Fermentation method and application of bacillus laterosporus

Abstract

The invention discloses a fermentation method and application of bacillus laterosporus, and belongs to the technical field of microbial fermentation engineering and biological control. In order to solve the technical problems of unstable fermentation level, low spore formation rate and the like of bacillus laterosporus, the invention provides a high-density fermentation method of bacillus laterosporus, which comprises the following steps: adding a nutrition-signal composite inducer in stages in the middle and later periods of fermentation, and coupling pH / DO two-stage control, so that the spore formation rate is greater than or equal to 95%; besides, fermentation liquor of bacillus laterosporus and the bentonite-trehalose-sodium glutamate composite carrier are mixed and then subjected to low-temperature spray drying, the viable bacterium yield of the prepared microbial agent is larger than or equal to 95%, the viable bacterium rate is larger than or equal to 90% after the microbial agent is stored for 12 months at the temperature of 25 DEG C, and the microbial agent shows a remarkable effect in tomato root-knot nematode prevention and treatment of livestock and poultry diarrhea and has good application prospects. The compound can completely or partially replace chemical pesticides and antibiotics, and has a wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation engineering and biological control technology, specifically relating to a fermentation method and application of Bacillus laterosporus. Background Technology

[0002] Lateral spores (Bacillus retroflexus) Brevibacillus laterosporus This strain is a valuable resource of biocontrol microorganisms and probiotics with significant application potential. It can synthesize a variety of novel and broadly active antimicrobial substances, mainly including: (i) laterosporamin, a cyclic antimicrobial peptide with a unique mechanism of action, exhibiting strong inhibitory effects against various Gram-positive bacteria; (ii) various cyclic lipopeptide compounds, exerting bactericidal functions by disrupting the cell membrane integrity of pathogenic microorganisms; and (iii) spore-forming agents and other antimicrobial proteins, showing significant antagonistic effects against plant pathogenic fungi and nematodes. Based on the complex antimicrobial spectrum produced by this strain, it shows broad prospects in the biological control of agricultural plant diseases (especially soil-borne diseases such as root-knot nematodes and Fusarium wilt) and as an alternative to antibiotics in livestock and poultry farming.

[0003] Although the antibacterial activity of Bacillus laterosporus is widely recognized, its transition from laboratory research to industrial application still faces a series of severe technical bottlenecks, hindering its market competitiveness and stable application effects. First, unstable fermentation levels and low spore formation rates are the most prominent problems. Under existing fermentation processes, the spore formation rate of Bacillus laterosporus is generally below 80%. Spores are crucial for resisting adverse environments and ensuring product shelf life; a low spore rate directly leads to a rapid decline in the number of viable bacteria during storage and transportation, resulting in poor product stability and a short effective period. Second, downstream processing results in significant viable bacteria loss. In the critical formulation stage—spray drying—existing technologies struggle to effectively protect the bacteria, leading to viable bacteria losses typically exceeding two orders of magnitude (>2log). Even if the initial fermentation broth meets viable bacteria count standards, the finished product after drying and powdering experiences a significant decrease in viable bacteria count, and the viable bacteria rate of the formulation is generally below 50% after six months of storage, far from meeting the basic requirements for commercial application. Third, there is a lack of compounding technology solutions closely integrated with actual field application scenarios. Single microbial agents often fail to fully realize their biocontrol potential in complex natural environments. Existing technologies are mostly limited to the fermentation optimization of the strains themselves, and have failed to systematically develop efficient and stable compound systems that can be used in conjunction with commonly used chemical pesticides, fertilizers or adjuvants, thus limiting their control effects and universality in actual agricultural production.

[0004] In summary, the existing Bacillus laterosporus production and application technology chain suffers from interconnected pain points such as "low and unstable fermentation levels, poor formulation survival rates, and difficulty in reproducing field effects." The industry urgently needs to overcome these limitations and develop an integrated fermentation and preparation technology that can efficiently improve spore formation rate, significantly enhance formulation processing and storage stability, and ensure stable and reliable field application effects. This would truly unleash the enormous commercial value and application potential of Bacillus laterosporus as an excellent biocontrol agent and probiotic. Summary of the Invention

[0005] To address the technical challenges of unstable fermentation levels, low spore formation rates, poor formulation survival rates, and difficulty in reproducing field effects of Bacillus laterosporus, this invention provides a high-density fermentation method for Bacillus laterosporus based on a composite induction-dynamic pH / DO coupling control. This method achieves high-density spore fermentation of Bacillus laterosporus by adding a nutrient-signal composite inducer in stages during the later stages of fermentation and coupling pH / DO control in two stages. Furthermore, this invention utilizes a composite carrier adsorption-drying technology to mix the fermentation broth of Bacillus laterosporus with a bentonite-trehalose-sodium glutamate composite carrier, followed by low-temperature spray drying, to obtain a high-survival-rate inoculum. This inoculum exhibits significant effects in controlling tomato root-knot nematodes and treating livestock and poultry diarrhea.

[0006] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a method for high-density spore fermentation of Bacillus laterosporus, the method comprising the following steps: The bacterial culture of Bacillus laterosporus was inoculated into a fermentation medium for fermentation. During the mid-log phase of the bacterial growth and the spore initiation phase, inducer A and inducer B were added to the fermentation medium, respectively. Inducer A contains glutamic acid or its acceptable salt, soluble iron salt, and soluble zinc salt. Inducer B contains cyclic dinucleotide and methyl ester.

[0007] In one embodiment of the present invention, the inducer A, based on the mass fraction of the fermentation broth, contains 0.15-0.25% monosodium glutamate, 0.05-0.15% ferric ammonium citrate, and 0.04-0.06% ZnSO4·7H2O, and is added in a single dose; the inducer B, based on the volume of the fermentation broth, contains 0.8-1.2 mmol / L c-di-GMP and 0.4-0.6% methyl jasmonate, and is added in a pulse.

[0008] Preferably, the inducer A contains 0.2% monosodium glutamate, 0.1% ferric ammonium citrate, and 0.05% ZnSO4·7H2O by mass fraction of the fermentation broth; the inducer B contains 1 mmol / L c-di-GMP and 0.5% methyl jasmonate by volume of the fermentation broth.

[0009] In one embodiment of the present invention, the high-density spore fermentation method involves inoculating a Bacillus laterosporus culture medium with a bacterial suspension and fermenting at 35-37°C and a tank pressure of 0.04-0.06 MPa for 32-40 h. The fermentation is divided into two stages. In the first stage, the pH is controlled at 7.2±0.2, the dissolved oxygen (DO) is 30%-40%, the stirring speed is 300-400 rpm, and the aeration rate is 1.1-1.3 vvm. In the second stage, the pH is controlled at 6.8±0.2, the aeration rate is 0.3-0.4 vvm, and the stirring speed is 180-220 rpm, so that the DO is stabilized at 10%-15%.

[0010] In one embodiment of the present invention, the fermentation medium comprises 23-27 g / L glucose, 13-17 g / L corn steep liquor powder, 6-10 g / L yeast powder, 0.4-0.6 g / L MgSO4·7H2O, 0.04-0.06 g / L MnSO4·H2O, 2-4 g / L CaCO3, with the balance being water, and a pH of 7.2±0.2.

[0011] Preferably, the fermentation medium consists of 25 g / L glucose, 15 g / L corn steep liquor powder, 8 g / L yeast powder, 0.5 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, 3 g / L CaCO3, with the balance being water, and a pH of 7.2.

[0012] In one embodiment of the present invention, the mid-logarithmic growth phase of the strain is during the fermentation period of 8-10 h.

[0013] In one embodiment of the present invention, the spore initiation period is the fermentation period of 14-16 hours.

[0014] In one embodiment of the present invention, the taxonomic name of the *Bacillus laterosporus* is... Brevibacillus laterite KN-705 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20232527, on December 11, 2023, at Wuhan University, Wuhan, China.

[0015] The second objective of this invention is to provide a fermentation broth obtained by the above-described high-density spore fermentation method.

[0016] A third objective of this invention is to provide a method for preparing a microbial agent, the method comprising the following steps: By mass, 5-7 parts bentonite, 7-9 parts trehalose, 1-3 parts monosodium glutamate, and 0.5-1.5 parts skim milk powder are mixed evenly to obtain a composite carrier; the fermentation broth described in claim 5 is mixed with the composite carrier at a mass ratio of 1:0.1-0.15, and continuously mixed by a static mixer, and the mixture is kept at 28-32℃ for 10-20 min to complete the adsorption; then, the mixture is spray-dried at low temperature to obtain the bacterial agent.

[0017] Preferably, the preparation method includes the following steps: mixing 6 parts bentonite, 8 parts trehalose, 2 parts monosodium glutamate and 1 part skim milk powder evenly by mass to obtain a composite carrier; mixing the fermentation broth of claim 5 with the composite carrier at a mass ratio of 1:0.12, continuously mixing by a static mixer, and keeping at 30°C for 15 min to complete the adsorption; and then performing low-temperature spray drying to obtain the bacterial agent.

[0018] In one embodiment of the present invention, the bentonite has a particle size of 200 mesh.

[0019] In one embodiment of the present invention, the conditions for low-temperature spray drying are an inlet air temperature of 110°C and an outlet air temperature of 65°C.

[0020] The fourth objective of this invention is to provide a microbial agent prepared by the above-described preparation method.

[0021] The fifth objective of this invention is to provide the application of the above-mentioned microbial agent in the control of tomato nematodes.

[0022] The sixth objective of this invention is to provide the application of the above-mentioned bacterial agent in the treatment of diarrhea in livestock and poultry.

[0023] The beneficial effects of this invention are: This invention provides a high-density fermentation method for Bacillus laterosporus based on a composite induction-dynamic pH / DO coupling control. By adding a nutrient-signal composite inducer in stages during the middle and late stages of fermentation and coupling pH / DO two-stage control, the spore formation rate is ≥95%. In addition, the fermentation broth of Bacillus laterosporus is mixed with a bentonite-trehalose-sodium glutamate composite carrier using composite carrier adsorption drying technology and then spray-dried at low temperature to obtain a bacterial agent. The prepared bacterial agent has a viable cell yield of ≥95%, and a viable cell yield of ≥90% after storage at 25℃ for 12 months. It shows significant effects in the control of tomato root-knot nematodes and the treatment of livestock and poultry diarrhea, and can completely or partially replace chemical pesticides and antibiotics, with broad market prospects.

[0024] A complete process for high-density spore fermentation, high-survival-rate formulation, and efficient field application of Bacillus laterosporus has been developed through a three-step integrated technology of "composite induction - dynamic pH / dissolved oxygen coupling control - composite carrier adsorption drying". Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be noted that the embodiments mentioned below are only for explaining this invention and are not intended to limit the scope of this invention. The embodiments mentioned below are only some embodiments of this invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of this invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, any embodiments obtained by other skilled personnel without creative effort are protected by this invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0027] The Bacillus laterosporus screened in this invention ( Brevibacillus laterosporus KN-705 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20232527, deposited on December 11, 2023, at Wuhan University, Wuhan, China.

[0028] The culture medium and its formulation used for fermenting Bacillus laterosporus in this invention are as follows: Fermentation medium: glucose 25 g / L, corn steep liquor powder 15 g / L, yeast powder 8 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.05 g / L, CaCO3 3 g / L, balance water, pH 7.2.

[0029] Example 1: High-density fermentation of Bacillus laterosporus KN-705 in a 15 L fully automated fermenter based on fermentation induction technology Bacillus laterosporus KN-705 was activated by inoculating it into fermentation medium. After activation, the bacterial culture of Bacillus laterosporus KN-705 was inoculated at a rate of 5% into a 15 L fully automated fermenter containing 7.5 L of fermentation medium. Fermentation was carried out at 37℃ and a pressure of 0.05 MPa for 36 h. During fermentation, pH and dissolved oxygen (DO) were controlled in stages. From 0 to 12 h of fermentation, the pH was 7.2±0.2, DO was 30%-40%, the stirring speed was 300-400 rpm, and the aeration was 1.2 vvm. From 12 h to the end of fermentation, 2 mol / L citric acid was automatically added to lower the pH to 6.8±0.2, while the aeration was reduced to 0.3-0.4 vvm and the stirring speed was 200 rpm to stabilize the DO at 10%-15%. At the same time, inducer A and inducer B were added to the fermentation medium during the logarithmic growth phase (10 h) and the spore initiation phase (16 h), respectively. Inducer A (nutritional type): 0.2% (w / v) sodium glutamate + 0.1% (w / v) ferric ammonium citrate + 0.05% (w / v) ZnSO4·7H2O, administered once during mid-log phase (8-10 h); Inducer B (signal type): 1.0 μmol / L cyclic dipeptide c-di-GMP + 0.5% (v / v) methyl jasmonate, administered in a pulse during spore initiation phase (14-16 h).

[0030] Comparative Example 1: The difference from Example 1 is that inducer A and inducer B were not added during the logarithmic phase and spore initiation phase, respectively.

[0031] After fermentation in Example 1 and Comparative Example 1, the biomass and spore formation rate of Bacillus laterosporus KN-705 were measured.

[0032] The results showed that at the end of fermentation, the biomass of Example 1 was 1.2 × 10⁻⁶. 10 With a CFU / mL concentration and a spore formation rate of 96%, Example 1 showed a 2.1-fold increase in biomass and a 20% increase in spore formation rate compared to the uninduced Comparative Example 1.

[0033] Comparative Example 2: The difference from Example 1 is that no segmented pH adjustment was performed, the pH value was controlled at 7.0±0.1 throughout the process, the stirring speed was 300-400 rpm, the aeration was 1.2 vvm, and the dissolved oxygen changed naturally.

[0034] After fermentation in Example 1 and Comparative Example 2, the biomass and spore formation rate of Bacillus laterosporus KN-705 were measured.

[0035] The results showed that at the end of fermentation, the biomass of Example 1 was 1.2 × 10⁻⁶. 10With CFU / mL and a spore formation rate of 96%, Example 1 showed a 3.0-fold increase in biomass and a 15-fold increase in spore formation rate compared to Comparative Example 2, which did not undergo pH / DO coupling control.

[0036] Example 2: Preparation method of bacterial agent containing Bacillus laterosporus KN-705 based on composite carrier adsorption and drying Preparation of composite carrier: Mix 6 parts (200 mesh) of bentonite, 8 parts of trehalose, 2 parts of sodium glutamate and 1 part of skim milk powder by mass.

[0037] Online mixing: The fermentation broth obtained in Example 1 was continuously mixed with the above composite carrier at a ratio of 1:0.12 (w / w) using a static mixer, and the mixture was kept at 30°C for 15 min to complete the adsorption.

[0038] Low-temperature spray drying: inlet air temperature 110℃, outlet air temperature 65℃, forming a "vitrified-porous" dual protective structure on the surface of the bacteria.

[0039] The bacterial agent was obtained by low-temperature spray drying, with a viable bacteria content of 5×10⁻⁶. 11 CFU / g, viable cell yield ≥95%, moisture ≤4%, water activity aw ≤0.15, viable cell yield ≥90% after 12 months of storage at 25℃, viable cell yield ≥95% after 24 months of storage at 4℃.

[0040] Example 3: Application of wettable powder containing Bacillus laterosporus KN-705 in the control of root-knot nematodes in tomatoes. Formulation combination: 0.3% microbial agent (prepared in Example 2), 0.1% chitosan oligosaccharide and 0.05% xanthan gum were mixed in mass fraction to form a suspension (solvent is water) for the control of root-knot nematodes in tomatoes.

[0041] Field design: The seedlings were randomly assigned to groups with three replicates per group. The chemical control group was treated with 10% thiazophos (granules) at a rate of 2 kg / mu, and tomato seedlings were transplanted after application. The fermentation liquid group was treated with the fermentation liquid obtained in Example 1 at a rate of 3 kg / mu, diluted 50 times, and tomato seedlings were transplanted after spraying. The treatment group was treated with a suspension at a rate of 3 kg / mu, diluted 100 times, and tomato seedlings were transplanted after spraying the soil. The control group was treated without any chemical agents or fungicides applied to the soil and tomato seedlings were transplanted directly.

[0042] Thirty days after tomato transplanting, nematode damage was assessed, and the disease index and relative control effect were calculated. Tomato plant height was measured, and tomato yield was determined at the tomato harvest. The results are shown in Table 1.

[0043] The nematode hazard rating is as follows: Level 0: There are no root nodes on any of the roots; Level 1: 0% < the number of roots with root knots accounts for < 10% of the total root system; Level 3: 10% ≤ the number of roots with root knots accounts for less than 25% of the total root system; Level 5: 25% ≤ the number of roots with root knots accounts for less than 50% of the total root system; Level 7: 50% ≤ the number of roots with root knots accounts for less than 75% of the total root system; Level 9: 75% ≤ the number of roots with root knots accounts for ≤ 100% of the total root system.

[0044] Methods for calculating disease index and relative efficacy: Disease index (%) = ∑(number of diseased plants at each level × disease level) × 100% / (total number of plants investigated × highest disease level).

[0045] Relative prevention and control effect (%) = (disease index of control area - disease index of treatment area) × 100% / disease index of control area.

[0046] Table 1. Statistical results of average disease index, relative control efficacy, plant height, and yield for each treatment group.

[0047] As shown in Table 1, in terms of control efficacy, the average disease index of the treatment group treated with suspension after 30 days was reduced by 72.4% compared to the CK group, and by 19.91% compared to the fermentation broth group treated with fermentation broth, which was comparable to the chemical drug group. In terms of growth promotion, the plant height of the treatment group treated with suspension was increased by 18.7% and the yield was increased by 15.3% compared to the CK group, and both plant height and yield were also improved compared to the chemical drug group.

[0048] Example 4: Application of bacterial agents containing Bacillus laterosporus KN-705 in livestock and poultry farming The microbial agent prepared in Example 2 was added to the feed at a dosage of 200 g / t and fed to 28-day-old weaned piglets for 42 days. After 42 days of feeding, the diarrhea rate of the weaned piglets was recorded, the levels of IgA and IgG in the serum of the weaned piglets were detected using a kit, the feed conversion ratio was calculated based on the weight of the weaned piglets, and the number of Escherichia coli and Salmonella in the feces of the weaned piglets was detected.

[0049] The results showed that compared with direct feeding without added microbial agents, feeding weaned piglets with feed containing the microbial agents prepared in Example 2 reduced the diarrhea rate by 56%, increased the serum IgA and IgG levels by 21% and 17%, respectively, reduced the feed conversion ratio by 0.11, increased weight gain by 8.9%, decreased the number of Escherichia coli in feces by 1.8 log, and did not detect Salmonella.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A high-density spore fermentation method of Bacillus laterosporus, characterized by, The method comprises the following steps: The bacterial liquid of Bacillus laterosporus is inoculated into a fermentation medium for fermentation, and inducer A and inducer B are supplemented into the fermentation medium at the middle logarithmic growth phase and the spore initiation phase of the strain respectively; the inducer A contains glutamic acid or an acceptable salt thereof, a soluble iron salt and a soluble zinc salt; the inducer B contains cyclic dinucleotides and methyl ester.

2. The high-density spore fermentation process of claim 1, wherein, The inducer A contains 0.15-0.25% sodium glutamate, 0.05-0.15% ferric ammonium citrate and 0.04-0.06% ZnSO4·7H2O by mass fraction of the fermentation liquid, and is supplemented at one time; the inducer B contains 0.8-1.2 mmol / L c-di-GMP and 0.4-0.6% methyl jasmonate by volume fraction of the fermentation liquid, and is supplemented in pulses.

3. The high-density spore fermentation process of claim 1, wherein, The bacterial liquid of Bacillus laterosporus is inoculated into a fermentation medium, and fermentation is carried out at 35-37°C and a tank pressure of 0.04-0.06 MPa for 32-40 h; the fermentation is divided into two stages, in the first stage, the pH is controlled to be 7.2±0.2, the DO is controlled to be 30%-40%, the stirring speed is controlled to be 300-400 rpm, and the aeration is controlled to be 1.1-1.3 vvm; in the second stage, the pH is controlled to be 6.8±0.2, the aeration is controlled to be 0.3-0.4 vvm, the stirring speed is controlled to be 180-220 rpm, and the DO is stabilized at 10%-15%.

4. The high-density spore fermentation process of claim 1, wherein, The fermentation medium comprises 23-27 g / L of glucose, 13-17 g / L of corn syrup dry powder, 6-10 g / L of yeast powder, 0.4-0.6 g / L of MgSO4·7H2O, 0.04-0.06 g / L of MnSO4·H2O, 2-4 g / L of CaCO3, and the balance is water, and the pH is 7.2±0.

2.

5. The high-density spore fermentation process of claim 1, wherein, The middle logarithmic growth phase of the strain is a period of 8-10 h of fermentation, and the spore initiation phase is a period of 14-16 h of fermentation.

6. A fermentation liquid obtained by the high-density spore fermentation method in claims 1-5.

7. A method for preparing a microbial agent, characterized by, The method comprises the following steps: 5-7 parts of bentonite, 7-9 parts of trehalose, 1-3 parts of sodium glutamate and 0.5-1.5 parts of skimmed milk powder are uniformly mixed to prepare a composite carrier; the fermentation liquid in claim 5 and the composite carrier are mixed at a mass ratio of 1:0.1-0.15, continuously mixed by a static mixer, and incubated at 28-32°C for 10-20 min to complete adsorption; Then, a bacterial agent is prepared by low-temperature spray drying.

8. The bacterial agent prepared by the preparation method in claim 7.

9. The bacterial agent in claim 8 is used for preventing and treating nematodes in tomatoes.

10. The bacterial agent in claim 8 is used for treating diarrhea in livestock and poultry.

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