic field 6 V / cm, magnesium ion 1.3 mmol / L), the static magnetic field and electrostatic field treatments are beneficial for the cells to absorb magnesium ions from the culture medium. Under the condition of the same magnesium ion concentration, the magnesium ions transferred from the fermentation broth into the cells after magnetoelectric coupling treatment increase by 28.23%. This may be related to the fact that the electromagnetic field changes the biofilm potential and affects its ion permeability, thus affecting microbial metabolism and the synthesis of surfactin.

[0112] All aspects, embodiments, features, and examples of the present invention should be considered illustrative in all respects and are not intended to limit the present invention. The scope of the present invention is only defined by the claims. Without departing from the spirit and scope of the claimed invention, those skilled in the art will understand other embodiments, modifications, and uses.

[0113] In addition, the inventors of this case also referred to the foregoing embodiments and conducted tests with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0114] Although the present invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions and / or additions can be made without departing from the spirit and scope of the present invention and elements of the embodiments can be replaced with substantially equivalent ones. Additionally, many modifications can be made to adapt a particular situation or material to the teachings of the present invention without departing from the scope of the present invention. Therefore, it is not intended that the present invention be limited to the particular embodiments disclosed for carrying out the present invention, but rather that the present invention will include all embodiments falling within the scope of the appended claims. Further, unless specifically stated, any use of the terms first, second, etc. does not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.

Claims

1. A method for promoting the fermentation of Bacillus subtilis to produce surfactin by magnetoelectric coupling, characterized in that, Comprising: Adding magnesium ions to the fermentation system of Bacillus subtilis, and subjecting the fermentation system to continuous static magnetic field treatment and electrostatic field treatment to obtain surfactin.

2. The method according to claim 1, characterized in that: The concentration of magnesium ions in the fermentation system is 0.5 - 1.5 mmol / L; And / or, the magnesium ions are derived from magnesium ion-containing compounds, and the magnesium ion-containing compounds include at least any one of anhydrous magnesium sulfate or magnesium chloride.

3. The method according to claim 1, wherein: The intensity of the static magnetic field treatment is 10 - 20 mT, and the time of the static magnetic field treatment is 72 h; And / or, the intensity of the electrostatic field treatment is 4 - 8 V / cm, and the time of the electrostatic field treatment is 72 h.

4. A cultivation method of Bacillus subtilis, characterized in that, Comprising: Activating and culturing the strain of Bacillus subtilis to obtain a seed solution; Adding the seed solution to a fermentation medium to form a fermentation system, adding magnesium ions, and subjecting the fermentation system to continuous static magnetic field treatment and electrostatic field treatment to obtain surfactin.

5. The cultivation method according to claim 4, wherein Comprising: Melting the preserved Bacillus subtilis ATCC21223 strain and inoculating it onto a solid agar medium for incubation, then inoculating a single colony into a seed medium, and then placing it in a shaker unit for activation and culture to obtain a seed solution; Preferably, the solid agar medium includes LB medium; Particularly preferably, the components of the solid agar medium include 10 - 12 g / L peptone, 5 - 7 g / L yeast powder, 10 - 12 g / L sodium chloride, and 0.2 - 0.4 g / L agar powder; Preferably, the seed medium includes liquid LB medium; Particularly preferably, the components of the seed medium include 10 - 12 g / L peptone, 5 - 7 g / L yeast extract, and 10 - 12 g / L sodium chloride.

6. The culturing method according to claim 5, wherein Further comprising: Adding the seed solution to a fermentation medium, placing it in a culture device for fermentation culture, and performing continuous static magnetic field treatment and electrostatic field treatment to obtain surfactin; Preferably, the fermentation medium includes a basal salt medium.

7. The cultivation method according to claim 6, characterized in that: The components of the fermentation medium include 40 g / L glucose, 20 g / L tryptone, 40 mmol / L disodium hydrogen phosphate, 30 mmol / L potassium dihydrogen phosphate, 0.007 mmol / L calcium chloride, 0.5 - 1.5 mmol / L magnesium ion-containing compound, 0.004 mmol / L ethylenediaminetetraacetic acid, 0.2 mmol / L ferrous sulfate heptahydrate, and 0.01 mmol / L manganese sulfate monohydrate.

8. The cultivation method according to claim 4, characterized in that: The content of surfactin in the fermentation system is 0.5 - 4.5 g / L.

9. The culturing method according to claim 4, wherein: The concentration of magnesium ions in the fermentation system is 0.5 - 1.5 mmol / L; And / or, the magnesium ions are derived from magnesium ion-containing compounds, and the magnesium ion-containing compounds include at least any one of anhydrous magnesium sulfate or magnesium chloride; And / or, the intensity of the static magnetic field treatment is 10 - 20 mT, and the time of the static magnetic field treatment is 72 h; And / or, the intensity of the electrostatic field treatment is 4 - 8 V / cm, and the time of the electrostatic field treatment is 72 h.

10. A culture device for Bacillus subtilis, characterized in that, For the culture method according to any one of claims 4 - 9, and the culture device includes: A sample chamber for at least accommodating a container loaded with the fermentation system; A magnetic field generating unit, at least for generating a static magnetic field in the sample chamber; An electric field generating unit, at least for generating an electrostatic field in the sample chamber; A shaker unit, at least for carrying a container and rotating it; the shaker unit is placed in the sample chamber; A temperature regulating unit, at least for regulating the temperature in the sample chamber; A control unit, connected to the magnetic field generating unit, the electric field generating unit, the shaker unit, and the temperature regulating unit, and at least for regulating the working states and working parameters of the magnetic field generating unit, the electric field generating unit, the shaker unit, and the temperature regulating unit.

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