Bacillus velezensis for producing broad-spectrum antibacterial substances and application of bacillus velezensis

Abstract

The invention provides bacillus velezensis H5 capable of producing broad-spectrum antibacterial substances and application of the bacillus velezensis H5. The preservation number of the bacillus velezensis H5 in the China General Microbiological Culture Collection Center is CGMCC No.32671. The bacillus velezensis H5 is separated from sea mud in a specific sea area, can generate antibacterial substances with broad-spectrum antibacterial activity, and mainly comprises bacitracin, surfactin and mycostatin. The antibacterial substance of the bacillus velezensis H5 is applied to preservation of perishable fruits such as grapes and the like and mildew prevention of cereals such as corn and the like, and can effectively inhibit spoilage of the perishable fruits and synthesis of mycotoxins such as aflatoxin and the like, and the action effect of the antibacterial substance is obviously superior to that of a food preservative natamycin. The invention provides a novel, green, safe and broad-spectrum antibacterial substance for preservation and fresh-keeping of fruits and mildew prevention of grains, and has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of food microbiology, and particularly relates to a Bacillus velezensis strain producing broad-spectrum antibacterial substances and its application. Background Art

[0002] Fruits such as grapes, strawberries, cherry tomatoes, and cherries are rich in nutrients, providing good nutritional conditions for the growth and reproduction of many natural microorganisms. Therefore, such berry fruits are extremely prone to spoilage and deterioration due to the growth and metabolic activities of microorganisms during storage, showing adverse changes such as color change, taste change, and surface mildew. These dominant flora mainly include the genera of molds, yeasts, Cladosporium, Gram-positive (G + ) and Gram-negative (G – ) etc. During the storage of grains, grains such as corn, peanuts, wheat, and rice are easily infected by filamentous fungi such as Aspergillus flavus, Fusarium graminearum, and Fusarium oxysporum, especially in an environment where humidity and temperature are not properly controlled. These fungi can metabolize and produce a series of mycotoxins, including but not limited to aflatoxin (a class I carcinogen), vomitoxin, and zearalenone, posing a serious threat to grain quality and human health.

[0003] A variety of Bacillus strains such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus brevis, and Bacillus polymyxa can produce extracellular antibacterial peptides, which have good antibacterial effects on common foodborne pathogenic and spoilage bacteria. Currently, domestic and foreign research shows that Bacillus-derived AMPs such as Fengycins, Surfactins, Iturins, and Lichenysins, etc., exhibit significant antibacterial activities against various pathogenic bacteria and fungi, especially G + bacteria and filamentous fungi, and have broad development potential and application prospects. Bacillus antibacterial peptides have the characteristics of wide source, strong antibacterial activity, and high safety, and are a type of green biogenic food antibacterial agent with broad development prospects. They can be applied to the anti-corrosion and preservation of perishable fruits such as grapes, strawberries, cherry tomatoes, and cherries; at the same time, they can also be used to inhibit the growth of filamentous fungi and the biosynthesis of mycotoxins during the storage of grains and oilseeds such as corn, peanuts, wheat, and rice.

[0004] Research has found that most Bacillus antibacterial peptides have antibacterial activities against G + bacteria, and only a few antibacterial peptides can inhibit G –Bacteria. In the research reports on the antibacterial mechanism of Bacillus - derived AMP, the membrane damage mechanism has always been the mainstream theory. When AMP exerts its antibacterial effect, the cell lipid membrane is the first barrier it contacts. Therefore, most AMPs target the cell lipid membrane. Due to electrostatic interaction, cationic antimicrobial peptides are more likely to aggregate on the surface of the lipid membrane of negatively charged phospholipid molecules, but have a weaker binding ability to neutral phospholipids. Therefore, cationic antimicrobial peptides (such as surfactin Surfactins) can selectively bind to the phospholipid molecules of bacterial cell membranes containing at least 15% negative charges, selectively kill bacteria, and do not produce cytotoxicity to normal mammalian cells. The Shai - Matsuzaki - Huang model can be used to explain how most AMPs interact with the membrane. Free AMP has no stable secondary structure in solution. After interacting with the membrane, it can form an amphiphilic secondary structure (such as α - helix, β - sheet). The positively charged side can directly interact with the lipid groups of the cell membrane, thus integrating into the outer half of the membrane, resulting in the thinning of the outer leaf of the cell membrane. Subsequently, AMP can form ion channels on the cell membrane or cause cell lysis. Based on this, researchers have successively proposed various models of the action mechanism of AMP, such as the barrel - stave model, the carpet model, the toroidal pore model, and the micelle aggregation channel model. In addition, due to the differences in the structure and functional groups of AMP, its action mechanism on bacterial cells is also different. For example, mersacidin inhibits the transglycosylation step of cell wall biosynthesis by binding to lipid II, thus achieving antibacterial effects; gramicidin A can embed in the bacterial cell membrane to form channels, increasing the permeability of the bacterial cell membrane, being more permeable to ions and small molecules, thus disrupting the ion balance inside and outside the cell until the bacteria are killed; tridecaptin A1 exerts its bactericidal effect by binding to the precursor lipid II of the bacterial cell wall and destroying the proton motive force of the bacteria.

[0005] In addition, the main substance used for inhibiting food - borne fungi in China at present is natamycin. However, natamycin only has antibacterial activity against fungi and has no antibacterial effect on G + and G – bacteria, which limits its application in food preservation. Given the country's high attention to food safety and the food safety risks of chemically synthesized preservatives, it is of great significance to develop new, green, safe, and broad - spectrum antibacterial peptides for fruit preservation, inhibiting the growth of microorganisms and the contamination of food - borne pathogenic bacteria. Summary of the Invention

[0006] The purpose of the present invention is to provide a Bacillus velezensis producing broad - spectrum antibacterial substances and its application in view of the deficiencies of the prior art.

[0007] To achieve the above - mentioned purpose, the present invention adopts the following technical solutions: The first aspect of the present invention provides a Bacillus velezensis H5 that produces broad-spectrum antibacterial substances. The Bacillus velezensis H5 was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024. It is classified and named as Bacillus velezensis, with a deposit number of CGMCC No. 32671, and the deposit address is No. 3, Courtyard 1, Beizhan West Road, Chaoyang District, Beijing.

[0008] The second aspect of the present invention provides the application of the above-mentioned Bacillus velezensis H5 in the preparation of fruit preservatives and / or grain mold inhibitors and / or broad-spectrum antibacterial agents.

[0009] The third aspect of the present invention provides a product for fruit preservation and / or grain mold prevention and / or broad-spectrum antibacterial. The product uses Bacillus velezensis H5 and / or the supernatant of the fermentation broth of Bacillus velezensis H5 and / or the crude extract of the antibacterial substance of Bacillus velezensis H5 as the active ingredient; the Bacillus velezensis H5 was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024. It is classified and named as Bacillus velezensis, with a deposit number of CGMCC No. 32671, and the deposit address is No. 3, Courtyard 1, Beizhan West Road, Chaoyang District, Beijing.

[0010] Furthermore, the preparation method of the supernatant of the fermentation broth of the above-mentioned Bacillus velezensis H5 is as follows: inoculate Bacillus velezensis H5 into LB liquid medium for cultivation, centrifuge, discard the precipitate, and take the supernatant to obtain the supernatant of the fermentation broth of Bacillus velezensis H5.

[0011] Furthermore, the preparation method of the crude extract of the antibacterial substance of the above-mentioned Bacillus velezensis H5 is as follows: inoculate Bacillus velezensis H5 into LB liquid medium for cultivation, centrifuge, discard the precipitate, and take the supernatant to obtain the supernatant of the fermentation broth of Bacillus velezensis H5; adjust the pH value of the supernatant of the fermentation broth of Bacillus velezensis H5 to 2, and let it stand at 4°C for acid precipitation for 12 h; after the acid precipitation is completed, centrifuge, discard the supernatant, take the precipitate, and then dissolve the precipitate in methanol to obtain a precipitate solution; centrifuge the precipitate solution, discard the precipitate, take the supernatant, and then adjust the pH value of the supernatant to 7. The obtained sample is separated and purified by Sephadex LH-20 column chromatography, and the eluate with antibacterial activity is collected and combined, and concentrated by rotary evaporation to obtain the crude extract of the antibacterial substance of Bacillus velezensis H5.

[0012] Furthermore, the mobile phase of the above-mentioned Sephadex LH-20 column chromatography is 80% methanol, and the flow rate of the mobile phase is 0.3 mL / min.

[0013] The fourth aspect of the present invention provides the application of the above-mentioned product in the preparation of fruit preservatives and / or grain mold inhibitors and / or broad-spectrum antibacterial agents.

[0014] The remarkable advantages of the present invention are as follows: The present invention screened and obtained a Bacillus velezensis H5 that produces broad-spectrum antibacterial peptides, which has a significant inhibitory effect on a variety of common fungi and bacteria. The antibacterial substance produced by Bacillus velezensis H5 is applied to the preservation and anti-mildew of fruits such as grapes and grains such as corn, and can effectively inhibit the spoilage of perishable fruits and the synthesis of mycotoxins such as aflatoxin. Its effect is significantly better than the food preservative natamycin used to inhibit fungi in the prior art. Brief Description of the Drawings

[0015] Figure 1 : Colony morphology and cell morphology of Bacillus velezensis H5.

[0016] Figure 2 : Phylogenetic tree of Bacillus velezensis H5 strain.

[0017] Figure 3 : Bacteriostatic effect of the supernatant of Bacillus velezensis H5 fermentation broth.

[0018] Figure 4 : Bacteriostatic activity of different fractions separated by Sephadex LH-20 column chromatography of the supernatant of Bacillus velezensis H5 fermentation broth against Bacillus cereus.

[0019] Figure 5 : Bacteriostatic activity of different fractions separated by RP-HPLC of the crude extract of antibacterial substance of Bacillus velezensis H5 against Bacillus cereus.

[0020] Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 : TOF / MS and MS / MS spectra of the crude extract of antibacterial substance of Bacillus velezensis H5.

[0021] Figure 13 : Effect of the crude extract of antibacterial substance of Bacillus velezensis H5 on grape preservation.

[0022] Figure 14 : Application of the crude extract of antibacterial substance of Bacillus velezensis H5 in corn anti-mildew.

[0023] Figure 15 : Inhibitory effect of the crude extract of antibacterial substance of Bacillus velezensis H5 on the synthesis of aflatoxin B1 during corn storage. Detailed implementation manners

[0024] To make the content of the present invention more understandable, the technical solutions of the present invention will be further described below in conjunction with specific implementation manners. However, the following examples are only examples of the present invention and do not represent the scope of the claimed protection of the present invention. The scope of the claimed protection of the present invention shall be subject to the claims.

[0025] Example 1: Bacillus velezensis H5 in the present invention was isolated from the marine mud at a depth of 0.5 m in the coastal waters of Touch Beach, Tangdong Village, Jinjing Town, Jinjiang City, Fujian Province. The specific isolation method is as follows: Take 10 g of marine mud and resuspend it with sterile physiological saline. After 10-fold serial dilution, spread it on an LB solid medium plate and statically culture it at 30 °C for 24 h; pick a single colony and place it in an LB liquid medium, and statically culture it at 30 °C for 72 h; boil it in water at 85 °C for 40 min, then re-spot the bacterial solution on an LB solid medium and culture it for 24 h; pick a single colony and place it in an LB liquid medium, and statically culture it at 30 °C for 36 h, centrifuge at 10000 g / min for 1 min, discard the supernatant, collect the bacterial cell precipitate, use a bacterial total DNA extraction kit, extract the total DNA according to the instruction manual, and perform PCR amplification of the 16S rRNA gene using the primers 5’-AGTTTGATCMTGGCTCAG-3’ and 5’-GGTTACCTTGTTACGACTT-3’. Take 5 μL of the PCR product for 1% agarose gel electrophoresis, purify and recover the PCR product with a kit, and perform 16S rRNA gene sequencing by Sangon Biotech (Shanghai) Co., Ltd. The sequencing result of the 16S rDNA gene sequence of strain H5 is shown in SEQ ID NO.1. After homology comparison and constructing a phylogenetic tree of strain H5 using MEGA X 10.1 Figure 1 )

[0026] Inoculate strain H5 into an LB liquid medium and activate and transfer it to the logarithmic growth phase at 30 °C. Take 1 - 2 mL of the logarithmic growth phase culture solution of strain H5 and centrifuge it at 10000 g / min for 1 min, discard the supernatant, collect the bacterial cell precipitate, use a bacterial total DNA extraction kit, extract the total DNA according to the instruction manual, and perform PCR amplification of the 16S rRNA gene using the primers 5’-AGTTTGATCMTGGCTCAG-3’ and 5’-GGTTACCTTGTTACGACTT-3’. Take 5 μL of the PCR product for 1% agarose gel electrophoresis, purify and recover the PCR product with a kit, and perform 16S rRNA gene sequencing by Sangon Biotech (Shanghai) Co., Ltd. The sequencing result of the 16S rDNA gene sequence of strain H5 is shown in SEQ ID NO.1. After homology comparison and constructing a phylogenetic tree of strain H5 using MEGA X 10.1 Figure 2 ), the result shows that strain H5 is Bacillus velezensis.

[0027] Biological material preservation information: Bacillus velezensis H5 was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 18, 2024, with the preservation number CGMCC No. 32671 and the preservation address at No. 3, Courtyard 1, Beizhan West Road, Chaoyang District, Beijing.

[0028] Example 2: Inoculate a single colony of Bacillus velezensis H5 into 50 mL of LB liquid medium, and perform shake flask fermentation culture at 37 °C and 180 rpm for 36 h. Centrifuge (at 4 °C, 10000 rpm, for 10 min), discard the precipitate, and take the supernatant to obtain the supernatant of the fermentation broth of Bacillus velezensis H5. Using Staphylococcus aureus, Escherichia coli, Enterohemorrhagic Escherichia coli, Methicillin-resistant Staphylococcus aureus, Cronobacter sakazakii, Listeria monocytogenes, Bacillus pumilus, Vibrio parahaemolyticus, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexneri, Proteus mirabilis, Pseudomonas fluorescens, Salmonella typhimurium, Micrococcus luteus, and Saccharomyces cerevisiae as indicator bacteria, the antibacterial effect of the supernatant of the fermentation broth of Bacillus velezensis H5 on the indicator bacteria was evaluated by the punching antibacterial method. Using Aspergillus flavus as the indicator bacteria, the antibacterial effect of the supernatant of the fermentation broth of Bacillus velezensis H5 on the indicator bacteria was evaluated by the plate confrontation method. The results showed ( Figure 3 ), the supernatant of the fermentation broth of Bacillus velezensis H5 had antibacterial activity against the above-mentioned indicator bacteria, indicating that the supernatant of the fermentation broth of Bacillus velezensis H5 contained substances with broad-spectrum antibacterial activity.

[0029] Example 3: Inoculate a single colony of Bacillus velezensis H5 into 50 mL of LB liquid medium, and perform static fermentation culture at 37 °C for 24 h. After the culture, centrifuge (at 4 °C, 10000 rpm, for 10 min), discard the precipitate, and take the supernatant to obtain the supernatant of the fermentation broth of Bacillus velezensis H5; slowly add concentrated hydrochloric acid to the obtained supernatant of the fermentation broth of Bacillus velezensis H5 and stir evenly until the pH value is adjusted to 2, then let it stand for acid precipitation at 4 °C for 12 h; after acid precipitation, centrifuge (at 4 °C, 10000 rpm, for 10 min), discard the supernatant, take the precipitate, then add 5 mL of methanol to the precipitate and stir evenly to fully dissolve it to obtain a precipitate solution; centrifuge the obtained precipitate solution (at 4 °C, 10000 rpm, for 10 min), discard the precipitate, take the supernatant, and then adjust the pH value of the supernatant to 7 with NaOH solution. The obtained sample was stored refrigerated at 4 °C for subsequent separation and purification of antibacterial active substances. The separation and purification steps are as follows: Take 50 g of Sephadex LH-20 dry powder and swell it in 65% ethanol by volume for 24 h, then pack it into a chromatography column with an inner diameter of 10 mm and a length of 100 cm; use ultrapure water as the mobile phase, set the flow rate at 0.3 mL / min, turn on the peristaltic pump, and equilibrate for 2 - 3 column volumes; after the sample passes through a 0.45 μm filter head, carefully and slowly load 2 mL of the sample along the column wall, use 80% chromatographic grade methanol by volume as the mobile phase, and perform isocratic elution at a flow rate of 0.3 mL / min, collecting the eluate every 5 min. Using Bacillus cereus as the indicator bacterium, detect the antibacterial activity of each tube of eluate. The results show ( Figure 4 ), the eluate from the 10th to 20th tubes has antibacterial activity against the indicator bacterium Bacillus cereus. Combine the eluate from the 10th to 20th tubes, and under the conditions of a heating bath temperature of 50 °C, a vacuum of -0.01 MPa, and a cooling medium temperature of 4 °C, perform rotary evaporation and concentration to one-half of the original volume to obtain a crude extract of the antibacterial substance of Bacillus velezensis H5, then freeze-dry it and store it at 4 °C for standby.

[0030] Example 4: Take 1 mL of the crude extract of the antibacterial substance of Bacillus velezensis H5 and filter it through a 0.22 μm filter head, and further separate it by RP-HPLC. Mobile phase A is ultrapure water, and mobile phase B is chromatographic grade acetonitrile. The gradient elution conditions are as follows: 0 - 5 min, 60% - 60.5% acetonitrile; 5 - 20 min, 60.5% - 60% acetonitrile; the flow rate is 0.8 mL / min; the injection volume is 10 μL; the column temperature is 40 °C. Collect each elution peak, use Bacillus cereus as the test bacterium, and measure the antibacterial activity of each active peak by the agar diffusion method. The results show ( Figure 5 ), the crude extract of the antibacterial substance of Bacillus velezensis H5 is further separated by reverse-phase HPLC to obtain different components, and the separated components corresponding to the nine peaks all have antibacterial activity.

[0031] Example 5: Collect Figure 5 the RP-HPLC active peaks 1, 5, and 9, and perform first-order mass spectrometry characterization using TOF / MS. Find antibacterial peptides with the same molecular weight as the antibacterial peptides from Bacillus in the first-order mass spectrometry, and then verify them using MS / MS. Perform mass spectrometry analysis on the antibacterial substance and analyze its molecular structure. The results are as Figures 6 - 12 shown.

[0032] The surfactin family consists of different surfactin subtypes, which are composed of a cyclic heptapeptide (the hydrophilic part of the molecule) and a β-hydroxy fatty acid side chain (constituting the hydrophobic part). The cyclic peptide chain of surfactin is composed of the following amino acid sequence: Glu-Leu-Leu-Val-Asp-Leu-Leu, with one or more amino acids replaced according to different subtypes.

[0033] The MS / MS fragment at m / z 685.4 [M+H]+ is characteristic of surfactin A, while m / z 653.3 is characteristic of surfactin B and m / z 681.3 is characteristic of [Leu4] surfactin. The LC-MS / MS spectrum of the precursor ion [M+H] at m / z 994 corresponds to surfactin containing a C12 β-hydroxy fatty acid chain. As shown, the b1–b6 and y1–y6 as well as b”’1–b”’6 and y”’1–y”’6 fragment ions represent surfactin A, the sequence of which is shown as such, while the series of b’1–b’6 and y’1–y’6 fragment ions represent surfactin B, the sequence of which is C12β-OH FA-Cyclic[E-I / L-I / L-V-D-L / I-V]. And the series of b”1–b”6 and y”1–y”6 fragment ions represent [Leu4] surfactin, the sequence of which is C12β-OH FA-Cyclic[E-I / L-I / L-L / I-D-L / I-I / L]. The fragments at m / z 895.4, 881.5, 784.4, 685.4, 654.4, etc. correspond to the loss of amino acid residues. The same is true for the LC-MS / MS spectra of [M+H]+ at m / z 1008, [M+H]+ m / z 1022, and [M+H]+ m / z 1036. Taking the precursor ion m / z 1022 as an example, fragment ions appeared at a retention time of 8.05 minutes, and a series of b and y ions could be easily assigned. This means that the protonated ester bond was initially cleaved. The series of b fragments of m / z 1022, 909, 796, 681, 568, 455, are consistent with the loss of Leu / Tle-Leu / Tle-Asp-Leu / Ile-Leu / Ile-Leu / Ile from the C-terminus. In addition, the second set of typical y fragment ions contains the peptide molecule within the C-terminal product ion. The m / z values of the C-terminal product ions 554, 441, 328 show the loss of the hydroxy fatty acid chain-Glu-Leu / Ile-Leu / Ile in the precursor ion. Based on these typical fragment ion fragments, the sequence can be deduced as Cyβ-hydroxy fatty acid chain-E-L / I-L / I-L / I-D-L / I-L / I.

[0034] Bacillomycin D, bacillomycin D, and mycosubtilin are all subtypes of Iturin C. When the cyclic peptide opens at the proline residue, proline occupies the position of AA#4 in bacillomycin D. Bacillomycin L lacks proline; therefore, it has a more uniform fragmentation pattern on β-amino acids.

[0035] When the peptide bond is cleaved at proline, a series of y - fragments are found at m / z 934.5, m / z 805.5, m / z 392.0, m / z 278.0, and m / z 115.0. The b - series fragments also correspond to the relevant amino acids. These findings confirm that the compound with m / z of 1031 is a bacillomycin D homolog. Its sequence is P - E - S - T - BAA - N - Y - N.

[0036] The interpretation of the MS / MS spectra obtained for the other detected ions can be carried out in a similar manner. Thus, for the parent ions at m / z 1021 and m / z 1043, the observed main peaks are assigned as described in the figure. Based on those typical fragments (diagnostic fragments), the sequence of m / z 1021 can be inferred to be S - E - S - T - BAA - N - Y - N. It is designated as a bacillomycin L homolog. The sequence of m / z 1043 can be inferred to be P - S - N - BAA - N - Y - N - Q. It is designated as a mycosubtilin homolog.

[0037] Example 6: The preparation method of the crude extract of the antibacterial substance of Bacillus velezensis H5 is the same as that in Example 3.

[0038] Weigh 500 g of fresh grapes and place them in a sterile sealed bag. Set different experimental groups, and add 500 μg / g of the crude extract of the antibacterial substance of Bacillus velezensis H5 or 500 μg / g of natamycin to different experimental groups according to the grape weight. After mixing evenly, seal the bag and store it at room temperature. At the same time, set the treatment with sterile normal saline as the blank control group, and sample at 1, 3, 5, 7, and 9 days respectively; after sampling, weigh 8.5 - 10 g of the grapes, add sterile normal saline at a ratio of 1:10, homogenize and mix, then dilute by gradient, spread on LB solid medium, and culture at 30 °C for 18 h and then perform colony counting. As Figure 13 shown, after treatment with the crude extract of the antibacterial substance of Bacillus velezensis H5 for 1 - 9 days, the spoilage process of the grapes is inhibited, and no spoilage occurs on the 9th day, maintaining good freshness; in the blank control group and the natamycin treatment group, the spoilage degree of the grapes shows a gradually increasing trend with the extension of the storage time. It shows that the crude extract of the antibacterial substance of Bacillus velezensis H5 can effectively inhibit the spoilage of grapes and extend their shelf life.

[0039] Example 7: Take 3 × 200 g of dry corn kernels, weigh them and place them in a sterile sealed bag. According to the weight of the corn, add the crude extract of the antibacterial substance of Bacillus velezensis H5 at 500 μg / g, or add natamycin at 500 μg / g. After mixing evenly, seal it and store it at room temperature. At the same time, set up a sterile saline treatment as a blank control group, and take samples at 0, 5, 9, 15, 20, and 30 days respectively; after sampling the corn, weigh 30 g, observe the number of mildews, and break the corn to prepare for measuring the toxin content. The results show ( Figure 14 ), the crude extract of the antibacterial substance of Bacillus velezensis H5 can effectively inhibit the mildew of corn and extend its shelf life; different degrees of mold growth can be seen in the blank control group and the natamycin treatment group at different storage periods. The content of aflatoxin B1 measured by an enzyme-linked immunosorbent assay kit shows ( Figure 15 ), the number of mildews and the toxin content of the corn treated with the crude extract of the antibacterial substance of Bacillus velezensis H5 are significantly lower than those of the saline and natamycin treatment groups, indicating that the crude extract of the antibacterial substance of Bacillus velezensis H5 can effectively inhibit the synthesis and accumulation of aflatoxin B1 in corn.

Claims

1. A Bacillus Velezii H5 producing a broad-spectrum antibacterial substance, characterized in that: The Velez subsp. velez H5 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 18, 2024, and was classified and named Velez subsp. velez Bacillus velezensis ), the deposit number is CGMCC No.32671, and the deposit address is No. 3, Yard 1, Beizhan West Road, Chaoyang District, Beijing.

2. Use of the Bacillus Velez H5 as claimed in claim 1 in the preparation of fruit preservatives and / or grain mildew preventives and / or broad-spectrum antibacterial agents.

3. A product for fruit preservation and freshness preservation and / or grain mildew prevention and / or broad-spectrum antibacterial, characterized in that: The product uses Bacillus Velez H5 and / or the supernatant of Bacillus Velez H5 fermentation broth and / or the crude extract of antibacterial substances of Bacillus Velez H5 as active ingredients; the Bacillus Velez H5 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 18, 2024, and is classified and named Bacillus Velez ( Bacillus velezensis ), the deposit number is CGMCC No.32671, and the deposit address is No. 3, Yard 1, Beizhan West Road, Chaoyang District, Beijing.

4. The product according to claim 3, characterized in that: The preparation method of the supernatant of the fermentation liquid of Bacillus Velez H5 is as follows: inoculating Bacillus Velez H5 into LB liquid culture medium for cultivation, centrifuging, discarding the precipitate, taking the supernatant, and obtaining the supernatant of the fermentation liquid of Bacillus Velez H5.

5. The product according to claim 3, characterized in that: The preparation method of the crude extract of the antibacterial substance of Bacillus Velez subtilis H5 is as follows: inoculating Bacillus Velez subtilis H5 into LB liquid culture medium for cultivation, centrifuging, discarding the precipitate, taking the supernatant, and obtaining the fermentation liquid supernatant of Bacillus Velez subtilis H5; adjusting the pH value of the fermentation liquid supernatant of Bacillus Velez subtilis H5 to 2, and allowing the fermentation liquid to settle at 4°C for 12 hours; centrifuging after the acid precipitation, discarding the supernatant, taking the precipitate, and then dissolving the precipitate in methanol to obtain a precipitate solution; centrifuging the precipitate solution, discarding the precipitate, taking the supernatant, and then adjusting the pH value of the supernatant to 7, separating and purifying the obtained sample through Sephadex LH-20 column chromatography, collecting and merging the eluate with antibacterial activity, and concentrating by rotary evaporation to obtain the crude extract of the antibacterial substance of Bacillus Velez subtilis H5.

6. The product according to claim 5, characterized in that: The mobile phase of the Sephadex LH-20 column chromatography is 80% methanol, and the flow rate of the mobile phase is 0.3 mL / min.

7. Use of the product as claimed in claim 3 in the preparation of fruit preservatives and / or grain mildew preventives and / or broad-spectrum antibacterial agents.

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