Fermentation process for improving yield of antibacterial peptide of bacillus subtilis

Abstract

The invention relates to the technical field of biological fermentation, and discloses a fermentation process for improving the yield of bacillus subtilis antibacterial peptides, which comprises the following steps: firstly, inoculating bacillus subtilis into a culture medium containing a magnetic carrier for adhesion growth to form a bacterium-carrying magnetic carrier; secondly, capturing the bacterium-carrying magnetic carrier through an external magnetic field and fixing the bacterium-carrying magnetic carrier in a production area in the fermentation tank to form a solid-phase production area with high cell density and a liquid-phase exchange area with low cell density; and then, orderly synergistic induction treatment is carried out on the solid-phase production area, namely anaerobic or micro-aerobic stress is applied firstly, and when it is monitored that the oxidation-reduction potential is reduced to a preset value, pulse type ultrasonic waves are applied. According to the method, the contradiction between thallus growth and product synthesis is relieved by constructing spatial partitions; through synergistic induction based on physiological state feedback, the product synthesis and secretion efficiency is improved; and the production life of the system is prolonged through asynchronous cycle operation, and the yield and purity of the antibacterial peptide and the production rate of the system are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation, in particular to a fermentation process for improving the yield of Bacillus subtilis antibacterial peptides. BACKGROUND

[0002] Antibacterial peptides, as a class of polypeptides with broad-spectrum antibacterial activity, have shown great application potential in the medical field. Bacillus subtilis is a commonly used engineering strain for producing various antibacterial peptides, and microbial fermentation is the main way to obtain these antibacterial peptides.

[0003] However, in the existing fermentation process, the traditional batch or fed-batch fermentation mode is usually adopted. In this mode, the growth and reproduction of the bacteria and the synthesis of the secondary metabolites are carried out in the same reaction system. The two physiological processes have different requirements for environmental conditions and even conflict with each other: the rapid growth of the bacteria requires sufficient nutrients and dissolved oxygen, while the synthesis of antibacterial peptides is often triggered in large quantities in the post-growth stage, under nutrient-limited or specific stress conditions. This inherent contradiction limits the final yield and production efficiency of antibacterial peptides. At the same time, since the bacteria and the product are mixed in the entire fermentation broth, the harvested product contains a large amount of bacterial proteins and other metabolic impurities, increasing the difficulty and cost of subsequent separation and purification.

[0004] In addition, in order to improve the yield, the existing technology often uses the addition of chemical inducers or the change of culture conditions for induction, but these methods often lack precision, are difficult to control according to the real-time physiological state of the bacteria, and have low induction efficiency. Moreover, long-term stress treatment can easily cause the bacteria to age prematurely and become inactive, resulting in a short production cycle and poor stability of the entire fermentation system, limiting the improvement of equipment utilization and total yield.

[0005] Therefore, it is a technical problem to be solved in the field to develop a new fermentation process that can effectively separate the growth of bacteria from the synthesis of products, achieve precise and efficient induction, and maintain long-term stable operation of the system. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a fermentation process for improving the yield of Bacillus subtilis antibacterial peptides, which solves the problems of mutual restriction of bacteria growth and product synthesis in traditional batch fermentation, low product purity and difficulty in subsequent separation and purification, lack of precise control in the induction process leading to low product synthesis efficiency, and poor production stability and short effective production cycle of the fermentation system.

[0007] To achieve the above purpose, the present application is implemented by the following technical scheme: a fermentation process for improving the yield of Bacillus subtilis antibacterial peptides, comprising: Step S1 adhesion growth: Bacillus subtilis is inoculated into a fermenter containing growth medium and magnetic carriers. The volume of the magnetic carriers added is 10-20% (v / v) of the working volume of the fermenter. The culture is carried out under suitable growth conditions to allow Bacillus subtilis to attach to the surface of the magnetic carriers and form biofilm, thus obtaining the magnetic carriers with bacteria.

[0008] Step S2: Partitioning and capturing: In the preset production zone of the fermenter, a magnetic field with a strength of 0.2-0.5 T is generated by an electromagnetic system arranged outside the fermenter. The magnetic field captures and fixes the magnetic carriers with bacteria obtained in step S1 in the production zone, thus separating a solid-phase production zone with high cell density and a liquid-phase exchange zone with low cell density in the fermenter.

[0009] Preferably, while the magnetic field is applied to capture the magnetic carriers with bacteria, a micro-perfusion system is used to perfuse a production perfusion liquid into the solid-phase production zone at a flow rate of 0.05-0.1 V (working volume) / h. The production perfusion liquid contains a slow metabolic carbon source (such as glycerol or citric acid) at a concentration of 30-50 g / L and precursor amino acids required for the synthesis of antimicrobial peptides at a total concentration of 2.0-9.0 g / L, to locally construct a nutrient environment suitable for product synthesis.

[0010] Step S3: Synergistic induction: The magnetic carriers with bacteria in the solid-phase production zone are subjected to multiple stress treatments. First, the magnetic carriers with bacteria are subjected to anaerobic or micro-aerobic stress treatment by temporarily stopping or reducing the local gas supply to the solid-phase production zone, or switching the local gas supply to the solid-phase production zone to an inert gas. The stress treatment process is controlled by monitoring the oxidation-reduction potential (ORP) in the solid-phase production zone. When the ORP value decreases by 50-150 mV relative to the aerobic state, it indicates that the preset stress level has been reached, at which point the subsequent ultrasonic application is triggered.

[0011] Subsequently, pulsed ultrasonic waves are applied to the solid-phase production zone. The frequency of the pulsed ultrasonic waves is 20-40 kHz, and the acoustic power density is 0.1-0.5 W / cm². Preferably, the pulsed ultrasonic waves work in an alternating mode with an on-time of 3-8 seconds and an off-time of 5-15 seconds. The stress treatment and ultrasonic application work together to synergistically induce the synthesis and secretion of antimicrobial peptides.

[0012] Step S4: Harvesting the product: From the liquid-phase exchange zone, fermentation supernatant containing the target product antimicrobial peptides is harvested by extraction or continuous flow.

[0013] In a preferred embodiment of the present application, the magnetic carrier is a functionalized magnetic porous carrier, which is prepared by a method comprising the following steps: Providing a carrier matrix with porous structure and magnetic responsiveness; Activating the carboxyl functional groups on the surface of the carrier matrix using a carbodiimide-based activator with a concentration of 10-50 mM and N-hydroxysuccinimide with a concentration of 10-50 mM; Reacting the activated carrier matrix with a quorum sensing signal peptide analogue solution with a concentration of 0.1-0.5 mg / mL at 4°C for 12-24 hours to form covalent connections on the surface of the carrier matrix.

[0014] In a preferred embodiment of the present application, steps S2 and S3 are performed in an asynchronous cycle. That is, periodically removing the magnetic field to allow the bacteria-carrying magnetic carriers to disperse in the fermentation broth for recovery, and then performing a new round of capture and induction on part or all of the bacteria-carrying magnetic carriers. The cycle of a single asynchronous cycle is set to 2-4 hours.

[0015] The present application provides a fermentation process for improving the yield of Bacillus subtilis antibacterial peptides. The following beneficial effects are achieved: 1. The present application forms a high-density cell solid-phase production area and a low-density cell liquid-phase exchange area in a single fermentation tank through magnetic field partition operation. This structure allows the accumulation of bacterial biomass and the synthesis of antibacterial peptides to be separated in space, and to be carried out in areas with different nutritional and environmental conditions, thereby eliminating the mutual constraints of the two physiological processes on the fermentation conditions.

[0016] 2. The present application produces a synergistic technical effect by sequentially combining multiple treatment methods. The quorum sensing signal produced by the high-density bacterial population formed by magnetic field capture, the pre-set anaerobic or micro-aerobic stress treatment, and the subsequent application of pulsed ultrasonic waves all work together in time to increase the synthesis rate and extracellular secretion efficiency of antibacterial peptides.

[0017] 3. Since most of the bacteria are physically fixed in the solid-phase production area, and the target product antibacterial peptides are secreted and diffuse to the liquid-phase exchange area, the supernatant harvested from the liquid-phase exchange area has a low content of impurities such as proteins, which reduces the technical difficulty of subsequent product separation and purification. In addition, the use of an asynchronous cycle operation mode can prolong the effective production time of the entire fermentation system, thereby improving the utilization rate of the equipment and the total yield of antibacterial peptides. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The process flowchart of the present application. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the specification of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0020] The main raw materials and reagents used in the following examples and comparative examples are as follows, and the reagents not specifically described are commercially available analytical pure or higher grade products.

[0021] Ferric chloride (III) hexahydrate, CAS number: 10025-77-1; Ferrous chloride (II) tetrahydrate, CAS number: 13478-10-9; Ammonia water, CAS number: 1336-21-6; Sodium alginate, CAS number: 9005-38-3; Anhydrous calcium chloride, CAS number: 10043-52-4; Chitosan, CAS number: 9012-76-4; Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, CAS number: 25952-53-8; N-hydroxysuccinimide, CAS number: 6066-82-6; D (+) -anhydrous glucose, CAS number: 50-99-7; Glycerol, CAS number: 56-81-5; Yeast extract, CAS number: 8013-01-2; Sodium chloride, CAS number: 7647-14-5; Dipotassium hydrogen phosphate, CAS number: 7758-11-4; Monopotassium phosphate, CAS number: 7778-77-0; Magnesium sulfate heptahydrate, CAS number: 10034-99-8; L-leucine, CAS number: 61-90-5; L-tyrosine, CAS number: 60-18-4; l-valine, CAS number: 72-18-4; L-asparagine, CAS number: 70-47-3; L-phenylalanine, CAS number: 63-91-2; Acetonitrile, CAS number: 75-05-8; Trifluoroacetic acid, CAS No.: 76-05-1; Nitrogen, CAS No.: 7727-37-9.

[0022] Examples 1 and 2; This example aims to illustrate in detail an integrated process for improving the production of Bacillus subtilis antimicrobial peptides by using functionalized magnetic carriers, zoned induction and asynchronous cycle mode.

[0023] 1. Preparation of functionalized magnetic porous carriers Preparation of magnetic core-shell carrier matrix 11.7 g of iron (III) chloride hexahydrate and 4.3 g of ferrous (II) chloride tetrahydrate were dissolved in 200 mL of deionized water, and heated to 80°C under nitrogen protection. Under vigorous mechanical stirring (600 rpm), 50 mL of 25% w / w ammonia water was added dropwise to make the pH value of the system reach 10.5, and the reaction was continued for 1 hour under this condition. After the reaction was completed, the black Fe3O4 magnetic nanoparticles were separated using a permanent magnet and washed with deionized water until neutral to obtain the magnetic core.

[0024] The above magnetic core was dispersed in 100 mL of a 2% (w / v) sodium alginate solution. The mixed solution was dropped into 500 mL of a 2% (w / v) calcium chloride solution at a flow rate of 20 mL / h using a syringe pump, and stirred at room temperature for 2 hours to form magnetic calcium alginate gel microspheres with a diameter of about 2-3 mm. The microspheres were collected and washed with sterile water three times to obtain a carrier matrix with a porous structure and magnetic responsiveness.

[0025] Functionalization of the surface of the carrier matrix The above-prepared carrier matrix (wet weight about 50 g) was suspended in 200 mL of MES buffer (0.1 M, pH 6.0). To the suspension, 30 mM of ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 30 mM of N-hydroxysuccinimide (NHS) were added, and the reaction was carried out for 30 minutes at room temperature with shaking to activate the carboxyl functional groups on the surface of the carrier.

[0026] After the reaction was completed, the carrier was quickly washed with sterile water three times to remove unreacted activators. Immediately, the activated carrier was immersed in 100 mL of a 0.3 mg / mL solution of quorum sensing signal peptide analog (ComXPheromone) and reacted for 18 hours at 4°C with slight shaking to achieve covalent attachment of the signal peptide to the surface of the carrier.

[0027] After the reaction, the functionalized magnetic porous carriers were washed three times with phosphate buffer solution (PBS, pH 7.4) containing 0.05% Tween-20 and three times with sterile water to remove the physically adsorbed signal peptide. The final functionalized magnetic porous carriers were stored at 4°C under sterile conditions for later use.

[0028] 2. Fermentation process for improving the yield of Bacillus subtilis antibacterial peptide; Seed liquid preparation A loop of Bacillus subtilis ATCC6633 was picked from the slant medium and inoculated into a 250 mL flask containing 50 mL of LB medium (10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride) and incubated at 37°C, 200 rpm for 12 hours to prepare the seed liquid.

[0029] Step S1: Adhesion growth phase A 5 L working volume fermenter was filled with 3 L of growth medium (20 g / L of proteose peptone, 20 g / L of D(+) anhydrous glucose, 10 g / L of yeast extract, 1 g / L of potassium phosphate dibasic, 0.5 g / L of magnesium sulfate heptahydrate). 450 mL (15% v / v of the working volume) of the functionalized magnetic porous carriers prepared above were added to the fermenter. The seed liquid was inoculated at a 10% (v / v) inoculation amount, and the adhesion growth culture was carried out at 37°C, pH 7.0 (automatically controlled by adding 2M NaOH and 2M HCl), stirring rate 300 rpm, sterile air aeration rate 1.0 VVM for 12 hours.

[0030] First execution of steps S2 and S3: zoned capture and synergistic induction After the adhesion growth phase, an external electromagnetic system was started on the pre-set production zone of the fermenter to apply a magnetic field strength of 0.4T to capture and fix the magnetic carriers in this area, forming a solid-phase production zone.

[0031] At the same time, a perfusion pump was started to perfuse the production perfusion liquid into the solid-phase production zone at a flow rate of 0.24 L / h (equivalent to 0.08 V / h). The formula of the production perfusion liquid was: glycerol 40 g / L, L-leucine 2.0 g / L, L-tyrosine 1.0 g / L, L-valine 1.5 g / L, L-asparagine 1.5 g / L, L-phenylalanine 1.0 g / L.

[0032] At the same time of starting the perfusion, the local gas supply to the solid-phase production zone was stopped and switched to sterile nitrogen gas. The oxidation-reduction potential was monitored in real time by the ORP electrode installed in the solid-phase production zone. When the ORP value decreased by 100 mV compared to the aerobic state, the ultrasonic generator was triggered to work. The ultrasonic generator applied a frequency of 30 kHz and a sound power density of 0.3 W / cm 2ultrasonic waves and works in a pulse mode with 5 seconds on and 10 seconds off.

[0033] Asynchronous cycle with execution of step S4: product harvest and system maintenance; After the combined operation of steps S2 and S3 lasts for 3 hours (constituting an induction period), the external magnetic field is removed and the stirring rate is increased to 400 rpm to re-disperse the magnetic carriers in the broth and restore for 1 hour (constituting a restoration period). The 3-hour induction period and the 1-hour restoration period together constitute a complete 4-hour asynchronous cycle period.

[0034] After the first restoration period, the electromagnetic system is re-started and the induction operation of steps S2 and S3 is repeated for the other half of the magnetic carriers. This cycle is repeated.

[0035] During the entire induction production phase (from the first execution of steps S2 and S3), 300 mL of fermentation supernatant is extracted from the liquid exchange zone on the other side of the fermenter every 4 hours for product analysis and collection, and an equal volume of fresh production perfusion liquid (glycerol base liquid without amino acids) is added to maintain the working volume.

[0036] Final product collection and recordation; The total fermentation time lasts for 72 hours, and all the supernatant samples extracted from the liquid exchange zone are collected for subsequent performance testing.

[0037] Comparative Examples 1-3; Comparative Example 1: traditional batch fermentation process; The difference compared with Example 1 is that the comparative example uses a traditional batch fermentation process, i.e., no magnetic porous carriers are added during the fermentation process, no magnetic field partition capture, perfusion and co-induction operation are performed, and only continuous batch fermentation in the growth medium for 72 hours is performed. The rest of the conditions such as strains, preparation of seed liquid and formula of basic medium are the same.

[0038] Comparative Example 2: process lacking co-induction; The difference compared with Example 1 is that, during the co-induction stage of step S3, no anaerobic or micro-aerobic stress treatment is performed (i.e., no local gas supply is stopped or switched to nitrogen), and only the same pulsed ultrasonic waves as in Example 1 are applied to the solid-phase production zone under continuous aerobic conditions. All other steps, conditions and parameters are the same.

[0039] Comparative Example 3: process using non-functionalized carriers; The difference compared with Example 1 is that the magnetic porous carrier used in this comparative example is an unfunctionalized carrier, i.e. the carrier has only completed the construction of the magnetic inner core-shell carrier matrix at the time of preparation, without the covalent connection step of quorum sensing signal peptide analog. All other fermentation process steps, conditions and parameters are the same.

[0040] Test Example 1-3; Test Example 1: Performance test and data comparison This test example aims to test the performance of the fermentation supernatant obtained in Example 1 and Comparative Examples 1-3, in order to quantify the data to prove the beneficial effects of the technical solutions of the present application.

[0041] (I) Test method Sample pretreatment: 5 mL of supernatant sample collected at the end of 72-hour fermentation of Example 1 and Comparative Examples 1-3 was centrifuged at 4°C, 10000 x g for 10 minutes to remove residual cells and solid particles. The supernatant was filtered using a polyether sulfone (PES) filter membrane with a pore size of 0.22 µm, and the obtained filtrate was used for subsequent analysis.

[0042] Antibacterial peptide yield determination was quantitatively analyzed by high performance liquid chromatography (HPLC).

[0043] Chromatographic conditions: Agilent 1260 Infinity II HPLC system was used, equipped with a C18 reversed-phase chromatographic column (4.6 mm x 250 mm, 5 µm). The mobile phase A was ultrapure water containing 0.1% (v / v) trifluoroacetic acid, and the mobile phase B was acetonitrile containing 0.1% (v / v) trifluoroacetic acid. Linear gradient elution was used: 0-5 minutes, 10% B; 5-35 minutes, 10%-70% B; 35-40 minutes, 70%-10% B; 40-45 minutes, 10% B. The flow rate of the mobile phase was 1.0 mL / min, the column temperature was maintained at 30°C, and the detection wavelength was 214 nm.

[0044] Quantitative method: A series of standard solutions with concentration gradients were prepared using antibacterial peptide standard (sequence ADLPFEF) with purity ≥98%, and injected into the HPLC system. The standard curve was drawn by peak area versus concentration. 20 µL of the treated sample filtrate was injected, and the concentration of antibacterial peptide in the sample (mg / L) was calculated according to the peak area of the corresponding retention time on the chromatogram through the standard curve, which was the antibacterial peptide yield.

[0045] Product purity analysis in fermentation supernatant was based on the above HPLC chromatogram, and peak area normalization method was used to calculate the product purity. The calculation formula is: product purity (%) = (area of antibacterial peptide target peak / total area of all peaks on the chromatogram) x 100.

[0046] System productivity (mg / L / h) was calculated according to the following formula: System productivity = Fermentation endpoint antimicrobial peptide yield (mg / L) / Total fermentation duration (h). In this test, the total fermentation duration was 72 hours.

[0047] (II) Test results The performance test data of Example 1 and Comparative Examples 1-3 are summarized in Table 1 below.

[0048] Table 1: Comparison of performance indicators of different fermentation processes The results obtained from Table 1 are analyzed as follows: From the data in Table 1, it can be seen that the process of Example 1 is higher than the processes of Comparative Examples 1, 2 and 3 in terms of antimicrobial peptide yield, product purity and system productivity. This result is attributed to the solid-phase production zone and liquid-phase exchange zone constructed by the magnetic field in the fermenter. This spatial partition structure allows cell growth and product synthesis to occur in their respective suitable microenvironments, solving the problem of metabolic competition in traditional batch fermentation (Comparative Example 1). At the same time, since most of the cells are fixed on the carrier, the content of impurities in the supernatant harvested from the liquid-phase exchange zone is low, so the product purity is significantly higher than that of Comparative Example 1 in which the cells are freely suspended.

[0049] Comparing the data of Example 1 and Comparative Example 2, in the absence of anaerobic or microaerobic stress treatment, even if ultrasonic waves are applied, the yield and productivity are much lower than those of Example 1. This indicates that there is a synergistic effect between the anaerobic or microaerobic stress achieved by stopping the local gas supply or switching to an inert gas and the subsequent application of pulsed ultrasonic waves. First, the stress conditions induce the bacteria to enter a specific secondary metabolic physiological state, and then the ultrasonic waves transiently increase the permeability of the cell membrane. This orderly combination treatment has an effect that exceeds that of a single physical field treatment.

[0050] Example 1 also shows advantages in yield and productivity compared to Comparative Example 3 using a non-functionalized carrier. This difference shows that covalently linking a quorum-sensing signal peptide analogue to the surface of the carrier can form a high-concentration local signal environment in the solid-phase production zone, which directly acts on the high-density attached cells, thereby producing an additional promoting effect on the expression of the antimicrobial peptide gene. This signal enhancement design based on carrier surface modification is another important technical component for achieving high yield.

[0051] Test Example 2: Effect of different induction modes on system production stability This test example aims to investigate the effect of the asynchronous cycle induction mode used in the present application and the traditional continuous induction mode on the stability of system production.

[0052] (I) Test method The experimental group and the control group were set up. The same strains, medium, functionalized magnetic carriers and the attachment growth stage (step S1) were used in both groups.

[0053] Experimental group: The asynchronous cycle mode described in Example 1 was completely followed. That is, after 12 hours of attachment growth, a 3-hour induction period (partitioned capture and synergistic induction) was performed, followed by a 1-hour recovery period (the magnetic field was removed and the carriers were redispersed), and this 4-hour cycle was continued until the end of fermentation (total duration of 72 hours).

[0054] Control group: The continuous induction mode was used. That is, after 12 hours of attachment growth, the same partitioned capture and synergistic induction operations (steps S2 and S3) as in Example 1 were performed, but no recovery period was set. Instead, the magnetic field, perfusion, anaerobic and ultrasonic treatment were continuously applied from the 12th hour until the end of fermentation (total duration of 72 hours).

[0055] Sample collection and interval productivity determination: Samples were collected from the liquid exchange zone of the fermentation tank at 24h, 36h, 48h, 60h, and 72h of fermentation. The HPLC method described in Test Example 1 was used to determine the concentration of antimicrobial peptides at each time point. According to the measured concentration data, the production rate of different time intervals was calculated.

[0056] Interval production rate calculation formula: Interval production rate ; Wherein is the measured antimicrobial peptide concentration at time t, and is the sampling time point.

[0057] (II) Test results The production rate data of the experimental group and the control group in different time intervals are summarized in Table 2 below.

[0058] Table 2: Comparison of production rates in different time intervals under different induction modes Analysis of the results obtained from Table 2: From the data in Table 2, it can be seen that the experimental group (asynchronous cycle mode) maintained a relatively stable interval production rate throughout the production cycle. The interval production rate of the control group (continuous induction mode) showed a continuous downward trend over time, especially in the later stage of fermentation. This indicates that the operation of periodically removing the physical field and redispersing the carrier-loaded magnetic carriers in the fermentation broth helps to maintain the production activity of the cell population.

[0059] ​In continuous induction mode, cells immobilized in the solid-phase production zone are continuously subjected to multiple environmental stresses, such as anaerobicity, ultrasound, and high concentration of metabolic products. This uninterrupted stress condition causes the physiological load of cells to exceed their regulatory capacity, resulting in irreversible damage to metabolic activity and premature cell senescence. This phenomenon directly reflects the rapid decay of system productivity.

[0060] In contrast, the recovery period set in the asynchronous cycle mode provides an opportunity for the bacteria-loaded magnetic carriers to escape from the strong stress zone. During the process of re-dispersing in the liquid-phase exchange zone, cells can contact a more moderate environment to repair necessary physiological functions and adjust metabolic balance. This rotating "induction-recovery" operation, through batch and alternating use of cell populations, prolongs the effective production life of the entire biological reaction system, ultimately achieving higher total yield and system productivity.

[0061] Test Example 3: Study on the effect of different ultrasound application timing on product synthesis The purpose of this test example is to investigate the effect of the timing relationship between anaerobic stress and ultrasound application on the yield of antimicrobial peptides, in order to verify the necessity of triggering ultrasound by monitoring the oxidation-reduction potential (ORP).

[0062] (I) Test method The experiment is divided into three experimental groups A, B, and C. All groups use the same strain, medium, functionalized magnetic carriers, attachment growth stage (step S1), and magnetic field partition capture (step S2) as in Example 1. The difference lies in the specific execution timing of step S3 in the simultaneous induction.

[0063] Group A (ORP-triggered mode): completely follow step S3 of Example 1. That is, after starting anaerobic stress, the ORP value of the solid-phase production zone is monitored in real time. When the ORP value decreases by 100 mV relative to the aerobic state, the pulsed ultrasonic generator is started.

[0064] Group B (synchronous application mode): immediately start the pulsed ultrasonic generator at the same time as starting anaerobic stress (switching to nitrogen gas). That is, anaerobic stress and ultrasound application start simultaneously.

[0065] Group C (anaerobic stress only mode): only perform anaerobic stress operation, without applying any ultrasound during the entire induction period.

[0066] Sample collection and concentration determination were performed at 0 h (start of induction phase), 1 h, 2 h, and 3 h from the liquid-phase exchange zone of each group's fermenter. The HPLC method described in Test Example 1 was used to determine the concentration of antimicrobial peptides in the samples at each time point.

[0067] (II) Test results The antibacterial peptide concentration data of groups A, B and C at different time points in the induction stage are summarized in Table 3 below.

[0068] Table 3: Changes in antibacterial peptide concentration under different ultrasonic triggering modes Analysis of Table 2 results As can be seen from the data in Table 3, at each time point in the induction stage, the antibacterial peptide concentration of group A is significantly higher than that of groups B and C. This result shows that correlating the application time of ultrasonic waves with the changes in the physiological state of the bacteria can achieve higher product synthesis and secretion efficiency. The decrease in oxidation-reduction potential (ORP) is an objective indicator of the shift of cells from aerobic metabolism to anaerobic metabolism, which is a prerequisite for inducing the synthesis of secondary metabolites.

[0069] In the process of group A, the adjustment of the antibacterial peptide synthesis pathway and the initial accumulation of the product are first induced in the cell interior by the anaerobic environment. When the ORP value reaches the preset threshold, it indicates that the cells are in a physiological state suitable for product synthesis. At this time, pulsed ultrasonic waves are applied, which mainly instantaneously increase the permeability of the cell membrane, thereby efficiently releasing the antibacterial peptides that have been accumulated in the cell into the fermentation broth. This orderly and physiological state feedback-based synergistic effect enables efficient connection between the synthesis and secretion processes of the product.

[0070] In contrast, the ultrasonic waves applied too early in group B act on cells that have not completed physiological state adjustment, and their effect on membrane permeability cannot be synchronized with high-level product secretion, and the simultaneous application of double stress can cause excessive damage to the cells. The results of group C show that without the aid of ultrasonic waves for secretion, even if the cells synthesize part of the product under anaerobic conditions, the efficiency of its release to the extracellular is greatly limited, resulting in a lower concentration of the product in the fermentation broth. This further confirms the clear synergistic relationship between anaerobic stress and the application of ultrasonic waves at a specific time.

Claims

1. A fermentation process for increasing the yield of a Bacillus subtilis antibacterial peptide, characterized in that, The method comprises the following steps: S1, inoculating Bacillus subtilis into a growth medium containing magnetic carriers for adherent growth to obtain bacteria-loaded magnetic carriers; S2, applying a magnetic field with a strength of 0.2-0.5T in a preset production area of a fermenter to capture and fix the bacteria-loaded magnetic carriers obtained in step S1 in the production area, thereby forming a solid-phase production area and a liquid-phase exchange area in the fermenter; S3, subjecting the bacteria-carrying magnetic carriers in the solid-phase production zone to anaerobic or micro-aerobic stress treatment, and after the stress treatment, applying pulsed ultrasound with a frequency of 20-40 kHz and a sound power density of 0.1-0.5 W / cm2to the solid-phase production zone to synergistically induce the synthesis and secretion of antibacterial peptides; 2 S3, subjecting the bacteria-carrying magnetic carriers in the solid-phase production zone to anaerobic or micro-aerobic stress treatment, and after the stress treatment, applying pulsed ultrasound with a frequency of 20-40 kHz and a sound power density of 0.1-0.5 W / cm2to the solid-phase production zone to synergistically induce the synthesis and secretion of antibacterial peptides; S4, harvesting fermentation supernatant containing antibacterial peptides from the liquid-phase exchange area.

2. The fermentation process for improving the production of Bacillus subtilis antibacterial peptide according to claim 1, characterized in that, The magnetic carrier is a functionalized magnetic porous carrier prepared by a method comprising the following steps: providing a carrier substrate with a porous structure and magnetic responsiveness; activating carboxyl functional groups on the surface of the carrier substrate using a carbodiimide-based activator with a concentration of 10-50mM and N-hydroxysuccinimide with a concentration of 10-50mM; reacting the activated carrier substrate with a quorum sensing signal peptide analogue solution with a concentration of 0.1-0.5mg / mL at 4°C for 12-24 hours to form covalent connections on the surface of the carrier substrate.

3. The fermentation process for improving the production of Bacillus subtilis antibacterial peptide according to claim 1, characterized in that, The anaerobic or micro-aerobic stress treatment in step S3 comprises temporarily stopping or reducing local gas supply to the solid-phase production area or switching the local gas supply to the solid-phase production area to an inert gas, and the anaerobic or micro-aerobic stress treatment is controlled by monitoring the redox potential of the solid-phase production area, and when the redox potential value decreases by 50-150mV relative to the aerobic state, the subsequent ultrasonic wave application is triggered.

4. The fermentation process for improving the production of Bacillus subtilis antibacterial peptide according to claim 1, characterized in that, The application of the magnetic field in step S2 comprises generating a magnetic field gradient by an electromagnetic system arranged outside the fermenter to apply the magnetic field, and at the same time, perfusing a production perfusion solution into the solid-phase production area.

5. The fermentation process for improving the production of Bacillus subtilis antibacterial peptide according to claim 4, characterized in that, The production perfusion solution contains a slow metabolic carbon source with a concentration of 30-50g / L and precursor amino acids required for antibacterial peptide synthesis with a total concentration of 2.0-9.0g / L.

6. The fermentation process for improving the production of Bacillus subtilis antibacterial peptide according to claim 4, characterized in that, The perfusion flow rate of the production perfusion solution is 0.05-0.1V / h.

7. The fermentation process for improved production of Bacillus subtilis antibacterial peptide according to claim 1, characterized in that, Steps S2 and S3 are performed in an asynchronous cycle, i.e., periodically removing the magnetic field to re-disperse the bacteria-loaded magnetic carriers in the fermentation broth, and then performing a new round of capture and induction on the bacteria-loaded magnetic carriers.

8. The fermentation process for improving the production of Bacillus subtilis antibacterial peptide according to claim 7, characterized in that, The single cycle period of the asynchronous cycle is 2-4 hours.

9. The fermentation process for improved production of Bacillus subtilis antibacterial peptide according to claim 1, characterized in that, The pulsed ultrasonic wave in step S3 alternately works in an on-time of 3-8 seconds and an off-time of 5-15 seconds.

10. The fermentation process for improved production of Bacillus subtilis antibacterial peptide as claimed in claim 1 wherein, In step S1, the addition volume of the magnetic carriers accounts for 10%-20% of the working volume of the fermenter.

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