Bacillus subtilis strain jbh-bk8 and application thereof

Abstract

The application provides a bacillus subtilis JBH-BK8 and application thereof, and relates to the technical field of microorganisms.The bacillus subtilis JBH-BK8 in the application is preserved in the China Center for Type Culture Collection on October 25, 2023, and the preservation number is CCTCC NO: M 20232018.The bacillus subtilis in the application is natural and safe, acid-resistant, alkali-resistant, high-temperature-resistant, and has strong abilities of producing protease, amylase, lipase and cellulase, and has inhibitory effects on escherichia coli, salmonella, staphylococcus aureus, clostridium perfringens, aeromonas hydrophila and vibrio parahaemolyticus.The bacillus subtilis can be used in the aquaculture industry, can not only purify aquaculture water bodies and reduce the nitrogen and phosphorus content, but also can regulate the intestinal flora balance of farmed animals, promote growth, improve disease resistance and increase the aquaculture benefit.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Bacillus subtilis JBH-BK8 and its applications. Background Technology

[0002] In recent years, with the rapid development of aquaculture, global fisheries and aquaculture production have reached record highs. However, it is undeniable that large-scale, intensive aquaculture has led to the accumulation of harmful substances such as residual feed, excrement, ammonia nitrogen, nitrite, nitrate, and sulfides in water bodies, resulting in water quality deterioration and frequent outbreaks of aquatic animal diseases. While chemical drugs are highly effective in controlling aquatic animal diseases and purifying water quality, they easily lead to environmental pollution, drug residues, and drug resistance. Because probiotics have many benefits, including not polluting the environment, promoting aquatic animal growth, enhancing immune responses, and purifying water quality, they are often used for the prevention and treatment of aquatic diseases. Bacillus subtilis, a probiotic among many microecological preparations, exists in spore form and possesses advantages in storage and transportation due to its strong resistance and high stability. Simultaneously, the various enzymes secreted by Bacillus subtilis, such as proteases, lipases, and amylases, can rapidly and effectively decompose organic matter in aquaculture water, providing nutrients for algae. Algae, in turn, can produce dissolved oxygen through photosynthesis for the respiration of aquatic animals, maintaining the balance of the microecological environment in the water. Bacillus subtilis can also produce various antibacterial active ingredients such as bacteriocins, iturobacterin, and fenbacterin, inhibiting the growth of pathogens in the water and achieving the effect of purifying water quality.

[0003] Currently, numerous Bacillus subtilis strains have been reported for water purification, but their effectiveness varies greatly. Few strains are truly effective in actual production, and their application is often limited. Furthermore, existing patents on Bacillus subtilis for water purification primarily focus on degrading ammonia nitrogen and nitrite nitrogen in water, with few reports on its efficient degradation of total phosphorus and sulfides. Therefore, developing and reserving Bacillus subtilis strains with broader applications and higher efficiency in water treatment is of great significance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a Bacillus subtilis strain JBH-BK8 and its applications. This strain has simple cultivation conditions, is resistant to high temperatures and acids / alkalis, and produces amylase, lipase, protease, and cellulase. It can effectively reduce COD, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus, and sulfides in water bodies, making it a multifunctional strain for improving water quality.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a strain of Bacillus subtilis ( Bacillus subtilisThe strain JBH-BK8 was isolated in 2022 from the silt of Pipa Temple Reservoir in Tangyin County, Anyang, Henan Province, and was classified as Bacillus subtilis. Bacillus subtilis JBH-BK8 was deposited at the China Center for Type Culture Collection on October 25, 2023, with accession number CCTCC NO: M 20232018.

[0006] The present invention also provides a Bacillus subtilis seed culture obtained by inoculating the Bacillus subtilis JBH-BK8 in a seed culture medium.

[0007] Preferably, the seed culture medium consists of the following components: 8-12 g / L peptone, 2-4 g / L beef extract, and 3-6 g / L sodium chloride; the culture conditions are: 35-40℃, 120-150 r / min overnight culture.

[0008] More preferably, the seed culture medium has the following components: 10 g / L peptone, 3 g / L beef extract, and 5 g / L sodium chloride.

[0009] The present invention also provides a Bacillus subtilis fermentation broth obtained by inoculating the Bacillus subtilis seed liquid into a fermentation medium at a volume ratio of 1-3% and culturing at 35-40°C and 120-150 r / min for 48-72 h.

[0010] Preferably, the carbon source of the fermentation medium is any one or more of glucose, sucrose, soybean meal, corn starch, and corn steep liquor; and the nitrogen source of the fermentation medium is any one or more of ammonium chloride, ammonium sulfate, potassium nitrate, yeast extract, peptone, and beef extract.

[0011] More preferably, the fermentation medium comprises the following components: soybean meal powder 5-7 g / L, corn starch 3-5 g / L, sucrose 0.5-1.5 g / L, calcium carbonate 0.3-0.5 g / L, yeast extract 0.04-0.06 g / L, ammonium sulfate 0.02-0.04 g / L, potassium nitrate 0.01-0.03 g / L, potassium dihydrogen phosphate 0.2-0.4 g / L, magnesium sulfate 0.2-0.3 g / L, corn steep liquor 0.2-0.3 g / L, sodium chloride 0.2-0.3 g / L, 5000 U / g neutral protease 0.1-0.2 g / L, and defoamer 0.1-0.2 g / L.

[0012] More preferably, the fermentation medium comprises the following components: 6 g / L soybean meal, 4 g / L corn starch, 1 g / L sucrose, 0.4 g / L calcium carbonate, 0.05 g / L yeast extract, 0.03 g / L ammonium sulfate, 0.02 g / L potassium nitrate, 0.3 g / L potassium dihydrogen phosphate, 0.23 g / L magnesium sulfate, 0.28 g / L corn steep liquor, 0.28 g / L sodium chloride, 0.17 g / L 5000 U / g neutral protease, and 0.15 g / L defoamer.

[0013] The present invention also provides a microbial agent prepared using the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth.

[0014] The present invention also provides the application of the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth in the purification of aquaculture water.

[0015] Preferably, the application is specifically in reducing the content of COD, nitrogen, phosphorus and / or sulfides in aquaculture water.

[0016] More preferably, the aquaculture species in the aquaculture water body include, but are not limited to, fish, shrimp, and crab.

[0017] The Bacillus subtilis JBH-BK8 of this invention achieves a denitrification rate of over 95% when using potassium nitrate, ammonium sulfate, and sodium nitrite as single nitrogen sources. Specifically, JBH-BK8 can perform denitrification within the concentration ranges of potassium nitrate (0.01~4500 PPM), ammonium sulfate (0.05~3000 PPM), and sodium nitrite (0.1~2500 PPM), respectively.

[0018] The Bacillus subtilis JBH-BK8 of this invention achieves a phosphorus removal rate of over 90% when using potassium dihydrogen phosphate, phosphorus pentoxide, and disodium hydrogen phosphate as single phosphorus sources. Specifically, JBH-BK8 can perform phosphorus removal within the concentration ranges of 0.05-3000 PPM for potassium dihydrogen phosphate, 0.01-4000 PPM for phosphorus pentoxide, and 0.05-2500 PPM for disodium hydrogen phosphate.

[0019] The Bacillus subtilis JBH-BK8 of this invention has a desulfurization rate of over 95% when using a sulfide standard solution as the sole sulfur source.

[0020] When the Bacillus subtilis JBH-BK8 of this invention is applied to aquatic environments with denitrification, dephosphorization, and desulfurization conditions, the pH of the aquaculture water is 6-8, the temperature is 0-50 ℃, and the dissolved oxygen is 0-15 mg / L. Within this pH, temperature, and dissolved oxygen range, its removal rate of nitrogen, phosphorus, and sulfur is all above 90%.

[0021] The inoculum size of Bacillus subtilis JBH-BK8 in this invention ranges from 10⁻¹⁰ to 10⁻¹⁰. 6 It exhibits good denitrification, dephosphorization, and desulfurization capabilities at CFU / mL.

[0022] The present invention also provides the application of the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth in the production of protease, amylase, lipase and / or cellulase.

[0023] The present invention also provides the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth in Escherichia coli (Escherichia coli). Escherichia coli ),salmonella( Salmonella Staphylococcus aureus ( Staphylococcus aureus, S. aureus Clostridium perfringens ( ) Clostridium perfringens Aeromonas hydrophila ( ) Aeromonas hydrophila ) and / or Vibrio parahaemolyticus ( Vibrio Parahaemolyticus Applications of inhibition.

[0024] Compared with the prior art, the present invention has the following technical effects: (1) In addition to having good denitrification ability, Bacillus subtilis JBH-BK8 in this invention also has efficient dephosphorization and desulfurization ability.

[0025] (2) The Bacillus subtilis JBH-BK8 in this invention has a strong tolerance to high concentrations of nitrogen, phosphorus and sulfur in water, and can achieve efficient denitrification, dephosphorization and desulfurization even with low concentrations of bacteria.

[0026] (3) The Bacillus subtilis JBH-BK8 in this invention also has the ability to efficiently remove nitrogen, phosphorus and sulfur in water bodies with low temperature and low dissolved oxygen environment.

[0027] (4) The Bacillus subtilis JBH-BK8 in this invention has a strong ability to produce protease, amylase, lipase and cellulase.

[0028] (5) The Bacillus subtilis JBH-BK8 in this invention has antibacterial effects against Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Aeromonas hydrophila and Vibrio parahaemolyticus.

[0029] (6) The Bacillus subtilis JBH-BK8 in this invention can be used in the aquaculture industry to not only purify the aquaculture water and reduce the nitrogen and phosphorus content, but also regulate the balance of intestinal flora of farmed animals, promote growth, improve disease resistance and increase aquaculture benefits.

[0030] Preservation certificate statement: Bacillus subtilis JBH-BK8: Preservation institution: China Center for Type Culture Collection; Accession number: CCTCC NO: M 20232018; Deposit date: October 25, 2023; Location of collection: Wuhan University, Wuhan, China; Taxonomic nomenclature: Bacillus subtilis . Attached Figure Description

[0031] Figure 1 This is a colony morphology diagram of Bacillus subtilis JBH-BK8 in Example 1 of the present invention; Figure 2 This is a microscopic image of Bacillus subtilis JBH-BK8 in Example 1 of the present invention; Figure 3 The graph shows the metabolic kinetics growth curve and pH change curve of Bacillus subtilis JBH-BK8 in Example 1 of this invention. Figure 4 This is a phylogenetic tree of the 16S rRNA gene of Bacillus subtilis JBH-BK8 in Example 1 of the present invention; Figure 5 This is a graph showing the protease activity of Bacillus subtilis JBH-BK8 in Example 1 of the present invention; Figure 6 This is a graph showing the amylase production capacity of Bacillus subtilis JBH-BK8 in Example 1 of the present invention; Figure 7 This is a graph showing the lipase activity of Bacillus subtilis JBH-BK8 in Example 1 of the present invention; Figure 8 This is a graph showing the cellulase activity of Bacillus subtilis JBH-BK8 in Example 1 of the present invention; Figure 9 This is a graph showing the change in viable count and survival rate of Bacillus subtilis JBH-BK8 over time during the high-temperature resistance test in Example 1 of this invention. Figure 10 This is a graph showing the change in the survival rate of Bacillus subtilis JBH-BK8 over time at different pH levels in the acid resistance test of Bacillus subtilis JBH-BK8 in Example 1 of the present invention. Figure 11 This is a graph showing the changes in the survival rate and viable count of Bacillus subtilis JBH-BK8 in bile salt tolerance testing at different pig bile salt concentrations in Example 1 of this invention. Figure 12 This is a diagram showing the antibacterial results of Bacillus subtilis JBH-BK8 in Example 1 of the present invention. Detailed Implementation

[0032] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The equipment and raw materials used in the following embodiments are commercially available, and the methods used in the embodiments, unless otherwise specified, are consistent with conventionally used methods.

[0033] This invention provides a strain of Bacillus subtilis ( Bacillus subtilis The strain JBH-BK8 was isolated in 2022 from the silt of Pipa Temple Reservoir in Tangyin County, Anyang, Henan Province, and was classified as Bacillus subtilis. Bacillus subtilis JBH-BK8 was deposited at the China Center for Type Culture Collection on October 25, 2023, with accession number CCTCC NO: M 20232018.

[0034] The present invention also provides a Bacillus subtilis seed culture obtained by inoculating the Bacillus subtilis JBH-BK8 in a seed culture medium.

[0035] Preferably, the seed culture medium consists of the following components: 8-12 g / L peptone, 2-4 g / L beef extract, and 3-6 g / L sodium chloride; the culture conditions are: 35-40℃, 120-150 r / min overnight culture.

[0036] More preferably, the seed culture medium has the following components: 10 g / L peptone, 3 g / L beef extract, and 5 g / L sodium chloride.

[0037] The present invention also provides a Bacillus subtilis fermentation broth obtained by inoculating the Bacillus subtilis seed liquid into a fermentation medium at a volume ratio of 1-3% and culturing at 35-40°C and 120-150 r / min for 48-72 h.

[0038] Preferably, the carbon source of the fermentation medium is any one or more of glucose, sucrose, soybean meal, corn starch, and corn steep liquor; and the nitrogen source of the fermentation medium is any one or more of ammonium chloride, ammonium sulfate, potassium nitrate, yeast extract, peptone, and beef extract.

[0039] More preferably, the fermentation medium comprises the following components: soybean meal powder 5-7 g / L, corn starch 3-5 g / L, sucrose 0.5-1.5 g / L, calcium carbonate 0.3-0.5 g / L, yeast extract 0.04-0.06 g / L, ammonium sulfate 0.02-0.04 g / L, potassium nitrate 0.01-0.03 g / L, potassium dihydrogen phosphate 0.2-0.4 g / L, magnesium sulfate 0.2-0.3 g / L, corn steep liquor 0.2-0.3 g / L, sodium chloride 0.2-0.3 g / L, 5000 U / g neutral protease 0.1-0.2 g / L, and defoamer 0.1-0.2 g / L.

[0040] More preferably, the fermentation medium comprises the following components: 6 g / L soybean meal, 4 g / L corn starch, 1 g / L sucrose, 0.4 g / L calcium carbonate, 0.05 g / L yeast extract, 0.03 g / L ammonium sulfate, 0.02 g / L potassium nitrate, 0.3 g / L potassium dihydrogen phosphate, 0.23 g / L magnesium sulfate, 0.28 g / L corn steep liquor, 0.28 g / L sodium chloride, 0.17 g / L 5000 U / g neutral protease, and 0.15 g / L defoamer.

[0041] The present invention also provides a microbial agent prepared using the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth.

[0042] The present invention also provides the application of the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth in the purification of aquaculture water.

[0043] Preferably, the application is specifically in reducing the content of COD, nitrogen, phosphorus and / or sulfides in aquaculture water.

[0044] More preferably, the aquaculture species in the aquaculture water body include, but are not limited to, fish, shrimp, and crab.

[0045] The Bacillus subtilis JBH-BK8 of this invention achieves a denitrification rate of over 95% when using potassium nitrate, ammonium sulfate, and sodium nitrite as single nitrogen sources. Specifically, JBH-BK8 can perform denitrification within the concentration ranges of potassium nitrate (0.01~4500 PPM), ammonium sulfate (0.05~3000 PPM), and sodium nitrite (0.1~2500 PPM), respectively.

[0046] The Bacillus subtilis JBH-BK8 of this invention achieves a phosphorus removal rate of over 90% when using potassium dihydrogen phosphate, phosphorus pentoxide, and disodium hydrogen phosphate as single phosphorus sources. Specifically, JBH-BK8 can perform phosphorus removal within the concentration ranges of 0.05-3000 PPM for potassium dihydrogen phosphate, 0.01-4000 PPM for phosphorus pentoxide, and 0.05-2500 PPM for disodium hydrogen phosphate.

[0047] The Bacillus subtilis JBH-BK8 of this invention has a desulfurization rate of over 95% when using a sulfide standard solution as the sole sulfur source.

[0048] When the Bacillus subtilis JBH-BK8 of this invention is applied to aquatic environments with denitrification, dephosphorization, and desulfurization conditions, the pH of the aquaculture water is 6-8, the temperature is 0-50 ℃, and the dissolved oxygen is 0-15 mg / L. Within this pH, temperature, and dissolved oxygen range, its removal rate of nitrogen, phosphorus, and sulfur is all above 90%.

[0049] The inoculum size of Bacillus subtilis JBH-BK8 in this invention ranges from 10⁻¹⁰ to 10⁻¹⁰. 6 It exhibits good denitrification, dephosphorization, and desulfurization capabilities at CFU / mL.

[0050] The present invention also provides the application of the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth in the production of protease, amylase, lipase and / or cellulase.

[0051] The present invention also provides the Bacillus subtilis JBH-BK8, the Bacillus subtilis seed liquid and / or the Bacillus subtilis fermentation broth in Escherichia coli (Escherichia coli). Escherichia coli ),salmonella( Salmonella Staphylococcus aureus ( Staphylococcus aureus , S. aureus Clostridium perfringens ( ) Clostridium perfringens Aeromonas hydrophila ( ) Aeromonas hydrophila ) and / or Vibrio parahaemolyticus ( Vibrio Parahaemolyticus Applications of inhibition.

[0052] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0053] Example 1: Screening and Identification of JBH-BK8 (1) Sample processing: Silt from Pipa Temple Reservoir in Tangyin County, Anyang, Henan Province was collected aseptically, rinsed and mixed three times with 0.85% physiological saline, centrifuged, and then diluted 10 times in sequence. Different dilution gradients were selected for use.

[0054] (2) Isolation and culture: Prepare nutrient agar medium with the following components: peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, and agar 18 g / L. Autoclave at 121℃ for 20 min, cool to about 50℃ and pour into petri dishes. After the medium in the petri dishes has cooled, take the dilutions of different dilution gradients in (1) and spread them on RCM agar medium plates. Place them in a constant temperature and humidity incubator at 37℃ overnight for culture.

[0055] (3) Purification: Select single colonies of different morphologies under a stereomicroscope and transfer them to nutrient agar plates for purification.

[0056] (4) Preservation: Pick a purified single colony and inoculate it into a nutrient broth liquid medium without agar and incubate overnight at 37°C. Take 500 μL of culture medium and 50% sterile glycerol at a ratio of 1:1 (V / V) and store it in an ultra-low temperature freezer at -80°C.

[0057] (5) Activation: Inoculate the strain isolated and preserved in step (4) into a nutrient broth liquid medium without agar, activate at 37°C for 14-16 h, and then inoculate it into a nutrient broth liquid medium at a 2% inoculation amount, and incubate at 37°C for 14-16 h to obtain Bacillus subtilis culture medium.

[0058] (6) Separation: Centrifuge the culture medium from step (5) to collect the supernatant, filter the supernatant through a 0.22 μm microporous membrane, and store it in a refrigerator at 2-8℃ for later use.

[0059] (7) Screening: Add the pathogen indicator bacteria after 24 h of culture to LB medium, mix well and pour into plates. After cooling and solidification, punch 9 mm wells, 3 wells per plate, add 200 μL of the filtrate prepared in step (5) to each well, and culture at 37℃ for 14-16 h. Measure the diameter of the inhibition zone with vernier calipers and screen strains with excellent antibacterial performance based on the size of the inhibition zone diameter.

[0060] (8) Colony morphology: The selected Bacillus subtilis JBH-BK8 was spread on nutrient agar plates, and the culture results are as follows. Figure 1 As shown. From Figure 1 As can be seen, the strain forms white, irregularly sized colonies on the plate, which are flat, have irregular edges, and are about 1-3 mm in diameter. Figure 2 The results showed that the sample was Gram-positive, had spores, and the spores were oval in shape.

[0061] (9) Physiological and biochemical tests: The target strain was subjected to physiological and biochemical tests using micro-biochemical tubes. The physiological and biochemical characteristics of the strain were analyzed with reference to the "Common Bacterial Systematic Identification Manual" and "Berge's Manual of Bacterial Identification" to determine the species of the target strain. The 24-hour culture medium of the test bacteria was inoculated into 12 kinds of micro-fermentation tubes, including contact enzyme, arabinose, glucose, and xylose, with the opening facing down. The tubes were placed in a constant temperature and humidity incubator at 37°C for 24 hours. The results were observed and recorded. The results are shown in Table 1.

[0062] Table 1. Results of physiological and biochemical tests on strain JBH-BK8

[0063] Note: + indicates positive; - indicates negative.

[0064] As can be seen from Table 1, Bacillus subtilis strain JBH-BK8 can ferment and utilize 10 reagents other than indole and propionic acid. Based on the search of the "Handbook of Systematic Identification of Common Bacteria", it was preliminarily identified as Bacillus subtilis.

[0065] (10) Metabolic kinetics of JBH-BK8: Single colonies of JBH-BK8 were picked and inoculated into nutrient broth medium and cultured at 37℃ and 220 r / min for 14-16 h. The next day, an appropriate amount of culture solution was transferred to 100 mL of sterile nutrient broth at a ratio of 1% and cultured anaerobically at 37℃ and 220 r / min. The absorbance and pH of the bacterial culture solution were measured at 600 nm every 2 h. The growth curve and pH change curve of JBH-BK8 in nutrient broth medium are shown below. Figure 3 As shown.

[0066] Depend on Figure 3 It can be seen that JBH-BK8 grows well in nutrient broth medium, entering the logarithmic growth phase after 3-4 hours. During this period, the bacterial count increases rapidly. After maintaining the logarithmic growth phase for about 5 hours, JBH-BK8 enters the stationary growth phase, after which the bacterial count no longer increases, and the OD of the culture medium... 600 The value remains basically unchanged at around 1.7, and its pH value is stable between 6.5 and 6.8.

[0067] (11) Molecular biological identification of JBH-BK8: Using the genome of strain JBH-BK8 as a template, the gene sequence of approximately 1500 bp, as shown in SEQ ID NO. 1, was amplified by PCR using universal primers for bacterial 16S rRNA. A construct was then constructed as shown in [the original text]. Figure 4 The phylogenetic tree shown is an example of this.

[0068] JBH-BK8 16S rRNA sequence (SEQ ID NO. 1): Based on homology comparison, strain JBH-BK8 is similar to... Bacillus subtillis The homology was 100%, belonging to Bacillus subtilis. The JBH-BK8 strain and the Bacillus subtilis Wa-7 strain from Wuhan, China (GenBank accession number: OQ954758.1) clustered together with a confidence level of 99%, indicating that the two are most closely related.

[0069] (12) Enzyme production capacity: Select a single colony of JBH-BK8 and inoculate it into nutrient broth. Incubate at 37℃ and 220 r / min for 14-16 h. The next day, transfer an appropriate amount of culture medium at a ratio of 1% to 100 mL of sterile nutrient broth and incubate at 37℃ and 220 r / min for 14-16 h. Centrifuge 2 ml of bacterial solution at 5000 r / min for 15 min for later use. Determine the production capacity of JBH-BK8 for protease, amylase, lipase, and cellulase using the Oxford cup method. Observe the ratio of the diameter of the clear zone (D) around the colony to the diameter of the colony (C). The enzyme production capacity of JBH-BK8 is as follows: Figures 5-8 And as shown in Table 2.

[0070] Table 2. Enzyme activity of JBH-BK8

[0071] Note: "D" represents the average diameter of the hydrolysis zone, and "C" represents the average diameter of the JBH-BK8 bacterial moss.

[0072] The results showed that strain JBH-BK8 could produce protease, amylase, lipase and cellulase.

[0073] (13) Stress resistance test High-temperature resistance test: Fresh culture medium of JBH-BK8 was placed in a 250 mL Erlenmeyer flask and heated in an 85℃ water bath for 10 min. Samples were taken before heating, at 5 min, and at 10 min after heating to measure the viable count and calculate the survival rate. The results are as follows: Figure 9 As shown.

[0074] Acid resistance test: JBH-BK8 was inoculated at a rate of 1% into test tubes containing sterile phosphate buffer at pH 1.0, pH 2.0, and pH 3.0, respectively. The tubes were placed in an anaerobic incubator at 37 ℃, and viable cell counts were performed at 0 h, 1 h, 2 h, and 3 h. The survival rate was calculated, and the results are as follows: Figure 10 As shown.

[0075] Bile salt tolerance test: JBH-BK8 was inoculated at a 1% inoculum into nutrient broth containing 0.10%, 0.20%, 0.30%, 0.50%, and 1.00% porcine bile salts, respectively. Survival rates were calculated after 36 hours of incubation. The results are as follows: Figure 11 As shown.

[0076] The results showed that the survival rate of strain JBH-BK8 was above 90% after treatment at 85℃ for 5 min and 10 min. Even in a strongly acidic environment with a pH of 1.0, the survival rate of strain JBH-BK8 remained above 90%. After treatment for 36 h at a bile salt concentration below 0.3%, the survival rate of JBH-BK8 was above 90%, and at a bile salt concentration of 0.3%, the survival rate was also above 90%. At higher bile salt concentrations of 0.5% and 1.0%, the survival rate of strain JBH-BK8 remained above 85%.

[0077] (14) Antibacterial effect test Six pathogenic bacteria strains (Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Aeromonas hydrophila, and Vibrio parahaemolyticus) were evenly spread onto nutrient agar plates using sterilized medical cotton swabs (high-temperature, high-pressure sterilization). Then, 1 ml pipette tip (outer diameter: 9 mm) was used to make holes in the nutrient agar, and the plates were flame-sealed for later use. 200 μL of JBH-BK8 fermentation broth was added to the corresponding wells. The culture dishes containing the samples were placed in a 4°C refrigerator and allowed to stand for 2 hours before being transferred to a 37°C incubator for anaerobic culture of Clostridium perfringens plates. After 12 hours, the diameter of the inhibition zone in the plates was measured and recorded to determine the antibacterial effect of the tested samples. The antibacterial effect of strain BH-BK8 against seven different animal-derived pathogens is shown below. Figure 12 As shown.

[0078] The results showed that JBH-BK8 had a significant inhibitory effect on all six different pathogens, with the inhibitory ability in the following order: Clostridium perfringens type C > Vibrio parahaemolyticus > Aeromonas hydrophila > Clostridium perfringens type A > Salmonella typhimurium > Escherichia coli > Staphylococcus aureus.

[0079] Example 2: Degradation effect of JBH-BK8 on COD, nitrogen, phosphorus, and sulfur in artificially prepared polluted water. The artificially prepared wastewater contained the following components: ammonium sulfate 0.1 g / L, potassium nitrite 0.2 g / L, ammonium nitrate 0.2 g / L, sucrose 10 g / L, sodium dihydrogen phosphate 0.2 g / L, sodium sulfite 0.1 g / L, and magnesium sulfate 0.1 g / L. It contained ammonia nitrogen 82 mg / L, nitrite nitrogen 58 mg / L, nitrate nitrogen 72 mg / L, total phosphorus 59 mg / L, and total sulfides (calculated as hydrogen sulfide) 42 mg / L.

[0080] Using Bacillus subtilis CGMCC NO.19440 as a control strain, strain JBH-BK8 and strain Bacillus subtilis CGMCC NO.19440 were mixed at a ratio of 10... 3The final concentration of CFU / mL was added to artificially prepared wastewater as the experimental group, and an equal volume of physiological saline was added to the control group. Each group was repeated in 3 groups.

[0081] The ammonia nitrogen was detected using the indophenol blue colorimetric method, the nitrite nitrogen was detected using the diazotization coupling spectrophotometric method, the nitrate nitrogen was detected using the ultraviolet spectrophotometric method, the sulfide was detected using the methylene blue spectrophotometric method, and the total phosphorus was detected using the ammonium molybdate spectrophotometric method. The degradation rates of these indicators were calculated.

[0082] Experiments were conducted on JBH-BK8 and Bacillus subtilis CGMCC NO.19440 strains at different nitrogen source tolerance concentration ranges of 0.01~0.05, 0.1~1.0, 5~10, 10~50, 50~200, 500~1000, 1500~2500, 3000~4000, and 4000~4500 PPM.

[0083] Under culture conditions of pH 6.8, temperature 27℃, and dissolved oxygen 7 mg / L, JBH-BK8 and Bacillus subtilis CGMCC NO.19440 strains were inoculated into 100 mL of the above-mentioned artificially prepared wastewater and cultured with shaking at 150 r / min. The contents of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen were measured every 12 h, and the measurement was continued until 72 h. The test results are shown in Table 3.

[0084] Table 3. Degradation capacity of nitrogen, phosphorus, and sulfur by strains JBH-BK8 and CGMCC NO.19440 under different nitrogen, phosphorus, and sulfur concentrations.

[0085] As shown in Table 3, JBH-BK8 has different performance in various COD levels. It exhibits good removal effects of nitrogen, phosphorus, and sulfur in the concentration ranges of nitrogen, phosphorus, and sulfur sources, with removal rates all above 90%. It can withstand COD 4000 PPM, ammonia nitrogen 3000 PPM, nitrate nitrogen 4500 PPM, nitrite nitrogen 2500 PPM, total phosphorus 3000 PPM, and hydrogen sulfide 4000 PPM.

[0086] Example 3: Degradation effect of JBH-BK8 on COD, nitrogen, phosphorus, and sulfur under different temperature conditions The artificially prepared wastewater contained the following components: ammonium sulfate 0.1 g / L, potassium nitrite 0.2 g / L, ammonium nitrate 0.2 g / L, sucrose 10 g / L, sodium dihydrogen phosphate 0.2 g / L, sodium sulfite 0.1 g / L, and magnesium sulfate 0.1 g / L. It contained ammonia nitrogen 82 mg / L, nitrite nitrogen 58 mg / L, nitrate nitrogen 72 mg / L, total phosphorus 59 mg / L, and total sulfides (calculated as hydrogen sulfide) 42 mg / L.

[0087] Under culture conditions of 0, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50℃, with Bacillus subtilis CGMCC NO.19440 as the control strain, strains JBH-BK8 and Bacillus subtilis CGMCC NO.19440 were cultured at 10℃. 3 The final concentration of CFU / mL was added to 100 mL of artificially prepared wastewater as the experimental group and cultured with shaking at 150 r / min. A control group with an equal volume of physiological saline was also included, with three replicates for each group. Ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen levels were measured every 12 h, continuing for up to 72 h. The results are shown in Table 4.

[0088] Table 4. Degradation capacity of JBH-BK8 strain and CGMCC NO.19440 for COD, nitrogen, phosphorus and sulfur under different temperature conditions.

[0089] The results in Table 4 show that strain JBH-BK8 exhibited a degradation capacity of over 90% for COD, nitrogen, phosphorus, and sulfur across temperature ranges of 0, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50℃, with the highest degradation capacity for all three at 25℃. This indicates that changes in water temperature have a relatively small impact on the degradation capacity of JBH-BK8 for COD, nitrogen, phosphorus, and sulfur. In contrast, CGMCC NO.19440 showed lower degradation capacity for COD, nitrogen, phosphorus, and sulfur compared to JBH-BK8 at different temperature gradients.

[0090] Example 4: Degradation effect of JBH-BK8 on COD, nitrogen, phosphorus, and sulfur under different dissolved oxygen conditions The artificially prepared wastewater contained the following components: ammonium sulfate 0.1 g / L, potassium nitrite 0.2 g / L, ammonium nitrate 0.2 g / L, sucrose 10 g / L, sodium dihydrogen phosphate 0.2 g / L, sodium sulfite 0.1 g / L, and magnesium sulfate 0.1 g / L. It contained ammonia nitrogen 82 mg / L, nitrite nitrogen 58 mg / L, nitrate nitrogen 72 mg / L, total phosphorus 59 mg / L, and total sulfides (calculated as hydrogen sulfide) 42 mg / L.

[0091] The dissolved oxygen content in artificially prepared wastewater was adjusted by controlling the rotation speed of the constant-temperature shaker and the volume of liquid in the Erlenmeyer flask, and the dissolved oxygen content was measured using a dissolved oxygen meter. Under different dissolved oxygen conditions, Bacillus subtilis CGMCC NO.19440 was used as a control strain, and strains JBH-BK8 and Bacillus subtilis CGMCC NO.19440 were respectively subjected to 10... 3 A final concentration of CFU / mL was added to 100 mL of artificially prepared wastewater as the experimental group and cultured by shaking at 150 r / min. The contents of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen were measured every 12 h, and the measurement was continued until 72 h. The test results are shown in Table 5.

[0092] Table 5. Degradation capacity of JBH-BK8 and CGMCC NO.19440 for COD, nitrogen, phosphorus, and sulfur under different dissolved oxygen levels.

[0093] The results in Table 5 show that within the dissolved oxygen range of 0–15 PPM, strain JBH-BK8 achieved degradation rates of over 90% for COD, nitrogen, phosphorus, and sulfur in wastewater. Furthermore, strain JBH-BK8 also maintained degradation rates of over 90% for COD, nitrogen, phosphorus, and sulfur in wastewater when dissolved oxygen levels were between 0 and 3 PPM. Moreover, the degradation rates increased slightly with increasing dissolved oxygen levels. Therefore, dissolved oxygen levels had a relatively small impact on strain JBH-BK8. In contrast, CGMCC NO.19440 exhibited lower degradation capabilities for COD, nitrogen, phosphorus, and sulfur than JBH-BK8 under different dissolved oxygen levels.

[0094] Example 5: Effects of different inoculum amounts of JBH-BK8 on the degradation of COD, nitrogen, phosphorus, and sulfur. The artificially prepared wastewater contained the following components: ammonium sulfate 0.1 g / L, potassium nitrite 0.2 g / L, ammonium nitrate 0.2 g / L, sucrose 10 g / L, sodium dihydrogen phosphate 0.2 g / L, sodium sulfite 0.1 g / L, and magnesium sulfate 0.1 g / L. It contained ammonia nitrogen 82 mg / L, nitrite nitrogen 58 mg / L, nitrate nitrogen 72 mg / L, total phosphorus 59 mg / L, and total sulfides (calculated as hydrogen sulfide) 42 mg / L.

[0095] The culture conditions for the reaction system were set as follows: pH 6.8, temperature 27℃, and dissolved oxygen 7 mg / L. Bacillus subtilis CGMCC NO.19440 was used as a control strain. JBH-BK8 strain and Bacillus subtilis CGMCC NO.19440 strain were inoculated at different amounts (10... 1 10 2 10 3 10 4 10 5 10 6Ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen content were added to 100 mL of the artificially prepared wastewater (CFU / mL) and cultured with shaking at 150 r / min. The levels of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen were measured every 12 h, continuing for up to 72 h. A control group with added physiological saline was also included, with three replicates per group. The results are shown in Table 6.

[0096] Table 6. Degradation capacity of JBH-BK8 and CGMCC NO.19440 for COD, nitrogen, phosphorus, and sulfur under different inoculum levels.

[0097] The results in Table 6 show that strain JBH-BK8 maintained a degradation rate of over 80% for COD, nitrogen, phosphorus, and sulfur within an inoculum size range of 101–106 CFU / mL. Therefore, strain JBH-BK8 can achieve good degradation rates for COD, nitrogen, phosphorus, and sulfur in wastewater even at relatively low inoculum sizes. Furthermore, the degradation rates for COD, nitrogen, phosphorus, and sulfur increased with increasing inoculum size. In contrast, CGMCC NO. 19440 showed lower degradation capabilities for COD, nitrogen, phosphorus, and sulfur than JBH-BK8 under different inoculum size gradients.

[0098] Example 6: The degradation effect of JBH-BK8 on COD, nitrogen, phosphorus, and sulfur in actual aquaculture production. Healthy, uniformly sized largemouth bass (initial weight 50 ± 0.5 g) of good condition were selected. They were randomly divided into two groups: a control group and an experimental group, with three replicates per group. They were housed in circular aquariums with a diameter of 1.5 m and a height of 1 m, at a density of 50 fish per tank.

[0099] Before the experiment, tilapia fry were pre-raised and observed in aquariums for one week. For the first three days of the experiment, COD, nitrogen, phosphorus, and sulfur levels were measured daily. Once the values ​​were relatively stable, the experiment could begin, and these values ​​were taken as the initial COD, nitrogen, phosphorus, and sulfur levels.

[0100] Using Bacillus subtilis CGMCC NO.19440 as a control strain, strains JBH-BK8 and Bacillus subtilis CGMCC NO.19440 were respectively diluted at 10... 3 A final concentration of CFU / mL was added to the experimental group's aquariums. The water quality was maintained without change during the experiment, and the fish were fed three times a day, with a 4-hour interval between feedings. The experiment lasted 10 days, and water samples were taken every other day to test the COD, nitrogen, phosphorus, and sulfur content. A control group without added bacteria was also included. The test results are shown in Table 7.

[0101] Table 7. Changes in COD, nitrogen, phosphorus, and sulfur content in the water of the experimental group after inoculation with JBH-BK8 strain and CGMCC NO.19440.

[0102] The results in Table 7 show that strain JBH-BK8 achieved degradation rates of over 80% for COD, nitrogen, phosphorus, and sulfur in the aquaculture water during actual aquaculture trials. Within a 10-day period, the levels of COD, nitrogen, phosphorus, and sulfur in the experimental water all exhibited a significant and stable decreasing trend. Furthermore, no fish fry mortality was observed. In contrast, CGMCC NO.19440, under the same experimental conditions, showed inferior degradation capabilities for COD, nitrogen, phosphorus, and sulfur compared to JBH-BK8. Therefore, strain JBH-BK8 possesses excellent degradation capabilities for COD, nitrogen, phosphorus, and sulfur, making it suitable for use in actual aquaculture.

[0103] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis JBH-BK8, characterized in that, The Bacillus subtilis JBH-BK8 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232018.

2. A Bacillus subtilis seed liquid, characterized in that, It was obtained by inoculating Bacillus subtilis JBH-BK8 as described in claim 1 into a seed culture medium.

3. The Bacillus subtilis seed liquid according to claim 2, characterized in that, The seed culture medium consists of the following components: 8-12 g / L peptone, 2-4 g / L beef extract, and 3-6 g / L sodium chloride; the culture conditions are: 35-40℃, 120-150 r / min overnight culture.

4. A Bacillus subtilis fermentation broth, characterized in that, The fermentation medium is obtained by inoculating the Bacillus subtilis seed liquid of claim 3 into the fermentation medium at a volume ratio of 1-3% and culturing at 35-40°C and 120-150 r / min for 48-72 h; the carbon source of the fermentation medium is any one or more of glucose, sucrose, soybean meal, corn starch, and corn steep liquor; the nitrogen source of the fermentation medium is any one or more of ammonium chloride, ammonium sulfate, potassium nitrate, yeast extract, peptone, and beef extract.

5. The Bacillus subtilis fermentation broth according to claim 4, characterized in that, The fermentation medium consists of the following components: soybean meal 5-7 g / L, corn starch 3-5 g / L, sucrose 0.5-1.5 g / L, calcium carbonate 0.3-0.5 g / L, yeast extract 0.04-0.06 g / L, ammonium sulfate 0.02-0.04 g / L, potassium nitrate 0.01-0.03 g / L, potassium dihydrogen phosphate 0.2-0.4 g / L, magnesium sulfate 0.2-0.3 g / L, corn steep liquor 0.2-0.3 g / L, sodium chloride 0.2-0.3 g / L, 5000 U / g neutral protease 0.1-0.2 g / L, and defoamer 0.1-0.2 g / L.

6. A microbial agent prepared using Bacillus subtilis JBH-BK8 as described in claim 1, Bacillus subtilis seed liquid as described in any one of claims 2 to 3, and / or Bacillus subtilis fermentation broth as described in any one of claims 4 to 5.

7. The application of Bacillus subtilis JBH-BK8 as described in claim 1, Bacillus subtilis seed liquid as described in any one of claims 2 to 3, and / or Bacillus subtilis fermentation broth as described in any one of claims 4 to 5 in the purification of aquaculture water.

8. The application according to claim 7, characterized in that, The specific application is in reducing the content of COD, nitrogen, phosphorus and / or sulfides in aquaculture water.

9. The use of Bacillus subtilis JBH-BK8 as described in claim 1, Bacillus subtilis seed liquid as described in any one of claims 2 to 3, and / or Bacillus subtilis fermentation broth as described in any one of claims 4 to 5 in the production of protease, amylase, lipase, and / or cellulase.

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