A method for improving the germination speed of bacillus spores and its application

Abstract

The present application belongs to the field of microbial technology and fermentation engineering, and particularly relates to a method for improving the spore germination speed of bacillus and application thereof. The method for improving the spore germination speed of bacillus comprises the following steps: spore production culture optimization stage: performing spore production culture in an optimized culture medium containing effective amount of germination precursor and germination enzyme system inducer to obtain mature spores; spore post-treatment activation stage: collecting and purifying the spores, and performing mild exosporium treatment or physical stimulation treatment to enhance the response of the spores to chemical germination agent without significantly damaging the stress resistance of the spores; reaction termination: immediately cooling and washing with deionized water after the treatment to terminate the treatment reaction. The present application significantly improves the spore germination speed, synchronism and efficiency by optimizing the culture medium composition and culture conditions of the spore formation stage in combination with mild physical or chemical post-treatment, and is suitable for the fields of probiotic preparation, biological control, feed additive and industrial fermentation start-up.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and fermentation engineering, and specifically relates to a method for improving the germination rate of Bacillus spores and its application. Background Technology

[0002] Bacillus spores possess exceptional resilience, surviving for extended periods in harsh environments, thus making them widely applicable in industry and agriculture. However, the dormancy characteristic of these spores also results in slow, asynchronous, and inefficient germination in practical applications, directly impacting the start-up speed and overall efficiency of downstream processes.

[0003] Currently, strategies to increase spore germination speed mainly fall into the following two categories: (1) Genetic modification: such as overexpression of specific nutrient germination receptors (Ger receptors). Studies have shown that overexpression of the gerA operon in Bacillus subtilis can increase the germination rate in L-alanine.

[0004] However, this method has obvious limitations: 1. Limited efficacy for complex germination agents that require a synergistic response from multiple receptors (such as AGFK).

[0005] 2. Genetic manipulation may interfere with the normal sporulation process, affecting spore yield and trait stability.

[0006] 3. The method has poor universality and is subject to regulatory restrictions, making it unsuitable for food-grade or probiotic strains.

[0007] (2) Optimization of germination conditions: This is mainly achieved by adding germination promoters or adjusting the germination environment (such as pH, ionic strength, and temperature). This method does not change the spore's own "germination potential," so the improvement effect is limited, and it may increase the complexity and cost of the process.

[0008] In recent years, research has revealed the intrinsic determinants of spore germination rate: 1. Germination receptor (GR) level: The abundance of GRs in spores is directly and positively correlated with germination rate. Spores formed in poor culture media have GR subunit levels that are only 1 / 3 to 1 / 8 of those formed in rich culture media, resulting in a significant prolongation of the germination delay period.

[0009] 2. Spore structural integrity: The structure of the spore coat and cortex affects the penetration and action of germination agents. The activity and localization of the cortex hydrolase CwlJ depend on the integrity of the coat structure, while Ca... 2+ -DPA germination pathways are particularly affected by surface conditions.

[0010] 3. Assembly of germination-related proteins: GerD proteins are crucial for signal transduction in nutrient germination, and their correct location and functional state affect germination initiation efficiency.

[0011] 4. Necessity of activation treatment: For example, thermophilic Bacillus spores often require heat activation to achieve rapid germination, mainly to shorten the germination delay period (Tlag).

[0012] Furthermore, recent research has revealed that Duf421 family proteins (such as YetF, YdfS, and their homologs) in the spore inner membrane (IM) play a crucial regulatory role in spore stress resistance and germination rate. These proteins influence germination initiation efficiency by stabilizing the inner membrane structure, reducing permeability, and protecting germination-related proteins (such as GRs and GerD) from heat damage. Simultaneously, the pH and water content of the spore core directly affect the accumulation and utilization of 3-phosphoglycerate (3PGA), a key energy reserve for ATP generation in the early stages of germination.

[0013] Existing technologies lack a non-genetically modified, systematic process that can optimize the internal germination architecture of spores during the spore formation stage and improve the accessibility of germination agents through mild post-processing, thereby synergistically activating multiple germination pathways and achieving a rapid, synchronous, and efficient germination response. Summary of the Invention

[0014] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a method for improving the germination speed of Bacillus spores and the technical solution for its application.

[0015] The present invention is specifically implemented using the following technical solutions: The first aspect of this invention provides a method for increasing the germination rate of Bacillus spores, comprising the following steps: S.1 Sporulation Culture Optimization Stage: Sporulation culture was carried out in an optimized culture medium containing an effective amount of germination precursor substances and germination enzyme inducers to obtain mature spores; S.2 Spore post-treatment activation stage: Collect and purify the spores, and perform mild desurfacing treatment or physical stimulation treatment to enhance the spore response to chemical germination agents without significantly impairing their stress resistance; S.3 Termination of reaction: Cool immediately after treatment and wash with deionized water to terminate the treatment reaction.

[0016] Furthermore, the Bacillus is Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) or thermophilic strobilurinary spores ( Geobacillus stearothermophilus ).

[0017] Furthermore, the germination precursor is Mn. 2+ and Ca 2+ .

[0018] Furthermore, the Mn 2+ It was added in the form of MnSO4 at a concentration of 0.05–0.5 mM; the Ca 2+ It is added in the form of CaCl2 at a concentration of 0.1–5 mM.

[0019] This invention Mn 2+ It is a cofactor of the key enzyme in DPA synthesis, Ca 2+ It is the main chelating ion of DPA, and optimizing its concentration can significantly increase the DPA pool capacity of the spore core. Low concentrations of germination agent analogs can induce the efficient synthesis and correct assembly of GRs and cortical hydrolases (such as SleB and CwlJ) without triggering premature germination, thereby enhancing the intrinsic germination potential of spores. A richer variety and quantity of germination receptors can increase germination speed. At the same time, regulating the pH of the sporulation environment helps acidify the prespore core (pH ~6.4), inhibits phosphoglycerate mutase (PGM) activity, promotes the accumulation of large amounts of 3PGA, and provides an immediate source of ATP in the early stages of germination.

[0020] Furthermore, the germination enzyme inducer is L-alanine or D-glucose at a concentration of 10–100 µM, added during the mid to late sporulation stage.

[0021] Furthermore, the pH of the optimized culture medium is adjusted to 6.0–6.5 during the mid-to-late sporulation stage.

[0022] Furthermore, the mild chemical desurfacing treatment includes the following steps: treating with an alkaline buffer containing 0.01%–0.1% SDS and 1–10 mM DTT at 25–37°C for 5–30 minutes, wherein the pH of the alkaline buffer is 9.0–10.5.

[0023] Furthermore, the mild physical stimulation treatment includes the following steps: heat shock at 60–75°C for 2–10 minutes, or pressure cycling at 50–150 MPa.

[0024] Mild chemical or physical treatments can partially modify or relax the spore surface structure, reducing the penetration barrier of germination agents (especially macromolecular or hydrophobic germination agents such as dodecylamine) to the inner membrane action sites (such as GRs and CwlJ), thereby synergistically enhancing the response efficiency of nutrient receptor pathways and non-nutrient germination pathways without significantly impairing spore stress resistance and the function of related germination proteins. Recent research further reveals that appropriate post-treatment (such as mild heat shock) can partially mimic the "inner membrane protein protection" mechanism by mildly altering the conformation or aggregation state of inner membrane proteins (such as YetF / YdfS), improving the stability and response efficiency of germination proteins such as GRs and GerD, without destroying the overall stress-resistant structure of the spore.

[0025] A second aspect of the present invention provides the application of any of the above methods in improving the fermentation efficiency of Bacillus.

[0026] The present invention has the following beneficial effects: (1) Non-GMO safety: No genetic modification is involved in the entire process. It is applicable to food-grade, probiotic and industrial strains and has no regulatory risks.

[0027] (2) Significant synergistic effect: By intervening in both the intrinsic potential of spore formation and the permeability of germination agents, the germination speed is significantly increased compared to single methods.

[0028] (3) Dual pathway activation: Simultaneously enhancing the trophic receptor pathway (GRs) and the non-trophic germination pathway (Ca). 2+ The response efficiency of -DPA is good for both single and compound germination agents.

[0029] (4) Performance can be quantified and improved: the germination delay period of spores treated by this process is shortened by 20%–50%, the half germination time (T50) is reduced by 30%–60%, and the germination rate can be increased by more than 2 times under the condition of germination agent concentration 50% lower than the conventional concentration.

[0030] (5) The process is controllable and easy to scale up: all step parameters are clear and easy to implement and control in industrial production.

[0031] (6) Energy reserve optimization: By regulating the pH environment during the sporulation period, 3PGA accumulation is promoted, ensuring rapid ATP generation in the early stage of germination, and improving the survival rate and metabolic initiation efficiency of spores after germination.

[0032] (7) Improved stability of inner membrane proteins: Mild post-treatment helps maintain the structural integrity of inner membrane germination protein complexes (such as Germinosomes), reduces protein damage during thermal activation, and further improves germination synchronization. Attached Figure Description

[0033] Figure 1 This is a process flow diagram of the present invention.

[0034] Figure 2 A schematic diagram of the synergistic activation mechanism of dual pathways in spore germination.

[0035] Figure 3 The germination kinetics curve of Bacillus subtilis induced by 1 ml of alanine is shown. Detailed Implementation

[0036] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0037] Example 1: Preparation and performance testing of highly germinating Bacillus subtilis spores 1. Optimization of sporulation culture: Control group: Sporulation occurred on standard 2xSG solid medium.

[0038] The invention group: In modified 2xSG liquid medium (with 0.1mM MnSO4 and 1mM CaCl2 added, and 50µM L-alanine added during the middle of sporulation), the culture was shaken at 37°C until the sporulation rate was >95%.

[0039] 2. Post-spore treatment: Collect spores and treat them with Tris-HCl buffer (pH=10.0) containing 0.05% SDS and 5 mM DTT at 30°C for 15 minutes, followed by washing with ice water to terminate the treatment.

[0040] The control group did not require post-spore treatment.

[0041] 3. Germination performance testing (as shown in Table 1) Figure 3 ): Germination was performed by adding 1 mM L-alanine to the culture medium as a germination agent, and OD was monitored. 600 decline.

[0042] The culture medium consisted of 50 mM potassium phosphate buffer (KPO4), pH 7.4, and the germination agent was 1 mM L-alanine.

[0043] Instructions: Suspend the purified spores in the above germination medium and adjust the initial OD. 600 The OD was monitored at 37℃, with a value ranging from 0.8 to 1.0. 600 decline.

[0044] Table 1

[0045] Using 0.5 mM L-alanine as a germination agent, OD was monitored. 600 Decrease (as shown in Table 2).

[0046] Table 2

[0047] Results: Under 1 mM L-alanine conditions, the germination delay period of spores in the present invention group was shortened by approximately 50% compared to the control group, with T50 decreasing from 25 minutes in the control group to 12 minutes in the present invention group (see Table 1). Under 0.5 mM low-concentration L-alanine conditions, the present invention group exhibited a significant advantage over low-concentration germination agents, with the germination rate at 60 minutes increasing dramatically from 32% in the control group to 88%. The spore OD of the present invention group... 600 The percentage dropped to 12%, while the control group only dropped to 68% (see Table 2).

[0048] 4. Energy metabolism detection: The 3PGA content in spores was detected by 31P-NMR. The 3PGA reserve of spores in the present invention group was increased by about 40% compared with the control group.

[0049] Example 2: Preparation and performance testing of *Bacillus stearothermophilus* spores 1. Optimization of sporulation culture: In a nutrient broth containing 0.2 mM MnSO4 and 2 mM CaCl2, the culture was statically incubated at 55°C until sporulation was completed.

[0050] 2. Post-spore treatment: Heat shock in a 60℃ water bath for 5 minutes, followed immediately by ice bath cooling.

[0051] 3. Germination performance testing: The germination agent was tested using a mixture of L-alanine and inosine.

[0052] Results: The spore T50 of the present invention group was shortened by about 40% compared with the untreated group, and the germination rate was significantly improved under low concentration of germination agent.

[0053] Example 3: Response test to the compound germination agent AGFK and comparison with genetic modification methods The spores prepared in Example 1 were used with 1×AGFK as the germination agent.

[0054] Bacillus subtilis spores overexpressing GerA and GerB receptors (prepared according to literature methods).

[0055] The T50 of AGFK germination in the spores of this invention was reduced by about 55%, and the final germination rate was close to 100%; the improvement in the overexpression receptor group was limited, which confirms the advantages of this process in multi-receptor synergistic germination.

[0056] Example 4: Comparison of the effects of combining different sporulation methods with this process The formulation was optimized according to Example 1, and sporulation was carried out on liquid culture medium and solid plates, respectively, followed by the same post-treatment.

[0057] Results: Liquid sporogenous spores germinated slightly faster initially, while plate sporogenous spores showed better germination synchronization after treatment and retained higher heat resistance, making them suitable for applications requiring sterilization.

Claims

1. A method for increasing the germination rate of Bacillus spores, characterized in that, Includes the following steps: S.1 Sporulation Culture Optimization Stage: Sporulation culture was carried out in an optimized culture medium containing an effective amount of germination precursor substances and germination enzyme inducers to obtain mature spores; S.2 Spore post-treatment activation stage: Collect and purify the spores, and perform mild desurfacing treatment or physical stimulation treatment to enhance the spore response to chemical germination agents without significantly impairing their stress resistance; S.3 Termination of reaction: Cool immediately after treatment and wash with deionized water to terminate the treatment reaction.

2. The method as described in claim 1, characterized in that, The Bacillus species is Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) or thermophilic strobilurinary spores ( Geobacillus stearothermophilus ).

3. The method as described in claim 1, characterized in that, The germination precursor substance is Mn 2+ and Ca 2+ .

4. The method as described in claim 3, characterized in that, The Mn 2+ It was added in the form of MnSO4 at a concentration of 0.05–0.5 mM; the Ca 2+ It is added in the form of CaCl2 at a concentration of 0.1–5 mM.

5. The method as described in claim 1, characterized in that, The germination enzyme inducer is L-alanine or D-glucose at a concentration of 10–100 µM, added during the mid to late sporulation stage.

6. The method as described in claim 1, characterized in that, The pH of the optimized culture medium was adjusted to 6.0–6.5 during the mid-to-late sporulation stage.

7. The method as described in claim 1, characterized in that, The mild chemical desurfacing treatment includes the following steps: treating with an alkaline buffer containing 0.01%–0.1% SDS and 1–10 mM DTT at 25–37°C for 5–30 minutes, wherein the pH of the alkaline buffer is 9.0–10.

5.

8. The method as described in claim 1, characterized in that, The mild physical stimulation treatment includes the following steps: heat shock at 60–75°C for 2–10 minutes, or pressure cycling at 5–150 MPa.

9. The application of the method according to any one of claims 1-8 in improving the fermentation efficiency of Bacillus.

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