Bacillus safensis and application thereof

Abstract

The application belongs to the technical field of microorganisms and relates to a Bacillus safensis and application thereof. The preservation number of the Bacillus safensis AK-SF1 is CCTCC NO: M 2025809, the Bacillus safensis AK-SF1 has good acid resistance, alkali resistance, bile salt resistance, trypsin resistance and high-temperature resistance, the adhesion rate of the Bacillus safensis AK-SF1 to duck enterocytes reaches 15.8%, the Bacillus safensis AK-SF1 is sensitive to common antibiotics, the safety of the Bacillus safensis AK-SF1 is high, the Bacillus safensis AK-SF1 can be prepared into a medicine or used as a feed additive in duck breeding, and the application provides a better microecological preparation reference for expanding a duck-derived probiotic resource library and application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Bacillus safranin and its applications. Background Technology

[0002] As people's living standards improve, their demand for and requirements in the breeding industry are gradually increasing, and the duck breeding industry is developing rapidly as a result. However, compared with the mature breeding model of chickens, duck breeding technology is relatively lagging behind, and is mostly characterized by problems such as: scattered and unregulated breeding, low efficiency, and frequent occurrence of bacterial diseases.

[0003] With the large-scale development of duck farming, the demand for antibiotic-free farming and green feed additives is becoming increasingly urgent. People are striving to find feed additives that can replace antibiotics. Bacillus, as a safe and highly effective probiotic, possesses characteristics such as acid resistance, salt resistance, and high temperature resistance. It can colonize the animal intestines and exert beneficial effects, thus being widely used in the treatment and prevention of digestive tract diseases in humans and animals. Bacillus has strong protease, lipase, and amylase activities, which can promote feed absorption and utilization efficiency; stimulate the development of immune organs, and enhance the body's immunity and resistance.

[0004] Current research on the screening and application of Bacillus strains specific to the duck gut is still relatively limited, especially lacking strains with good tolerance, adhesion ability, and safety. Therefore, isolating high-efficiency Bacillus strains from the duck gut is of great significance for developing microecological preparations suitable for duck farming. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art in that there are few duck-derived lactic acid bacteria strains available for screening and isolation, and that it is difficult to combine tolerance, adhesion ability and safety, so as to provide a strain of Bacillus sabolicii with strong stress resistance.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: This invention discloses a Bacillus safensis AK-SF1, with accession number: CCTCC NO: M 2025809.

[0007] The present invention also discloses the culture of the above-mentioned Bacillus sabovellatus AK-SF, wherein the culture is a fermentation broth.

[0008] The method for preparing the above-mentioned culture of the present invention is as follows: the *Bacillus sabolicii* AK-SF1 is inoculated into a medium containing LB broth, shaken and cultured for 20-24 hours, and the bacterial cells are removed to obtain the culture of *Bacillus sabolicii* AK-SF1.

[0009] The present invention also provides a drug, the active ingredient of which is the *Bacillus sabolicii* AK-SF1 or a culture thereof.

[0010] The present invention also provides a feed additive, wherein the active ingredient of the feed additive is Bacillus sabolicii AK-SF1 or the culture thereof.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The strains disclosed in this invention are duck-derived strains with probiotic effects screened from the intestines of healthy ducks. These strains exhibit good resistance to acid, alkali, bile salts, trypsin, and high temperatures, and achieve an adhesion rate of 15.8% to duck intestinal epithelial cells, allowing them to colonize the intestines and exert their probiotic effects. Furthermore, the strains of this invention are sensitive to most antibiotics, demonstrating high safety, and can be prepared into drugs or used as feed additives in duck farming, providing a better reference for expanding the duck-derived probiotic resource bank and its applications. Information on the preservation of biological materials

[0012] The *Bacillus safensis* AK-SF1 provided in this invention is classified as *Bacillus safensis*. Its accession number is CCTCC NO: M 2025809. The depositary institution is the China Center for Type Culture Collection (CCTCC); address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is April 17, 2025. Attached Figure Description Figure 1 Characteristic image of Bacillus sabinatus AK-SF1 cultured on MRS agar. Figure 2 Image of Gram staining microscopic examination of Bacillus sabovella AK-SF1. Figure 3 Thermoresistant bar chart of Bacillus sabovella AK-SF1 Figure 4 Phylogenetic tree of Bacillus sabovella AK-SF1 16S rRNA. Figure 5 This is a 24-hour growth curve of Bacillus sabinatus AK-SF1. Figure 6 The pH change curve of Bacillus sabovella AK-SF1 over 16 h is shown. Figure 7 Diagram of hemolysis experiment of Bacillus sarcodactylis AK-SF1 Figure 8 Acid-resistant bar chart of Bacillus sarcodactylis AK-SF1 Figure 9 Bar chart of alkali-resistant Bacillus sabovella AK-SF1 Figure 10Bar chart of trypsin-resistant Bacillus sabovella AK-SF1 Figure 11 Bar chart of bile salt resistant Bacillus sabovella AK-SF1. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0014] Example 1 1. Isolation, culture and purification of bacterial strains In a clean bench, a segment of mucosa from the intestine of a healthy duck was scraped using the back of a sterile scalpel. The scraped mucosa was placed in a sterile test tube containing physiological saline, shaken well, and immediately serially diluted with physiological saline. 100 drops of each dilution (10⁻⁴, 10⁻⁵, 10⁻⁶) were added to LB agar plates and spread onto plates. The plates were incubated overnight at 37°C. Plates with good growth and sparse colonies were selected for further screening. Colony selection criteria included: good growth, white color, slightly convex center, and slight surface wrinkles, consistent with the morphology of *Bacillus sabethricus*. Qualified colonies were picked up with an inoculation loop and streaked onto LB agar plates for incubation, repeating this cycle at least 5 times. This process preliminarily screened several pure bacterial strains, which were named AK-SF1. Colonies on LB agar plates are shown in the image. Figure 1 As shown.

[0015] 2. Staining and microscopic examination of smears Single purified colonies were picked and smeared, Gram-stained, and observed under an oil immersion microscope. The results showed that AK-SF1 bacteria were purple rod-shaped, arranged in pairs or chains, and were Gram-positive. (See attached image). Figure 2 .

[0016] 3. Biochemical tests Fermentation tests were conducted separately for glucose, lactose, maltose, and sucrose, as well as for peptone water, glucose phosphate peptone water, and Simon's citrate tests. The test bacteria were inoculated into peptone water, glucose phosphate peptone water, Simon's citrate, and sugar fermentation tubes (glucose, lactose, maltose, and sucrose) using an inoculation loop or inoculation needle and incubated at 37°C for 24-48 h.

[0017] The results, shown in Table 1, indicate that AK-SF1 can break down glucose, maltose, sucrose, and lactose; it is positive for glucose phosphate peptone water, but negative for peptone water, glucose phosphate peptone water, and Simon's citrate. This suggests that AK-SF1 has a strong carbohydrate metabolism capacity, consistent with the physiological and biochemical characteristics of Bacillus spp.

[0018] Table 1 Biochemical Tests AK-SF1 + + + + - + - 4. PCR identification: PCR amplification was performed using universal primers 27F and 1452R for the bacterial 16S rRNA gene.

[0019] (1) Nucleic acid extraction: A single colony of the bacterium was picked up with an inoculation loop and inoculated into LB liquid medium and cultured at 37°C and 120 rpm for 24 h with shaking. Nucleic acid was then extracted using the Ezup column-type bacterial genomic DNA extraction kit from Shanghai Sangon Biotech Co., Ltd.

[0020] (2) PCR amplification and sequencing analysis of 16S rRNA: The PCR reaction system was 50 μL: PCR Master Mix 25 μL, upper primer 2 μL, lower primer 2 μL, DNA template 5 μL, and sterile water 16 μL. PCR reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 10 min. The PCR products were detected by agarose gel electrophoresis and then sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO: 1.

[0021] See evolutionary tree Figure 4 Sequencing results were compared and analyzed in the NCBI nucleic acid database. The 16S rRNA gene of AK-SF1 strain and Bacillus sabolicii C4 strain (GenBank accession number: OR924281.1) showed 100% homology, confirming the tested bacterium as Bacillus sabolicii, and a phylogenetic tree was constructed. The results are shown in Table 2 and... Figure 4 As shown.

[0022] Table 2. Results of comparison analysis in nucleic acid databases

[0023] 5. Resistance to acid, alkali, bile salts, and trypsin inhibitors. Add 100 μL of AK-SF1 bacterial suspension to 10 mL of LB medium and incubate at 37℃ with shaking at 120 rpm for 24 h. Collect bacterial cells by centrifugation at 8000 r / min for 10 min. Resuspend the bacterial cells in LB medium with pH values ​​of 2, 3, 4, 8, and 9, bile salt concentrations of 0.1%, 0.2%, and 0.3%, and trypsin concentrations of 1%, 1.2%, and 1.4%, respectively. Incubate at 37℃ with shaking for 2 hours, then sample and plate for counting. See Table 3 for details. Figure 8 , Figure 9 , Figure 10 , Figure 11 .

[0024] Table 3 Results of acid resistance, bile salt resistance, and pancreatic enzyme resistance tests pH=2 52% pH=3 72% pH=4 84% pH=8 85% pH=9 76% The bile salt concentration was 0.1%. 100% The bile salt concentration was 0.2%. 81% The bile salt concentration was 0.3%. 76% The concentration of trypsin is 1%. 65% The trypsin concentration was 1.2%. 84% The trypsin concentration was 1.4%. 92% As can be seen from Table 3 and the figure, AK-SF1 exhibits good survival ability in the pH range of 4 to 8, especially with better tolerance under neutral to alkaline conditions; it is completely tolerant at a bile salt concentration of 0.1%, and even in a high bile salt environment of 0.3%, the survival rate still reaches 76%; it also has strong adaptability to pancreatic enzymes, indicating that it has excellent tolerance characteristics to the gastrointestinal simulated environment and has potential value for probiotic development.

[0025] 6. High temperature resistance test 100 μL of AK-SF1 bacterial suspension was added to 10 mL of LB medium and cultured at 37℃ with shaking at 120 rpm for 24 h. Subsequently, 200 μL of AK-SF1 bacterial suspension was incubated at 40℃, 50℃, 60℃, 70℃, and 80℃ for 20 min, respectively. The results showed that the survival rates of each group were 100%, 99%, 93%, 85%, and 67%, respectively. (See details...) Figure 3 This indicates that the bacterium has good heat resistance.

[0026] 7. Analysis of growth characteristics and lactic acid / base production capacity The AK-SF1 strain was inoculated into LB medium and cultured at 37°C with shaking at 120 rpm. Samples were taken every 2 hours, using sterile LB medium as a control. The OD value and pH value at 600 nm were measured, and curves were plotted with culture time on the x-axis and OD value and pH value on the y-axis, respectively. See Figure 5 , Figure 6 As shown in the figure, AK-SF1 bacteria enter the logarithmic growth phase at 4 h and begin to increase rapidly, then enter the plateau phase around 18 h with little concentration change. The pH of AK-SF1 bacteria drops to 6.60 at 4 h (end of acid production), and gradually rises to 8.01 from 4 to 16 h, indicating that its metabolic process first produces acid and then alkali, thus regulating the intestinal acid-base environment.

[0027] 8. Drug sensitivity test 100 μL of AK-SF1 bacterial suspension was added to 10 mL of LB medium and incubated at 37℃ with shaking at 120 rpm for 24 h. The bacterial suspension was then evenly spread onto LB agar plates using a cotton swab, and drug sensitivity test strips were attached to the plates. The plates were incubated at 37℃ for 24 h. The results showed that AK-SF1 was sensitive to CIP, NORLEV, FFC, IPM, DO, CPZ, CC, CN, PIP, S, AMK, and GEN; moderately sensitive to VAN, TET, E, MI, AZI, C, and CZ; and resistant to MY, AMP, XO, CTR, CAZ, SXT, PB, and PEN. These results indicate that AK-SF1 is relatively sensitive to common antibiotics and has good safety.

[0028] Table 4. Drug susceptibility testing CIP (Ciprofloxacin) S NOR (Norfloxacin) S VAN (vancomycin) I MY (Merubenen) R AMP (Ampicillin) R XO (Furazoline) R TET (Tetracycline) I E (erythromycin) I MI (minocycline) I AZI (Azithromycin) I LEV (levofloxacin) S FFC (florfenicol) S IPM (Imipenem) S DO (doxycycline) S CXM (Cefuroxime) R CPZ (Cefoperazone) S CTR (Ceftriaxone) R CAZ (ceftazidime) R SXT (Sulfamethoxazole / Trimethoprim) R PB (Polymycin B) R C (chloramphenicol) I CC (Clarithromycin) S CN (Gentamicin) S CZ (Cefazolin) I PIP (piperacillin) S PEN (penicillin) R S (Streptomycin) S AMK (Amikacin) S GEN (gentamicin) S Note: S = Sensitive; R = Tolerant; I = Moderately Sensitive 9. Hemolysis test Fresh mycelial growth of AK-SF1 bacteria was streaked onto sheep blood agar plates and incubated at 37°C for 24 hours. The results are as follows. Figure 7 This indicates that AK-SF1 is a gamma hemolytic agent and has good safety.

[0029] 10. Cell adhesion The experimental cells were duck intestinal epithelial cells. 200 μL of bacteria were inoculated into a 24-well plate containing duck intestinal epithelial cells and incubated for 1 h. Cell counts were then performed on the plate. The AK-SF1 cell adhesion rate was 15.8%, indicating that it has good adhesion ability in the intestine and can exert a probiotic function.

[0030] Table 5 AK-SF1 cell adhesion rate 14.7% 16.8% 15.9% 15.8% In this invention, a high-performance lactic acid bacteria strain AK-SF1 was isolated from the cecum of ducks and identified as *Bacillus sabolicii*. Experiments showed that the isolated AK-SF1 strain exhibited good resistance to acid, alkali, bile salts, and trypsin.

[0031] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A type of Bacillus safranin, characterized in that, The *Bacillus safortus* mentioned is *Bacillus safortus* AK-SF1 with the accession number: CCTCC NO: M2025809.

2. A culture based on *Bacillus sabovellatus* as described in claim 1, characterized in that, The culture of Bacillus sabovella AK-SF1 is a fermentation broth culture.

3. A method for preparing a culture of *Bacillus saboides* according to claim 2, characterized in that, The process includes the following steps: inoculating the *Bacillus sabolicii* AK-SF1 into LB medium, shaking and culturing for 20-24 h, removing the bacterial cells, and obtaining a culture of *Bacillus sabolicii* AK-SF1.

4. A drug, characterized in that, The active ingredient of the drug comprises the Bacillus sarcodactylis AK-SF1 of claim 1 and / or the culture of claim 2.

5. A feed additive, characterized in that, The active ingredient of the feed additive comprises the Bacillus sabriophyidae AK-SF1 as described in claim 1 and / or the culture as described in claim 2.

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