Bacillus paralicheniformis strain bsl-10 and application thereof

Abstract

The application discloses a bacillus paralicheniformis BSL-10 and application thereof, and belongs to the technical field of microorganisms.A bacillus paralicheniformis BSL-10 is preserved in the China Center for Type Culture Collection on May 13, 2026, and the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 2026946.The BSL-10 has high acetic acid yield and excellent comprehensive denitrification capacity, especially high nitrite degradation efficiency.The bacteria can be made into various dosage forms of bacterial agents, applied to livestock and aquaculture, can promote animal growth, improve feed conversion rate, enhance body immunity, replace antibiotics in breeding, and can stably reduce ammonia nitrogen and nitrite nitrogen in breeding water, and purify water quality, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus paralichrysum BSL-10 and its applications, and more specifically, to a strain of Bacillus paralichrysum BSL-10 that can produce high levels of acetic acid, efficiently degrade nitrite, and simultaneously degrade ammonia nitrogen and nitrate while reducing total nitrogen in the system, as well as the application of this strain in animal husbandry and aquaculture water purification. Background Technology

[0002] Bacillus paralicheniformis is a species of Bacillus that is distributed in natural environments such as soil and plant roots. Some strains can secrete antibacterial active substances and various extracellular enzymes, showing good application potential in the field of biological feed. Acetic acid, as an important short-chain fatty acid, can provide energy for animal intestinal epithelial cells, lower intestinal pH, inhibit the proliferation of harmful pathogenic bacteria in the intestine, and is a high-quality microbial metabolic product.

[0003] The long-term overuse of antibiotics in large-scale livestock, poultry, and aquaculture easily induces the growth of drug-resistant bacteria and excessive drug residues in livestock and poultry products. Developing probiotic feed additives that can replace antibiotics has become a hot research topic in the industry. Currently, commercially available Bacillus strains are mainly Bacillus subtilis and Bacillus licheniformis, but most have the drawback of weak acetic acid production. Meanwhile, Bacillus coagulans, which produces high levels of lactic acid, lacks denitrification capabilities in aquatic environments, resulting in limited functional diversity and difficulty in simultaneously addressing animal gut health and aquaculture water quality control.

[0004] During aquaculture, the continuous decomposition of uneaten feed and animal feces leads to the accumulation of ammonia nitrogen and nitrite in the water. Nitrite is highly toxic and can impair the oxygen-carrying capacity of fish and shrimp blood, significantly reducing the survival rate of aquaculture. Most existing denitrifying Bacillus species are only adapted to single aerobic or anaerobic environments. Under anaerobic conditions, the efficiency of nitrite degradation decreases significantly, resulting in obvious limitations in their application. Summary of the Invention

[0005] Therefore, this invention provides a strain of Bacillus paralicheniformis BSL-10 and its application to solve the problem that existing Bacillus strains cannot simultaneously achieve the dual functions of high acetic acid production and efficient degradation of nitrite and ammonia nitrogen in water.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides a strain of Bacillus paralicheniformis BSL-10, which was deposited on May 13, 2026 at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2026946.

[0008] According to a second aspect of the present invention, the present invention provides a microbial inoculant containing Bacillus paralicheniformis and / or its fermentation metabolites as described above.

[0009] Furthermore, the microbial agent is a liquid fermentation broth, a freeze-dried powder, or a solid microbial agent, with the live count of the liquid microbial agent being ≥1×10^9 CFU / mL and the live count of the solid microbial agent being ≥2×10^10 CFU / g.

[0010] According to a third aspect of the present invention, the present invention provides a method for preparing the microbial agent as described above, the method comprising: inoculating the BSL-10 strain as described above into a liquid seed culture medium and culturing it at 37°C and 200 rpm for 12 h to obtain a seed liquid; transferring the seed liquid into a fermentation culture medium at a volume inoculation rate of 5% and fermenting at 37°C for 36-48 h; terminating fermentation when the spore formation rate is ≥90% to obtain a fermentation broth with a viable count exceeding 1×10^9 CFU / mL, wherein the liquid seed culture medium comprises: 10 g / L peptone, 5 g / L yeast extract, 15 g / L NaCl, 2 g / L glucose, pH 7.0; and the fermentation culture medium comprises: 20 g / L glucose, 15 g / L soybean meal powder, 2 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, pH 7.0.

[0011] According to a fourth aspect of the present invention, the present invention provides the use of Bacillus paralichrysogenum as described above, or the microbial agent as described above, in the preparation of animal feed additives.

[0012] Furthermore, the animals include livestock, poultry, and aquatic animals; the feed additives are used to improve animal gut health, inhibit intestinal pathogens, improve feed conversion rate, promote animal growth, and enhance animal immunity.

[0013] Furthermore, the dosage of solid microbial agents added to livestock and poultry feed is 200-400 g / t of basal diet; the dosage of solid microbial agents applied to aquaculture water is 0.3-0.5 g / m³. 3 Spray once every 3 days.

[0014] According to a fifth aspect of the present invention, the present invention provides the use of Bacillus paralichrysum as described above, or the microbial agent as described above, in the preparation of products that degrade nitrite in water.

[0015] Furthermore, the nitrite-degrading product is suitable for seawater and freshwater aquaculture waters.

[0016] According to a sixth aspect of the present invention, the present invention provides the application of Bacillus paralicheniformis as described above, or microbial agents as described above, in the improvement of saline-alkali soil.

[0017] The embodiments of the present invention have the following advantages:

[0018] This invention relates to Bacillus paralichrysogenus BSL-10, which produces high levels of acetic acid, improving the intestinal microenvironment of animals and regulating the pH of water. It also possesses excellent comprehensive denitrification capabilities, degrading ammonia nitrogen, nitrite, and nitrate while reducing total nitrogen. Its nitrite degradation efficiency is particularly high, exceeding 99.9% under both aerobic and anaerobic conditions within 24 hours. This bacterium can be formulated into various formulations for use in livestock and aquaculture. It can promote animal growth, improve feed conversion rates, enhance immunity, replace antibiotics in aquaculture, and steadily reduce ammonia nitrogen and nitrite nitrogen in aquaculture water, purifying the water quality. It has promising application prospects. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0020] Figure 1 The colony morphology diagram of BSL-10 provided by this invention;

[0021] Figure 2 The phylogenetic tree of BSL-10 provided by this invention;

[0022] Figure 3 The liquid chromatogram of the lactic acid standard solution provided by this invention;

[0023] Figure 4 The liquid chromatogram of the acetic acid standard solution provided by this invention;

[0024] Figure 5 The liquid chromatogram of BSL-10 fermentation products provided by this invention;

[0025] Figure 6 The figure shows the growth and denitrification results of BSL-10 in nitrite medium provided by this invention;

[0026] Figure 7 The figure shows the growth and denitrification results of BSL-10 in ammonia nitrogen medium provided by this invention;

[0027] Figure 8 The figure shows the growth and denitrification results of BSL-10 in potassium nitrate medium provided by this invention;

[0028] Figure 9The figure shows the growth and denitrification results of BSL-10 in a mixed nitrogen source medium provided by this invention;

[0029] Figure 10 The graph shows the changes in total nitrogen, intracellular nitrogen, and pH of BSL-10 provided by this invention when cultured in a mixed nitrogen source medium.

[0030] Figure 11 The effect of BSL-10 provided by this invention on ammonia nitrogen in Litopenaeus vannamei culture water;

[0031] Figure 12 The effect of BSL-10 provided by this invention on nitrite nitrogen in Litopenaeus vannamei culture water;

[0032] Figure 13 The effect of BSL-10 provided by this invention on nitrate nitrogen in Litopenaeus vannamei culture water. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1 Isolation and Identification of Strains BSL-10

[0035] The strain of this invention was isolated from the leaf litter and silt surrounding the Bali Hot Spring in Houqiao Village, Houqiao Town, Tengchong City, Baoshan City, Yunnan Province. The silt sample was serially diluted and plated onto LB agar plates with calcium carbonate (culture medium formulation: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 2 g / L glucose, 5 g / L calcium carbonate, pH 6.8), and incubated at 37°C for 24 h. The single colony with the largest transparent calcium dissolution zone was selected and streaked three times to obtain pure cultured candidate Bacillus.

[0036] Each purified strain was inoculated into liquid fermentation medium (LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, glucose 2 g / L, pH 6.8), and fermented at 37℃ and 200 rpm for 72 h with shaking. An appropriate amount of fermentation broth was centrifuged at 5000 r / min for 10 min, and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the contents of organic acids and lactic acid were detected by high-performance liquid chromatography. Strains with acetic acid content more than 10 times that of lactic acid were selected as high-acetic acid production target strains.

[0037] The strain grew well in LB medium, producing white colonies with extracellular secretions; under a light microscope, the bacteria were rod-shaped and capable of forming spores. Figure 2 ).

[0038] The 16S rRNA gene sequence of the strain is as follows (sequencing by Wuhan Tianyi Huayu Gene Technology Co., Ltd.):

[0039]

[0040] A Neighbor-Joining phylogenetic tree was constructed using Marinococcus halophilus DSM 20408 (GenBank accession number X90835.1) as the outgroup. Further genome-wide homology analysis was performed between the tested strain and the model strain of Bacillus paralicheniformis KJ-16. The results showed an ANI value of 99.31%, higher than the homology threshold of 96%; and a dDDH value of 94.3%. Therefore, this strain was identified as Bacillus paralicheniformis and named BSL-10.

[0041] This strain was deposited on May 13, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2026946.

[0042] Example 2 Preparation of microbial agent

[0043] BSL-10 strain was inoculated into liquid seed culture medium (10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 2 g / L glucose, pH 7.0) and cultured at 37℃ and 200 rpm for 12 h to obtain seed culture. The seed culture was then transferred to fermentation medium (20 g / L glucose, 15 g / L soybean meal, 2 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, pH 7.0) at a 5% volume inoculation rate and fermented at 37℃ for 36-48 h. Fermentation was terminated when the spore formation rate in the fermentation system was ≥90%, yielding a fermentation broth with a viable cell count exceeding 1 × 10^9 CFU / mL.

[0044] This fermentation broth can be used directly, or it can be centrifuged, freeze-dried into powder, or made into solid inoculum using bran, maltose, etc. as adsorbent carriers.

[0045] Test Example 1: Determination of the ability of Bacillus paralichrysiforme BSL-10 to produce acetic acid and lactic acid

[0046] 1. Materials and Methods

[0047] 1.1 Test strains

[0048] Bacillus paralicheniformis BSL-10, Bacillus coagulans BSL-9, and Bacillus licheniformis BSL-3 were all provided by Hubei Lvtiandi Biotechnology Co., Ltd.

[0049] 1.2 Culture Media and Main Reagents

[0050] NA liquid culture medium, refer to national standard GB20287-2006;

[0051] MRS liquid culture medium, referencing Hubei Lvtiandi Enterprise Standard Q / LTD09-2014;

[0052] 0.1% phosphoric acid solution: Accurately pipette 1.00 mL of phosphoric acid, dissolve it in deionized water, and dilute to 1000 mL;

[0053] Acetic acid standard solution: Accurately weigh 0.2 g of glacial acetic acid solution into a 500 mL volumetric flask to prepare a standard solution with a concentration of 400 mg / L;

[0054] Lactic acid standard solution: Weigh approximately 3.0 g of lactic acid into a 500 mL round-bottom flask, add 270 mL of water, attach a spherical condenser, connect to cooling water, heat under reflux for 15 h, and cool to room temperature. The lactic acid content is determined according to Appendix A of GB / T23877-2009 to be 9.13 g / L.

[0055] Acetonitrile was purchased from Honeywell, and other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0056] 1.3 Main Instruments

[0057] Balance, Ohaus Instruments (Shanghai) Co., Ltd.; Visible spectrophotometer, Shanghai Precision Scientific Instruments Co., Ltd.; Shaker, Harbin Donglian Electronic Technology Development Co., Ltd.; Centrifuge, Eppendorf; Liquid Chromatograph, Shimadzu Corporation, Japan.

[0058] 1.4 Preparation of Fermentation Products

[0059] Bacillus paralichrysogenus BSL-10 was inoculated into 100 mL of NA liquid medium, while Bacillus coagulans BSL-9 and Bacillus lichrysogenus BSL-3 were inoculated into two separate 100 mL aliquots of MRS liquid medium. All cultures were incubated at 37°C and 200 rpm in a shaker for 48 h. The OD values ​​of each bacterial suspension were measured using a visible spectrophotometer. 600 The bacterial suspension was then inoculated into 100 mL of the corresponding liquid culture medium, and the inoculation amount was determined according to the OD value to ensure that the bacterial content in the culture medium was approximately the same. The culture was carried out at 37℃ and 200 rpm, and samples were taken at 72 h and 120 h, respectively. The samples were centrifuged at 12000 rpm and filtered through a 0.22 μm filter membrane before analysis.

[0060] 1.5 Detection methods for acetic acid and lactic acid

[0061] The determination of acetic acid content in bamboo pre-hydrolyzed solution was performed using high-performance liquid chromatography (HPLC) according to the standard GB / T23877-2009 "Determination of Citric Acid, Fumaric Acid and Lactic Acid in Feed Acidifiers" and the reference Li Guang et al., 2013. HPLC conditions were as follows: column: C18 column, 300 mm long, 4.6 mm inner diameter, 5 μm particle size (polar column); column temperature: 35℃; mobile phase: acetonitrile and 0.1% phosphoric acid solution at a volume ratio of 2.5:97.5; flow rate: 0.5 mL / min; detection wavelength: 210 nm; injection volume: 20 μL. Standard solutions and sample solutions were taken separately and analyzed by HPLC under the above conditions.

[0062] 2 Results and Analysis

[0063] 2.1 Spectra of Standard Solutions and Sample Solutions

[0064] The liquid chromatograms of the standard solutions of lactic acid and acetic acid are shown below. Figure 3 and Figure 4 The liquid chromatogram of the BSL-10 fermentation products is shown below. Figure 5 .

[0065] 2.2 Acid production capacity of the strain

[0066] After culturing and fermenting the three strains for 72 h and 120 h, samples were taken to determine the content of lactic acid and acetic acid in the fermentation broth. The results are shown in Table 1 and Table 2.

[0067] Table 1. Lactic acid production capacity of strains (g / L)

[0068]

[0069] Table 2 Acetic acid production capacity of strains (g / L)

[0070]

[0071] The results showed that the dominant metabolite of Bacillus paralichrysum BSL-10 was acetic acid, and its acetic acid production at 72 h was significantly higher than that of other strains; the dominant metabolite of Bacillus coagulans BSL-9 was lactic acid, and Bacillus paralichrysum BSL-3 had a balanced ability to produce both lactic acid and acetic acid.

[0072] Test Example 2: Denitrification Performance Determination of Bacillus paralichrysiforme BSL-10

[0073] 1. Materials and Methods

[0074] 1.1 Test strains

[0075] The Bacillus paralicheniformis BSL-10, Bacillus bereaves BSL-1, Bacillus licheniformis BSL-3, Bacillus subtilis BSL-5, Bacillus coagulans BSL-12, and Bacillus pumilus BSL-44 were all provided by Hubei Lvtiandi Biotechnology Co., Ltd.

[0076] 1.2 Test Equipment

[0077] Constant temperature shaker, constant temperature incubator, spectrophotometer, pipette.

[0078] 1.3 Culture medium

[0079] 1.3.1 Microbial slant culture medium

[0080] LB medium: Tryptone 10g / L, yeast extract 5g / L, sodium chloride (NaCl) 10g / L, distilled water to a final volume of 1000mL, pH adjusted to 7.0-7.2, dispensed into 500mL Erlenmeyer flasks, 50mL per flask, sealed with 12 layers of gauze, and sterilized at 121℃ for 30min.

[0081] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and distilled water to a final volume of 1000 ml. Adjust the pH to 7.0-7.2, dispense 50 mL into 500 mL Erlenmeyer flasks, cover each flask with 12 layers of gauze, and sterilize at 118°C for 20 min.

[0082] 1.3.2 Denitrification media with different nitrogen sources

[0083] Basic denitrification medium: sodium succinate 2.0 g / L, potassium nitrate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 1 g / L, pH 7.0.

[0084] Single nitrogen source medium: When testing the degradation of nitrate nitrogen by the strain, replace the 500 mg / L potassium nitrate in the basic denitrification medium with 432 mg / L potassium nitrate; when testing the degradation of nitrite nitrogen by the strain, replace the 500 mg / L potassium nitrate in the basic denitrification medium with 300 mg / L sodium nitrite; when testing the degradation of ammonia nitrogen by the strain, replace the 500 mg / L potassium nitrate in the basic denitrification medium with 210 mg / L ammonium chloride.

[0085] Replace the 500 mg / L potassium nitrate in the basic denitrification medium with 144 g / L potassium nitrate, 100 g / L sodium nitrite, and 70 g / L ammonium chloride.

[0086] All culture media were dispensed into 500mL Erlenmeyer flasks, with 50mL dispensed into each flask. The flasks were then sealed with 12 layers of gauze and sterilized at 121℃ for 20 minutes.

[0087] 1.4 Inoculation and Culture Methods

[0088] 1.4.1 Inoculation and culture method of the test strains: Bacillus coagulans was inoculated into YDP shake flasks and cultured at 45℃ and 200rpm for about 6 hours; the remaining Bacillus strains were inoculated into LB shake flasks and cultured at 30℃ and 200rpm to the logarithmic phase (about 8 hours).

[0089] 1.4.2 Method for inoculating denitrification media with different nitrogen sources: Transfer 0.5 mL of the bacterial culture of various test strains cultured in shake flasks to nitrate / nitrite / ammonium chloride / mixed nitrogen source shake flasks using a sterile pipette. Inoculate 4 shake flasks for each strain.

[0090] The various test bacteria were divided into two groups (4 bottles in total). Two bottles were placed on a shaker at 30℃ and cultured at 200 rpm for 24 hours (aerobic culture). The other two bottles were placed in an incubator at 30℃ and cultured statically for 24 hours (anaerobic culture).

[0091] 1.5 Determination methods for ammonia nitrogen, nitrite, nitrate, and total nitrogen

[0092] Ammonia nitrogen was determined using the indophenol blue spectrophotometric method, nitrate nitrogen using the phenol disulfonic acid spectrophotometric method (GB / T 7480-1987), and nitrite nitrogen using the naphthylethylenediamine spectrophotometric method (GB / T 7493-1987). The operating procedures were performed in accordance with the "Marine Monitoring Specifications" (GB17378-2007) and the "Marine Survey Specifications" (GB / T 12763-2007).

[0093] 1.5.1 Growth and denitrification function of Bacillus paralichrysiforme BSL-10 in nitrite medium

[0094] Bacillus paralichrysogenum BSL-10 was cultured in a denitrification medium with sodium nitrite as the sole nitrogen source. Samples were taken at 12h, 24h, 48h, 72h, and 96h to determine the OD of the bacterial culture. 600 The residual nitrite content in the supernatant was measured after centrifugation at 5000 r / min for 5 min, and the degradation rate was calculated.

[0095] 1.5.2 Growth and denitrification function of Bacillus paralichrysiforme BSL-10 in ammonia nitrogen medium

[0096] Bacillus paralichrysogenum BSL-10 was cultured in a denitrification medium with ammonium chloride as the sole nitrogen source. Samples were taken at 12h, 24h, 48h, 72h, and 96h to determine the OD of the bacterial culture. 600 The ammonia nitrogen content in the supernatant was measured after centrifugation at 5000 r / min for 5 min, and the degradation rate was calculated.

[0097] 1.5.3 Growth and denitrification function of Bacillus paralichrysogenum BSL-10 in nitrate medium

[0098] Bacillus paralichrysogenum BSL-10 was cultured in shake flasks in a denitrification medium with potassium nitrate as the sole nitrogen source. Samples were taken at 12h, 24h, 48h, 72h, and 96h to determine the OD of the bacterial culture. 600 The residual nitrate content in the supernatant was measured after centrifugation at 5000 r / min for 5 min, and the degradation rate was calculated.

[0099] 1.5.4 Denitrification characteristics of Bacillus paralichrysiformis BSL-10 in mixed nitrogen sources

[0100] Bacillus paralichrysum BSL-10 was cultured in a denitrification medium with a mixed nitrogen source in shake flasks. Samples were taken at 12h, 24h, 48h, 72h, and 96h to measure the pH and OD values ​​of the medium. 600 The total nitrogen content (including bacterial cells) was measured. The contents of total nitrogen, nitrite, ammonia nitrogen, and nitrate in the supernatant after centrifugation at 5000 rpm for 5 min were determined. The degradation rates of nitrite, ammonia nitrogen, nitrate, and total nitrogen in the culture medium, as well as the pH and OD values, were calculated. 600 Changes in values ​​and intracellular nitrogen.

[0101] The denitrification performance of Bacillus belyssus BSL-1, Bacillus licheniformis BSL-3, Bacillus subtilis BSL-5, Bacillus coagulans BSL-12, and Bacillus pumilus BSL-44 was tested according to the above method, with uninoculated culture medium as the control (NA).

[0102] 1.6 Data Statistics and Analysis

[0103] Minitab 21.3.1 statistical software was used to perform one-way ANOVA and LSD multiple comparisons on each indicator.

[0104] 2 Results and Analysis

[0105] 2.1 The ability of Bacillus paralicheniformis BSL-10 and other Bacillus species to degrade nitrite

[0106] The results in Table 3 show that under aerobic (shaking culture) conditions, Bacillus paralichrysum BSL-10 degraded nitrite at a rate of 99.994%; under anaerobic (static culture) conditions, Bacillus paralichrysum BSL-10 degraded nitrite at a rate of 99.942%, demonstrating significantly better degradation performance than other Bacillus species.

[0107] Table 3. Degradation of nitrite by different Bacillus species

[0108]

[0109] Note: Different lowercase letters after the data in the same column indicate significant differences between groups (P < 0.05), while the same lowercase letter indicates no significant differences between groups (P > 0.05).

[0110] 2.2 Growth and denitrification function of Bacillus paralichrysiforme BSL-10 in a single nitrogen source

[0111] 2.2.1 Growth and denitrification function of Bacillus paralichrysiforme BSL-10 in nitrite medium

[0112] Bacillus paralichrysogenum BSL-10 was cultured in nitrite medium with shaking, and the results were as follows: Figure 6 The strain grows rapidly from 0 to 24 hours, with an OD of [value missing] at 12 hours. 600 The value was 0.531; OD at 24h 600 It was 1.125. Then it reached a plateau, OD 600 The degradation rate of nitrite by Bacillus paralichrysogenum BSL-10 remained basically unchanged. The trend of nitrite degradation rate was consistent with the growth status of the strain. Nitrite was rapidly degraded within 0-24h, reaching 42.13% at 12h and 99.93% at 24h.

[0113] 2.2.2 Growth and denitrification function of Bacillus paralichrysiforme BSL-10 in ammonia nitrogen medium

[0114] Bacillus paralichrysogenum BSL-10 was cultured in ammonia nitrogen medium with shaking, and the results were as follows: Figure 7 Bacillus paralichrysogenum BSL-10 grows slowly from 0 to 12 hours, with the fastest growth rate occurring between 12 and 48 hours, reaching a plateau at 48 hours. Under aerobic conditions, the ammonia nitrogen degradation rate is 79.65% after 24 hours of cultivation, reaching 98.65% after 48 hours. Once the ammonia nitrogen is depleted, the strain's growth also reaches a plateau. OD 600 It reached 1.063, and then gradually declined.

[0115] 2.2.3 Growth and denitrification function of Bacillus paralichrysum BSL-10 in nitrate nitrogen medium

[0116] Bacillus paralichrysogenum BSL-10 was cultured in potassium nitrate medium with shaking, and the results were as follows: Figure 8 Bacillus paralichrysogenum BSL-10 grew slowly and steadily over 12-96 hours, with an OD of [value missing] at 96 hours. 600 The peak value was 1.079. The degradation trend of nitrate by Bacillus paralichrysogenum BSL-10 increased steadily in sync with the growth of the strain. The degradation rate of nitrate was 18.65% at 12h and reached the highest value of 82.16% at 96h.

[0117] 2.2.4 Growth and denitrification function of Bacillus paralichrysiformis BSL-10 in mixed nitrogen sources

[0118] Bacillus paralichrysogenum BSL-10 was cultured with shaking in a medium containing sodium nitrite, ammonium chloride, and potassium nitrate. Figure 9 This indicates that Bacillus paralichrysum BSL-10, when cultured under aerobic conditions, exhibits rapid ammonia nitrogen degradation within 12-24 hours, reaching 89.33% at 12 hours and 96.35% at 24 hours, at which point the OD... 600 It also reached 0.821; nitrite degradation was rapid from 24 to 48 hours, reaching 91.53% after 48 hours, OD 600 The concentration reached 0.972; nitrate degradation was slow, with a degradation rate of only 62.35% after 96 hours. The experimental results show that Bacillus paralichrysum BSL-10 preferentially utilizes ammonia nitrogen, followed by nitrite, and finally nitrate when three ionic nitrogen sources are present simultaneously.

[0119] Changes in total nitrogen, intracellular nitrogen, and pH in the culture medium are as follows: Figure 10 When Bacillus paralichrysiformis BSL-10 was cultured with shaking in a medium containing sodium nitrite, ammonium chloride, and potassium nitrate for 96 hours, the pH of the medium gradually changed from neutral (pH 7) to alkaline (pH 8.3), indicating that the strain underwent aerobic denitrification and produced hydroxyl groups during the denitrification process.

[0120] During the culture process, the total nitrogen content in the culture medium, initially 330 mg / L, gradually decreased, reaching 179 mg / L (including intracellular nitrogen) at 96 h, with a degradation rate of 45.76%. Some of the degraded nitrogen was released into the air in the form of gases. The intracellular nitrogen of *Bacillus paralichrysogenus* BSL-10 increased from 0 mg / L to 109 mg / L, indicating that *Bacillus paralichrysogenus* BSL-10 assimilated some ionic nitrogen into intracellular organic nitrogen during its growth. At 96 h of culture, approximately 55.94 mg / L of the three ionic nitrogens remained in the culture medium, while about 14 mg / L of total nitrogen in the culture medium was still in an unknown form, indicating that the strain also formed other forms of nitrogen during the conversion of the three ionic nitrogens.

[0121] 3. Summary

[0122] Bacillus paralichrysogenum BSL-10 exhibits superior nitrite degradation capabilities compared to other tested Bacillus species under both aerobic and anaerobic conditions. Under a single nitrogen source, BSL-10 degrades nitrite most rapidly, achieving a degradation rate of 99.93% in 24 hours; ammonia nitrogen is the next fastest, with a degradation rate of 98.65% in 48 hours; nitrate is the slowest, with a degradation rate of 82.16% in 96 hours. Under mixed nitrogen source conditions, BSL-10 preferentially utilizes ammonia nitrogen, followed by nitrite, and finally nitrate, when all three ionic nitrogen sources are present simultaneously. The nitrogen sources degraded by BSL-10 ultimately undergo three fates: assimilation for the synthesis of intracellular organic nitrogen, denitrification releasing nitrogen gas into the environment, and a small amount being converted into other forms of nitrogen.

[0123] Application Example 1: Application of Bacillus paralichrysiformis BSL-10 in the breeding of weaned Enshi black pigs

[0124] 1. Materials and Methods

[0125] 1.1 Experimental animals and Bacillus paralichrysiforme BSL-10 preparation

[0126] 72 weaned Enshi black pigs, provided by Enshi Dida Agriculture and Animal Husbandry Co., Ltd.

[0127] Bacillus paralichrysogenus BSL-10 preparation: Fermentation broth with a viable count exceeding 1×10^9 CFU / mL was obtained according to the method in Example 2. After centrifugation, the slurry was spray-dried to obtain a content ≥3×10^9 CFU / mL. 12 The original powder with cfu / g was then mixed with maltose to obtain a live bacteria count ≥2×10⁻⁶. 10 Solid dosage form with cfu / g.

[0128] 1.2 Experimental Design

[0129] A completely randomized controlled trial design was used to randomly divide 72 healthy weaned Enshi black pigs (35 days old, initial weight 10.47±1.12 kg) into 4 groups (experimental groups 1-3 and a control group), with 3 replicates per group and 6 pigs per replicate. The control group was fed a basal diet; experimental groups 1, 2, and 3 were supplemented with 100, 200, and 400 g / t of Bacillus paralichrysiformis BSL-10 formulation, respectively, to their basal diet. The dietary composition and nutrient levels were the same for all groups. The trial period was 35 days.

[0130] 1.3 Experimental Diets and Nutrient Levels

[0131] The experimental basal diet was based on Hubei Provincial Local Standard DB42 / T 929-2024, mainly consisting of local natural feed. Its formula was: 55% corn, 20% soybean meal, 15% wheat bran, 8% sweet potato / potato, and 2% minerals. It contained no antibiotics and no growth promoters.

[0132] 1.4 Feeding and Management

[0133] Each group of black pigs is fed twice a day, with free access to food and water. The pigsty is well-ventilated, and other feeding procedures are carried out in accordance with the Enshi Black Pig Feeding and Management Guidelines.

[0134] 1.5 Measurement Items

[0135] 1.5.1 Production Performance

[0136] At the start of the experiment, the initial weight of each group of pigs was measured. Feeding was stopped at 20:00 one day before the end of the experiment, and the weight of each group of pigs was measured on an empty stomach at 08:00 on the second day. The feed intake of each group of pigs was recorded throughout the experiment. The average daily weight gain, average daily feed intake, and feed conversion ratio of each group of pigs during the experiment were calculated.

[0137] 1.5.2 Serum Biochemical Indicators

[0138] At the end of the experiment, 5 mL of blood was collected from the anterior vena cava of 2 pigs randomly selected from each replicate. The blood was centrifuged at 3500 r / min for 10 min, and the serum was separated and stored at -20℃ for the determination of blood biochemistry items (total protein, albumin, blood urea nitrogen, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, etc.) (using a kit purchased from Solarbio).

[0139] 1.5.3 Serum immune indicators

[0140] The levels of IgA, IgM and IgG in porcine serum were detected by immunoturbidimetry (the kits used were purchased from IDEXX, USA).

[0141] 1.6 Data Statistical Analysis

[0142] Minitab 21.3.1 statistical software was used to perform one-way ANOVA and LSD multiple comparisons on each indicator.

[0143] 2 Results and Analysis

[0144] 2.1 Effects of dietary supplementation with Bacillus paralicheniformis BSL-10 on the production performance of weaned Enshi black pigs

[0145] Table 4. Effects of Bacillus paralichrysiformis BSL-10 on the production performance of Enshi black pigs.

[0146]

[0147] Note: Different lowercase letters after the data in the same column indicate significant differences between groups (P < 0.05), while the same lowercase letter indicates no significant differences between groups (P > 0.05).

[0148] Table 4 shows that adding 200-400 g / t of Bacillus paralichrysum BSL-10 to the diet significantly improved the average daily weight gain, reduced the average daily feed intake, and decreased the feed conversion ratio in weaned piglets (P < 0.05). However, when the addition amount was 200 g / t, there was no significant difference compared to the control.

[0149] 2.2 Effects of dietary supplementation with Bacillus paralicheniformis BSL-10 on serum biochemical parameters of weaned Enshi black pigs

[0150] Bacillus paralichrysum BSL-10 can significantly increase the serum albumin content and alkaline phosphatase activity of weaned Enshi black pigs, reduce the activities of alanine aminotransferase and aspartate aminotransferase, and reduce the serum urea nitrogen content.

[0151] Table 5. Effects of Bacillus paralichrysiforme BSL-10 on serum biochemical parameters of Enshi black pigs.

[0152]

[0153] Note: Different lowercase letters after the data in the same column indicate significant differences between groups (P < 0.05), while the same lowercase letter indicates no significant differences between groups (P > 0.05).

[0154] Table 5 shows that the effects of adding Bacillus paralichrysum BSL-10 to the diet on the total protein and urea nitrogen content in pig serum were not statistically significant (P > 0.05). Adding Bacillus paralichrysum BSL-10 to the basal diet significantly increased the serum albumin content of weaned Enshi Black piglets (P < 0.05), and the serum albumin content increased with increasing Bacillus paralichrysum BSL-10 dosage, but remained within the normal range. Alkaline phosphatase activity was also significantly increased (P < 0.05), but there was no significant difference between low addition levels (100 g / t, 200 g / t), while the alkaline phosphatase activity in piglets was significantly higher at 400 g / t than at low addition levels. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly decreased (P < 0.05), but there was no significant difference between different Bacillus paralichrysum BSL-10 addition levels. Adding Bacillus paralicheniformis BSL-10 to the diet significantly reduced the serum urea nitrogen content in weaned Enshi black pigs (P<0.05), but there was no significant difference between different addition amounts.

[0155] 2.3 Effects of dietary supplementation with Bacillus paralichrysum BSL-10 on serum immune markers in weaned Enshi black pigs

[0156] Table 6. Effects of Bacillus paralichrysiforme BSL-10 on serum immune indicators in weaned Enshi black pigs (g / L)

[0157]

[0158] Note: Different lowercase letters after the data in the same column indicate significant differences between groups (P < 0.05), while the same lowercase letter indicates no significant differences between groups (P > 0.05).

[0159] Table 6 shows that the addition of Bacillus paralichrysum BSL-10 to the diet did not significantly affect the serum IgM content in pigs, but it significantly increased the levels of IgA and IgG. This indicates that Bacillus paralichrysum BSL-10 can significantly enhance the immunity of weaned black pigs.

[0160] 3. Summary

[0161] Adding 200-400 g / t of Bacillus paralichrysum BSL-10 to the diet of weaned Enshi black pigs can reduce the daily feed intake of weaned piglets, increase daily weight gain, and significantly reduce the feed conversion ratio; it can also significantly increase the serum albumin content and alkaline phosphatase activity of weaned Enshi black pigs, reduce the activities of alanine aminotransferase and aspartate aminotransferase, and reduce serum urea nitrogen; and it can significantly increase the content of serum IgA and IgG in pigs. This indicates that Bacillus paralichrysum BSL-10 has the functions of promoting livestock growth, improving livestock feed utilization, and enhancing livestock immunity.

[0162] Application Example 2: Application of Bacillus paralicheniformis BSL-10 in Litopenaeus vannamei shrimp farming

[0163] 1. Materials and Methods

[0164] 1.1 Shrimp used in the experiment

[0165] The shrimp larvae used in the experiment were purchased from Haimao Seed Industry Technology Group (Zhanjiang) Co., Ltd., and the experiment was conducted at CIMC Fishery Smart Aquaculture Base in Da'ao Town, Xinhui District, Jiangmen City, Guangdong Province.

[0166] Bacillus paralichrysogenus BSL-10 preparation: Fermentation broth with a viable count exceeding 1×10^9 CFU / mL was obtained according to the method in Example 2. After centrifugation, the slurry was spray-dried to obtain a content ≥3×10^9 CFU / mL. 12 The original powder with cfu / g was then mixed with maltose to obtain a live bacteria count ≥2×10⁻⁶. 10 Solid dosage form with cfu / g.

[0167] 1.2 Test Conditions

[0168] The experiment was conducted in an aquarium measuring 100cm long × 50cm wide × 45cm high, with the temperature controlled at 29℃, salinity at 25‰, and pH at 8.0.

[0169] 1.3 Experimental Grouping

[0170] Bacillus paralicheniformis BSL-10 was added to the aquaculture water at concentrations of 0 (control group) and 0.3 g / m³. 3(Experimental Group 1), 0.5g / m 3 (Experimental Group 2), 3 replicates. Add the supplement to the aquaculture water every 3 days.

[0171] Before spraying, Bacillus paralichrysogenum BSL-10 needs to be activated. The activation water is prepared by mixing fresh water and seawater in a volume ratio of 4:1. A small amount of brown sugar is added to the bacterial solution, and rice bran is added as an attachment substrate. After aeration and incubation for 24 hours, the OD value steadily increases. Impurities are then removed by sieving through a 200-mesh sieve.

[0172] Select healthy and active shrimp larvae with an average weight of 0.04±0.01g, and stock each tank with 200 larvae. Place an inflatable stone in each aquarium. The seawater used for aquaculture is disinfected through two-stage sand filtration and ultraviolet light.

[0173] 1.4 Daily Management

[0174] The experiment lasted 50 days, with feeding five times a day, starting at 6:00 AM, every four hours, and ending at 10:00 PM. The feed was purchased from Guangdong Nanbao Group Co., Ltd. The feeding time for the shrimp larvae was controlled to 1 hour, and 10% of the water volume was changed daily.

[0175] 1.5 Shrimp growth measurement

[0176] Specific growth rate (SGR) = 100 × (lnW) 末 -lnW 始 ) / T;

[0177] Feed conversion ratio = Total feed intake (g) / Shrimp weight gain (g);

[0178] Survival rate (%) = N 末 / N 始 ×100.

[0179] W 末 Final average weight (g); W 始 Initial average weight (g); N 末 Final shrimp larvae quantity; N 始 : Initial number of shrimp larvae; T: Number of days of rearing.

[0180] 1.6 Water quality testing

[0181] Sample analysis included testing for temperature, salinity, pH (pH meter method), dissolved oxygen (iodometric method), ammonia nitrogen (indophenol blue spectrophotometry), nitrite nitrogen (naphthylethylenediamine spectrophotometry), and nitrate nitrogen (ultraviolet spectrophotometry). The procedures were performed in accordance with the "Marine Monitoring Standard" (GB17378-2007) and the "Marine Survey Standard" (GB / T 12763-2007).

[0182] 1.7 Data Statistics and Analysis

[0183] Minitab 21.3.1 statistical software was used to perform one-way ANOVA and LSD multiple comparisons on each indicator.

[0184] 2 Results and Analysis

[0185] 2.1 Effects of Bacillus paralicheniformis BSL-10 on the growth performance of Litopenaeus vannamei

[0186] The addition of *Bacillus paralichrysum* BSL-10 to the aquaculture water significantly promoted the growth of *Litopenaeus vannamei*, as shown in Table 7. *Bacillus paralichrysum* BSL-10 significantly increased the specific growth rate of *Litopenaeus vannamei* (P < 0.05), but there was no significant difference between different addition levels. *Bacillus paralichrysum* BSL-10 significantly improved the survival rate of *Litopenaeus vannamei*, and the higher the dosage, the higher the survival rate (P < 0.05). *Bacillus paralichrysum* BSL-10 significantly reduced the feed conversion ratio of *Litopenaeus vannamei* (P < 0.05), but there was no significant difference between different addition levels.

[0187] Table 7. Effects of Bacillus paralichrysiformis BSL-10 on the growth performance of Litopenaeus vannamei.

[0188]

[0189] Note: Different lowercase letters after the data in the same column indicate significant differences between groups (P < 0.05), while the same lowercase letter indicates no significant differences between groups (P > 0.05).

[0190] 2.2 Effects of Bacillus paralichrysogenum BSL-10 on water quality

[0191] 2.2.1 Effects on temperature, salinity, dissolved oxygen, and pH

[0192] As shown in Table 8, the addition of Bacillus paralichrysum BSL-10 to the culture water of Litopenaeus vannamei had no significant effect on the temperature and salinity of the water. With increasing addition and dosage of Bacillus paralichrysum BSL-10, dissolved oxygen in the water showed a decreasing trend, but the differences between treatments were not significant. However, the addition of Bacillus paralichrysum BSL-10 significantly reduced the pH of the water (P<0.05), and the higher the dosage, the more pronounced the decrease.

[0193] Table 8. Effects of Bacillus paralichrysiformis BSL-10 on water salinity, dissolved oxygen, and pH in Litopenaeus vannamei.

[0194]

[0195] Note: Different lowercase letters after the data in the same column indicate significant differences between groups (P < 0.05), while the same lowercase letter indicates no significant differences between groups (P > 0.05).

[0196] 2.2.2 Impact on ammonia nitrogen in aquaculture water

[0197] Figure 11 As shown, adding Bacillus paralichrysum BSL-10 to the culture water of Litopenaeus vannamei significantly controlled the concentration of ammonia nitrogen in the culture water. During the experiment, the concentration of ammonia nitrogen in the culture water of all groups showed an increasing trend. The control group increased to 0.45 mg / L at 20 days and continued to increase to 1.13 mg / L at 50 days. In the Bacillus paralichrysum BSL-10 treatment group, the concentration of ammonia nitrogen in the culture water was consistently lower than that in the control group, but there was no difference between different dosage treatments in the first 20 days. Starting from 30 days, the high-dose treatment of Bacillus paralichrysum BSL-10 resulted in a significantly lower concentration of ammonia nitrogen in the culture water than the low-dose treatment group. By 50 days, the concentration of ammonia nitrogen in the culture water of the high-dose treatment group was only 0.73 mg / L, while the concentration in the culture water of the low-dose treatment group reached 0.91 mg / L. The differences between the two groups and between the two groups and the control were significant (P<0.05).

[0198] 2.2.3 Impact on nitrite nitrogen in aquaculture water

[0199] Figure 12 As shown, adding Bacillus paralichrysum BSL-10 to the culture water of Litopenaeus vannamei can significantly control the increase of nitrite nitrogen concentration in the culture water. During the experiment, the nitrite nitrogen concentration in the culture water of each group showed an increasing trend. In the Bacillus paralichrysum BSL-10 treatment group, the nitrite nitrogen concentration in the culture water was consistently significantly lower than that in the control group (P<0.05), but there was no significant difference between different dosage treatments.

[0200] 2.2.4 Effects on nitrate nitrogen in aquaculture water

[0201] Figure 13 As shown, Bacillus paralicheniformis BSL-10 has no effect on nitrate nitrogen in the water used for Litopenaeus vannamei farming.

[0202] 3. Summary

[0203] The Bacillus paralichrysogenum BSL-10 preparation provided by this invention is 0.3~0.5g / m³. 3 It can significantly improve the survival rate and specific growth rate of shrimp larvae, and reduce the feed conversion ratio; at the same time, it can significantly inhibit the increase in the concentration of ammonia nitrogen and nitrite nitrogen in the water.

Claims

1. A strain of Bacillus paralicheniformis BSL-10, characterized in that, It was deposited on May 13, 2026 at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2026946.

2. A microbial inoculant, characterized in that, Contains Bacillus paralicheniformis as described in claim 1 and / or its fermentation metabolites.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a liquid fermentation broth, a freeze-dried powder, or a solid microbial agent. The viable count of the liquid microbial agent is ≥1×10^9 CFU / mL, and the viable count of the solid microbial agent is ≥2×10^10 CFU / g.

4. The method for preparing the microbial inoculant according to claim 2, characterized in that, The method comprises: inoculating the BSL-10 strain as described in claim 1 into a liquid seed culture medium and culturing it at 37°C and 200 rpm for 12 h to obtain a seed liquid; transferring the seed liquid into a fermentation culture medium at a volume inoculation rate of 5% and fermenting at 37°C for 36-48 h; terminating fermentation when the spore formation rate is ≥90% to obtain a fermentation broth with a viable count exceeding 1×10^9 CFU / mL, wherein the liquid seed culture medium comprises: 10 g / L peptone, 5 g / L yeast extract, 15 g / L NaCl, 2 g / L glucose, pH 7.0; and the fermentation culture medium comprises: 20 g / L glucose, 15 g / L soybean meal, 2 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, pH 7.

0.

5. The use of Bacillus paralichrysogenum according to claim 1, or the microbial agent according to claim 2, in the preparation of animal feed additives.

6. The application according to claim 5, characterized in that, The animals include livestock, poultry and aquatic animals; the feed additives are used to improve animal intestinal health, inhibit intestinal pathogens, improve feed conversion rate, promote animal growth and enhance animal immunity.

7. The application according to claim 6, characterized in that, The dosage of solid microbial agents added to livestock and poultry feed is 200-400 g / t of basal diet; the dosage of solid microbial agents applied to aquaculture water is 0.3-0.5 g / m³. 3 Spray once every 3 days.

8. The application of Bacillus paralichrysogenum according to claim 1, or the microbial agent according to claim 2, in the preparation of products that degrade nitrite in water.

9. The application according to claim 8, characterized in that, The nitrite-degrading product is suitable for use in seawater and freshwater aquaculture waters.

10. The application of Bacillus paralichrysogenum according to claim 1, or the microbial agent according to claim 2, in the improvement of saline-alkali soil.

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