Bacillus velezensis and application thereof in degradation of antibiotics

Abstract

The invention provides bacillus velezensis and an application of the bacillus velezensis in degradation of antibiotics. The bacillus velezensis is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the bacillus velezensis is GDMCC No: 66277. The bacillus velezensis is named as bacillus velezensis. The invention also provides a bacillus velezensis preparation or a bacillus velezensis liquid containing the strain. The invention also provides an application of the bacillus velezensis or the microbial inoculum thereof or the bacterial liquid thereof in degradation of kanamycin antibiotics. The application comprises the following steps: degrading the residual kanamycin antibiotics in soil or sewage, or repairing the soil polluted by the residual kanamycin antibiotics.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus belye and its application in the degradation of antibiotics. Background Technology

[0002] Kanamycin is an aminoglycoside antibiotic that exerts its antibacterial effect primarily by binding to the 30S ribosomal subunit of bacteria, inhibiting protein synthesis. Clinically, it is mainly used to treat infectious diseases caused by susceptible bacteria, such as intestinal infections, urinary tract infections, and respiratory infections. In the animal industry, kanamycin can be used to treat respiratory infections in animals caused by susceptible bacteria, such as swine enzootic pneumonia and chicken pullorum disease; it also has good therapeutic effects on gastrointestinal infections such as enteritis and peritonitis caused by Escherichia coli and Salmonella. In terms of bacterial infection prevention, kanamycin can be used to prevent certain bacterial diseases, such as porcine reproductive and respiratory syndrome (PRRS).

[0003] Kanamycin has a significant damaging effect on the cochlear nerve, leading to symptoms such as hearing loss, tinnitus, and a feeling of fullness in the ear. Kanamycin is also highly toxic to the kidneys, primarily manifesting as acute tubular necrosis; it can also block the neuromuscular junction, causing respiratory muscle paralysis and respiratory depression. Antibiotic residues may affect the safety of animal-derived foods, therefore, strict adherence to withdrawal periods is crucial during use. Its release into the environment during production and use poses a serious threat to animal and human health, requiring safe and reliable treatment. Therefore, screening microbial strains with strong kanamycin-degrading capabilities from nature and utilizing them for biological treatment of antibiotic-contaminated soil can completely or substantially degrade residual antibiotic components, eliminating their health hazards to humans and animals, mitigating potential environmental risks, and demonstrating significant social, environmental, and economic implications. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a strain of Bacillus belyssus (B. belyssus). Bacillus velezensis This strain forms round, milky-white, smooth, raised colonies with neat edges. It is opaque, Gram-positive, and rod-shaped. The optimal growth temperature is 37℃, with a maximum growth temperature of 45-47℃.

[0005] This *Bacillus belye* strain can utilize sucrose, glucose, galactose, maltose, tannic acid, and mannitol, but cannot utilize citric acid. It tests positive for nitrate reduction, nitrite reduction, catalase reaction, and gelatin liquefaction. The full-length 16S rRNA sequence of this strain is 1162 bp. Bacillus velezensis The 16S rRNA sequence showed 99% homology. In summary, based on the colony morphology, culture conditions, physiological and biochemical characteristics, and molecular biological identification results, this strain belongs to *Bacillus belyesense*, and is named... Bacillus velezensis ice1 .

[0006] strains Bacillus velezensis ice1 This drug, capable of degrading the antibiotic kanamycin, was screened from peanut soil samples contaminated with kanamycin. It was deposited on May 9, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 66277). Its taxonomic name is... Bacillus velezensis Address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China.

[0007] Another object of the present invention is to provide the above-mentioned Bacillus cereus bacterial suspension, which is obtained by inoculating strain Bacillus sp. ice1 into LB medium and culturing it at 32℃~40℃ for 12~36 h. The bacterial suspension concentration is (0.1~5)×10⁻⁶. 9 cfu / mL.

[0008] Another object of the present invention is to provide an application of the above-mentioned Bacillus belyssus in the degradation of antibiotics, wherein the antibiotics include kanamycin.

[0009] Furthermore, the applications include: degrading residual kanamycin antibiotics in soil or wastewater, or remediating soil contaminated with residual kanamycin antibiotics.

[0010] Furthermore, to further improve the degradation rate of kanamycin, achieving a degradation rate of over 50% for residual kanamycin antibiotics in the soil, the application includes: using a 10% concentration... 8 ~10 9 A CFU / mL *Bacillus belycera* bacterial suspension is inoculated into soil containing residual kanamycin at an inoculum size of 8%–12%. Water is added at a solid-liquid ratio of 0.65–0.8:1 g / mL and mixed thoroughly. The mixture is then incubated at a constant temperature of 32–40°C and 150–200 r / min. Preferably, the residual kanamycin content in the soil is 150–250 ng / g, and the incubation time is 3–5 days.

[0011] Furthermore, the application includes: adjusting the concentration to 10 8 ~10 9 An equal volume of *Bacillus belycera* bacterial suspension (cfu / mL) is inoculated into wastewater containing residual kanamycin antibiotics, and then fermented at a constant temperature of 32℃–40℃ and 150–200 r / min. Preferably, the concentration of residual kanamycin antibiotics in the wastewater is 50–80 ng / L, and the constant temperature fermentation time is 3–5 days. Attached Figure Description

[0012] Figure 1 The colony status (left) and Gram staining diagram (right) of Bacillus belyssus provided for this invention; Figure 2 pH value and salt concentration affect the strain described in this invention. Bacillus velezensis ice1 The impact; Figure 3 It is a strain Bacillus velezensis ice1 Growth curve; Figure 4 It is constructed based on 16S rRNA. Bacillus velezensis Phylogenetic tree diagram of the strain; Figure 5 This is a standard curve used in Example 2 of the present invention to detect the content of kanamycin, an antibiotic residue in peanut soil. Detailed Implementation

[0013] The technical solution of the present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the terminology used in the present invention is a common term in the relevant field. Unless otherwise specified, the screening methods, cultivation methods, identification methods, detection methods, etc. used in the various embodiments are conventional means well known to those skilled in the art, and the raw materials, culture media and equipment used can all be obtained from publicly available commercial channels.

[0014] In the following examples, kanamycin was kanamycin sulfate; the kanamycin enzyme-linked immunosorbent assay kit was purchased from Jiangsu Enzyme Immunoassay Co., Ltd.

[0015] The components of LB culture medium include: 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract.

[0016] Example 1 Bacillus velezensis ice1 Acquisition and identification Enrichment, isolation and purification of bacterial strains On September 17, 2024, peanut soil samples were collected in Yanling District, Xuchang City, Henan Province. The samples were diluted with an appropriate amount of sterile water, mixed thoroughly, and allowed to stand. The resulting suspension was then inoculated into LB medium containing kanamycin sulfate using a stepwise increasing concentration enrichment method. This resulted in a turbid, high-concentration target culture. Specifically, 50 grams of soil sample were collected, diluted with 100 ml of sterile water, mixed thoroughly, and allowed to stand. The suspension was then inoculated into LB medium containing 300 mg / L kanamycin sulfate and cultured at 37°C with constant temperature shaking at 220 rpm for 3 days. After the culture became turbid, it was sequentially transferred to LB medium at concentrations of 600 mg / L, 900 mg / L, and 1200 mg / L every 3 days. The target culture was enriched by stepwise concentration increase in LB broth containing mg / L kanamycin sulfate to obtain a turbid high-concentration target culture. This high-concentration target culture was then transferred to LB broth diluted 10 times with water and cultured to obtain a turbid culture. The turbid culture was then serially diluted with 10, 100, 1000, 10000, 100000, and 1000000 times sterile water, and then spread onto LB agar plates and incubated at 37°C. After the colonies grew on the LB agar plates, single colonies were isolated by streak plating to obtain strains with different colony morphologies.

[0017] Validation of aminoglycoside antibiotic-degrading bacteria Different bacterial colony morphologies were obtained and inoculated into a sterile aqueous solution containing only 0.4 mg / L aminoglycoside antibiotics. The mixture was then fermented at 37°C with constant temperature shaking at 220 r / min for 15–72 h. Since the sterile aqueous solution contained only aminoglycoside antibiotics, only strains capable of degrading aminoglycosides could utilize this sole nutrient for growth and reproduction, causing the sterile aqueous solution to become turbid due to cell proliferation. Strains unable to degrade aminoglycosides could not grow because they could not utilize them as nutrients, and the sterile aqueous solution remained clear. The strains that caused the sterile aqueous solution to become turbid were selected, thus obtaining the aminoglycoside-degrading bacterium *Bacillus velezensis* ice1 described in this invention. This bacterium exhibits extremely strong degradation and digestion capabilities for aminoglycoside antibiotics.

[0018] 1. Identification of strains Colony morphology: Bacillus velezensis ice1 The colony state is as follows Figure 1 As shown on the left, milky white, smooth, raised, well-defined, and opaque circular colonies were produced on LB medium.

[0019] Take 10 μL of the concentration as 10 8 ~10 9A bacterial suspension of CFU / mL was placed on a sterilized glass slide and heated with an alcohol lamp until the water evaporated. One to two drops of crystal violet dye were added to cover the smear, and staining was performed for approximately 1 minute. The slide was then rinsed with a small stream of distilled water until the wash water was colorless. Approximately 300 μL of iodine solution was added to cover the smear, and staining was performed for approximately 1 minute. The slide was then rinsed with distilled water until the wash water was colorless. The slide was tilted, and ethanol solution was added to decolorize the slide until the ethanol no longer showed a purple hue. One to two drops of safranin solution were added, and staining was performed for approximately 1 minute. The slide was then rinsed with a small stream of distilled water until the wash water was colorless. After the specimen dried, it was observed under a microscope. The results are as follows: Figure 1 As shown on the right, it is Gram-positive and straight.

[0020] Physiological and biochemical characteristics: Bacillus velezensis ice1 Physiological and biochemical identification was performed with reference to the "Handbook for Systematic Identification of Common Bacteria" (Dong Xiuzhu and Cai Miaoying, 2001). Physiological and biochemical indicators such as carbon source utilization, nitrogen source utilization, nitrate reduction, nitrite reduction, gelatin liquefaction, and catalase test were measured. The results are shown in Table 1.

[0021] Table 1 Bacillus velezensis ice1 Physiological and biochemical characteristics Identification indicators Bacillus velezensis ice1 Identification indicators Bacillus velezensis ice1 Growth temperature range 15℃～42℃ Urea - Salt concentration ≤8% peptone + Glucose oxidation, fermentation, and motility Fermented type, sports type ammonium chloride + sucrose + ammonium sulfate + glucose + Calcium nitrate - Galactose + ammonium nitrate - maltose + nitrate reduction reaction + Tannic acid - nitrite reduction reaction + Citric acid - Gelatin liquefaction + Mannitol + catalase test + As can be seen from Table 1: Bacillus velezensis ice1 The strain became turbid after being cultured in LB medium for 24 h; the growth temperature range was 15–42℃, the optimal growth temperature was 37℃, and the maximum salt tolerance was 8%. Bacillus velezensis ice1 The strain can ferment sucrose, glucose, galactose, maltose, mannitol, peptone, ammonium chloride, and ammonium sulfate, but cannot ferment tannic acid, citric acid, urea, calcium nitrate, and ammonium nitrate; it is positive for nitrate reduction, nitrite reduction, catalase reaction, and gelatin liquefaction.

[0022] 16S rRNA sequence homology identification: DNA was extracted from bacterial samples using a silica matrix adsorption column method. The extracted DNA was amplified by PCR using 2×Taq Master Mix. The results were entered into the NCBI website for homology comparison. Simultaneously, homologous sequences were downloaded and imported into MEGA 12 software to construct a phylogenetic tree, such as... Figure 4 As shown. The full-length 16S rRNA sequence of this strain is 1162 bp. Bacillus velezensis The 16S rRNA sequence showed 99% homology. In summary, based on the colony morphology, culture conditions, physiological and biochemical characteristics, and molecular biological identification results, this strain belongs to *Bacillus velezensis*, and is named... Bacillus velezensis ice1 .

[0023] 2. Biological characteristics of the strain 2.1 Determination of Growth Curve: The screened strain Bacillus sp.ice1 was inoculated into LB liquid medium and cultured at 37 ℃ and 180 r / min for 12 h. The cultured bacterial solution was then inoculated into LB liquid medium at a 1% inoculum and cultured for another 12 h to prepare the seed fermentation broth. The cultured bacterial solution was then transferred to LB medium at a 1% inoculum, shaken to mix, and placed in a constant temperature shaker at 37 ℃ and 180 r / min for 36 h. Every 2 h, the culture was removed, and the OD600 nm value was measured using sterile deionized water as a blank control. A growth curve was plotted with culture time on the x-axis and OD600 nm value on the y-axis. The results are shown below. Figure 3 As shown.

[0024] from Figure 3 It can be seen that the growth trend of strain Bacillus sp.ice1 is first increased and then decreased, reaching its peak at around 24 hours.

[0025] 2.2 Salt Tolerance Test: Different masses of NaCl were added to 100 mL of liquid LB medium to prepare salt solutions with different mass percentages of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 11%. 1 mL of bacterial culture was added to each of these salt solutions, and the mixtures were incubated at 37 ℃ and 180 r / min for 24 h using a constant temperature shaker. The absorbance was measured at 600 nm using a UV spectrophotometer. A curve was plotted with the NaCl solution concentration on the x-axis and the OD600 nm value on the y-axis. The results are shown below. Figure 2 As shown on the right.

[0026] from Figure 2 As can be seen in the right image, the growth of Bacillus sp.ice1 cells is inhibited as the concentration of the salt solution increases. The cell concentration drops rapidly when the mass percentage concentration is 7% to 9%, and the strain is completely inhibited when the salt solution concentration is 9%.

[0027] 2.3 Acid and Alkali Resistance Test: The pH of 10 mL of liquid LB medium was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 using 1 mol / L NaOH and 1 mol / L HCl solutions, respectively. 100 μL of bacterial culture was added to each of these salt concentrations, and the medium was incubated at 37 ℃ and 180 r / min for 24 h using a constant temperature shaker. The absorbance was measured at 600 nm using a UV spectrophotometer. A curve was plotted with pH on the x-axis and OD600 nm value on the y-axis. The results are shown below. Figure 2 As shown on the left.

[0028] from Figure 2 As can be seen from the left, growth is more vigorous at pH 5-9 and the optimal pH is 6; growth is hindered at pH 2-4 and 10-13.

[0029] from Figure 3 As can be seen, the OD600nm value of strain Bacillus velezensis ice1 first increases and then decreases with time, reaching its peak at around 24 hours. Example 2 Bacillus velezensis ice1 Application in degrading kanamycin antibiotic residues in peanut soil This embodiment provides a Bacillus velezensis ice1 The bacterial solution, which is provided in Example 1 Bacillus velezensis ice1 The strain was uniformly dispersed in LB broth and cultured at 37℃ with constant temperature shaking at 180 r / min for 24 h to prepare a concentration of 4×10⁻⁶. 8 Bacillus velezensis ice1 bacterial culture at CFU / mL.

[0030] The kanamycin antibiotic content in peanut soil before and after inoculation fermentation was analyzed using a kanamycin enzyme-linked immunosorbent assay (ELISA) kit. The standard curve for kanamycin antibiotics is shown below. Figure 5 As shown in the figure. Among them, the kanamycin content in the peanut soil before inoculation was 220.80 ng / g, as tested.

[0031] 10 g of peanut soil containing 220.80 ng / g kanamycin was inoculated with Bacillus velezensis ice1 bacterial culture provided in this example. The inoculation amount was 10% of the soil weight. Water was added and mixed well. The solid-liquid ratio was controlled at 0.7:1 g / mL. After fermentation at 37 ℃ for 72 h, the kanamycin antibiotic content was detected to be 70.90 ng / g. The calculated degradation rate of kanamycin antibiotic in peanut soil was 67.89%.

[0032] Example 3 Bacillus velezensis ice1 Application in the degradation of residual kanamycin sulfate in wastewater This embodiment provides a Bacillus velezensis ice1 The bacterial solution, which is provided in Example 1 Bacillus velezensis ice1 The strain was uniformly dispersed in LB broth and cultured at 37℃ with constant temperature shaking at 180 r / min for 30 h to prepare a concentration of 2×10⁻⁶. 9 Bacillus velezensis ice1 bacterial culture at CFU / mL.

[0033] The kanamycin sulfate content in wastewater before and after inoculation fermentation was analyzed using a kanamycin enzyme-linked immunosorbent assay (ELISA) kit. The kanamycin sulfate content in the wastewater before inoculation was found to be 63.9 ng / L.

[0034] Take 10 mL of Bacillus velezensis ice1 bacterial culture provided in this example and inoculate it into 10 mL of wastewater with a kanamycin sulfate content of 63.9 ng / L. After incubation at 37℃ and 180 r / min for 3 days, the kanamycin sulfate content in the wastewater was measured to be 38 ng / L. The calculated degradation rate of kanamycin sulfate in the wastewater was 40.53%.

[0035] Therefore, the Bacillus berberis obtained using the embodiments of the present invention can rapidly degrade kanamycin antibiotics without the need for other nutrients, resulting in low cost and simple operation.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis ), characterized in that, The Bacillus belye is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66277.

2. A Bacillus belyceae preparation, characterized in that, Includes the Bacillus berberis as described in claim 1.

3. A Bacillus belesiensis bacterial suspension, characterized in that, Including concentrations of (0.1~5)×10 9 Bacillus berberis as claimed in claim 1, cfu / mL.

4. A method for culturing Bacillus belye bacterial suspension as described in claim 3, characterized in that, The Bacillus berreatus of claim 1 was inoculated into LB medium and cultured at 32°C to 40°C for 12 to 36 h.

5. The use of Bacillus belyssus of claim 1 or the Bacillus belyssus preparation of claim 2 in the degradation of kanamycin antibiotics.

6. The application according to claim 5, characterized in that, The applications include: degrading residual kanamycin antibiotics in soil, sewage, or bacterial residue; Or remediate soil contaminated with residual kanamycin antibiotics.

7. The application of the Bacillus belyssus culture of claim 3 in the degradation of antibiotics, characterized in that, The application includes: [the following is a description of a specific application method] with a concentration of (0.1–5) × 10 [units]. 9 Inoculate a CFU / mL Bacillus belye bacterial suspension into soil containing residual kanamycin antibiotics at an inoculum of 8%–12%. Add water at a solid-liquid ratio of 0.65–0.8:1 g / mL and mix well. Then, ferment at a constant temperature of 32–40℃ and 150–200 r / min.

8. The application according to claim 7, characterized in that, The residual kanamycin antibiotic content in the soil is 150-250 ng / g, and the isothermal fermentation culture time is 3-5 days.

9. The application of the Bacillus berberis bacterial suspension according to claim 3 in the degradation of antibiotics, characterized in that, The application includes: [the following is a description of a specific application method] with a concentration of (0.1–5) × 10 [units]. 9 An equal volume of Bacillus belye bacterial culture (cfu / mL) was inoculated into wastewater containing residual kanamycin antibiotics, and then fermented at a constant temperature of 32℃~40℃ and 150~200 r / min.

10. The application according to claim 9, characterized in that, The concentration of residual kanamycin antibiotic in the wastewater is 50–80 ng / L, and the isothermal fermentation culture time is 3–5 days.

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