Bacillus velezensis obtained by space mutation and application thereof

Abstract

The invention discloses bacillus velezensis obtained through space mutation and application of the bacillus velezensis. The technical problem to be solved by the invention is how to improve the yield of the antibacterial lipopeptide, and the provided bacillus velezensis strain has the serial number of Bv1-b39, is preserved in the China General Microbiological Culture Collection Center (CGMCC), has the preservation number of CGMCC No.32879, and is a strain obtained by carrying out space mutagenesis treatment by taking Bv1 as an original strain. After the Bv1-b39 strain is fermented, the lipopeptide antibacterial compound iturin A can be produced at high yield, the shake flask yield can reach 195.95 mg / L, which is increased by 26% compared with that of an original strain, and the Bv1-b39 strain has good hereditary stability, is a strain with biocontrol application and antibacterial lipopeptide industrial production value, and has huge application potential in the field of agricultural production.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a Bacillus velezinis strain obtained by spaceflight mutagenesis and applications thereof. Background Art

[0002] Pesticides are an indispensable means of production in modern agriculture, playing a vital role in controlling crop pests and diseases, ensuring national food security, and ensuring the effective supply of key agricultural products. However, unscientific and irrational use can negatively impact agricultural product quality and the ecological environment. Therefore, there is an urgent need to shift from an over-reliance on chemical pesticides for disease and pest control, vigorously promote green pest control, and implement integrated and sustainable pest management.

[0003] Bacillus is an important biocontrol microbial resource. Its growth and metabolism produce a variety of antimicrobial substances, including non-ribose-synthesized lipopeptide antibiotics, surfactants, fengycins, and iturins. These substances have applications in agriculture, aquaculture, biocontrol, medicine, and environmental remediation. Antimicrobial lipopeptides exhibit broad-spectrum antifungal activity, rapid and potent action, and resistance to drug resistance. They are not only environmentally friendly, safe, and healthy, but also improve the growing environment and have long-lasting effects. They play a significant role in promoting agricultural production and sustainable development. They are widely used to control fungal diseases of various crops, vegetables, and fruits. They can inhibit soil-borne diseases, improve soil quality, and contribute to soil remediation. Due to the difficulty of artificially producing antimicrobial lipopeptides, Bacillus is often used as a base cell and extracted from its fermentation products. However, the low natural production of antimicrobial lipopeptides has been a bottleneck hindering their widespread application.

[0004] Iturins, one of the three major families of lipopeptides, are highly effective in preventing and controlling bacterial leaf spot and soft rot of cucumber (Cucumis sativus) and fusarium head blight of wheat (Triticum aestivum). Furthermore, iturins have a strong hemolytic effect, but their physiological activity differs from that of surfactin, resulting in a strong antagonistic effect against fungi (including yeasts). Studies have shown that iturins have a strong inhibitory effect against Trichophyton rubrum. Iturins have a broad antimicrobial spectrum, high stability, low toxicity, and low allergenicity, thus offering a broad range of applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to increase the yield of antimicrobial lipopeptides and carry out large-scale production.

[0006] To this end, the present invention provides a Bacillus sp., which is Bacillus velezensis Bv1-b39, which is deposited in the China General Microbiological Culture Collection Center (CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with a deposit number of CG MCC No. 32879.

[0007] The present invention also provides the use of the bacillus in preparing antibacterial products and in preparing products producing antibacterial lipopeptides.

[0008] In the above application, the antimicrobial lipopeptide may be surfactin or iturin A.

[0009] The product may or may not contain the Bacillus. The product may be a fermentation product of the Bacillus or an extract of the fermentation product. The fermentation product may be a substance obtained by culturing the Bacillus in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the Bacillus and a substance secreted into a liquid culture medium, or a solid fermentation product containing the Bacillus and a substance secreted into a solid culture medium).

[0010] The fermentation product may also be a substance obtained by removing the Bacillus from the fermentation broth and collecting the remaining components of the fermentation broth.

[0011] The fermentation product extract is a product obtained by subjecting the fermentation broth of Bacillus velezensis to acid precipitation; the fermentation product extract contains fengycin A and / or iturin A.

[0012] Furthermore, the fermentation product extract includes C 14 -C 16 One or more of Iturin A.

[0013] The present invention also provides a composition containing the Bacillus.

[0014] The above-mentioned composition may be a culture, and the culture is a substance obtained by culturing the Bacillus in a microbial culture medium.

[0015] The above composition may be a bacterial agent.

[0016] The above-mentioned bacterial agent refers to a live bacterial preparation made by expanding the target microorganisms and using a carrier as an adsorbent to adsorb the fermentation liquid or solid fermentation product of the bacteria.

[0017] The above-mentioned microbial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0018] If necessary, the bacterial agent may further include a carrier, which may be a solid carrier or a liquid carrier.

[0019] The composition can be any one of the following: A1) a composition for antibacterial use, A2) a composition for producing an antibacterial lipopeptide compound.

[0020] The present invention also provides a method for preparing a composition, wherein the composition is the above composition, and the method comprises the step of using the Bacillus as a component of the composition.

[0021] The present invention also provides the use of the composition in preparing a product, wherein the product has at least one of the following properties: B1) antibacterial, B2) production of antibacterial lipopeptide compounds.

[0022] The present invention also provides an antibacterial method, comprising the step of contacting the composition with bacteria.

[0023] In the above, the antibacterial agent may be an antifungal agent. The fungus may be a fungus. The fungus may be a fungus of the subdivision Deuteromycotina.

[0024] The high-yield antimicrobial lipopeptide-producing space-bred Bacillus Velez subtilis strain described herein is derived from a starting strain of antimicrobial lipopeptide-producing Bacillus Velez subtilis strain, which was subjected to space-induced mutagenesis and then screened using a plate standoff method. Bv1-b39 demonstrated 50% and 38.33% higher antibacterial rates in primary and secondary plate standoff screening experiments, respectively, compared to the original strain, and the in vitro antibacterial rate of the fermentation broth was 60% higher than that of the original strain. After fermentation, the Bv1-b39 strain achieved an iturin A yield of 195.95 mg / L, a 26% increase compared to the starting strain. LC-MS confirmed that the type of iturin produced by the strain remained unchanged after space-induced mutagenesis, and that iturin A could be produced. Continuous fermentation and passage of the strain to the eighth generation revealed an iturin A yield of 130.52 mg / L, a decrease of only 15% from the first generation's 153.93 mg / L. Therefore, Bv1-b39 is a strain with biocontrol applications and industrial production value for antimicrobial lipopeptides, and is superior to other positive mutant strains, with great application potential in the field of agricultural production.

[0025] The beneficial effects achieved by the present invention are:

[0026] (1) The high-yield antimicrobial lipopeptide space-bred Bacillus velezensis of the present invention has a strong inhibitory effect on a variety of plant pathogenic fungi and plays a very important role in the biological control of plant diseases by antagonizing bacteria.

[0027] (2) The high-yield antimicrobial lipopeptide space-bred Bacillus velezensis of the present invention has simple culture conditions, high yield of the antimicrobial lipopeptide - iturin, is easy to preserve, and can be easily used as a chassis cell for industrial production of antimicrobial lipopeptides, and has good development and application prospects.

[0028] One of the objectives of the present invention is to provide a highly effective biological control strain, reducing reliance on chemical control for plant disease prevention and reducing environmental harm. The present invention provides a high-yield antimicrobial lipopeptide space-bred Bacillus velezinis strain, obtained by screening after space-induced mutagenesis of the starting strain. The use of the antagonistic bacteria of the present invention can effectively reduce the incidence of various soil-borne plant diseases, improve the health of the soil-plant system, and achieve sustainable agricultural development.

[0029] The second purpose of the present invention is to use Bacillus as a chassis cell to effectively increase the yield of antimicrobial lipopeptides produced by Bacillus, reduce the production cost of lipopeptides, and help achieve large-scale production and widespread application of lipid molecules of the lipopeptide family represented by iturin.

[0030] Therefore, we specifically isolated a strain of Bacillus velezensis from soil that exhibits antagonistic effects against plant pathogenic fungi and produces antimicrobial lipopeptides. We used this strain as the starting strain for space mutagenesis. Using methods such as plate standoff and punch-hole assays, we screened a library of mutants with significantly enhanced antimicrobial activity and increased antimicrobial lipopeptide production. We then evaluated their biocontrol potential and potential for industrial lipopeptide production, including antimicrobial activity, fermentation product antimicrobial activity, antimicrobial lipopeptide production and type, and genetic stability. From these strains, we selected a high-yield lipopeptide strain with strong antagonistic activity.

[0031] The present invention discloses a space breeding strain of high-yield antimicrobial lipopeptide Bv1-b39 (Bacillus velezensis) and its application. The space breeding strain of high-yield lipopeptide of the present invention belongs to the genus Bacillus, and is deposited in the China General Microbiological Culture Collection Center with a microorganism deposit number of CGMCC No.32879. The high-yield antimicrobial lipopeptide Bacillus velezensis of the present invention was originally an antagonistic bacterium isolated from the soil, and after fermentation, it was found by mass spectrometry (MS) to be able to produce antimicrobial lipopeptide compounds. The strain was used as the starting strain for space mutagenesis treatment, and after returning to the ground, it was obtained by experimental screening using the plate standoff method and the fermentation liquid punching method, and its yield and lipopeptide type were determined by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). The Bv1-b39 strain can produce a high yield of the antimicrobial lipopeptide iturin A after fermentation, reaching 195.95 mg / L, a 26% increase compared to the starting strain. The positive mutant strain was continuously fermented and passaged through ten generations, and strain Bv1-b39 maintained good genetic stability after eight passages. Therefore, it is a strain with potential for biocontrol applications and industrial production of antimicrobial lipopeptides, and has great potential for application in agricultural production.

[0032] Preservation Instructions

[0033] Bacteria name: Bacillus velezensis

[0034] Latin name: Bacillus velezensis

[0035] Strain ID: Bv1-b39

[0036] Depository: General Microbiology Center of China Culture Collection Administration

[0037] Abbreviation of depository institution: CGMCC

[0038] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0039] Deposit date: December 2, 2024

[0040] CGMCC registration number: CGMCC No.32879 BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Figure 2 is a heat map analysis of the ANI values ​​of strain Bv1 and its related strains.

[0042] Figure 2 MALDI-TOF-MS analysis of strain Bv1 after fermentation in (a) YPG medium and (b) fermentation medium.

[0043] Figure 3 (a) Initial screening by plate confrontation (b) Improved inhibition rate of plate confrontation rescreening for aerospace strains (c) Some strains with reduced antibacterial activity.

[0044] Figure 4 The punch method was used to evaluate the antibacterial effect of the antibacterial mutant strains. (a) Control plate (b) Positive mutant strain plate (arrows point to Bv1-b39), (c) The antibacterial improvement rate of b39 (Bv1-b39) relative to Bv1.

[0045] Figure 5 (a) OD of each strain at different fermentation times 600 (b) Iturin A concentration curve.

[0046] Figure 6A1 This is the LC-MS detection result of the fermentation broth of strain Bv1 with a retention time of 6.569 min.

[0047] Figure 6A2 This is the LC-MS detection result of the fermentation broth of strain Bv1 with a retention time of 11.344 min.

[0048] Figure 6B1 This is the LC-MS detection result of the fermentation broth of strain Bv1-a48 with a retention time of 5.766 min.

[0049] Figure 6B2 This is the LC-MS detection result of the fermentation broth of strain Bv1-a48 with a retention time of 7.635 min.

[0050] Figure 6C1 This is the LC-MS detection result of the fermentation broth of strain Bv1-b39 with a retention time of 5.806 min.

[0051] Figure 6C2 This is the LC-MS detection result of the fermentation broth of strain Bv1-b39 with a retention time of 11.114 min.

[0052] Figure 7 The OD corresponding to ten subcultures of the spaceflight strain 600 .

[0053] Figure 8 (a) Red oil-draining circles of standard solutions of surfactant with different concentrations; (b) Linear fitting of the diameter of the oil-draining circle and the surfactant concentration.

[0054] Figure 9 (a) Oil-draining ring diameter (b) Surfactant concentration (c) Iturin A concentration of the strain after ten subcultures. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0056] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0057] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0058] Example 1. Screening and identification of strains

[0059] 1. Identification of an antagonistic bacterium strain Bv1 and the type of antimicrobial lipopeptide it produces

[0060] 1.1 Test strains:

[0061] (1) Bacillus velezensis Bv1 (referred to as strain Bv1): Bacillus velezensis, isolated from soil infected with tomato wilt in Pinggu District, Beijing, is a strain with the deposit number CGMCC No. 20317 in the October 4, 2022, authorization announcement CN112680380B. The public may obtain the biological materials described in the Examples from the applicant to replicate the present experiments. The biological materials are for use solely in replicating the experiments of the present invention and may not be used for any other purpose.

[0062] 1.2 Experimental Materials

[0063] (1) YPG liquid medium: 10.0 g yeast extract, 20.0 g peptone, 20.0 g glucose, 1000 mL distilled water, adjust the pH to 7.0.

[0064] (2) Fermentation medium: glucose 15 g / L, soluble starch 1 g / L, peptone 30 g / L, yeast powder 0.2 g / L, KH2PO4 1.5 g / L, K2HPO4 3 g / L, MgSO4·7H2O 0.5 g / L, CaCO3 0.1 g / L, FeSO4·7H2O 0.1 g / L.

[0065] 1.3 Experimental instrument: SCIEX4800Plus mass spectrometer

[0066] 1.4 Experimental methods

[0067] (1) Identification of strain Bv1

[0068] The purified Bv1 strain was sent to Shanghai Meiji Biotechnology Co., Ltd. for whole-genome sequencing.

[0069] Whole-genome sequencing sequences were submitted to http: / / www.ezbiocloud.net / ezgenome / ani for alignment, and cluster analysis was performed using the Morpheus online tool (https: / / software.broadinstitute.org / morpheus) to create an ANI heat map. Digital DNA-DNA hybridization analysis was performed using the Model Strain Genome Service Platform https: / / tygs.dsmz.de.

[0070] (2) Identification of lipopeptide types produced by strain Bv1 by MALDI-TOF MS

[0071] Bv1 seed solution was inoculated into YPG medium and fermentation medium at a 5% inoculum, respectively, and cultured at 37°C, 200 rpm, for 48 hours. The fermentation broth was centrifuged at 6000 rpm at 4°C for 20 minutes, and the precipitate was discarded. The supernatant was adjusted to pH 2.0 with concentrated hydrochloric acid, sealed with parafilm, and incubated at 4°C overnight. The acid precipitate was centrifuged at 12,000 rpm at 4°C for 15 minutes, the supernatant discarded, and the precipitate collected. The precipitate was frozen at -80°C for 2 hours and then freeze-dried in a freeze dryer for 24 hours to obtain a crude lipopeptide extract.

[0072] 0.1 g of crude extract powder was dissolved in a mixed solution of 50 μL of deionized water and 50 μL of methanol, and the analysis was performed using a SCIEX4800Plus mass spectrometer with α-cyano-4-hydroxycinnamic acid (CHA) as the matrix, a 337 nm nitrogen laser beam excitation, an excitation voltage of 2-20 kV, and a scanning detection of the nucleus-to-cytoplasm ratio range of 800-2000 m / z to verify the target lipopeptide ion peak and accumulate the target lipopeptide ions.

[0073] 1.5 Experimental Results

[0074] (1) Identification of strain Bv1

[0075] ANI calculation results and cluster analysis ( Figure 1) showed that Bv1 clustered with Bacillus velezensis JS25R, Bacillus siamensis KCTC 13613, and Bacillus amyloliquefaciens GKT04, with ANI values ​​of 99.87%, 99.68, and 99.61%, respectively. Digital DNA-DNA hybridization analysis showed that Bv1 strain was closely related to Bacillus velezensis NRRL B - The dDDH value of strain 41580 (G CF_002117165.1) was 84.5%. Hybridization values ​​with Bacillus siamensis KCTC 13613, Bacillus amyloliquefaciens DSM 7, and Bacillus nakamurai NRRL B-41091 were 56.6%, 55.4%, and 30.8%, respectively. Hybridization values ​​with other Bacillus species were all less than 30%. Based on the generally accepted thresholds for species classification, strain Bv1 should be assigned to Bacillus velezensis.

[0076] (2) Identification of lipopeptide types produced by strain Bv1 by MALDI-TOF MS

[0077] MALDI-TOF MS was used to determine the molecular weight of the crude lipopeptide mixture extracted from the fermentation broth. Screening ensured that the hydrogen, sodium, or potassium ion adducts of the lipopeptides all carried a single positive charge, and that the nucleotope-to-mass ratio on the mass spectrum was equal to the adduct molecular weight. The nucleotope-to-mass ratios of the surfactin and iturin families were distributed in the 1000-1100 m / z range, while those of the fenmucin family were distributed in the 1400-1600 m / z range.

[0078] After fermentation in YPG medium, the crude lipopeptide extract of Bv1 showed ion peaks at 1065.7980m / z, 1079.8081, and 1081.78m / z ( Figure 2 In (a), the peaks corresponding to the sodium ion addition of C14 and C15 IturinA, and the potassium ion addition peak of C14 IturinA, respectively, indicate that strain Bv1 can produce IturinA homologs.

[0079] Since the type of lipopeptide produced by the strain is greatly affected by the culture conditions, after the fermentation medium was changed to fermentation medium, the fermentation broth of strain Bv1 had ion peaks at 1463.507m / z, 1485.493m / z, and 1499.4794m / z ( Figure 2 b) corresponds to the hydrogenation and sodium ion peaks of C16Fengycin A, and the sodium ion peak of C17Fengycin A, respectively, indicating that strain Bv1 can also produce homologues of Fengycin A.

[0080] 2. Space breeding

[0081] 2.1 Test strain: Bacillus velezensis Bv1

[0082] 2.2 Experimental conditions

[0083] (1) Vehicle: manned spacecraft;

[0084] (2) Time: June 5, 2022 - December 4, 2022 (i.e., launch on June 5, return on December 4, 183 days in orbit);

[0085] (3) Launch site: Jiuquan Satellite Launch Center.

[0086] 2.3 Experimental methods

[0087] The starting strain, Bacillus velezensis Bv1, was prepared into freeze-dried powder. The samples were then collected and loaded into test chambers. The spacecraft was launched into space for in-orbit experiments. The return capsule was recovered, opened, and the test chambers were unpacked before the samples were distributed.

[0088] 2.4 Experimental Results

[0089] A total of about 200 mg of freeze-dried bacterial powder of the mutagenic strain was obtained. After the strain was returned, it was preserved in this laboratory for subsequent experiments.

[0090] 3. Screening of Bv1-b39 and other positive mutants

[0091] 3.1 Test strains:

[0092] (1) Rhizoctonia solani Kühn, preserved in our laboratory.

[0093] (2) Bacillus velezensis Bv1: Bacillus velezensis, deposited in our laboratory.

[0094] (3) Using the aerial mutagenesis strain obtained in step 2, thousands of single colonies were selected for a plate standoff primary screening experiment, and then a plate standoff rescreening experiment was performed on the strains with better results in the primary screening.

[0095] 3.2 Experimental Materials

[0096] Landy medium: glucose 20 g / L, L-glutamic acid 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, FeSO4·7H2O 0.15×10 -3g / L, MnSO4 5×10 -3 g / L, CuSO4·5H2O 0.16×10 -3 g / L, and the culture medium needs to be sterilized at 115℃ for 30min after preparation.

[0097] 3.3 Experimental methods

[0098] (1) Preparation of single colonies of space-breeding mutant strains

[0099] Add the freeze-dried bacterial powder returned in step 2 to LB liquid medium and 30% glycerol to prepare a space breeding bacterial solution for use. Dilute the bacterial solution with sterile water and apply it to LB solid medium to form a single colony as a space breeding sample plate.

[0100] (2) Initial screening by plate confrontation method

[0101] Under sterile conditions, Rhizoctonia solani was inoculated in the center of a PDA plate as an indicator pathogen. Three different single colonies were selected from a pre-prepared space breeding sample plate and spot-inoculated 2.0 cm from the center of the culture medium. The starting strain was also spot-inoculated 2.0 cm from the center of the culture medium. The plates were placed upright in a 25°C incubator and incubated. After the control strain formed a clear inhibition zone, the presence and size of each mutant zone were observed. The width of the inhibition zone of the colony was measured, and the improvement in antibacterial activity was calculated. The percentage of improvement in inhibition rate (%) = (M - WT) / WT × 100% (WT: width of the inhibition zone of the wild-type strain; M: width of the inhibition zone of the space-bred strain).

[0102] (3) Flat plate confrontation method rescreening

[0103] Mutants with an inhibition rate of 50% or more in the initial plate-to-plate screening were rescreened. Under sterile conditions, Rhizoctonia solani was inoculated in the center of a PDA plate as an indicator pathogen. Three locations of the same strain were inoculated 2.0 cm from the center of the medium. The starting strain was also inoculated 2.0 cm from the center of the medium. The plates were placed upright in a 25°C incubator. Once a clear zone of inhibition formed on the control strain, the size of the zone of inhibition on the mutant strain was observed. The inhibition rate and improvement in antibacterial activity were calculated using the same method as above.

[0104] (4) Punching method for rescreening

[0105] Mutants with an inhibition rate of 50% or more in the initial plate-based screening were rescreened using the punch method. Seed liquid from different mutants was inoculated into Landy medium at a 5% inoculum and cultured at 37°C, 200 rpm, for 48 hours. The fermentation broth was centrifuged at 4°C, 6000 rpm, and 40°C for 20 minutes. The precipitate was discarded, and the supernatant was adjusted to pH 2.0 with concentrated hydrochloric acid. The broth was sealed with parafilm and incubated at 4°C overnight. The acid precipitate was centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant was discarded and the precipitate was collected. The precipitate was frozen at -80°C for 2 hours and then freeze-dried in a freeze dryer for 24 hours to obtain a crude lipopeptide extract.

[0106] The crude lipopeptide extract was dissolved in chromatography-grade methanol (methanol:water = 1:1) and the pH was adjusted to 7.0 with 10 g / L NaHCO₃. The crude lipopeptide extracts from different mutants were dissolved in the same volume of chromatography-grade methanol, adjusted to pH 7.0, and then made up to the same volume with water. The volume of solution used for final dissolution and pH adjustment was minimized to ensure a high concentration and a more pronounced antibacterial effect. The final solution was aseptically filtered through a 0.22 μm filter to obtain a sterile filtrate for antibacterial activity testing.

[0107] Under aseptic conditions, a pathogen agar block was inoculated in the center of a PDA flat plate and punched (approximately 0.4 cm in radius) 2.0 cm away from the center. In the control plate, two holes were added with 80 μL of the starting strain crude extract sterile filtrate, and the other two holes were added with 80 μL of sterile water and 80 μL of 10 g / L NaHCO 3 to regulate the pH to 7.0 of chromatographic grade methanol. In the experimental plate, four holes were added with 80 μL of different mutant crude extract sterile filtrate. The flat plate was placed in an incubator at 25°C for cultivation. The pathogen in the control group was grown to the punched hole, and each mutant was observed to have an inhibition zone. The inhibition zone radius was measured and the antibacterial activity improvement rate was calculated as follows: improvement rate = (mutant inhibition zone radius-hole radius) / (wild bacteria inhibition zone radius-hole radius)-1 × 100%.

[0108] 3.4 Experimental Results

[0109] (1) Initial screening by plate confrontation method

[0110] The experimental results show that ( Figure 3 In the screening of thousands of aerospace strains, the antibacterial rates of the aerospace strains Bv1-a48, Bv1-b39, Bv1-c58, Bv1-12, and Bv1-106 were respectively increased by 100%, 50%, 150%, 100%, and 100% compared with the original strains, all of which were increased to 50% or above. Some of these strains also lost their antibacterial activity or had reduced antibacterial activity. Figure 3 Bv1 is the control strain on the left side of center c, and the others are space-induced mutagenesis strains.

[0111] These results preliminarily indicate that these five strains are positive mutants with significantly improved inhibition rates, performing well among all the strains initially screened. However, the initial screening experiments were not replicated, so the positive mutants with good initial screening results were rescreened using the same method in three replicates on the opposite plates.

[0112] (2) Flat plate confrontation method rescreening

[0113] The above five aerospace strains and other aerospace strains with an antibacterial rate of 50% or more were screened on a plate. The experimental results showed that ( Figure 3 In middle b), the antibacterial improvement rates of aerospace strains Bv1-a48, Bv1-b39, Bv1-c58, Bv1-12, and Bv1-106 were 30%, 38.33%, 73.33%, 70%, and 47.78%, respectively.

[0114] The results showed that in the plate confrontation rescreening experiment, the antibacterial rates of the above five aerospace strains increased by 30% or more. Although the experimental results were lower than the initial screening results, the antibacterial improvement rates were still higher than those of other rescreened strains.

[0115] (3) Punching method for rescreening

[0116] The antibacterial activity of 35 strong antibacterial positive mutants, including the five aerospace strains mentioned above, was evaluated to verify the antibacterial activity of their fermentation filtrates. The experimental results showed that the sterile water control group and the chromatographic methanol control group had no antibacterial activity ( Figure 4 a), among which the spaceflight strain Bv1-b39 ( Figure 4 b) The antibacterial rate increased by 60% ( Figure 4 In c), the antibacterial activity was significantly enhanced compared with other strains.

[0117] These results further indicate that the spaceflight strain Bv1-b39 is a positive mutant with enhanced antibacterial activity. However, the punch method cannot reflect the real-time antibacterial effect, blocking other possible antibacterial mechanisms. The antibacterial activity was assessed solely by extracting antibacterial substances, but some mutants showed a change in antibacterial strength that was unrelated to lipopeptides. Furthermore, the growth of each strain in the plate standoff assay was complex and difficult to control, resulting in significant differences in the amount of lipopeptides produced. Therefore, high-performance liquid chromatography (HPLC) was required to identify and quantify each component.

[0118] Example 2: Yield Identification of Bv1 Strain and Its Space Breeding Strain

[0119] 1. Iturin A production determination

[0120] 1.1 Test strains:

[0121] Bacillus velezensis Bv1 and the space-induced mutagenesis strains obtained by screening in step 3 of Example 1: Bv1-a48, Bv1-b39, Bv1-c58, Bv1-12, and Bv1-106.

[0122] 1.2 Experimental Materials

[0123] (1) Fermentation medium: 7 g corn flour, 1 g soluble starch, 45 g peptone, 0.2 g yeast powder, 1.5 g KH2PO4, 3 g K2HPO4, 0.5 g MgSO4·7H2O, 0.1 g CaCO3, 0.1 g FeSO4, 2 g glutamic acid dissolved in deionized water, filtered and sterilized, and added to 1000 mL distilled water.

[0124] (2) LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, add distilled water to 1000 mL, and adjust the pH to 7.4.

[0125] (3) Other drugs: Iturin A standard product (chromatographically pure, ≥98.0%, MCE, America).

[0126] 1.3 Experimental equipment

[0127] (1) Liquid chromatography system: LC-20A HPLC system (Shimadzu, Kyoto, Japan), with a C18 column (5 μm, 250 × 4.6 mm);

[0128] (2) Microplate reader: Model Infinite M200Pro (Tecan, Switzerland).

[0129] 1.4 Experimental methods

[0130] 1.4.1 Fermentation of Bv1 and its Space Bred Strains

[0131] (1) Pick a single colony with a sterile toothpick and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of LB liquid medium. Incubate at 28°C and 180 rpm for 12 h to obtain seed solution.

[0132] (2) In a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium (natural pH), inoculate the seed solution at a 5% inoculum volume and incubate at 28°C, 180 rpm, and shake for 72 h. Repeat three times.

[0133] (3) At each time period, 200 μL of fermentation liquid was placed in a 96-well plate, and the OD value of each strain was measured using a microplate reader at a wavelength of 600 nm.

[0134] 1.4.2 Measurement of iturin concentration in fermentation broth by HPLC

[0135] (1) At 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, 1 mL of fermentation broth was centrifuged at 12,000 rpm for 10 min and the supernatant was collected;

[0136] (2) The fermentation supernatant was filtered through a 0.22 μm PES filter and transferred to a liquid phase bottle for detection. The mobile phase was 0.035% formic acid / acetonitrile (55:45, v / v) and the detection wavelength was 220 nm.

[0137] (3) Diluting a 1000 mg / L standard iturin A solution with a 1% NaHCO3 aqueous solution to prepare a 75-250 mg / L iturin A solution, and establishing a chromatographic absorption peak area-iturin A concentration standard curve;

[0138] (4) The fermentation broth was centrifuged at 12,000 rpm for 10 min to remove bacterial cells and then filtered through a 0.22 μm filter. The iturin A concentration in the solution was calculated.

[0139] 1.5 Experimental Results

[0140] (1) Growth curves of Bv1 strain and its space-breeding strain

[0141] Five space-induced mutant strains and the wild-type strain Bv1 with strong antibacterial activity were obtained by screening. They were cultured in fermentation medium for 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, respectively. 200 μL of fermentation liquid was taken in a 96-well plate at each time point, and the OD of each strain was measured at a wavelength of 600 nm using a microplate reader. 600 value( Figure 5 (a)

[0142] The experimental results showed that, except for strain Bv1-c58, the OD values ​​of the above five space-breeding strains gradually increased with the increase of fermentation time, and the OD values ​​after 24 hours of fermentation were 600 The values ​​were always higher than those of the original strain. At the end of fermentation, the OD of strain Bv1-c58 was 600 The highest value was 2.2454, followed by strain Bv1-b39, OD 600 The value finally reached 1.9011. The above results show that the growth of the four mutant strains after space breeding is significantly different from that of the original strain, and the growth rate is higher than that of the original strain.

[0143] (2) Determination of iturin A concentration produced by Bv1 strain and its space-breeding strain

[0144] The iturin A content in 108 samples was determined using high-performance liquid chromatography (HPLC), and the chromatographic peak area of ​​each sample was calculated. The concentration of iturin A in each sample was calculated based on the resulting standard curve to determine the iturin A concentration of each strain at different fermentation times.

[0145] The experimental results showed that after 72 hours of fermentation, the iturin A production of strains Bv1-c58 and Bv1-12 reached 133.29 mg / L and 126.04 mg / L, respectively, both lower than the 155.62 mg / L of the original strain Bv1. The final iturin A concentrations of strains Bv1-a48, Bv1-b39, and Bv1-106 were 194.27 mg / L, 195.97 mg / L, and 183.32 mg / L, respectively, representing increases of 25%, 26%, and 18% compared to the original strain.

[0146] During the critical fermentation period of 24h to 72h ( Figure 5 In b), iturin A production in strain Bv1-b39, a natural secondary metabolite of lipopeptides, began to increase significantly after 36 hours, reaching 163.06 mg / L at 48 hours of fermentation, a 48.5% increase compared to the original strain. During this period, iturin A concentration increased rapidly, and the strain also grew well. After 48 hours of fermentation, iturin A production in strain Bv1-b39 continued to increase with fermentation time, ultimately reaching 194.27 mg / L. During this period, the strain OD 600 It also increased significantly, and the growth rate accelerated.

[0147] Results showed that strain Bv1-b39 produced the highest iturin A yield of the five space-farming strains. Its yield increased by 48.5% after 48 hours of fermentation and by 26% after 72 hours of fermentation compared to the original strain. Its growth rate was also significantly increased compared to the original Bv1 strain, demonstrating its potential as a positive space-farming mutant capable of producing high levels of antimicrobial lipopeptides.

[0148] 2. Identification of lipopeptide types produced by space-breeding strains

[0149] 2.1 Test strains:

[0150] (1) Bacillus velezensis Bv1: Bacillus velezensis, deposited in our laboratory.

[0151] (2) Space-induced mutagenesis strains obtained by screening in step 3 of Example 1: Bv1-a48 and Bv1-b39.

[0152] 2.2 Experimental Materials

[0153] Fermentation medium: corn flour 7 g, soluble starch 1 g, peptone 45 g, yeast powder 0.2 g, KH2PO4 1.5 g, K2HPO4 3 g, MgSO4·7H2O 0.5 g, CaCO3 0.1 g, FeSO4 0.1 g, glutamic acid 2 g, distilled water 1000 mL.

[0154] 2.3 Experimental instruments

[0155] (1) SCIEX4800Plus mass spectrometer;

[0156] (2) MALDI-TOF / TOF mass spectrometer (AXIMA-Performance MA, Shimadzu Corporation, Japan).

[0157] 2.4 Experimental methods

[0158] (1) LC-MS sample preparation

[0159] Centrifuge the 72-hour fermentation broth of the Bv1 strain, the 60-hour fermentation broth of the Bv1-a48 strain, and the 60-hour fermentation broth of the Bv1-b39 strain in step 1.4.2 of Example 2 at 12,000 rpm for 10 minutes, and collect the supernatant. Filter the fermentation supernatant through a 0.22 μm PES filter membrane to prepare the sample.

[0160] (2) LC-MS identification of the antimicrobial lipopeptide types produced by mutant strains

[0161] MALDI-TOF-MS was used to determine the molecular weight of the solute using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. An AXIMA-Performance MA mass spectrometer was used, with excitation at 2-20 kV using a 337 nm nitrogen laser beam, and scanning over the mass-to-charge ratio range of 300-2000 m / z.

[0162] 2.5 Experimental Results

[0163] According to the high performance liquid chromatography results of step 1.5 in Example 2, representative peaks were selected for LC-MS detection, namely strains Bv1, Bv1-a48, and Bv1-b39.

[0164] The experimental results showed that the original strain Bv1 had a retention time of 6.569 min ( Figure 6A1 ) contains two mass spectrometry peaks at 1043m / z and 1071m / z, corresponding to the hydrogen ion adducts of C14 and C16 of Iturin A, respectively. At retention time 11.344min ( Figure 6A2 ) showed a mass spectrum peak of 1095 m / z, which was the potassium ion adduct of C15Iturin A. This showed that the original strain Bv1 could produce Iturin A with a carbon chain length of 14-16.

[0165] The strain Bv1-a48 was detected at 5.766 min ( Figure 6B1 ) appears at a retention time of 1043m / z peak, corresponding to the hydrogen ion adduct of C14 of Ituri n A. And at a retention time of 7.635min( Figure 6B2 ), corresponding to the C15 potassium ion adduct of iturin A. This indicates that the space-breeding strain Bv1-b39 can still produce iturin A with carbon chain lengths of C14 and C15.

[0166] The strain Bv1-b39 was detected at 5.806 min ( Figure 6C1 ) appears at the retention time of 1043m / z peak, corresponding to the hydrogen ion adduct of C14 of Itur in A. And at the retention time of 11.114min( Figure 6C2 ), two peaks at 1071 m / z and 1095 m / z appeared, representing the hydrogen ion adduct of C16 and the potassium ion adduct of C15 of Iturin A, respectively. This indicates that the space-breeding strain Bv1-a48 can still produce Iturin A with a carbon chain length of 14-16.

[0167] The above experimental results show that the strains bred through space-induced mutagenesis are the same as the starting strains and can still produce Iturin A with different carbon chain lengths.

[0168] Iturin A (CB Number: CB1163737) has 8 isomers, named A1-A8. According to the mass spectrometry results, the molecular weight of iturin in the fermentation broth is 1042Da, 1056Da, and 1070Da. Therefore, the reference Iturin: cyclic lipopeptide with multifunction biological potential Figure 2 , in the present invention, it should specifically be iturin A2 configuration.

[0169] 3. Verification of genetic stability of space breeding strains

[0170] 3.1 Test strains

[0171] (1) Bacillus velezensis Bv1: Bacillus velezensis, deposited in our laboratory.

[0172] (2) Space-induced mutagenic strains obtained by screening in step 1.4 of Example 2: Bv1-a48, Bv1-b39, and Bv1-106.

[0173] 3.2 Experimental Materials

[0174] (1) Culture medium

[0175] LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, add distilled water to 1000 mL, and adjust the pH to 7.4;

[0176] Fermentation medium: 7 g corn flour, 1 g soluble starch, 45 g peptone, 0.2 g yeast powder, 1.5 g KH2PO4, 3 g K2HPO4, 0.5 g MgSO4·7H2O, 0.1 g CaCO3, 0.1 g FeSO4, 2 g glutamic acid dissolved in deionized water, filtered and sterilized, and added to 1000 mL distilled water.

[0177] (2) Other medicines

[0178] Iturin A standard product (chromatographically pure, ≥98.0%, MCE, America);

[0179] Surfactin standard (chromatographically pure, ≥98.0%, MCE, America);

[0180] Oil red solution: Add 0.5 g of oil red to 100 mL of n-dodecane, shake thoroughly to dissolve, and filter with a 0.45 μm filter membrane. The filtrate is the oil red solution.

[0181] 3.3 Experimental instruments

[0182] (1) Liquid chromatography system: LC-20A HPLC system (Shimadzu, Kyoto, Japan), with a C18 column (5 μm, 250 × 4.6 mm).

[0183] (2) Microplate reader: Model Infinite M200Pro (Tecan, Switzerland).

[0184] 3.4 Experimental methods

[0185] 3.4.1 Subculture of Bv1 and its Space Breeding Strains

[0186] (1) Pick a single colony with a sterile toothpick and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of LB liquid medium. Incubate at 28°C and 180 rpm for 12 h to obtain seed solution.

[0187] (2) In a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium (natural pH), inoculate the seed solution at a 5% inoculum volume and culture at 28°C, 180 rpm, and shake for 48 h in triplicate.

[0188] (3) Each generation was cultured for 48 h, for a total of ten generations. At the end of each generation, 200 μL of the fermentation broth was placed in a 96-well plate and the OD value of each strain was measured using a microplate reader at a wavelength of 600 nm.

[0189] 3.4.2 Determination of surfactant by oil ring method

[0190] (1) Establishment of oil drain ring standard curve

[0191] To determine the diameter of the oil ring, use a surfactant standard solution with a concentration of 0.4-1.4 g / L. Place a 9 cm diameter glass Petri dish on graph paper. Add 40 mL of deionized water. Once the water surface stabilizes, add 200 μL of oil red solution. Once the oil red has spread evenly, add 2.5 μL of surfactant standard solution to the center of the oil red surface. Once the oil ring stabilizes, take a photo and record its diameter. Create a standard curve using the surfactant concentration as the horizontal axis and the oil ring diameter as the vertical axis, and derive the equation for the standard curve.

[0192] (2) Determination of the ability of strains to produce surfactant

[0193] 1 mL of fermentation broth was taken from each generation and centrifuged at 12,000 rpm for 10 min. The supernatant was collected and the diameter of the oil-discharge red circle was determined by the oil-discharge red circle method. The lipopeptide content of the strain was calculated based on the surfactin standard curve.

[0194] 3.4.3 Measurement of iturin concentration in fermentation broth by HPLC

[0195] (1) At the end of each generation, 1 mL of fermentation broth was centrifuged at 12,000 rpm for 10 min and the supernatant was collected;

[0196] (2) The fermentation supernatant was filtered through a 0.22 μm PES filter and transferred to a liquid phase bottle for detection. The mobile phase was 0.035% formic acid / acetonitrile (55:45, v / v) and the detection wavelength was 220 nm.

[0197] (3) The fermentation broth was centrifuged at 12,000 rpm for 10 min to remove bacterial cells and then filtered through a 0.22 μm filter. The iturin A concentration in the solution was calculated.

[0198] 3.5 Experimental Results

[0199] 3.5.1 Growth of Space Breeding Strains During Subculture

[0200] Bv1-a48, Bv1-b39, and Bv1-106, which had a higher iturin A yield than the original strain obtained in step 1.4 of Example 2, were selected for subculture and cultured for ten generations. At the end of each generation, 200 μL of the fermentation liquid was placed in a 96-well plate and the OD value of each strain was measured at a wavelength of 600 nm using a microplate reader ( Figure 7 ).

[0201] The experimental results showed that in the first eight generations, the OD of strain Bv1-106 was 600 The value was the highest among the four strains, but at the ninth generation, OD 600 The decline is large, which may be due to the excessive number of passages, the decline in the activity of the strain, and the poor genetic stability. 600 Slightly lower than Bv1-106, but its OD 600 The values ​​are always slightly different, and the growth is more stable.

[0202] 3.5.2 Establishment of oil drain ring standard curve

[0203] Lipopeptides, as surfactants, are a class of molecules with amphiphilic structures that can spontaneously aggregate and assemble at the water-oil interface, effectively reducing the interfacial tension between the two phases. Therefore, the oil ring method, which measures the diameter of the oil ring, can quickly and roughly quantify the lipopeptide content in fermentation broth samples.

[0204] First, we used surfactin as the lipopeptide standard to measure the oil-discharging ring and obtain the standard curve. We used 0.4-1.4g / L lipopeptide standard solution to measure the oil-discharging ring diameter ( Figure 8 a) Regression analysis showed that in the range of 0.4-1.4 g / L, the oil drain ring diameter Dr (mm) and the lipopeptide concentration Csrf (g / L) showed a good linear relationship: Dr = 22 × Csrf + 0.2, R 2 =0.97919( Figure 8 b).

[0205] 3.5.3 Determination of Lipopeptide Yield by the Oil Ring Method in Space-Borne Breeding Strains

[0206] The oil-discharging red circle method was used to determine the diameters of the oil-discharging red circles of the three strains Bv1-a48, Bv1-b39 and Bv1-106 from the first to the tenth generation. Figure 8 b) Calculate the content of lipopeptide in the strain. The experimental results show that the diameter of the oil ring corresponding to ten transfer cultures ( Figure 9 After calculating the lipopeptide standard curve in (a) and (b), it can be seen that the lipopeptide content of the three strains decreased significantly after the eighth generation ( Figure 9 (b) Strain Bv1-a48 had the lowest lipopeptide content overall during the first eight subcultures. Strain Bv1-106 showed a significant decrease in lipopeptide production after the seventh subculture, indicating poor genetic stability and inability to consistently produce lipopeptides. Strain Bv1-b39 showed a slight decrease in lipopeptide production after the eighth subculture, but the decrease was minimal, indicating good genetic stability.

[0207] 3.5.4 Determination of iturin A concentration in spaceflight strains

[0208] The iturin A content of the first and eighth generation fermentation broths of the strains was determined by high performance liquid chromatography. The chromatographic peak area of ​​each sample was calculated, and the concentration of iturin A in each sample was calculated to obtain the iturin A concentration of the first and eighth generation fermentation broths of each strain ( Figure 9 (c)

[0209] Compared to other strains, strain Bv1-a48 exhibited relatively low iturin A production, dropping to 81.6 mg / L after the eighth subculture. Strain Bv1-106 exhibited significantly lower iturin A production after the eighth subculture compared to the first generation, dropping from 186.51 mg / L to 79.81 mg / L, indicating poor genetic stability. In contrast, strain Bv1-b39 exhibited no significant change in iturin A production between generations, reaching 130.52 mg / L after the eighth subculture, a mere 15% decrease compared to the first generation, demonstrating good genetic stability.

[0210] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Bacillus, characterized in that The bacillus is Bacillus velez ( Bacillus velezensis ) Bv1-b39, which is deposited in the China General Microorganism Culture Collection (CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No. 32879.

2. Use of the Bacillus according to claim 1 in the preparation of antibacterial products.

3. Use of the Bacillus according to claim 1 in the preparation of products producing antimicrobial lipopeptides.

4. A composition characterized in that The composition contains the Bacillus according to claim 1.

5. The composition according to claim 4, characterized in that: The composition is a culture, and the culture is a substance obtained by culturing the Bacillus in a microbial culture medium.

6. The composition according to claim 4, characterized in that: The composition is a bacterial agent.

7. The composition according to any one of claims 4 to 6, characterized in that The composition is any one of the following: A1) Compositions for antibacterial use, A2) Compositions for producing antimicrobial lipopeptide compounds.

8. A method for preparing a composition, characterized in that The composition is the composition according to any one of claims 4 to 7, and the method comprises the step of using the Bacillus according to claim 1 as a component of the composition.

9. Use of the composition according to claims 4-7 in preparing a product, characterized in that: The product has at least one of the following properties: B1) antibacterial, B2) Production of antimicrobial lipopeptide compounds.

10. An antibacterial method, characterized in that: The method comprises the step of contacting the composition according to claims 4 to 7 with bacteria.

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