A non-culturable strain CBS2, a co-culture system of the strain and bacillus subtilis, and a method for efficiently producing antifungal subtilin

Abstract

The application discloses a bacterial strain which cannot be cultured alone Candidatus Bacillus cooperans CBS2, and a mixed fermentation system for improving the fermentation efficiency of anti-mildew subtilin by co-culturing CBS2 and bacillus subtilis BS-Z15. The preservation number of CBS2 is CCTCC NO: M 20251893, and the strain cannot be cultured alone. The preservation number of BS-Z15 is CCTCC NO: M 20251892. When the bacterial strain CBS2 which cannot be cultured alone is co-cultured with bacillus subtilis BS-Z15, the fermentation characteristics of BS-Z15 for producing anti-mildew subtilin can be changed extremely significantly, the yield peak of anti-mildew subtilin is brought forward from 60-80 hours to 18-26 hours, and the production efficiency is improved by several times. The application further provides a co-culturing system composed of the two strains, a method for efficiently producing anti-mildew subtilin by using the system and application of the method in the fields of agricultural biological control and the like. The application provides a disruptive technology for green and efficient production of antibiotics, and opens up a new way for developing microbial resources which cannot be cultured alone.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and microbial fermentation engineering, specifically relating to microbial co-culture systems and the technical field of using such co-culture systems to efficiently produce antifungal and antimicrobial agents. Background Technology

[0002] Bacillus subtilis ( Bacillus subtilis Fungal microorganisms are important industrial microorganisms whose metabolic products, including lipopeptide antibiotics, possess broad antifungal activity. Among them, the iturin family comprises cyclic lipopeptides consisting of 7-8 amino acid residues and a β-amino fatty acid chain. Mycosubtilin is one of the most important and potent members of this family, and its structure is highly similar to iturin A. Mycosubtilin can efficiently disrupt the integrity of fungal cell membranes and exhibits strong inhibitory effects against various plant pathogenic fungi, thus demonstrating significant application potential in the field of biological control in green agriculture.

[0003] However, the industrial-scale, efficient production of Mycosubtilin using Bacillus subtilis still faces significant challenges. First, the fermentation cycle of Bacillus subtilis is long: cell growth and the synthesis of secondary metabolites require a considerable amount of time, typically over 60 hours, to reach peak yield, resulting in low production efficiency, high energy consumption, and low equipment turnover. Second, the yield of Bacillus subtilis needs improvement: although strain selection and fermentation optimization can increase yield to some extent, they often encounter bottlenecks, and the process is complex and costly. Furthermore, the control methods for Bacillus subtilis are limited: genetic engineering modification is subject to biosafety regulations, and the addition of chemical inducers may increase the difficulty of downstream separation and purification and raise concerns about residues.

[0004] Microbial co-culture technology, by mimicking natural microbial communities and utilizing interspecific mutualistic symbiotic relationships, can effectively activate silent gene clusters and optimize metabolic pathways, making it an effective strategy for increasing the yield of target products. However, there are currently no reports of using microorganisms that cannot be cultured alone as key symbiotic factors to significantly shorten the fermentation cycle and specifically enhance the antibiotic synthesis capacity of Bacillus subtilis. Summary of the Invention

[0005] To address the problems in existing technologies for producing antifungal subtilisin using Bacillus subtilis fermentation, such as long fermentation cycles, delayed product accumulation peaks, limited yield increases, and a lack of effective means to utilize unculturable rhizosphere strains for synergistic yield promotion, the inventors have screened an unculturable strain, CBS2, from cotton rhizosphere. They discovered that CBS2 can be co-cultured with Bacillus subtilis BS-Z15, and this co-culture can advance the peak accumulation time for antifungal subtilisin production from BS-Z15 from 60-80 hours to 18-26 hours, significantly improving efficiency. This provides a novel technical solution to the aforementioned industry challenges.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] The primary objective of this invention is to provide a non-culturable bacterial strain CBS2 with specific functions. This application provides a non-culturable bacterial strain CBS2, which is classified and named as follows: Candidatus Bacillus cooperans The accession number is CCTCC NO: M 20251893, and its 16S rRNA gene sequence is shown in SEQ ID NO: 1.

[0008] This application also provides the use of the aforementioned strain CBS2 for shortening the fermentation cycle of Bacillus subtilis to produce antibiotics and / or improving its fermentation efficiency.

[0009] The second objective of this invention is to provide a co-culture system consisting of the strain CBS2 and Bacillus subtilis BS-Z15.

[0010] Furthermore, this application also provides a microbial preparation comprising the strain described in claim 1. Candidatus Bacillus cooperans CBS2 and Bacillus subtilis ( Bacillus subtilis BS-Z15.

[0011] The ratio of viable Bacillus subtilis BS-Z15 to strain CBS2 is 1:50 to 50:1, and it is available in the form of liquid fermentation broth, solid fermentation product, cryopreservation solution, or lyophilized powder.

[0012] This application also provides a method for regulating the proportion of strains in a microbial preparation, characterized by using a selective culture medium: using NA medium to maintain a stable proportion, using TSB medium to significantly enrich CBS2, and using LB or PDA medium to significantly reduce the content of CBS2.

[0013] This application also provides the use of the aforementioned microbial preparation in the preparation of antifungal drugs, biopesticides, biofertilizers, food preservatives, and other pharmaceuticals requiring fermentation for antifungal and antimicrobial purposes.

[0014] A third objective of this invention is to provide a method for the efficient and rapid production of antifungal subtilisin using the aforementioned co-culture system.

[0015] The method employs the above-mentioned microbial preparation for fermentation culture, wherein the fermentation culture is carried out in a liquid culture medium at a temperature of 28-37°C for 12-48 hours.

[0016] Preferably, the fermentation broth or target product is harvested 18-26 hours after the fermentation culture has been carried out.

[0017] The antifungal agents mentioned mainly refer to one or more of Mycosubtilin and similar cyclic lipopeptides such as Iturin, Fengycin, and Surfactin.

[0018] Furthermore, this application also provides a method for preventing and controlling plant fungal diseases, wherein the fermentation broth or extract prepared by the above-mentioned strain CBS2 or by the method for producing antifungal subtilisin is applied to plants, plant seeds or plant growth media.

[0019] Compared with the prior art, the present invention has the following features: (1) The co-culture system of strain CBS2 and Bacillus subtilis BS-Z15 provided in this application and the method for producing antifungal subtilisin can disruptively shorten the production cycle, reducing the production cycle from 60-80 hours to 18-26 hours, increasing efficiency by more than 300%, and greatly reducing time cost, energy cost and equipment cost.

[0020] (2) The technical solution provided in this application has opened up a new way to utilize microbial resources that cannot be cultured alone. For the first time, a microbial strain CBS2 that cannot be cultured alone has been successfully developed into a highly efficient "biological inducer", providing an innovative paradigm for exploring the huge treasure trove of unknown microbial resources.

[0021] (3) The technical solution provided in this application is green and safe. The method is based entirely on biological process regulation and does not involve exogenous gene modification or the addition of chemical inducers.

[0022] (4) The technical solution provided in this application reveals a novel microbial interaction mechanism. The strain CBS2 can accumulate under nutrient-rich conditions, but still strictly depends on the characteristics of the strain BS-Z15, which clarifies a specific symbiotic relationship and has significant academic value.

[0023] (5) The method for producing antifungal subtilisin provided in this application has strong controllability. By selecting different culture media (such as LB or TSB), the proportion of strains in the co-culture system can be flexibly adjusted, providing a precise control method for industrial application. Attached Figure Description

[0024] Figure 1 The image shows the CBS2 phylogenetic tree based on the 16S rRNA gene sequence.

[0025] Figure 2 The figures show the colonies, cells, and relative quantifications of BS-Z15 and CBS2 co-cultured at different ratios.

[0026] Figures A, B, C, and D show pure cultures of BS-Z15 and mixed cultures with CBS2 content ranging from low to high, respectively. The first column shows colony morphology, the second column shows bacterial cell staining and optical microscopy observation, the third column shows bacterial cell electron microscopy observation, and the fourth column shows the relative content of BS-Z15 and CBS2 in the colonies based on quantitative PCR analysis using the 16S rRNA gene sequence.

[0027] Figure 3 The figure shown is a comparison of the yield of antifungal subtilisin during the culture of Bacillus subtilis BS-Z15 alone and co-culture of BS-Z15 and CBS2.

[0028] Figures A and B show the inhibitory effect of fermentation supernatants from co-culturing BS-Z15:CBS2 at different ratios on yeast growth after 24 h. (A, B) represent four fermentation systems with progressively increasing CBS2 content (pure BS-Z15, CBS2, etc.). C1 CBS2 C2 CBS2 C3 Figures 1-2 show the comparison of yeast inhibition zone formation in the 24-hour fermentation supernatant of BS-Z15 culture (Figure A); Figures E and G show the HPLC detection and statistical analysis of antifungalin in the fermentation broth of different proportions of BS-Z15 culture after 24 hours; Figures C and D show the comparison of yeast inhibition by pure culture of BS-Z15 culture after 12-72 hours of fermentation; Figures F and H show the inhibition of yeast by CBS2 culture. C1 HPLC analysis of antifungal subtilisin in mixed culture fermentation for 24 hours and pure culture fermentation for 12-72 hours on BS-Z15.

[0029] Figure 4 The image shows the colony screening and fluorescence analysis results for bacteria with high BS-Z15 content.

[0030] Figures A–C show three representative colonies; each row, from left to right, shows colony morphology, bright field, 523nm excitation fluorescence, and overlay. The green signal represents the autofluorescence of BS-Z15.

[0031] Figure 5 The image shows the colony screening and fluorescence analysis results for bacteria with moderate BS-Z15 content.

[0032] Figures A–C show three representative colonies; each row, from left to right, shows colony morphology, bright field, 523nm excitation fluorescence, and overlay. The green signal represents the autofluorescence of BS-Z15.

[0033] Figure 6 The image shows the colony screening and fluorescence analysis results for colonies with low BS-Z15 content.

[0034] Figures A–C show three representative colonies; each row, from left to right, shows colony morphology, bright field, 523nm excitation fluorescence, and overlay. The green signal represents the autofluorescence of BS-Z15.

[0035] Figure 7 The figure shows the antibacterial activity and its time dynamics of the fermentation supernatant in different BS-Z15:CBS2 co-culture systems.

[0036] Figure A shows the position and corresponding relationship of the filter paper pieces: the center is 1:0; the outermost parts are BS-Z15:CBS2; including 1:50, 50:1 and CBS2. C1 CBS2 C2 CBS2 C3 Figures B–F show the inhibition zones at different time points (0, 12, 18, 24, 30h).

[0037] Figure 8 The figure shows the effect of freezing time on the antibacterial activity of the samples.

[0038] Figure A shows the paper disc layout and corresponding plate results for the control group, and for the groups frozen for 24 hours and 7 days. Figure B shows the paper disc layout and corresponding plate results for the control group, and for the groups frozen for 30 days and 90 days. Red dots: control group; green dots: treatment groups with different freezing times. Detailed Implementation

[0039] The following examples are provided to further illustrate the content of this invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of the invention are within the scope of this invention.

[0040] In this application, the following embodiments are described. Candidatus Bacillus cooperans CBS2 strains are all abbreviated as "CBS2"; Bacillus subtilis ( Bacillus subtilis BS-Z15 is abbreviated as "BS-Z15".

[0041] The Bacillus subtilis BS-Z15 strain used in this application is from our research group, with accession number CCTCC NO: M20251892. Detailed information on the characteristics of the strain has been disclosed in the invention patent application number: 202511916175.1.

[0042] The LB medium, NA medium, and TSB medium used in this application are conventional media in this technical field.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] Example 1: Isolation, Identification and Preservation of Strains 1. Strain CBS2: All bacterial strains used in this embodiment were derived from rhizosphere soil of crops in Xinjiang Uygur Autonomous Region. The sampling and isolation of the strains were completed between April 2023 and June 2024. Strain CBS2 was derived from cotton field soil. The collected rhizosphere soil samples were serially diluted and spread on soil extraction agar (SEA) plates, incubated at 37℃ for 3-5 days, and typical colonies were selected and streaked multiple times to obtain the strain.

[0045] To accurately identify the above-mentioned strain, the 16S rRNA sequence of strain CBS2 was identified. The strain was inoculated into LB liquid medium and cultured with shaking until the logarithmic growth phase. Genomic DNA was extracted using a microbial genomic DNA extraction kit, and its purity and concentration were confirmed by Nano Drop assay before being used as a PCR template. The 16S rRNA fragment of strain CBS2 was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR reaction conditions were: 95℃ for 5 min; then 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 60 s, for a total of 35 cycles; and a final extension at 72℃ for 10 min. After confirming the correct band size by 1% agarose gel electrophoresis, the amplified products were sent to a sequencing company for bidirectional sequencing. Its 16S rRNA gene sequence is shown in SEQ ID NO: 1.

[0046] The 16S RNA gene sequence of SEQ ID NO: 1, CBS2 is as follows: The obtained 16S rRNA sequences were homology-aligned with the GenBank database using the NCBI BLASTn program. A neighbor-joining phylogenetic tree was constructed using phylogenetic analysis software (such as MEGA), and a comprehensive identification was performed based on colony morphology and related physiological and biochemical characteristics. See the appendix for results. Figure 1 As shown, the sequence of CBS2 is highly similar to uncultured bacterial clones such as Uncultureddbacterium clone NOD and Uncultured organism clone ELU0021, suggesting it may belong to a bacterial species that has not yet been cultured or characterized. Because it depends on BS-Z15 for survival, it is named CBS2. Candidatus Bacillus cooperans .

[0047] The characteristics of this strain are: (1) Promote the synthesis of antifungal subtilisin by BS-Z15: When it is physically coexisting with Bacillus subtilis BS-Z15, it can significantly promote the physiological metabolism of BS-Z15 through interspecies interaction, specifically and efficiently activate its antifungal subtilisin synthesis, and significantly advance the peak period of product synthesis.

[0048] (2) Selectivity of culture medium: In NA medium, the growth ratio of CBS2 to BS-Z15 is relatively stable; in LB and PDA medium, BS-Z15 is more favorable for enrichment but not CBS2; in TSB medium, CBS2 is more favorable for enrichment.

[0049] (3) Absolute dependence: It cannot grow alone on conventional laboratory culture media. Even after co-culturing with BS-Z15 in TSB medium, its quantity is significantly enriched, and pure culture cannot be obtained by extreme dilution (repeated hundreds of times), proving that its growth is strictly dependent on the specific signal or microenvironment provided by BS-Z15.

[0050] (4) Non-heat resistant: The strain CBS2 is a non-spore-forming bacillus that can be completely killed by boiling water bath treatment (e.g., 100℃, 10-30 minutes), while the spores of BS-Z15 can survive.

[0051] In summary, this invention provides a bacterial strain that cannot be cultured alone. Candidatus Bacillus cooperans strain CBS2. This strain was deposited on August 26, 2025, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The accession number is CCTCC NO: M20251893. Its 16S rRNA gene sequence is shown in SEQ ID NO: 1.

[0052] Example 2: Co-culture system / microbial preparation A co-culture system consisting of the aforementioned strain CBS2 and Bacillus subtilis BS-Z15. This system can be a live microbial composition existing in the form of liquid fermentation broth, solid fermentation product, cryopreservation solution (such as 15%-30% glycerol tubes), or lyophilized powder. By selecting different culture media (such as common bacterial media like LB, NB, and TSB), the ratio of CBS2 to BS-Z15 in this system can be adjusted to meet the needs of different application scenarios (such as stable production or bacterial propagation).

[0053] Example 3: Method for producing mycosubtilin A method for efficiently producing antifungal subtilisin, characterized by fermentation using the aforementioned co-culture system, comprising the following steps: (1) Seed culture preparation: Activate BS-Z15, activate the mixed bacteria with CBS2 as the dominant strain (by co-culturing with BS-Z15), and then mix the two in proportion to prepare co-culture seed culture.

[0054] (2) Fermentation culture: The co-cultured seed liquid is inoculated into the fermentation medium and fermented under suitable conditions (temperature 28-37℃, pH 6.0-7.5, aeration or shaking culture).

[0055] (3) Product harvesting: The fermentation broth is harvested 18-26 hours after fermentation, at which time the yield of antifungal subtilisin reaches its peak. The target product can then be extracted and purified from the fermentation broth using conventional methods in the field, such as centrifugation, filtration, acid precipitation, and chromatographic separation.

[0056] Example 4: Comparative Experiment of Co-culture and Solo Culture for Producing Antifungal Subtilisin This embodiment provides a co-culture system of the non-culturable bacterial group CBS2 and Bacillus subtilis BS-Z15 and its application in promoting the rapid synthesis of antifungal subtilisin. The strains used include: isolated Bacillus subtilis BS-Z15 and the mixed bacterial group preservation product CBS2. CBS2 cannot grow independently when inoculated on conventional artificial culture media, but it maintains good activity under co-culture conditions with BS-Z15. Conventional LB liquid and NB solid media were used, both autoclaved at 121℃ for 20 min before use. The antifungal subtilisin content in the fermentation broth was quantitatively analyzed by high-performance liquid chromatography (HPLC) using a C18 reversed-phase column, a 210 nm UV detection wavelength, and an acetonitrile-0.1% phosphate aqueous solution gradient elution system. The experiment was conducted using a constant-temperature shaking incubator (capable of shaking culture at 28-37℃ and 120-200 rpm), a clean bench, an autoclave, a benchtop centrifuge, an optical microscope, and a scanning electron microscope, among other conventional microbiological and characterization equipment. Antifungal subtilisin standards are used to establish HPLC standard curves. HPLC-grade methanol, acetonitrile, and acidifying reagents such as hydrochloric acid or phosphoric acid are selected as organic solvents. Gram staining kits are used for bacterial morphology observation.

[0057] First, a CBS2-BS-Z15 co-culture system was constructed. The mixed preservation of BS-Z15 and CBS2 was inoculated into LB liquid medium and co-activated at 28-37℃ and 120-200 rpm for 12-24 h. Afterward, it was diluted and plated onto LB solid plates and incubated at 28-37℃ until colonies grew. Preliminary screening was performed by picking and continuous subculturing based on differences in colony size, edge, and surface morphology. Then, colony morphology screening, microscopic observation, scanning electron microscopy (SEM), and qPCR relative abundance analysis were used to determine the characteristics of the co-culture community. First, colony morphology was classified by observing the size, edge, and surface morphology of samples on different culture media. Second, cell morphology was observed using an optical microscope to further record morphological differences. Then, scanning electron microscopy (SEM) was used for more detailed analysis of the cell microstructure and surface morphology. Finally, qPCR was used to quantitatively analyze the relative abundance of BS-Z15 and CBS2 in the samples, verifying the microbial composition of different samples at the molecular level. Three representative co-culture communities, CBS2C1, CBS2C2, and CBS2C3, were ultimately obtained. (CBS2 was the most representative co-culture community.) C1 CBS2 C2 and CBS2 C3 The BS-Z15 single strain and the three co-culture communities mentioned above were respectively plated on LB agar plates. After incubation for 24-48 hours, the colony morphology was recorded. Figure 1 Chinese Library A- Figure 1As shown on the left side of Figure D in the middle: BS-Z15 forms typical milky white, opaque colonies when cultured alone, while CBS2... C1 CBS2 C2 and CBS2 C3 The plates showed mixed colonies of varying sizes and edge morphologies, indicating the coexistence of two types of bacteria in the system. Further preparation of smears and Gram staining observations are detailed in the appendix. Figure 2 As shown in Figures A-D, the BS-Z15 single strain appears as a uniformly morphologically short bacillus, while two types of bacilli with different morphologies and staining patterns are observed in the three co-culture communities, indicating that CBS2 can proliferate in the culture medium in the presence of BS-Z15. Subsequently, the bacterial cells were fixed with glutaraldehyde, dehydrated in a gradient of ethanol, critical point dried, and gold-sprayed, and then observed under a scanning electron microscope (see Appendix). Figure 2 As shown in Figures A-D (right two), both types of bacteria have smooth and intact surfaces, indicating that they maintained good cell morphology under co-culture conditions. To quantitatively analyze the bacterial community composition under different culture conditions, samples from each group of cultures were collected by centrifugation, and total DNA was extracted. Real-time quantitative PCR was performed using specific primers for Bacillus subtilis BS-Z15 and CBS2, respectively. The copy number of each strain in each sample was calculated based on the standard curve or Ct value conversion formula, thus determining the relative proportion of BS-Z15 and CBS2 in the total bacterial count. The quantitative results are shown in the attached bar chart. Figure 2 As shown on the right side of Figures A–D: Under BS-Z15 monoculture conditions, only BS-Z15-specific amplification signals were detected, while the corresponding CBS2 qPCR signal was close to background levels, indicating that the bacterial community composition was almost entirely composed of BS-Z15. Under different co-culture conditions with CBS2, the qPCR quantitative results of the two strains showed significant changes in their relative abundance, gradually shifting from BS-Z15 dominance to a balance between both or CBS2 dominance, providing quantitative evidence for subsequent screening of the bacterial community composition most conducive to mycotoxin resistance synthesis. In CBS2… C1 BS-Z15 has a clear advantage, while CBS2 has a lower market share; in CBS2 C2 The proportions of the two types of bacteria are roughly equal in CBS2. C3 In the middle, CBS2 was dominant, while the proportion of BS-Z15 decreased significantly. This indicates that continuous co-culture with CBS2 can yield a variety of relatively stable co-culture communities with different compositions, providing a basis for subsequent screening of combinations most conducive to the synthesis of mycotoxin.

[0058] Based on this, the effects of the co-culture system on the synthesis and antibacterial activity of mycotoxin were further investigated. The control group consisted of BS-Z15 fermentation alone: ​​BS-Z15 was activated in LB liquid medium at 28-37℃ and 120-200 rpm for 12-24 h, then inoculated into the fermentation medium at a 5-20% (v / v) inoculation rate. Fermentation was carried out at 28-37℃ and 120-200 rpm with shaking, and samples were taken at 0, 12, 24, 36, 48, 60, and 72 h. The experimental group consisted of co-culture fermentation: a mixed culture of BS-Z15 and CBS2 was co-activated in LB liquid medium for 12-24 h, then transferred to a fermentation medium with the same composition at the same 5-20% inoculation rate. Cultured under the same conditions as the control group, samples were taken at the same time points. First, the antibacterial ability of different co-culture communities against pathogenic fungi at 24 h was compared: BS-Z15 and CBS2 were respectively... C1 CBS2 C2 With CBS2 C3 The supernatant from 24 hours of fermentation was centrifuged to remove bacterial cells, and then inoculated onto pre-spread fungal and yeast plates using a plate-punching method. Antimicrobial activity was evaluated by measuring the diameter of the inhibition zone. (See appendix) Figure 3 Figure A shows the morphology of the inhibition zone formed in the supernatant after 24 hours of fermentation. The four perforations correspond to BS-Z15 and CBS2, respectively. C1 CBS2 C2 and CBS2 C3 See appendix Figure 3 Figure B in the middle shows the statistical results of the relative diameters of the corresponding inhibition zones. The data indicate that CBS2 C1 CBS2 C3 and CBS2 C2 The inhibition zone of CBS2 was significantly larger than that of BS-Z15 culture alone, indicating that co-culturing with CBS2 significantly enhanced the inhibitory effect of the fermentation broth on fungi. Further, the antibacterial activity of the fermentation supernatant at different fermentation time points was determined using a pure BS-Z15 culture fermentation system. (See Appendix) Figure 3 Chinese Figure C and Figure 3 As shown in Figure D, during the 0-18h stage, the BS-Z15 fermentation supernatant produced almost no visible inhibition zones, which only began to appear at 24h. Although the diameter of the inhibition zones increased slightly between 24-72h, the overall inhibition level was significantly lower than that of the co-culture system during the same time period. These results indicate that compared to co-culture with CBS2, BS-Z15 fermentation alone not only produced antimicrobial substances with a significantly delayed time but also exhibited weaker antagonistic strength.

[0059] To quantitatively compare the yield of antimicrobial inoculin, the fermentation broth at each time point was centrifuged at 10,000 rpm for 10 min. The supernatant was collected, and the pH was adjusted to approximately 2.0-4.0. The mixture was allowed to stand for sufficient precipitation of metabolites (acid precipitation). The acid-precipitated fermentation broth was centrifuged (4℃, 8000 r / min, 20 min) to collect the precipitate. The crude metabolite was dissolved, filtered through a 0.22 μm organic filter membrane, and then separated and prepared by HPLC. HPLC was performed using a C18 reversed-phase column with a gradient elution of acetonitrile-aqueous solution containing 0.1% phosphoric acid. The characteristic peaks were determined by comparing the retention times with antimicrobial inoculin standards, and the concentration of antimicrobial inoculin in the samples was calculated based on the standard curve. (See Appendix) Figure 3 As shown in Figure E, after 24 hours of fermentation, BS-Z15 cultured alone and CBS2 cultured alone... C1 CBS2 C2 and CBS2 C3 The chromatograms of all three co-culture systems showed characteristic peaks against subtilisin within the 12-15 min range (marked with red boxes), but the peak height and peak area of ​​the three co-culture systems were significantly higher than those of BS-Z15 culture alone; the corresponding peak area statistics at 24 h are shown in the appendix. Figure 3 G, CBS2 C1 With CBS2 C2 CBS2 had the highest peak area of ​​antifungal subtilisin. C3 Secondly, both were significantly higher than BS-Z15, indicating that co-cultivation can significantly increase the yield of mycotoxin resistance in the early stage of fermentation. Further, CBS2... C1 Using BS-Z15 monoculture as an example, the dynamics of antifungal subtilisin synthesis at different time points from 12 to 72 h were compared between co-culture and BS-Z15 monoculture. The chromatographic overlay results are shown in the appendix. Figure 3 In Figure F, the red-boxed area represents the characteristic peak of antifungalin. It can be seen that the peak height of the co-culture system is close to its maximum at 24 hours, and the increase in peak height is limited after fermentation is extended to 72 hours. In contrast, the peak height of antifungalin in BS-Z15 culture alone gradually increases with fermentation time, reaching its peak at 72 hours. Converting the peak area to concentration at each time point and comparing them, the co-culture system reaches its peak antifungalin yield at 24 hours, with a concentration of 798.4 mg / L, while BS-Z15 culture alone reaches its peak at 72 hours, with a concentration of 852.6 mg / L. Although the peak concentrations are similar, co-culture significantly shortens the time required to reach the peak. The volumetric productivity of co-culture at 24 hours is 33.27 mg / (L·h), approximately 3.3 times that of BS-Z15 culture alone at 72 hours.

[0060] In summary, by co-culturing with the non-culturable bacterial group CBS2, CBS2 can be obtained. C1 CBS2 C2and CBS2 C3 A stable co-culture system composed of multiple microbial communities allows CBS2 to be maintained for a long period under artificial culture conditions. Furthermore, the co-culture system significantly enhances the antibacterial activity of the fermentation broth and greatly improves the efficiency of Bacillus subtilis BS-Z15 in synthesizing antifungal subtilisin. This allows antifungal subtilisin to reach its peak yield within 24 hours, approaching the level achieved by monoculture for 72 hours, with a volumetric productivity increase of approximately 3.3 times. This example demonstrates that the CBS2-BS-Z15 co-culture strategy can significantly shorten the fermentation cycle of antifungal subtilisin and improve production efficiency, effectively overcoming the technical bottlenecks of long fermentation cycles and low yields in existing technologies. This provides a new technical solution for the industrial production of antifungal subtilisin-based biopesticides.

[0061] Example 5: Verification of CBS2 strain dependence This embodiment verifies whether CBS2 can be cultured independently and its dependence on co-culture with BS-Z15. BS-Z15 and CBS2 were co-cultured in NA and TSB media, respectively. After culturing, total DNA was extracted from samples, and real-time quantitative PCR was used to quantify both to obtain their relative proportions in the co-culture system. Subsequently, the co-culture enriched in TSB was serially diluted (10T). -7 10 -8 10 -9 100 μL of each colony was spread onto TSB solid plates. After incubation, hundreds of single colonies were randomly selected for streak purification, and the colony / cell morphology was observed under a microscope. During purification, significant differences in colony morphology were observed among different single colonies (e.g., colony size, edge regularity, surface roughness / smoothness, and overall density). Given that BS-Z15 exhibits stable autofluorescence under 523 nm excitation, this embodiment further used this autofluorescence as a rapid tracer signal to perform bright-field, 523 nm excitation fluorescence, and superimposed imaging on morphologically different colonies to evaluate the relative content of BS-Z15 in each colony. The results showed ( Figure 4 – Figure 6The proportion of green fluorescent positive cells corresponding to colonies with different morphologies also varied, and they could be classified into three categories: high / medium / low BS-Z15 content. However, even in colonies with low BS-Z15 content, a small number of spontaneously green fluorescent positive bacilli could still be observed, indicating that the "dilution plating-single colony picking-stripe purification" procedure used in this example did not yield colonies completely free of BS-Z15 (i.e., no independent pure colonies of CBS2 were obtained). Representative colonies were then identified by 16S rRNA amplification and sequencing. The results showed that CBS2 could be detected by qPCR in the co-culture samples, but according to the conventional "dilution plating-single colony picking-stripe purification" isolation procedure, all single colonies that could grow stably and be passaged were identified as Bacillus subtilis BS-Z15, or a mixture of BS-Z15 and CBS2; no independently growing pure CBS2 colonies were ever obtained. This confirms that CBS2 cannot be cultured alone under the aforementioned culture conditions and requires a co-culture system with BS-Z15 to maintain its survival and amplification.

[0062] Example 6: Optimization Experiment with Different Inoculation Ratios This embodiment optimizes different initial inoculation ratios of Bacillus subtilis BS-Z15 and enriched CBS2 mixed cultures based on the strain isolation and co-culture system construction methods described in Examples 1 and 2, in order to screen for the optimal combination that promotes the rapid and efficient production of the target fermentation product (fermentation metabolites with antibacterial activity). The strains used in the experiment were the BS-Z15 identified and preserved in Example 1 and the CBS2 mixed culture obtained through multiple rounds of co-culture enrichment. LB liquid and LB solid media were used as culture media, and PDA plates and standard yeast strains were used to determine antibacterial activity. The main instruments included a constant temperature shaking incubator, a biosafety laminar flow hood, a high-speed centrifuge, micropipettes, and Vernier calipers, all used according to standard microbiological operating conditions.

[0063] The specific method is as follows: BS-Z15 and enriched CBS2 were inoculated separately into LB liquid medium and cultured at 28-37℃ and 120-200 rpm with shaking until the logarithmic growth phase. The bacterial suspension was then measured and adjusted to OD600≈0.1-1.0. Subsequently, BS-Z15 and enriched CBS2 were mixed at a volume ratio, and different inoculation combinations were set up, including pure BS-Z15 (without CBS2) and BS-Z15:CBS2 at ratios of 1:50 to 50:1. The mixed bacterial suspension was inoculated into Erlenmeyer flasks. Co-culture fermentation was carried out at 28-37℃ and 120-200 rpm. Samples were taken at different time points from 12 h to 30 h of fermentation, the bacterial cells were removed by centrifugation, and the fermentation supernatant was collected for the determination of antibacterial activity.

[0064] Antibacterial activity was determined using the paper disc diffusion method: a yeast suspension was evenly spread onto the surface of a PDA plate, and then... Figure 7 -A layout: Sterile filter paper discs are placed and an equal volume of fermentation supernatant is added; the central disc contains BS-Z15 fermentation supernatant alone (1:0), and the remaining discs contain fermentation supernatants from different co-culture systems (all in a ratio of BS-Z15:CBS2, with 1:50, 50:1, and CBS2 ratios). C1–C3 All were different co-culture systems obtained through CBS2 enrichment. The diameter of the inhibition zone was recorded and measured at 0, 12, 18, 24, and 30 hours after incubation; each treatment was repeated in triplicate. Figure 7 As shown, no clear inhibition zones were observed around the paper discs in any of the treatments from 0 to 18 hours. By 24 hours, clear inhibition zones began to appear in the co-culture treatments, particularly CBS2. C1 The inhibition zone was most prominent around the bacteria, while BS-Z15 fermentation alone showed almost no inhibition zone. After fermentation was extended to 30 hours, the inhibition zones in most co-culture treatments further expanded and became clearer, with overall antibacterial activity significantly stronger than the BS-Z15 system alone. This indicates that the introduction of CBS2 (especially CBS2) is beneficial. C1–C2 Related systems can accelerate the accumulation of antibacterial active substances. In contrast, when BS-Z15 is used in an initial inoculation ratio of enriched CBS2, the CBS2 content is significantly higher. C1 At that time, the fermentation supernatant produced obvious inhibition zones in 20-24 hours, and the diameter of the inhibition zones reached its maximum at 24 hours, which was significantly higher than that of pure BS-Z15 and other co-culture systems with different inoculation ratios. This indicates that this ratio can not only significantly shorten the time required for the accumulation of antibacterial substances, but also obtain a higher level of effective products in a shorter fermentation cycle.

[0065] In summary, by changing the ratio of BS-Z15 to enriched CBS2 in the initial inoculation, this embodiment determined the optimal ratio of CBS2. C1 This is the optimal inoculation combination. Fermentation at this ratio for 20-24 hours yields fermentation products with high antibacterial activity, which is significantly superior to pure BS-Z15 and other co-culture systems with different inoculation ratios. This provides optimized process parameters for subsequent fermentation process scale-up and formulation development.

[0066] Example 7: Cryopreservation and Application of Co-cultures This embodiment, based on the aforementioned co-culture system construction and inoculation ratio optimization, further investigates the storability of the optimal co-culture system under cryopreservation conditions and its application effect after thawing. Bacillus subtilis BS-Z15 obtained and identified in Example 1 and the CBS2 mixed bacterial group enriched in Examples 2 and 6 were selected and co-cultured in LB liquid medium at 28-37℃ and 120-200 rpm for 12-24 hours according to the optimal initial inoculation ratio determined in Example 6, to obtain a co-culture fermentation broth with stable antibacterial activity. The co-culture fermentation broth was mixed with sterile glycerol in a clean bench to achieve a final glycerol volume fraction of 15-30%. After thorough mixing, it was dispensed into sterile cryovials and immediately stored in an ultra-low temperature freezer at -80℃. Multiple time points were set for subsequent thawing evaluation, including 1 day, 1 week, 1 month, and 3 months of storage.

[0067] After the co-culture was cryopreserved for the predetermined time, the cryovials were removed from -80℃ and rapidly thawed in a 28-37℃ water bath. A small amount of the cryopreserved solution was immediately inoculated into Erlenmeyer flasks containing LB medium and incubated with shaking to allow the cells to fully recover. After recovery, samples were taken, centrifuged to remove the cells, and the supernatant was collected for antibacterial activity determination. Simultaneously, a fresh, unfrozen co-culture system was used as a control, treated using the same method. Antibacterial activity was determined using the filter paper disc method: the yeast suspension was evenly spread on the surface of a PDA plate, a certain volume of fermentation supernatant was added to the filter paper disc, and the diameter of the inhibition zone was observed and measured. This process was repeated in parallel.

[0068] Experimental results showed that after cryopreservation of co-cultures at -80℃ using sterile glycerol as a cryoprotectant, the yeast inhibitory activity of the revived co-culture systems after different storage times was basically consistent with that of the fresh co-culture system. Figure 8 As shown, the diameter of the inhibition zone formed by the fermentation supernatant after thawing from frozen storage for 1 day and 1 week was not significantly different from that of the unfrozen control. After 1 month and 3 months of frozen storage, the diameter of the inhibition zone remained above 90% of that of the control, indicating that the production capacity of key antimicrobial metabolites was not significantly weakened. These results demonstrate that when the co-culture system is mixed with glycerol and frozen at -80℃, its growth capacity, microbial composition, and antimicrobial function are well maintained after thawing. The effectiveness is not substantially different from that of co-cultures obtained through continuous subculturing, verifying the storage convenience and stability of this co-culture system in practical applications.

[0069] The above embodiments are merely examples to clearly illustrate the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A bacterial strain that cannot be cultured alone. Candidatus Bacillus cooperans CBS2, characterized in that, The strain was deposited at the China Center for Type Culture Collection (CCTCC) on August 26, 2025, with accession number CCTCCNO: M 20251893, and its 16S rRNA gene sequence is shown in SEQ ID NO:

1.

2. A microbial preparation, characterized in that, The microbial preparation comprises the strain according to claim 1. Candidatus Bacillus cooperans CBS2 and Bacillus subtilis ( Bacillus subtilis BS-Z15, of which Bacillus subtilis ( Bacillus subtilis The accession number of BS-Z15 is CCTCC NO: M 20251892.

3. The microbial preparation according to claim 2, characterized in that, The ratio of viable Bacillus subtilis BS-Z15 to strain CBS2 is (1-50):(50-1), and it is available in the form of liquid fermentation broth, solid fermentation product, cryopreservation solution, or lyophilized powder.

4. A method for producing antifungal subtilisin, characterized in that, The method employs the fermentation culture step of the microbial preparation described in any one of claims 2-3, wherein the fermentation culture is carried out in a liquid culture medium at a temperature of 28-37°C for 12-48 hours.

5. The method as described in claim 4, characterized in that, The fermentation broth or target product is harvested after 18-26 hours of fermentation culture.

6. The method as described in claim 4, characterized in that, The antifungal agents mentioned mainly refer to one or more of Mycosubtilin and similar cyclic lipopeptides such as Iturin, Fengycin, and Surfactin.

7. The use of strain CBS2 as described in claim 1 for shortening the fermentation cycle of Bacillus subtilis to produce antibiotics and / or improving its fermentation efficiency.

8. The use of the microbial preparation as described in any one of claims 2-3 in the preparation of antifungal drugs, biopesticides, biofertilizers, food preservatives, and other pharmaceuticals requiring fermentation for antifungal and antimicrobial purposes.

9. A method for regulating the proportion of bacterial strains in a microbial preparation as described in claim 2, characterized in that, The following methods were used to achieve this: NA medium was used to maintain a stable ratio, TSB medium was used to significantly enrich CBS2, and LB or PDA medium was used to significantly reduce the content of CBS2.

10. A method for controlling plant fungal diseases, characterized in that, The fermentation broth or extract prepared by any of the methods described in claims 5-8 is applied to plants, plant seeds, or plant growth media.

Images