Application of Bacillus altitudinis Q7

Abstract

The application discloses application of Bacillus altitudinis Q7. The application also discloses application of a fermentation product of the Bacillus altitudinis Q7 in inhibiting pathogenic bacteria. The preparation method of the fermentation product comprises the following steps: (1) preparing a seed solution of the Bacillus altitudinis Q7; (2) fermentation culture; (3) crude extraction of lipopeptides in the fermentation liquor; and (4) purification of the lipopeptides. The preparation method is low in cost, simple in operation and short in production cycle. The prepared cyclic lipopeptide biosurfactant has inhibiting effect on various pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to the application of Bacillus altitudinis Q7, specifically to the application of Bacillus altitudinis Q7 in the biosynthesis of cyclic lipopeptides and the inhibition of pathogens. Background Technology

[0002] Lipopeptides, also known as acyl peptides, are a class of low-molecular-weight antimicrobial peptides derived from amino acids. They are also important biosurfactants. Lipopeptides are mainly biosynthesized through non-ribosomal pathways and polyketide hybrid pathways in microorganisms. Natural lipopeptides are widely available, primarily derived from microorganisms, with bacteria being the most prevalent source. With ongoing research, lipopeptides have been isolated from many Gram-positive bacteria, Gram-negative bacteria, Gram-mutant bacteria, and various fungi. Lipopeptides are amphiphilic molecules containing both polar and nonpolar parts in their structure. The polar part is a cyclic peptide, while the nonpolar part consists of straight-chain or branched fatty acids of varying lengths and oxidation levels. Their unique molecular structure endows lipopeptides not only with antimicrobial functions but also with excellent surfactant properties.

[0003] The discovery and research of lipopeptides can be traced back to the late 1970s. In 1968, Arima et al. reported the first discovery of a lipopeptide crystalline surfactant in the fermentation broth of Bacillus subtilis strain, naming it surfactant. Although these substances were found to possess surfactant properties and have potential applications in medicine and bioengineering, they did not receive sufficient attention due to technological and cognitive limitations. It wasn't until 1980, when lipopeptides were seen as a potential alternative to chemically synthesized surfactants for addressing ecological issues, that interest in them gradually increased. With continued research, the application potential of lipopeptides in environmental remediation, oil extraction, biomedicine, agricultural production, and food processing has gradually emerged. How to utilize modern biotechnology for large-scale industrial production of lipopeptides has become one of the core issues in the field of lipopeptide research.

[0004] Numerous studies have confirmed that lipopeptides are synthesized via nonribosomal peptide synthases or hybrid polyketide synthases, which are essentially large iterative functional units (modules) of proteins containing multiple structural domains. These modular proteins are responsible for the biosynthesis of hundreds of bioactive compounds and are giant enzymes that catalyze polyketation or peptide conversion reactions. Most of the discovered nonribosomal peptide synthases are found in bacteria, such as Proteobacteria, Actinobacteria, Firmicutes, and Cyanobacteria. Furthermore, genetic mechanisms for the synthesis of nonribosomal peptides have been found in various bacterial phyla, including Anomala – Thermomycetes, Chlorophytes, Verrucous Microbes, and Blastomonas. Bioinformatic analysis of nonribosomal peptide synthesis operons can reveal detailed differences in the synthesis process of lipopeptide homologues. By integrating modern biotechnology, such as homologue modeling, sequence alignment, and biosynthesis, targeted differential modification of nonribosomal peptide synthases can be performed, thereby achieving prediction of synthetic products, precise control of synthetic pathways, and rapid improvement in synthetic yield.

[0005] Endophytic bacteria are microorganisms that colonize the space of plant tissue cells and do not produce any adverse or pathological effects on the plant. Endophytic bacteria can promote the accumulation of secondary metabolites by enhancing their own growth, adaptability, and tolerance to abiotic or biotic stresses. They are an important type of bioreactor for accumulating metabolites and have the potential for large-scale production. Summary of the Invention

[0006] The main objective of this invention is to overcome the shortcomings of existing biosurfactant preparation processes, such as high cost and complex procedures, and to provide a method for preparing lipopeptide biosurfactants. This invention provides the application of Bacillus altitudinis Q7 in the biosynthesis of lipopeptide biosurfactants and demonstrates the efficacy of this biosurfactant in inhibiting pathogenic bacteria.

[0007] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0008] This invention provides an application of Bacillus altitudinis Q7, the strain of which has the preservation number CCTCC NO: M 20221567 and the preservation date is October 14, 2022; and the application of the fermentation product of Bacillus altitudinis Q7 in inhibiting pathogenic bacteria.

[0009] Furthermore, in the above technical solution, the Bacillus altitudinis Q7 fermentation product is a cyclic lipopeptide, which is composed of lysine lysozyme, surfactant, pseudofactor I, and pseudofactor II.

[0010] Furthermore, in the above technical solution, the peptide chain of the lysine lysozyme is composed of glutamine (Gln), leucine (Leu), isoleucine (Ile), aspartic acid (Asp), and valine (Val); the peptide chain of the surfactant is composed of glutamic acid (Glu), leucine (Leu), valine (Val), and aspartic acid (Asp); the peptide chain of pseudofactor I is composed of glycine (Gly), serine (Ser), threonine (Thr), leucine (Leu), and valine (Val); and the peptide chain of pseudofactor II is composed of glycine (Gly), serine (Ser), threonine (Thr), and leucine (Leu).

[0011] Furthermore, in the above technical solution, the pathogens include bacteria and fungi.

[0012] Furthermore, in the above technical solution, the bacteria include one or more of Staphylococcus aureus, Micrococcus occulta, Bacillus subtilis, Salmonella or Escherichia coli; the fungi include one or two of Alternaria alternata or Botrytis cinerea.

[0013] Furthermore, in the above technical solution, the method for preparing the Bacillus altitudinis Q7 ferment includes the following steps:

[0014] (1) Fermentation culture of Bacillus altitudinis Q7 seed culture;

[0015] (2) Crude extraction of lipopeptides from fermentation broth;

[0016] (3) Lipopeptide purification.

[0017] Furthermore, in the above technical solution, the fermentation conditions are as follows: fermentation temperature 31±3℃, aeration rate 1.5~2L / min, stirring speed 200~300rpm, fermentation pH 6.0~7.5, and fermentation time 68~75h.

[0018] Furthermore, in the above technical solution, the method for crude extraction of lipopeptides from the fermentation broth includes: centrifuging the fermentation broth to remove microbial cells, adjusting the pH of the cell-free filtrate to 1.5–2.3 with 5.5–6.5 mol / L hydrochloric acid solution, and allowing it to stand at 4±3℃ for 12–16 h; centrifuging to recover the precipitate; washing the collected precipitate with acidified water at pH 1.9–2.2 and air-drying it, and extracting the biosurfactant from the precipitate with anhydrous methanol to remove the solvent.

[0019] Furthermore, in the above technical solution, the method for purifying the lipopeptide includes: purification using a macroporous resin.

[0020] The beneficial effects of this invention are as follows: Bacillus altitudinis Q7, isolated from Ginkgo biloba leaves, exhibits strong stress tolerance and a short culture cycle. This invention utilizes Bacillus altitudinis Q7 for fermentation to prepare lipopeptide biosurfactants, which is low-cost, simple to operate, and has a short production cycle, possessing broad industrial application value. It is truly a novel, progressive, and practical new design. Attached Figure Description

[0021] Figure 1 The images show the LC-MS chromatograms of Bacillus altitudinis Q7 lipopeptide. a) Total ion current chromatogram by LC-MS; b) Ion peak at retention time 3.38 min; c) Ion peak at retention time 3.59 min; d) Ion peak at retention time 4.19 min; e) Ion peak at retention time 4.49 min.

[0022] Figure 2 The total ion current chromatograms (a) and (b) of fatty acid methyl esters in lipopeptides are shown.

[0023] Figure 3 The mass spectra are those of fatty acid methyl esters (ac) and amino acid silane derivatives (dl). Detailed Implementation

[0024] The following specific embodiments will provide a complete and detailed description of the technical solution of the present invention, but the present invention is not limited thereto. The following embodiments are merely examples illustrating the technical solution of the present invention, and therefore do not limit the present invention in any way.

[0025] Example 1

[0026] The Bacillus altitudinis Q7 strain described in this invention was isolated from Ginkgo biloba leaves.

[0027] The *Bacillus altitudinis* Q7 strain was Gram-negative and exhibited rod-shaped cell morphology. In plate culture, colonies were regular round dots with a smooth, relatively transparent surface. In physiological and biochemical experiments, Q7 showed weak hydrolytic ability towards starch and fats, causing litmus milk to darken, the fermentation broth to become slightly alkaline, and it failed to liquefy gelatin. API 50Ch test results showed the following: ONPG(+), VP(-), Aescin(+), Salicin(-), Sucrose(+), Rhamnose(+), Citrate(-), Urease(-), Thiamine vinegar(-), Lactose(-), Aceculin(-), Hippurate hydrolysis(+), Maltose(-), Raffinose(+), D-arabia sugar(+), L-arabia sugar(+), Trisaccharide iron(-), Inulin(+), Melezitose(+), etc. Based on 16S rRNA identification and phylogenetic analysis, the Ginkgo endophytic bacterium Q7 is identified as Bacillus altitudinis Q7.

[0028] The sequence of Bacillus altitudinis Q7 is shown in SEQ ID NO.1.

[0029] The described Bacillus altitudinis Q7 strain is deposited at the China Center for Type Culture Collection (CCTCC, located at Luojia Mountain, Wuchang, Wuhan, 430072, China). The strain number of Bacillus altitudinis Q7 is CCTCC NO: M 20221567, the deposit date is October 14, 2022, and it is classified as Bacillus altitudinis, with the strain name Q7.

[0030] Bacillus altitudinis Q7, stored in a 4℃ incubator, was activated. A loopful of colonies was scraped and inoculated into activation medium (peptone (10 mg / mL), beef extract (5 mg / mL), glucose (5 mg / mL), sodium chloride (5 mg / mL), pH 7.3). Activation was performed at 28℃ and 160 rpm for 24 h in a shaker. The activated culture was then inoculated at 2% (v / v) into shake flasks (shake flask medium: peptone (10 g / L), beef extract (5 g / L), glucose (5 g / L), sodium chloride (5 g / L), pH 7.2) and pre-cultured for 16 h. The seed culture was then diluted with sterile shake flask medium in a clean bench. 600 Adjust the pH to approximately 0.8. Inoculate the prepared seed culture into a fermenter (the culture medium in the fermenter consisted of glucose (10 g / L), soybean peptone (10 g / L), beef extract (5 g / L), sodium chloride (5 g / L), and pH 7.0), with an inoculation volume of 3% (by volume). Adjust the fermentation temperature to 31°C, aeration rate to 1.5 L / min, stirring speed to 200 rpm, fermentation pH to 6.6, and fermentation time to 72 h.

[0031] The fermentation broth was then centrifuged at 8000 rpm for 15 min to remove microbial cells. The cell-free filtrate was adjusted to pH 2.0 with 6 mol / L hydrochloric acid solution and allowed to stand overnight at 4°C. The precipitate was then recovered by centrifugation at 12000 rpm for 20 min. The collected precipitate was washed twice with acidified water at pH 2.0 and air-dried. The biosurfactant (crude lipopeptide) was extracted from the precipitate with anhydrous methanol, concentrated by rotary evaporation, and freeze-dried to obtain the lipopeptide biosurfactant.

[0032] The lipopeptides were then purified using X-5 macroporous resin. 10 mL of a 50 mg / mL crude lipopeptide solution in anhydrous methanol was loaded onto the column using a peristaltic pump. The column was equilibrated with deionized water until the eluent was odorless. Elution was then performed using an 80% ethanol aqueous solution at a flow rate of 1 mL / min. The eluent was collected using a DBS-100 automatic collector at a frequency of one test tube every 5 minutes. The eluent was concentrated, lyophilized, and stored at -20°C.

[0033] Example 2

[0034] The instrument used was a Waters ACQUITY H-Class SQD2 ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS / MS). The sample concentration was 4 mg / mL, and the injection volume was 2 μL. The eluent for lipopeptide separation was acetonitrile and water (90:10 v / v). The column temperature was set to 35℃, and the elution time was 10 min. An ACQUITY UPLCr BEH C18 reversed-phase column (2.1 × 100 mm, 1.7 μm) was used, with a flow rate of 0.4 mL / min. A DAD detector was used, and the detection wavelength was set to 210 nm. Electrospray ionization (ESI) was performed using the SQD2 mass spectrometer, and the mass of the ionized components was analyzed in full scan mode, with a scan range of 50–1500 m / z. The purified lipopeptides were obtained.

[0035] LC-MS analysis of Bacillus altitudinis Q7 lipopeptide revealed that the main elution time was after 2.94 min; the pseudo-molecular ion peaks with retention times of 3.38 min, 3.59 min, 4.19 min, and 4.49 min had m / z values ​​of 1035.3, 1049.4, 1063.2, and 1077.3, respectively, corresponding to lichenysin A (15 Cs), surfactin (16 Cs), pseudoofactin I (16 Cs), and pseudoofactin II (16 Cs), respectively. Figure 1 ). Figure 1 In the image, a is the total ion chromatogram by LC-MS; b is the ion peak with a retention time of 3.38 min; c is the ion peak with a retention time of 3.59 min; d is the ion peak with a retention time of 4.19 min; and e is the ion peak with a retention time of 4.49 min.

[0036] Example 3

[0037] Weigh 50 mg of the purified lipopeptide and transfer it to a 20 mL ampoule. Add 5.0 mL of 6.0 mol / L hydrochloric acid solution, seal the ampoule by melting the mouth with an alcohol torch, and place it in a 90 °C oven for constant temperature hydrolysis for 24 h. Dilute the hydrolysate with 15.0 mL of deionized water, then add 5.0 mL of anhydrous diethyl ether to extract the fatty acids. Perform three extractions consecutively, combine the organic phases from the three extractions, and then evaporate the diethyl ether in a fume hood. The resulting extract is the fatty acid component of the lipopeptide sample.

[0038] Add 5.0 mL of 10% sulfuric acid / methanol solution to the anhydrous ether extract and react at 55 °C for 6 h in an oven to perform fatty acid methyl esterification. After the reaction is completed, cool to room temperature, add 15.0 mL of deionized water to dilute, add 3.0 mL of anhydrous ether to the diluent for extraction, and extract three times consecutively. Combine the organic phases, evaporate the organic solvent to obtain fatty acid methyl esters, add 2.0 mL of methanol to dissolve by sonication, transfer to a GC-MS vial, and perform gas chromatography-mass spectrometry using a SCION 456-GC-SQ system with a Scion-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm). High-purity helium (99.999%) was used as the carrier gas. The injection port temperature was set to 250℃, the flow rate was 1 mL / min, splitless, and automatic injection was used with an injection volume of 1.0 μL. The column oven parameters were set as follows: initial temperature 120℃, hold for 3 min, then increase to 260℃ at a rate of 10℃ / min and hold for 5 min. The mass spectrometry detection parameters were: solvent delay of 3 min, full scan positive ion mode, scan time of 200 ms, scan range m / z 45-500, ion source temperature of 230℃, and ionization voltage of 70 eV.

[0039] The acid-hydrolyzed lipopeptides were analyzed for amino acids. The reaction solution was transferred to a gas chromatography vial, dried, and then placed in an oven at 105°C for 2 hours to ensure complete removal of moisture. 1.5 mL of acetonitrile was added, mixed thoroughly, and then 1.0 mL of BSTFA reagent was added. The vial was sealed and placed in an oven at 60°C for 20 minutes for silanization. After the reaction, the amino acid components of the lipopeptides were analyzed using an autosampler. The column oven parameters were set as follows: initial temperature 60°C, hold for 3 minutes, then increase to 250°C at a rate of 10°C / min and hold for 5 minutes.

[0040] To verify the composition of the lipopeptide mixture, GC-MS analysis was performed on the lipopeptide hydrolysate. The free amino acids generated from the high-temperature hydrolysis of lipopeptides with hydrochloric acid reacted with BSTFA reagent to form amino acid silane derivatives. The total ion spectrum of the amino acid silane derivatives was obtained by GC-MS detection. Figure 2Nine ion peaks appeared in the 10-20 min range, named peaks A through I. According to the NIST mass spectrometry database search results of the detection system, the nine ion peaks with retention times of 10.9 min (peak A), 11.8 min (peak B), 12.3 min (peak C), 13.1 min (peak D), 13.3 min (peak E), 14.4 min (peak F), 15.3 min (peak G), 16.2 min (peak H), and 17.1 min (peak I) correspond to silanized derivatives of isoleucine (Ile), valine (Val), leucine (Leu), aspartic acid (Asp), glycine (Gly), glutamine (Gln), glutamic acid (Glu), serine (Ser), and threonine (Thr), respectively. Figure 3 ).

[0041] Example 4

[0042] The antibacterial activity of the surfactant (crude lipopeptide) obtained in Example 1 was preliminarily evaluated using the disk agar diffusion method. Common pathogenic bacteria Staphylococcus aureus, Micrococcus occulta, Bacillus subtilis, Escherichia coli, and Salmonella, and pathogenic fungi Alternaria alternata and Botrytis cinerea were selected as pathogens. The crude lipopeptide was dissolved in 0.01 mol / L PBS buffer to a concentration of 200 mg / L. 6 mm diameter paper disks were immersed in the lipopeptide solution and then placed on broth plates (bacteria) coated with 100 μL of pathogenic bacterial suspension or PDA plates (fungi) coated with 100 μL of spore suspension. A negative control was prepared using 0.01 mol / L PBS buffer, and a positive control was prepared using 200 mg / L streptomycin. Finally, after placing the paper disks, the microbial plates were incubated at 37°C for 24 h. After incubation, the inhibitory activity was assessed by the diameter of the inhibition zone around the disk, and the experiment was repeated three times.

[0043] The inhibitory activity of crude lipopeptides against five bacteria and two fungi was determined by measuring the diameter of the inhibition zone (Table 1). The lipopeptides produced by Bacillus altitudinis Q7 exhibited broad-spectrum antibacterial activity against common pathogenic microorganisms, showing some inhibitory effect against bacteria, but significantly lower than their inhibitory ability against fungi. Compared with the positive control streptomycin, the inhibitory effect of the lipopeptides against bacteria was weaker. Among the two common crop pathogenic fungi used in this study, Alternaria alternata was more sensitive to the lipopeptides of Bacillus altitudinis Q7. At a concentration of 200 mg / L, the diameter of the inhibition zone was 43.2 ± 0.35 mm. In summary, the lipopeptides produced by Bacillus altitudinis Q7 showed significantly better inhibitory activity against fungi than streptomycin, and exhibited excellent antibacterial activity against Alternaria alternata, making them a promising biocontrol agent for agricultural production.

[0044] Table 1. Antibacterial activity of B. altitudinis Q7 lipopeptide

[0045]

[0046] In Table 1, different letters indicate significant differences (p < 0.05, Duncan's test), and the values ​​in the table are the average of three replicates (n = 3).

[0047] The above embodiments provide a detailed description of the present invention. For any person skilled in the art, modifications can be made to the embodiments of the present invention, or equivalent embodiments can be substituted, without departing from the scope of the invention or its technical solution. However, all such modifications that do not depart from the technical principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A type of highland Bacillus ( Bacillus saltitudinis Q7, characterized in that, The accession number of the Bacillus hygroscopicus Q7 is CCTCC NO: M 20221567, and the accession date is October 14, 2022.

2. The Bacillus hygroscopicus Q7 according to claim 1, characterized in that, The *Bacillus hygroscopicus* Q7 can ferment to produce cyclic lipopeptides, which are composed of lichenysin A, surfactant, pseudofactor I, and pseudofactor II. The peptide chain of lichenysin A is composed of glutamine, leucine, isoleucine, aspartic acid, and valine. The peptide chain of surfactant is composed of glutamic acid, leucine, valine, and aspartic acid. The peptide chain of pseudofactor I is composed of glycine, serine, threonine, leucine, and valine. The peptide chain of pseudofactor II is composed of glycine, serine, threonine, and leucine.

3. The application of the fermentation product of Bacillus hygroscopicus Q7 as described in claim 2 as a biological control agent for agricultural production in inhibiting pathogens, characterized in that... The fermentation product of the Bacillus hygroscopicus Q7 is a cyclic lipopeptide, and the pathogen is one or both of Alternaria alternata or Botrytis cinerea.

4. The application according to claim 3, characterized in that, The method for preparing the ferment of Bacillus hygroscopicus Q7 includes the following steps: (1) Fermentation culture of Bacillus hygroscopicus Q7 seed culture; (2) Crude extraction of cyclic lipopeptides from fermentation broth; (3) Purification of cyclic lipopeptides.

5. The application according to claim 4, characterized in that, The fermentation conditions were as follows: fermentation temperature 31±2℃, aeration rate 1.5~2L / min, stirring speed 200~300rpm, fermentation pH 6.3~7.0, and fermentation time 70~75h.

6. The application according to claim 4, characterized in that, The method for crude extraction of cyclic lipopeptides from the fermentation broth includes: centrifuging the fermentation broth to remove microbial cells, adjusting the pH of the cell-free filtrate to 1.9-2.2 with 5.8-6.4 mol / L hydrochloric acid solution, and allowing it to stand at 4±2℃ for 12-16 h; centrifuging to recover the precipitate; washing the collected precipitate with acidified water at pH 1.9-2.2 and air-drying it, extracting the crude cyclic lipopeptides from the precipitate with anhydrous methanol, and removing the solvent.

7. The application according to claim 4, characterized in that, The method for purifying the cyclic lipopeptide includes purification using a macroporous resin.

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