Bacillus velezensis TG1 and application thereof

Abstract

The invention discloses bacillus velezensis TG1 and application thereof. The bacillus velezensis TG1 is classified and named as bacillus velezensis, and the preservation number of the bacillus velezensis TG1 is GDMCC 66317; the preservation date is May 12, 2025, and the preservation unit is Guangdong Microbial Culture Collection Center. The bacillus velezensis TG1 provided by the invention has broad-spectrum antibacterial activity, high environmental adaptability and high safety, provides a green, sustainable, safe and efficient microbial resource for aquaculture, and has important ecological and economic values.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Bacillus Velezii TG1 and applications thereof. Background Art

[0002] Aquaculture, the fastest-growing food production sector globally, supplies nearly 50% of human aquatic food. However, outbreaks of aquatic diseases (such as vibriosis and streptococcal disease) caused by intensive aquaculture have resulted in significant economic losses. Traditional antibiotic therapy is severely restricted by concerns about drug resistance and environmental pollution. Existing commercial probiotics (such as Bacillus subtilis) suffer from strong host specificity, poor seawater adaptability (survival rate <30% in high-salt environments), and a narrow antibacterial spectrum (covering only two to three pathogens), making them unable to meet industry needs. Therefore, there is an urgent need to develop new probiotics that combine broad-spectrum antibacterial activity, environmental adaptability, and safety. Summary of the Invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a Bacillus Velez TG1 and its application to solve the disease problems caused by pathogen infection and antibiotic abuse in aquaculture, and at the same time provide a safe and efficient probiotic resource for antibiotic-free aquaculture.

[0004] The present invention provides a Bacillus velezensis TG1. The Bacillus velezensis TG1 is classified as Bacillus velezensis, has a deposit number of GDMCC 66317, and is deposited on May 12, 2025, with the Guangdong Provincial Center for Microbial Culture Collection being the depository.

[0005] The Bacillus Velez TG1 provided by the present invention has both broad-spectrum antibacterial properties and high environmental adaptability. Specifically, the broad-spectrum antibacterial properties are specifically reflected in: it has antibacterial activity against 11 aquatic pathogens, including Aeromonas veronii, Aeromonas hydrophila, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Photobacterium damselae, Citrobacter freundii, Morganella morganii, Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi, among which the antibacterial effect against Aeromonas veronii is the best (the diameter of the inhibition zone reaches 21.10±0.66 mm); its high environmental adaptability is demonstrated by a survival rate of >70% in seawater with a salinity of 20‰ and tolerance to 0.4% bile salts (survival rate of 20% within 4 hours), significantly outperforming existing technologies. Its high safety is demonstrated by a negative hemolysis test. Compared to existing technologies, the Bacillus Velezii TG1 of the present invention overcomes the survival bottleneck of traditional probiotics in marine aquaculture, providing a safe and effective alternative to antibiotics. This provides a green, sustainable, safe, and efficient microbial resource for aquaculture, with significant ecological and economic value.

[0006] Preferably, the 16S rRNA gene sequence of the Bacillus velezensis TG1 is shown as SEQ ID NO: 1.

[0007] Preferably, the Bacillus Velez TG1 is isolated from the body surface mucus of pearl grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂).

[0008] The present invention provides a microecological preparation, which comprises the Bacillus Velezii TG1.

[0009] The present invention provides a feed additive, which comprises the microecological preparation.

[0010] The present invention provides a feed, which comprises the feed additive.

[0011] Preferably, in the feed, the addition amount of the Bacillus Velez TG1 is (1×10 8 )~(1×109 )CFU / kg.

[0012] In the feed of this solution, the addition amount of the Velez Bacillus TG1 is (1×10 8 )~(1×10 9 ) CFU / kg, which can improve the intestinal microecology of aquatic products, enhance immunity and promote growth.

[0013] Preferably, the feed is used in aquaculture of marine fish.

[0014] More preferably, the marine fish includes grouper.

[0015] The present invention provides a water body improver, which includes the microecological preparation.

[0016] Preferably, the concentration of the Bacillus Velez TG1 in water is (1×10 5 )~(1×10 9 ) CFU / L.

[0017] The concentration of Bacillus Velez TG1 in water in this scheme is (1×10 5 )~(1×10 9 ) CFU / L, which can effectively inhibit the growth of aquatic pathogens.

[0018] Further preferably, the concentration of the Bacillus Velez TG1 in water is (1×10 5 ) ~(5×10 6 ) CFU / L.

[0019] Further preferably, the concentration of the Bacillus Velez TG1 in the water is 1×10 6 CFU / L.

[0020] The present invention provides a probiotic, which includes the Bacillus Velez subtilis TG1. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the morphological observation diagram of Bacillus Velez TG1.

[0022] Figure 2 This is the result of hemolytic identification of Bacillus Velez TG1.

[0023] Figure 3 This is the growth curve of Bacillus Velez TG1.

[0024] Figure 4 This is the standard curve of Bacillus velez TG1.

[0025] Figure 5 This is a graph showing the salinity tolerance of Bacillus Velez TG1.

[0026] Figure 6 This is the pH tolerance diagram of Bacillus Velez TG1.

[0027] Figure 7 This is a graph showing the bile salt tolerance of Bacillus Velez TG1.

[0028] Figure 8 This is a diagram for evaluating the film-forming ability of Bacillus Velez TG1. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the drawings in the embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0030] Example 1 Isolation and Identification of Bacillus velezensis TG1 1. Main instruments and reagents Sterile operating table, constant temperature incubator, centrifuge, PCR instrument, electrophoresis system, LB medium, Columbia CNA blood agar plate, Gram staining kit, Chelex-100 DNA extraction reagent, 16S rRNA universal primer (27F / 1492R).

[0031] 2. Experimental Methods 2.1 Strain isolation Mucus from the body of Epinephelus spp. was collected aseptically and treated in a 70°C water bath for 20 min to remove contaminants. The mucus was inoculated into LB liquid medium, incubated at 37°C and 220 rpm, and then single colonies were isolated by streaking. Morphology was observed by Gram staining, and hemolytic activity was verified by blood agar plates. Genomic DNA was extracted, and the 16S rRNA gene was amplified by PCR and sequenced.

[0032] 3. Experimental Results Figure 1 This is the morphological observation diagram of Bacillus Velez TG1. Figure 1 It can be seen that the Bacillus velezensis TG1 isolated in Example 1 is a Gram-positive short rod-shaped strain.

[0033] Figure 2 This is the result of hemolytic identification of Bacillus Velez TG1. Figure 2It can be seen that when the fermentation liquid of Bacillus Velez TG1 isolated in Example 1 was inoculated onto a Columbia CNA blood agar plate, the hemolytic activity was negative.

[0034] 16S rRNA sequencing showed >99% homology with Bacillus velezini (NCBI alignment).

[0035] The 16S rRNA gene sequence (SEQ ID NO: 1) of Bacillus velezensis TG1 isolated in Example 1 is as follows: The Bacillus velezensis TG1 obtained in the above example was sent to the Guangdong Provincial Microbial Culture Collection Center (the collection address is: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences) for preservation on May 12, 2025, and was classified and named: Bacillus velezensis, and the collection number is: GDMCC66317.

[0036] Example 2 Growth characteristics and standard curve establishment 1. Main instruments and reagents Microplate reader, shaker, colony counter, LB liquid culture medium, sterile PBS buffer.

[0037] 2. Experimental Methods Bacillus velezensis TG1 was inoculated into LB medium at a 2% inoculum size and cultured continuously at 37°C for 72 h. The OD600nm value was measured every 2-12 h to draw a growth curve. The logarithmic phase bacterial solution was serially diluted and the plate counts were used to establish an OD600nm-CFU standard curve.

[0038] 3. Experimental Results Figure 3 is the growth curve of Bacillus velez TG1, Figure 3 It can be seen that Bacillus Velez TG1 entered the logarithmic phase at 4 h and reached the stable phase at 24 h, and grew rapidly.

[0039] Figure 4 is the standard curve of Bacillus velez TG1, Figure 4 It can be seen that the standard curve equation is: .

[0040] Example 3 Antibacterial activity detection 1. Main instruments and reagents Aseptic punch, vernier caliper, LB agar plate, pathogenic bacteria - Aeromonas veronii (AV), Aeromonas hydrophila (AH), Staphylococcus aureus (Sa), Vibrio cholerae (CHO), Escherichia coli (EC), Photobacterium damselae (PD), Citrobacter freundii (Cit), Morganella morganii (MM), Vibrio alginolyticus (ALG), Vibrio parahaemolyticus (VP), Vibrio harveyi (VH), Vibrio fluvialis (VF) 2. Experimental method Pathogenic bacteria suspension (10 ) was coated on LB plate; 60 μL of Bacillus velezensis TG1 fermentation broth (10 7 CFU / mL) was added by punch (the diameter of the hole was 6 mm), and it was cultured at 37 ℃ for 24 h; the diameter of the inhibition zone (mm) was measured by vernier caliper, and the measurement was repeated for 3 times.

[0041] 3. Experimental results The test results of Example 3 are shown in Table 1: Table 1: Analysis of broad-spectrum antibacterial activity of TG1

[0042] As shown in Table 1, the inhibition zone of Bacillus velezensis TG1 against Aeromonas veronii was 21.10±0.66 mm; it had broad-spectrum inhibition against 11 kinds of pathogenic bacteria, including Aeromonas veronii (21.10±0.66 mm), Vibrio harveyi (17.67±0.42 mm), Aeromonas hydrophila (17.13±1.79 mm), Staphylococcus aureus (18.00±0.26 mm), Vibrio cholerae (14.33±0.86 mm), Escherichia coli (14.53±1.81 mm), Photobacterium damselae (11.77±0.49 mm), Citrobacter freundii (14.40±0.89 mm), Morganella morganii (12.80±0.44 mm), Vibrio alginolyticus (13.43±0.84 mm), and Vibrio parahaemolyticus (11.77±0.15 mm).

[0043] Example 4 Environmental tolerance test 1. Main instruments and reagents pH meter, microplate reader, LB culture medium with different pH (2-12), and LB culture medium containing bile salts (0.4%).

[0044] 2. Experimental Methods 2.1 Test of strain salt (NaCl) tolerance LB liquid culture medium containing 20‰, 30‰, and 40‰ sodium chloride was prepared, and the bacterial solution in the logarithmic growth phase was inoculated into the LB liquid culture medium at 10% (v / v). After incubation at 37°C and 220 rpm for 4 h, the OD600nm was measured. Three parallels were set up for each group.

[0045] 2.2 Test of strain acid and alkali resistance To determine the acid and alkali tolerance of strain TG1, the pH of LB liquid medium was adjusted to 2.0, 4.0, 6.0, 7.0, 8.0, 10.0, and 12.0 using 2 mol / L hydrochloric acid and 2 mol / L NaOH solutions, then sterilized and used. A 10% (v / v) inoculum of the logarithmic growth phase bacteria was inoculated into the above medium and cultured at 37°C and 220 rpm for 4 h. OD600nm was then measured. Three replicates were set up for each group.

[0046] 2.3 Bile salt (ox bile salt) tolerance test Prepare LB liquid medium containing 0.4% (w / v) ox bile salts. Inoculate 10% (v / v) of the bacterial solution in the logarithmic growth phase into the LB liquid medium. Take the initial bacterial solution and the bacterial solution after culturing at 37°C and 220 rpm for 2 h and 4 h to spread on the plate, and calculate the survival rate. Set up three parallel plates for each group.

[0047] 3. Experimental Results Figure 5 is the salinity tolerance diagram of Bacillus velez TG1, Figure 5 It can be seen that when the salinity is 20‰, Bacillus Velez TG1 has the strongest adaptability.

[0048] Figure 6 is the pH tolerance diagram of Bacillus Velez TG1, Figure 6 It can be seen that Bacillus Velez TG1 can tolerate extreme environments with a pH of 2-12, and has a survival advantage in animal intestines and aquaculture water. Bacillus Velez TG1 grows best at a pH of 6-8, and the survival rate of Bacillus Velez TG1 is >30% at pH=2.

[0049] Figure 7 is the bile salt tolerance diagram of Bacillus velez TG1, Figure 7 It can be seen that after 4 h of treatment with 0.4% bile salt, the survival rate of Bacillus Velezii TG1 was 20%.

[0050] Example 5 Analysis of probiotic properties 1. Main instruments and reagents 96-well plate, microplate reader, crystal violet stain (0.1%), skim milk powder medium (protease activity), soluble starch medium (amylase activity), cellulose medium (cellulase activity), Congo red, Biolog GEN Ⅲ MicroPlate TM .

[0051] 2. Experimental Methods 2.1 Determination of biofilm formation in 96-well plates by crystal violet staining (OD570nm) Bacteria in the logarithmic growth phase were collected by centrifugation at 5000 g for 10 minutes, washed twice with sterile PBS buffer, and resuspended in LB liquid medium. The OD600 value of the cell suspension was adjusted to 0.8. 200 μL of this cell suspension was added to a 96-well plate, with three replicates per group. The plates were sealed with sealing film and incubated at 37°C for 24 hours. After the incubation period, the medium was gently aspirated, and the plates were washed three times with sterile PBS. 200 μL of 0.1% crystal violet solution was added to each well and stained for 15 minutes. After staining, the crystal violet solution was gently aspirated, and the plates were washed three times with sterile water to remove unbound dye. After washing, 200 μL of 95% ethanol was added to each well to dissolve the crystal violet bound to the biofilm. The OD570 nm was measured to assess the biofilm-forming ability of the strain.

[0052] 2.2 Determination of hydrolysis circle diameter (D / d) by point connection method and calculation of enzyme activity index Protease activity assay: The strain was inoculated into LB liquid medium and cultured at 37°C, 220 rpm for 24 h. 1 μL of the fermentation broth was then spotted onto the surface of solid medium containing 1.5% skim milk powder and cultured at 37°C overnight. The colonies were observed for the formation of a transparent zone. The colony diameter (d) and the hydrolysis zone diameter (D) were measured with a vernier caliper. The protease activity index (Ap = D / d) was calculated.

[0053] Amylase activity assay: Using the spot method, 1 μL of fermentation broth was spotted onto the surface of a soluble starch culture medium and incubated at 37°C for 24 h. Lugol's iodine solution was sprayed evenly over the surface of the culture medium. The formation of a transparent zone was observed, and the colony diameter (d) and the hydrolysis zone diameter (D) were measured with a vernier caliper. The amylase activity index (Aa = D / d) was calculated.

[0054] Cellulase activity assay: Cellulase activity was assessed using cellulase culture medium. 1 μL of fermentation broth was spotted onto the surface of the culture medium and incubated at 37°C for 48 h. The plate was incubated with 1 mmol / L NaCl solution for 10 min and then with 1 g / L Congo red solution for 30 min. The colonies were observed for the formation of a transparent zone. The colony diameter (d) and the hydrolysis zone diameter (D) were measured with a vernier caliper, and the cellulase activity index (Ac = D / d) was calculated. Each experiment was performed in triplicate.

[0055] 2.3 Biolog GEN III microplate assay for carbon source utilization and chemical sensitivity Streak fresh bacterial culture onto LB solid medium overnight. Following the instructions in the BIOLOG GEN III MicroPlate user manual, gently scrape an appropriate amount of colonies using a sterile disposable cotton swab and suspend them in IF-A inoculum. Vortex the suspension for 2 minutes to thoroughly mix. Add 100 μL of the suspension to each well of a GEN III identification plate. Incubate the plate at 37°C for 3–36 hours. Measure the absorbance of each well at 590 nm using a microplate reader to analyze the physiological and biochemical characteristics of the strain.

[0056] 3. Experimental Results 3.1 Figure 8 ( Figure 8 SH39 is a known Velez subspecies of grouper origin) is an evaluation chart of the film-forming ability of Velez subspecies TG1, Figure 8 It can be seen that Bacillus Velez TG1 has a significant biofilm-forming ability, which helps it to colonize in the host or the environment.

[0057] 3.2 Table 2 is an analysis table of digestive enzyme activity index of Bacillus Velez TG1. It can be seen from Table 2 that Bacillus Velez TG1 has protease, cellulase and amylase activities. The protease activity index (D / d) is 1.35±0.23; the cellulase activity index (D / d) is 1.91±0.20; and the amylase activity index (D / d) is 1.92±0.15. The enzyme activity is high, and adding it to the feed can help improve feed utilization.

[0058] Table 2 TG1 digestive enzyme activity index

[0059] Note: Digestive enzyme activity index is expressed as mean ± standard deviation.

[0060] 3.3 Table 3 shows the results of the carbon source utilization analysis of Bacillus velezensis TG1 on the BiologGEN III plate. In this table, “+” indicates a positive reaction and “-” indicates a negative reaction.

[0061] Table 4 shows the results of chemical sensitivity analysis of Bacillus velez TG1 to chemicals on the BiologGEN III plate. In this table, “+” indicates a positive reaction; “-” indicates a negative reaction.

[0062] Table 3 Carbon source utilization ability analysis results

[0063] Table 4 Chemical sensitivity analysis results

[0064] Example 6 Safety evaluation 1. Main instruments and reagents Constant temperature incubator, microplate reader, electronic balance, drug-susceptibility paper, vernier caliper, PBS, grouper, Vibrio harveyi.

[0065] 2. Experimental Methods 2.1 Safety assessment of live grouper juveniles Healthy groupers of similar size were selected for safety evaluation and divided into experimental groups (injected with 1×10 8 CFU / mL of TG1) and the control group (injected with 100 μL sterile PBS), three parallel groups of 10 each were set up, and each group was intraperitoneally injected with 100 μL sterile PBS (control group), 1×10 8 CFU / mL of Bacillus Velez TG1 (experimental group). The groupers were observed for one week after injection, and the growth and mortality of each group were observed and counted.

[0066] 2.2 Simulated challenge experiment against pathogenic bacteria in grouper aquaculture water Experimental subjects: healthy grouper (body length 10±2cm), 10 fish per group, 3 parallel Group setting: Control group (Group C): No bacteria were added to the water; Pathogen challenge group (Group P): Vibrio harveyi was added to the water Protection group (B+P group): first add Bacillus Velez TG1 ( ), and attack the poison 3 days later.

[0067] Experimental operation: Day 1-3: Bacillus Velez TG1 was added to the water of group B+P ( ), Day 4: P group and B+P group were added with Vibrio harveyi ( ), observe and record the number of dead fish in each group within 7 days, and calculate the survival rate.

[0068] 2.3 Antibiotic sensitivity analysis The susceptibility of potential probiotics to antimicrobial drugs was tested by the disc agar diffusion method (KB method), and the results were interpreted according to the 2021 standard of the Clinical Laboratory Standards Institute (CLSI). Nine common antibiotics, including enrofloxacin, florfenicol, sulfamethoxazole, neomycin, doxycycline, ciprofloxacin, sulfamethoxazole, thiamphenicol, and flumequine, were used for drug sensitivity testing. The test bacteria solution was diluted to 200 μL of the solution was evenly spread on the surface of an LB agar plate. Using tweezers, a drug-sensitive paper sheet was gently placed on the plate surface. The plate was then incubated in a 37°C incubator for 24 hours. After incubation, the diameter of the inhibition zone was measured using a vernier caliper. The size of the inhibition zone was used to determine the sensitivity of the strain to the drug. Three replicates were set up for each drug group.

[0069] 3. Experimental Results 3.1 Inject 100 μL PBS, During the entire subsequent experimental process, the grouper in both the control group and the experimental group were active, with no disease or death.

[0070] 3.2 The survival rate (7 days) of the control group (C) was 100%, the survival rate of the pathogenic bacteria challenge group (P) was 60%, and the survival rate of the Bacillus Velezii protection group (B+P) was 80%.

[0071] 3.3 Table 4 (“S” stands for sensitive) shows the test results of the antibiotic sensitivity test. As shown in Table 5, Bacillus velezensis TG1 is sensitive to eight commonly used aquatic antibiotics, including enrofloxacin and florfenicol, meeting the requirements for antibiotic-free aquaculture and demonstrating good biosafety.

[0072] Table 4 TG1 antibiotic sensitivity analysis

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Bacillus Velezii TG1, characterized in that The classification name of the Bacillus velezensis TG1 is Bacillus velezensis, and the preservation number is GDMCC 66317; the preservation date is May 12, 2025, and the preservation unit is Guangdong Provincial Microbiological Culture Collection Center.

2. The Bacillus Velezii TG1 according to claim 1, characterized in that The 16S rRNA gene sequence of the Bacillus Velezii TG1 is shown in SEQ ID NO:

1.

3. The Bacillus Velezii TG1 according to claim 1, characterized in that The Bacillus Velez TG1 is isolated from the mucus on the body surface of pearl grouper.

4. A microecological preparation, characterized in that: The probiotic preparation comprises the Bacillus Velezii TG1 according to any one of claims 1 to 3.

5. A feed additive, characterized in that The feed additive includes the probiotic preparation as claimed in claim 4.

6. A feed, characterized in that The feed comprises the feed additive according to claim 5.

7. The feed according to claim 6, characterized in that In the feed, the addition amount of the Velez Bacillus TG1 is (1×10 8 )~(1×10 9 ) CFU / kg.

8. A water improver, characterized in that: The water improver includes the microecological preparation as claimed in claim 4.

9. The water improver according to claim 8, characterized in that: The concentration of the Velez Bacillus TG1 in water is (1×10 5 )~(1×10 9 ) CFU / L.

10. A probiotic, characterized in that The probiotics include the Bacillus Velezii TG1 according to any one of claims 1 to 3.

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