Bacillus velezensis SE1318 and application thereof

Abstract

The invention provides bacillus velezensis SE1318 and application thereof, and belongs to the technical field of microorganisms. The bacillus velezensis SE1318 provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the bacillus velezensis SE1318 is CGMCC No. 35354. The strain has a remarkable inhibition effect on various common food-borne pathogenic bacteria such as staphylococcus aureus, listeria monocytogenes, escherichia coli, salmonella and botrytis cinerea.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a bacillus velezensis SE1318 and application thereof. BACKGROUND

[0002] During production and processing, and during storage and transportation before consumption, food spoilage bacteria and toxin-producing fungi can contaminate food and cause food spoilage or cause infection and disease. Food and agricultural product contamination caused by some foodborne pathogens such as Staphylococcus aureus, Salmonella, Listeria monocytogenes, Escherichia coli, and Botrytis cinerea, which can resist inactivation and survive in strict processing during food processing, is one of the key threats to public health. At present, chemical preservatives are still the main measure to prevent food deterioration and prolong shelf life, such as potassium sorbate, benzoic acid, sulfur dioxide, etc., but the frequent use of these chemical preservatives often leads to drug residues, toxicological risks, and environmental pollution.

[0003] In this study, a bacillus velezensis strain with antagonistic effect on common foodborne pathogens was isolated, and its morphology, physiology, biochemistry, and antibacterial active substances were studied. The strain and its fermentation supernatant have good inhibitory effect on common foodborne pathogens, and are expected to be developed as new feed, food preservatives, and biocontrol bacteria. SUMMARY

[0004] The present application aims to provide a bacillus velezensis SE1318 which can significantly inhibit a plurality of foodborne pathogens and spoilage fungi.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a bacillus velezensis (Bacillus velezensis) SE1318. Bacillus velezensis The bacillus velezensis SE1318 is deposited with the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on July 23, 2025, and has a deposit number of CGMCC No. 35354.

[0006] The present application also provides a microbial agent comprising the bacillus velezensis SE1318, and at least one of its culture, fermentation broth, or metabolite.

[0007] The present application also provides an application of the microbial agent in inhibiting pathogenic bacteria.

[0008] Preferably, the pathogenic bacteria include at least one of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella, and Botrytis cinerea.

[0009] The application also provides the application of the Bacillus velezensis SE1318 in producing enzymes.

[0010] Preferably, the enzyme is one or more of protease, amylase, cellulase or chitinase.

[0011] The application also provides the application of the Bacillus velezensis SE1318 in producing siderophores.

[0012] Beneficial effects

[0013] The application screens a Bacillus velezensis SE1318 with broad-spectrum antibacterial activity, which has a significant inhibitory effect on a plurality of common foodborne pathogenic bacteria (such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella, Botrytis cinerea, etc.), effectively reduces the application of chemical preservatives, reduces the risk of drug residues and environmental pollution, and meets the requirements of green and sustainable development. It can also synthesize various enzymes used in industrial and agricultural fields, such as protease, amylase, cellulase and chitinase, which can degrade different types of biological macromolecules. It can also synthesize siderophores, which can inhibit the growth of iron-dependent pathogenic bacteria on the one hand and assist the host such as plants in absorbing iron elements on the other hand, providing a basis for the development of agricultural biocontrol or biological fertilizers. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A phylogenetic tree of the Bacillus velezensis SE1318 in Example 1; Figure 2 An electrophoresis map of the 16S rRNA gene amplification product of the Bacillus velezensis SE1318 in Example 1; Figure 3 Physiological and biochemical activity detection results of the Bacillus velezensis SE1318 in Example 1; Figure 4 Antibacterial kinetics curve of the fermentation supernatant of the Bacillus velezensis SE1318 in Example 1; Figure 5 Inhibition result graph of the Bacillus velezensis SE1318 on Botrytis cinerea H405 in Example 1; Figure 6 Influence result graph of different temperatures on the antibacterial activity of the strain in Example 2; Figure 7 Influence result graph of different pH values on the antibacterial activity in Example 2; Figure 8 Influence result graph of different proteases on the antibacterial activity in Example 2; Figure 9 Influence result graph of different metal ions on the antibacterial activity in Example 2; Figure 10Figure for the results of the SE1318 cell population motility ability in Example 3; Figure 11 Micrograph for the SE1318 biofilm formation ability in Example 3.

[0015] Deposit certificate

[0016] Bacillus velezensis (Bacillus velezensis) Bacillus velezensis SE1318, which is deposited with the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on July 23, 2025, and has the deposit number of CGMCC No. 35354. DETAILED DESCRIPTION

[0017] The technical solutions provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0018] Example 1

[0019] 1. Preparation of Bacillus velezensis SE1318 seed liquid and fermentation broth

[0020] Bacillus velezensis SE1318 was cultured in NB (Nutrition Broth) medium (10.0 g / L of proteose peptone, 5.0 g / L of yeast extract powder, and 10.0 g / L of sodium chloride) at 37°C and 180 rpm for 12 h to obtain a seed liquid. 2% of the seed liquid was inoculated into NB medium, and the medium was cultured at 37°C and 180 rpm for 48 h to obtain a Bacillus velezensis SE1318 fermentation broth. The fermentation broth was centrifuged at 5000 rpm for 5 min at room temperature to obtain a fermentation supernatant.

[0021] 2. Bacillus velezensis SE1318 16S rRNA gene sequencing

[0022] The Bacillus velezensis SE1318 fermentation broth prepared above was centrifuged at 5000 rpm for 5 min at room temperature, and the supernatant was discarded. DNA was extracted, and PCR amplification was performed using bacterial 16S rRNA gene universal primers: 27F (SEQ ID NO. 1: 5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (SEQ ID NO. 2: 5'-TACGGYTACCTTGTTACGACTT-3'). The amplified sequence (the 16S rRNA sequence is shown as SEQ ID NO. 3) was sent to the Bioengineering Company for sequencing. The sequencing results were uploaded to the NCBI website to query the related strains, and a strain phylogenetic tree was constructed using MEGA11 (the results are shown as Figure 1

[0023] ​SEQ ID NO. 3: 5 μl of the PCR amplification product was taken, mixed with 1 μl of loading buffer, and spotted on a 2% agarose gel. After electrophoresis at 100 V for 60 min, the amplification product was detected (the result is shown in Fig. 1, wherein M is a DNA marker, and 1 is the 16S rRNA gene amplification product of Bacillus velezensis SE1318). Figure 2

[0024] 3. Exploration of physiological and biochemical activities of Bacillus velezensis SE1318

[0025] ​Bacillus velezensis SE1318 fermentation broth was prepared according to 1. The strain's protease production ability was detected using casein medium, a sterile Oxford cup was placed in the center of the plate, 200 μL of Bacillus velezensis SE1318 fermentation broth was added to the Oxford cup, and after 48 h of incubation at 37°C, observation was made. If a transparent circle appeared, it indicated that Bacillus velezensis SE1318 produced proteolytic enzymes. The strain's amylase production ability was detected using soluble starch-containing nutrient broth medium, a sterile Oxford cup was placed in the center of the plate, 200 μL of Bacillus velezensis SE1318 fermentation broth was added to the Oxford cup, and after 48 h of incubation at 37°C, observation was made. If a transparent circle appeared, it indicated that Bacillus velezensis SE1318 produced amylolytic enzymes. The strain's phosphate solubilizing ability was detected using inorganic phosphate solid medium (Pikovskaya's agar), a sterile Oxford cup was placed in the center of the plate, 200 μL of Bacillus velezensis SE1318 fermentation broth was added to the Oxford cup, and after 48 h of incubation at 37°C, observation was made. If a transparent circle appeared, it indicated that Bacillus velezensis SE1318 could solubilize phosphate. The strain's cellulase production ability was detected using cellulose Congo red medium, a sterile Oxford cup was placed in the center of the plate, 200 μL of Bacillus velezensis SE1318 fermentation broth was added to the Oxford cup, and after 48 h of incubation at 37°C, observation was made. After incubating the plate with 1 mg / mL Congo red for 15 min, the Congo red was discarded, and 1 M NaCl solution was used to remove unbound Congo red solution. If a transparent circle appeared, it indicated that Bacillus velezensis SE1318 could decompose cellulose. The strain's urease production ability was detected using agar medium containing 20% urea, a sterile Oxford cup was placed in the center of the plate, 200 μL of Bacillus velezensis SE1318 fermentation broth was added to the Oxford cup, and after 48 h of incubation at 37°C, observation was made. Negative results were observed for 4 days. If the medium was pink, it was urease positive, and if the color of the medium did not change, it was negative. The strain's siderophore production ability was detected using CAS medium, a sterile Oxford cup was placed in the center of the plate, 200 μL of Bacillus velezensis SE1318 fermentation broth was added to the Oxford cup, and after 48 h of incubation at 37°C, observation was made. If an orange halo formed around the Oxford cup, it indicated that Bacillus velezensis SE1318 had siderophore production ability. The strain's chitin degradation ability was detected using medium containing 0.45% chitin, a sterile Oxford cup was placed in the center of the plate, 200 μL of Bacillus velezensis SE1318 fermentation broth was added to the Oxford cup, and after 48 h of incubation at 37°C, observation was made. If a halo formed around the Oxford cup, it indicated chitin degradation. The results are shown in a-g. Figure 3

[0026] Figure 3 ​​It can be seen that after adding SE1318 fermentation broth, obvious transparent circles appeared on casein plate (a), amylase detection plate (b), cellulose Congo red plate (d) and chitinase (g) detection plate, indicating that Bacillus velezensis SE1318 produced protease, amylase, cellulase and chitinase in the plate, which decomposed protein, starch, cellulose and chitin in the culture medium to produce degradation circles; On phosphate plate (c) and urease detection plate (e), no degradation circle appeared around the Oxford cup of Bacillus velezensis SE1318, indicating that SE1318 did not produce organic acid and urease in the plate; On siderophore detection medium (f), an orange light ring was formed around the Oxford cup of SE1318 fermentation broth, indicating that SE1318 could produce siderophore.

[0027] 4. Inhibition spectrum determination of Bacillus velezensis SE1318 fermentation supernatant

[0028] The pathogenic indicator bacteria Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 26003, Staphylococcus aureus ATCC 6538 and Staphylococcus aureus CVCC 1882 were activated by culturing in BPY medium at 37°C for 12 h to obtain seed liquid, which was inoculated into BPY and cultured at 37°C for 20 h, then the bacterial liquid was diluted to 10 7 CFU / mL, and 200 μL was uniformly coated on the BPY plate; Listeria monocytogenes ATCC 19115 was activated by culturing in BHI medium at 37°C for 12 h to obtain seed liquid, which was inoculated into BHI and cultured at 37°C for 20 h, then the bacterial liquid was diluted to 10 7 CFU / mL, and 200 μL was uniformly coated on the BHI plate; Escherichia coli K88, Escherichia coli K99, Escherichia coli O 157 H7, Salmonella ATCC 14028 and Salmonella CVCC 541 were activated by culturing in LB medium at 37°C for 12 h to obtain seed liquid, which was inoculated into LB and cultured at 37°C for 20 h, then the bacterial liquid was diluted to 10 7 CFU / mL, and 200 μL was uniformly coated on the LB plate; Lactobacillus plantarum and Paracasei lactobacillus were activated by culturing in MRS medium at 37°C for 24 h to obtain seed liquid, which was inoculated into MRS and cultured at 37°C for 24 h, then the bacterial liquid was diluted to 10 7CFU / mL, 200 μL of the uniform sample was spread on MRS plate; Botrytis cinerea was inoculated on PDA plate, and cultured at 28°C for 7 days, and then a 5 mm diameter mycelium cake was placed in the center of PDA plate, and a sterile Oxford cup was placed at a distance of 2.5-3 cm from the center of the mycelium cake. The fermentation supernatant of Bacillus velezensis SE1318 was inoculated into the Oxford cup of the above-mentioned plate containing the indicator bacteria, and sterile normal saline was used as a control, and the plates were cultured at 37°C for 24 h (bacteria) and at 28°C for 96 h (Botrytis cinerea), respectively, and the diameter of the inhibition zone was measured. The inhibition rate of Bacillus velezensis SE1318 on Botrytis cinerea was calculated as follows: (diameter of control colony-diameter of treated colony) / diameter of control colony x 100%, and the results are shown in Table 1.

[0029] Table 1: Detection of antibacterial activity of fermentation supernatant of Bacillus velezensis SE1318

[0030] As shown in Table 1, the fermentation supernatant of the strain has obvious antibacterial activity against common pathogenic bacteria and Botrytis cinerea.

[0031] 5. Antibacterial kinetics curve of fermentation supernatant of Bacillus velezensis SE1318

[0032] Bacillus velezensis SE1318 was inoculated in LB liquid medium, and cultured at 37°C with 200 rpm shaking, and samples were taken every 4 h, and the OD 600 nm was measured to determine the absorbance, which represented the biomass of the strain. Meanwhile, the samples taken every 4 h were centrifuged at 5000 rpm and room temperature for 5 min, and the supernatant was detected for antibacterial activity against Salmonella CVCC 541 by the Oxford cup method. The culture time was used as the abscissa, and the OD 600 absorbance and the inhibition zone diameter of the fermentation supernatant against Salmonella CVCC 541 were used as the ordinate, and the antibacterial kinetics curve of the strain was drawn. The results are shown in Figure 4 .

[0033] As shown in Figure 4 , SE1318 entered the logarithmic growth phase at about 4 h, entered the stationary phase after 20 h, and entered the decline phase at 44-48 h. The fermentation supernatant of SE1318 had no antibacterial effect on Salmonella CVCC 541 within 28 h, and the fermentation supernatant showed antibacterial activity after 28 h of growth, and the antibacterial activity increased with the increase of growth time within 28-48 h, indicating that the antibacterial substance produced by the strain was a secondary growth metabolite of the strain.

[0034] 6. Inhibition of Botrytis cinerea by Bacillus velezensis SE1318

[0035] The inhibition effect of Bacillus velezensis SE1318 on Botrytis cinerea was detected by using plate confrontation method with Botrytis cinerea as an indicator strain. A Botrytis cinerea cake was inoculated in the center of a PDA plate, and Bacillus velezensis SE1318 was inoculated in the plate at a distance of 3 cm from the center of the cake. After 5 days of culture at 28°C, the growth of Botrytis cinerea was observed. The results are shown in Figure 5 .

[0036] It can be seen from Figure 5 that the growth of Botrytis cinerea was obviously inhibited by Bacillus velezensis SE1318 on both sides. The growth of mycelium of Botrytis cinerea in the horizontal direction was most inhibited, which was obviously different from the growth of mycelium in the vertical direction, indicating that SE1318 had an inhibitory activity on Botrytis cinerea.

[0037] Example 2 Influence of external environment on the inhibitory activity of strain SE1318

[0038] The SE1318 fermentation supernatant used in the following was prepared in Example 1

[0039] 2.1 Influence of different temperatures on the inhibitory activity

[0040] The inhibitory activity of the fermentation supernatant treated at 30, 40, 50, 60, 70, 80, 90 and 100°C was detected by Oxford cup method, and the results are shown in Figure 6 .

[0041] It can be seen from Figure 6 that the inhibitory activity of the fermentation supernatant of strain SE1318 completely disappeared at 100°C, indicating that the inhibitory activity of the fermentation supernatant of the strain was affected by high temperature.

[0042] 2.2 Influence of different pH on the inhibitory activity

[0043] The pH of the fermentation supernatant of the strain was adjusted to 2.0, 4.0, 6.0, 8.0 and 10.0, respectively. After incubation at room temperature for 1 h, the inhibitory activity was detected by Oxford cup method with the fermentation supernatant of the strain without treatment as a blank control. The results are shown in Figure 7 .

[0044] It can be seen from Figure 7 that different pH had a great influence on the inhibitory activity of strain SE1318. The inhibitory activity was better at pH 6.0-8.0 (neutral), and slightly decreased compared with the control group. The inhibitory activity decreased greatly at pH 2.0, 4.0 and 10.0, indicating that the inhibitory activity was easily affected by acidic and alkaline environments.

[0045] 2.3 Influence of different proteases on the inhibitory activity

[0046] The fermentation broth was treated with 1 mg / mL trypsin (human-derived, the same below), proteinase K, and pepsin, and incubated at 37°C for 1 h. The untreated fermentation supernatant was used as a control. The antibacterial activity of the supernatant was tested, and the results are as follows: Figure 8 As shown.

[0047] Depend on Figure 8 It can be seen that trypsin, proteinase K and pepsin do not affect the antibacterial activity of strain SE1318, indicating that the antibacterial activity of the fermentation supernatant treated with proteases is not significantly affected.

[0048] 2.4 Effects of different metal ions on antibacterial activity

[0049] Add 1 mol / L of K+ containing metal ions to the samples respectively + Na + Mg 2+ Ca 2+ Cu 2+ The antibacterial activity of the supernatant of the strain was tested after standing for 1 h, with untreated fermentation supernatant as a blank control. The results are as follows: Figure 9 As shown.

[0050] Depend on Figure 9 It can be seen that the antibacterial activity of the fermentation supernatant of the strain treated with metal ions was not significantly affected, indicating that the fermentation supernatant of this bacterium has a certain degree of stability against common metal ions and can be applied in a wide range of environments.

[0051] Example 3

[0052] 3.1 Cell clustering and movement ability

[0053] The aggregation and motility of the strains were assessed using semi-solid LB agar medium. The semi-solid LB agar medium consisted of: 10g sodium chloride, 10g tryptone, 5g yeast extract, 5g agar, and 1000ml distilled water. Strain SE1318 was inoculated onto semi-solid LB agar medium and incubated at 37℃ for 48 h, with three replicates.

[0054] When a strain exhibits diffusion during growth on a culture medium, it indicates that the strain possesses the ability to aggregate and move cells. The results are as follows: Figure 10 As shown (the three figures represent the results of three repetitions).

[0055] Depend on Figure 10 It can be seen that when SE1318 was cultured on semi-solid LB agar medium, the central colony of the plate was observed after the culture was completed, and the central colony showed a diffusion phenomenon, indicating that the SE1318 strain has the ability of cell aggregation and movement during the growth process.

[0056] 3.2 Biofilm formation ability

[0057] 0.1% Crystal Violet Solution: 0.1 g crystal violet, 100 mL distilled water.

[0058] PBS buffer solution: 0.08 g sodium chloride, 0.002 g potassium chloride, 0.0144 g disodium hydrogen phosphate, 0.0024 g potassium dihydrogen phosphate, 10 mL distilled water.

[0059] The SE1318 fermentation broth (prepared from Example 1) was diluted to OD. 600 The concentration was 0.1. 100 μL of diluted fermentation broth was added to the center of a glass slide, and the slide was incubated at 37°C for 24 h. After washing with PBS phosphate buffer, 200 μL of 0.1% crystal violet solution was added, and the slide was treated at room temperature for 30 min. The formation of a biofilm on the slide was observed under a 40x objective lens using an optical microscope. Four replicates were performed. Results are shown below. Figure 11 As shown.

[0060] Depend on Figure 11 As shown in the four figures (representing the results of four repeated experiments), observation of the slide under a 40x objective lens using an optical microscope revealed the formation of a biofilm on the slide, indicating that strain SE1318 possesses the ability to form a biofilm.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis SE1318, characterized in that, The Bacillus belyss SE1318 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 23, 2025, with accession number CGMCC No. 35354.

2. A microbial inoculant, characterized in that, It includes at least one of Bacillus belyssus SE1318 as described in claim 1, its culture, fermentation broth, or metabolites.

3. The application of the microbial agent according to claim 2 in inhibiting pathogens.

4. The application as described in claim 3, characterized in that, The pathogens include at least one of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella, and Botrytis cinerea.

5. The use of Bacillus belye SE1318 as described in claim 1 in enzyme production.

6. An application as described in claim 5, characterized in that, The enzyme is one or more of protease, amylase, cellulase or chitinase.

7. The use of Bacillus belye SE1318 as described in claim 1 in a siderogenic carrier.

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