A strain of Brevibacillus laterosporus, its composition, preparation and application

Abstract

The present invention provides a strain of Brevibacillus laterosporus, a composition, a preparation and an application thereof, belonging to the field of microorganisms. Specifically, the strain is named Brevibacillus laterosporus ( Brevibacillus laterite )BLCC1‑1219, which was deposited in the China Type Culture Collection at Wuhan University, Wuhan, China on September 29, 2024, with the deposit number CCTCC NO: M 20242118. The strain has high urease inhibition activity, significant ammonia emission inhibition effect, broad-spectrum antibacterial ability and good environmental adaptability. These characteristics make it have important application value in many fields such as environmental protection, agricultural production, animal husbandry management and pollution control.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Brevibacillus laterosporus, a composition, a preparation and an application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Urease is an enzyme widely found in bacteria, fungi, plants, and algae. It can hydrolyze urea into ammonia and nitrogen dioxide, which can then be absorbed and utilized by plants and ruminants, reducing fertilization and feed costs. However, urease's high efficiency in catalyzing the hydrolysis of urea also brings a series of negative effects: too fast ammonia production in the soil will lead to nitrogen loss; urease-producing microorganisms in livestock and poultry manure accelerate ammonia emissions and cause atmospheric environmental pollution; too fast ammonia production in the rumen of ruminants leads to decreased urea nitrogen utilization and even ammonia poisoning. Urease inhibitors can inhibit the reaction of urea to produce ammonia, which helps solve the problems of soil nitrogen loss, manure ammonia release, and low rumen urea nitrogen utilization caused by strong urease activity. At present, the types of urease inhibitors that the industry is concerned about are mainly chemical synthesis and microbial. The former mainly includes thiourea derivatives, hydroxamic acid derivatives (such as acetohydroxamic acid), phosphoramide and sulfonamide derivatives (such as NBPT), flavonoid derivatives, coumarin derivatives, indole derivatives, metal complexes (such as Schiff bases), etc. Among them, acetohydroxamic acid is the only approved pharmaceutical urease inhibitor, but it has large side effects and unstable properties; phosphoramide compounds are a kind of urease inhibitor with good inhibitory effect, but due to poor stability, they cannot be used in actual production. There are also some new inhibitors such as Schiff base compounds, but the development of these compounds is still not mature enough. Summary of the invention

[0004] In view of the technical problems that the existing urease inhibitors are toxic and harmful to livestock and poultry, cause environmental pollution, are structurally unstable, and have a low inhibition rate on ammonia, the present invention provides a strain of Brevibacillus laterosporus ( Brevibacillus laterosporus ) BLCC1-1219, fermentation products of the strain, compositions or preparations containing the strain, and applications of the strain. The Brevibacillus laterosporus provided by the present invention has high efficiency in inhibiting urease activity and can significantly reduce ammonia emissions; it has broad-spectrum antibacterial activity, can inhibit a variety of pathogens, and has strong environmental adaptability, can maintain good activity under a variety of conditions, and as a microbial preparation, it is highly safe and environmentally friendly. It has a wide range of applications and can be used for ammonia emissions and pollution control in multiple fields, making it have important application value in multiple fields such as environmental protection, agricultural production, animal husbandry management and pollution control.

[0005] Specifically, the present invention provides the following technical features, and a combination of one or more of the following technical features constitutes the technical solution of the present invention.

[0006] In a first aspect of the present invention, a strain of Brevibacillus laterosporus is provided, which is named Brevibacillus laterosporus ( Brevibacillus laterosporus )BLCC1-1219 was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on September 29, 2024, with the deposit number CCTCC NO: M 20242118.

[0007] The Brevibacillus laterosporus of the present invention is cultured at 37°C for 20 hours, and the colonies are round, colorless, raised, viscous, and have neat edges. Under an optical microscope, spores grow laterally, are Gram-positive, can become Gram-negative, and are rod-shaped.

[0008] In a second aspect of the present invention, a fermentation product of Brevibacillus laterosporus described in the first aspect is provided.

[0009] In the present invention, "fermentation product" refers to the general term for all products produced by the fermentation process of Brevibacillus laterosporus BLCC1-1219 under suitable conditions. The fermentation product may include, but is not limited to, the following components: living cells, dead cells, extracellular metabolites, and culture medium components. In the present invention, the fermentation product may be in liquid, semi-solid, or solid form after treatment.

[0010] In some embodiments of the present invention, the method for obtaining the fermentation product of Brevibacillus laterosporus BLCC1-1219 includes: strain activation, seed liquid preparation, fermentation culture and post-fermentation treatment.

[0011] Activation of strains: Inoculate the Brevibacillus laterosporus BLCC1-1219 strain into LB solid medium, culture at 30-37°C for 24-48 hours, and select single colonies for subsequent culture.

[0012] Preparation of seed solution: Inoculate the activated single colony into 100 mL of liquid LB medium and culture it in a shaking incubator at 30-37°C and 180 rpm for 18-24 hours. When the spore rate under microscopic examination is greater than 80%, it can be used as seed solution.

[0013] Fermentation culture: inoculation amount: 1-10% (v / v), temperature: 30-37°C, pH: 6.5-7.5, dissolved oxygen: 30-50%, stirring speed: 150-200 rpm, fermentation time: 18-48 hours.

[0014] Post-fermentation treatment: Different types of fermentation products can be obtained by using different post-treatment methods, including but not limited to the following types:

[0015] (1) Whole fermentation product: For example, the fermentation liquid is directly collected, which is the whole fermentation product.

[0016] (2) Fermentation supernatant: For example, the fermentation broth is centrifuged at 5000-10000 rpm for 10-15 minutes. The supernatant is collected as the fermentation supernatant.

[0017] (3) Bacteria: For example, centrifuge the fermentation broth in the same manner as above. Collect the precipitate and wash it 2-3 times with sterile water or buffer to obtain bacteria.

[0018] (4) Concentrating the fermentation product: For example, using an ultrafiltration membrane (e.g., with a molecular weight cutoff of 10-50 kDa) to concentrate the whole fermentation product or the fermentation supernatant to obtain a concentrated fermentation product. The concentration multiple can be adjusted according to demand, usually 2-10 times.

[0019] (5) Drying the fermented product: for example, freeze-drying or spray-drying the whole fermented product, fermentation supernatant or bacterial cells.

[0020] For example, freeze drying: pre-freezing (-20°C to -80°C, 4-12 hours), main drying (-60°C to 30°C, 24-48 hours). For example, spray drying: inlet temperature 150-200°C, outlet temperature 60-100°C.

[0021] By adjusting the above fermentation conditions and post-treatment methods, fermented products with different characteristics can be obtained to meet various application requirements. In practical applications, the most suitable fermentation conditions and treatment methods can be selected according to the target product and application scenario.

[0022] In the third aspect of the present invention, there is provided a use of a fermentation product of Brevibacillus laterosporus described in the first aspect or Brevibacillus laterosporus described in the second aspect in the preparation of a preparation for inhibiting urease activity.

[0023] In a fourth aspect of the present invention, there is provided a use of a fermentation product of the Brevibacillus laterosporus described in the first aspect or the Brevibacillus laterosporus described in the second aspect in the preparation of an antibacterial preparation.

[0024] In some embodiments, the antimicrobial formulation is used to inhibit at least one of Vibrio parahaemolyticus, Salmonella enteritidis, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus, Staphylococcus aureus, Escherichia coli of pigs, and Clostridium perfringens.

[0025] In a fifth aspect of the present invention, there is provided a use of the fermented product of the Brevibacillus laterosporus described in the first aspect or the Brevibacillus laterosporus described in the second aspect in the preparation of a preparation for reducing ammonia emissions. In particular, the Brevibacillus laterosporus described in the present invention can inhibit, reduce or eliminate ammonia emissions from substances containing ammonia or that may produce ammonia, such substances including but not limited to livestock and poultry manure, sewage, compost raw materials or organic waste, etc.

[0026] In a sixth aspect of the present invention, a composition is provided, comprising a fermentation product of the Brevibacillus laterosporus described in the first aspect or the Brevibacillus laterosporus described in the second aspect.

[0027] In some embodiments of the present invention, the composition may optionally include one or more of the following ingredients:

[0028] Protective agents: such as skimmed milk powder, sucrose, sodium alginate, etc., used to improve the stability of strains or fermentations.

[0029] Nutrients: such as glucose, yeast extract, etc., used to maintain strain activity.

[0030] Buffers: such as phosphate buffer, used to maintain appropriate pH.

[0031] Carrier: such as corn cob powder, bran, etc., used for the preparation of solid preparations.

[0032] In some embodiments of the present invention, there is provided a method for preparing a composition, comprising:

[0033] The live bacterial composition is prepared by culturing the Brevibacillus laterosporus BLCC1-1219 strain in a suitable culture medium to a logarithmic growth phase, collecting the bacterial cells by centrifugation, washing with sterile physiological saline, mixing the bacterial cells with a protective agent, and freeze-drying or spray-drying the cells.

[0034] Fermentation composition: Brevibacillus laterosporus BLCC1-1219 strain is cultured in a fermentation medium for an appropriate period of time. The fermentation broth is separated by centrifugation, and the supernatant is collected. The supernatant can be used directly or after being concentrated, dried, or the like.

[0035] In a seventh aspect of the present invention, a preparation is provided, comprising the fermentation product of Brevibacillus laterosporus described in the first aspect or Brevibacillus laterosporus described in the second aspect or the composition described in the sixth aspect.

[0036] In an embodiment of the present invention, the Brevibacillus laterosporus BLCC1-1219 or the fermentation product of Brevibacillus laterosporus BLCC1-1219 or the composition comprising Brevibacillus laterosporus BLCC1-1219 can be referred to as an active ingredient when preparing a preparation.

[0037] The preparations of the present invention may be of the following types:

[0038] Solid preparations: powders, granules, tablets, etc.; liquid preparations: suspensions, emulsions, solutions, etc.; gel preparations: hydrogels, oil gels, etc.

[0039] In some embodiments of the present invention, the following excipients may be optionally included depending on the type of preparation:

[0040] Dispersants: such as povidone K30, sodium carboxymethyl cellulose, etc.; binders: such as microcrystalline cellulose, hydroxypropyl methylcellulose, etc.; fillers: such as lactose, starch, etc.; preservatives: such as potassium sorbate, sodium benzoate, etc.

[0041] In some embodiments of the present invention, a method for preparing the above-mentioned preparation is also provided, which comprises:

[0042] For example, to prepare solid preparations: the active ingredient is mixed with an appropriate amount of excipients. Granules are obtained through granulation, drying, and granulation. Tablets can be further compressed.

[0043] For example, to prepare a liquid preparation: dissolve or disperse the active ingredient in the aqueous phase, add an appropriate amount of emulsifiers, stabilizers and other auxiliary materials, and then prepare the preparation through homogenization, filtration and other processes.

[0044] For example, to prepare a gel preparation: mix the active ingredient with an appropriate amount of thickener (such as carbomer), adjust the pH value to a suitable range, and add excipients such as moisturizers and preservatives.

[0045] The compositions and preparations of the present invention as described above can be applied to agriculture, for example, as biofertilizers, soil conditioners or biopesticides; can be applied to animal husbandry, for example, as feed additives or livestock and poultry manure treatment agents; can be used for environmental protection, for example, for sewage treatment, composting or environmental deodorization; can be used for aquaculture, for example, for improving the environment of aquaculture water bodies; can be used in industry, for example, for industrial wastewater treatment or waste gas treatment.

[0046] In some embodiments of the present invention, methods for using the above-mentioned compositions or preparations are also provided. Depending on different application fields, the methods of use may include but are not limited to: spraying, such as diluting the liquid preparation and spraying it on the area to be treated. Mixing, such as mixing the solid preparation with feed or fertilizer. Placing, such as directly placing the preparation into the water body or waste to be treated. Irrigation, such as diluting the liquid preparation and applying it through an irrigation system.

[0047] In some embodiments of the present invention, the preparation is a feed additive, a biological fertilizer, a biological pesticide, a biological deodorant, an environmental management agent, a soil conditioner or a water purifier.

[0048] In the eighth aspect of the present invention, a method for reducing ammonia emissions is provided, comprising adding the fermentation product of Brevibacillus laterosporus described in the first aspect or the Brevibacillus laterosporus described in the second aspect or the composition described in the sixth aspect or the preparation described in the seventh aspect to a substance to be treated, wherein the substance to be treated is a substance containing ammonia or that may produce ammonia, including but not limited to livestock and poultry manure, sewage, compost raw materials or organic waste, etc.

[0049] In some embodiments of the present invention, the method for reducing ammonia emissions comprises:

[0050] (1) preparing a fermentation broth of Brevibacillus laterosporus BLCC1-1219;

[0051] (2) Mixing the fermentation liquid with the substance to be treated.

[0052] In the present invention, "fermentation broth" specifically refers to the entire liquid fermentation system that has not been separated after the fermentation process is completed. The fermentation broth usually contains: suspended live bacteria, suspended dead bacteria, extracellular metabolites dissolved in the liquid, and remaining liquid culture medium components. In some embodiments of the present invention, the fermentation broth of Brevibacillus laterosporus BLCC1-1219 can be obtained by the following method: inoculating Brevibacillus laterosporus BLCC1-1219 in a liquid culture medium, then culturing under conditions suitable for the growth of the strain, and then obtaining the fermentation broth. In the present invention, the suitable growth conditions include: temperature 30-37°C, rotation speed 150-200 r / min, and culture time 18-48 hours.

[0053] For example, in one embodiment, step (1) includes: inoculating Brevibacillus laterosporus BLCC1-1219 into LB liquid culture medium, culturing at 37° C. and 180 r / min for 24 hours to obtain fermentation broth.

[0054] In some embodiments, in step (2), the mixing ratio of the fermentation liquid to the substance to be treated is 1%-25% of the volume or weight of the substance to be treated by the fermentation liquid, and the mixing ratio is preferably 5%-20%.

[0055] After the fermentation liquid of the strain is mixed with the material to be treated, it can be treated at a temperature of 20-45° C., preferably 25-40° C., for a treatment time of 30 minutes to 10 days. Preferably, the method further comprises stirring or turning the compost during the treatment process.

[0056] The method of the present invention can be applied in the following scenarios: livestock and poultry farm waste treatment, sewage treatment, organic fertilizer production, farmland fertilization, aquaculture, public environment management or industrial waste gas treatment, etc.

[0057] Compared with the prior art, the advantages of the present invention include:

[0058] The Brevibacillus laterosporus BLCC1-1219 of the present invention has significant urease inhibition activity. Test results show that the inhibition rate of the strain on urease can reach 92.52% within 30 minutes, 92.61% within 60 minutes, and 97.07% within 120 minutes, which is much higher than that of the control strain. This efficient urease inhibition activity can effectively reduce the generation of ammonia during urea hydrolysis.

[0059] The Brevibacillus laterosporus BLCC1-1219 of the present invention shows excellent effects in reducing ammonia emissions from livestock and poultry manure. In the chicken manure treatment test, the inhibition rate of ammonia emissions by Brevibacillus laterosporus BLCC1-1219 reached 82.65%. It also showed excellent ammonia emission inhibition effects in other types of livestock and poultry manure (such as pig manure and cow manure and urine mixture). This characteristic can significantly improve the breeding environment and reduce ammonia pollution to the atmosphere. And it shows significant ammonia nitrogen removal ability in sewage treatment. This characteristic makes the strain have potential application value in the fields of sewage treatment and water purification. And it can also effectively reduce ammonia emissions during the composting process. This characteristic can significantly improve environmental problems in the composting process and improve the production quality of organic fertilizers.

[0060] The Brevibacillus laterosporus BLCC1-1219 of the present invention has a broad-spectrum antibacterial activity. Test results show that Brevibacillus laterosporus BLCC1-1219 has an inhibitory effect on a variety of pathogens, among which the best antibacterial effect is on Vibrio parahaemolyticus, with an inhibition zone diameter of 14.00 mm. The antibacterial effect on Salmonella enteritidis and avian Escherichia coli is also good, with an inhibition zone diameter of ≥13.50 mm. This broad-spectrum antibacterial property makes the strain have potential application value in disease prevention and biological control.

[0061] As a biological agent, the Brevibacillus laterosporus BLCC1-1219 of the present invention has higher safety and environmental friendliness than chemical agents. It can solve the problems of ammonia emission and pathogenic bacteria pollution in a biological way, does not produce chemical residues, and meets the requirements of sustainable development and green production.

[0062] In summary, the Brevibacillus laterosporus BLCC1-1219 provided by the present invention has efficient urease inhibition activity, significant ammonia emission inhibition effect, broad-spectrum antibacterial ability and good environmental adaptability. These characteristics make it have important application value in multiple fields such as environmental protection, agricultural production, animal husbandry management and pollution control, and can provide new technical means for solving ammonia pollution, improving resource utilization efficiency and promoting sustainable development. The development and application of this strain not only has significant economic value, but also can bring huge environmental and social benefits, which is of great significance to promoting green development and ecological civilization construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

[0064] Figure 1 The results of the initial plate screening of non-urease producing strains are shown.

[0065] Figure 2 The graph shows the change of urease activity inhibition rate of the tested strains with reaction time.

[0066] Figure 3 The colony morphology of strain BLCC1-1219 is shown.

[0067] Figure 4 The bacterial morphology of strain BLCC1-1219 is shown (observed under oil microscope).

[0068] Figure 5 The phylogenetic tree of strain BLCC1-1219 is shown. DETAILED DESCRIPTION

[0069] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0070] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0071] Example 1: Screening of strains that do not produce urease

[0072] 1 Materials and methods

[0073] 1.1 Medium formulation

[0074] Plate screening culture medium: 20 g urea, 0.2 g phenol red, 10 g sucrose, 10 g peptone, 3 g beef powder, 5 g sodium chloride, 20 g agar, 1000 mL distilled water, pH 6.4. Sterilize at 121℃ for 30 min. Since high temperature sterilization will decompose urea, urea is not added during the preparation of the culture medium and it is sterilized normally. Before use, heat it in a microwave oven to melt and cool it to 40℃-50℃. Under sterile conditions, pass the urea solution through a 0.22 µm microporous filter membrane and mix it with other components of the culture medium before use.

[0075] Shake flask primary screening culture medium: 20 g urea, 10 g sucrose, 5 g potassium chloride, 0.5 g magnesium sulfate, 0.5 g calcium chloride, 1 g sodium dihydrogen phosphate, 1 g disodium hydrogen phosphate, 1000 mL distilled water, adjust pH to 6-8, sterilize at 121℃ for 30 min, the method of adding urea is the same as above.

[0076] NA medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 20 g agar, 1000 mL distilled water, adjust pH to 7.2-7.4, and sterilize at 121°C for 30 min.

[0077] 1.2 Initial screening of non-urease producing strains

[0078] Take fresh livestock and poultry feces samples (such as fresh broiler and pig feces), place them in a 30℃ incubator for 24 hours to allow the Bacillus in them to fully form spores. Weigh 5 g of the cultured livestock and poultry feces sample, place it in a 250 mL sterile conical bottle containing 95 mL of 0.85% sterile saline (with an appropriate number of sterile glass beads pre-placed in the bottle), shake on a shaker at 180 r / min for 30 minutes, and place in a water bath at 80℃ for 10 minutes to obtain a concentration of 10 -1 Then, the bacterial suspension was diluted 10-fold using sterile saline to obtain concentrations of 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Take a bacterial suspension with a concentration of 10 -4 , 10 -5 , 10 -6 and 10 -7The bacterial suspension was spread on 5 NA plate culture media, with 0.1 mL on each plate, and inverted cultured at 30℃-37℃ for 36 h.

[0079] Use an inoculation loop to pick the colonies growing on the nutrient agar (NA) plate medium, make a "cross" streak inoculation on the plate screening medium plate, and observe the color change around the "cross" bacterial lawn after inverted culture at 37℃ for 24 hours. Screen and pick the colonies with yellow surroundings (not red) that are non-urease-producing strains on the NA slant medium, and culture at 37℃ for 48 hours before preservation. Screen and pick the colonies with red surroundings that are urease-producing strains on the NA slant medium, and culture at 37℃ for 48 hours before preservation.

[0080] 1.3 Shake flask screening

[0081] Use an inoculation loop to pick up the slant of the non-urease-producing strain obtained from the initial screening of the 1.2 plate, inoculate it into a 50 mL shake flask screening medium (250 mL triangular flask), and culture it in a shaker at 37 ° C and 180 r / min for 48 hours, and observe the turbidity of the medium. The fermentation broth that is not turbid is the fermentation broth of the non-urease-producing strain, which is stored for later use. A total of 3 strains that do not produce urease were screened from more than 30 strains.

[0082] 2 Test results

[0083] 2.1 Initial screening of non-urease producing strains

[0084] Depend on Figure 1 It can be seen that the area around the "cross" fungus of strains BLCC1-1219, BLCC1-0014 and BLCC1-0168 is still yellow, which means they are strains that do not produce urease; the area around the "cross" fungus of strains BLCC1-1203 and BLCC1-0054 turns red, which means they are strains that produce urease.

[0085] 2.2 Initial screening of non-urease producing strains in shake flasks

[0086] The slant bacterial lawns of strains BLCC1-1219, BLCC1-0014 and BLCC1-0168 were picked up with an inoculation loop and placed in a shake flask screening medium. The culture was incubated at 37 °C and 180 r / min for 48 h in a shaking incubator. No turbidity of the fermentation broth was observed, indicating that the three strains did not produce urease.

[0087] Example 2: Screening of urease-producing strains

[0088] 1 Materials and methods

[0089] 1.1 Test strains

[0090] The urease-producing strains BLCC1-0054 and BLCC1-1203 screened in Example 1 of the present invention.

[0091] 1.2 Reagents and culture medium formulations

[0092] Urea: analytical grade.

[0093] Phenol red indicator: Weigh 0.1 g of phenol red, add 1.43 mL of 0.1 mol / L sodium hydroxide solution, grind in a mortar to promote dissolution, then transfer to a 250 mL volumetric flask, add distilled water to the scale, shake well and set aside.

[0094] NA medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 20 g agar, 1000 mL distilled water, adjust pH to 7.2-7.4, and sterilize at 121°C for 30 min.

[0095] NB medium: peptone 10g, beef extract 3g, sodium chloride 5g, distilled water 1000mL, adjust pH to 7.2-7.4, sterilize at 121℃ for 30 min.

[0096] 1.3 Screening of urease-producing strains

[0097] The colonies that turned red around the bacterial lawn after the "cross" inoculation on the plate screening medium plate in Example 1 were selected, that is, the bacterial lawns of strains BLCC1-0054 and BLCC1-1203 were selected and placed on the NA slant medium, cultured at 37 ° C for 36 h, and then stored for later use. The strains were inoculated into 250 mL Erlenmeyer flasks containing 50 mL NB medium, and cultured in a shaking incubator at 150 r / min for 24 h to prepare the fermentation broth.

[0098] Take 1.0 mL of the above fermentation broth, add it to a 25 mL colorimetric tube, add 0.02 g urea (1.0 mL of freshly prepared 2% m / v urea aqueous solution), add 2 drops of phenol red indicator, add 20 mL of water, and shake well for 15 seconds. Record the time when the pink color appears, and judge the urease activity based on the length of time. Set up a sample blank (without urea) and a reagent blank (without sample). The sample blank and the reagent blank do not change color after adding phenol red indicator, indicating that the test results are reliable. Record the fermentation broth with the shortest pink appearance time (less than 15 min), which corresponds to the highest urease activity of the strain, and store it for later use.

[0099] 2 Test results

[0100] Table 1 Color change time of urease-producing strains

[0101]

[0102] As shown in Table 1, the time for the two strains to turn pink was less than 15 min, and the pink color of BLCC1-0054 was observed after 5 seconds of reaction, indicating that the urease activity of this strain was high.

[0103] Example 3: Screening of urease inhibitor producing strains

[0104] 1 Materials and methods

[0105] 1.1 Test strains

[0106] The non-urease-producing strains BLCC1-0014, BLCC1-0168 and BLCC1-1219 screened in Example 1 of the present invention and the urease-producing strain BLCC1-0054 were used as test strains.

[0107] 1.2 Reagents and culture medium formulations

[0108] Urea solution: Weigh 0.15 g of urea, dissolve it in deionized water and make up to 50 mL.

[0109] 50 mmol / L HEPES buffer (pH 7.5): Weigh 1.19 g of sodium 4-hydroxyethylpiperazine sulfonate (HEPES), dissolve it in deionized water and make up to 100 mL, and adjust the pH to 7.5 with NaOH.

[0110] LB liquid medium: 10 g peptone, 5 g yeast extract, 5 g sodium chloride, 2 g glucose, 1000 mL distilled water, pH 7.0, sterilized at 121 °C for 30 min.

[0111] 1.3 Preparation of fermentation supernatant

[0112] The test strains were inoculated into 250 mL Erlenmeyer flasks containing 50 mL LB liquid medium, and cultured at 37 °C and 180 r / min for 24 h on a shaking table. The fermentation broth was centrifuged at 5000 r / min for 10 min, and the supernatant was collected for later use.

[0113] 1.4 Screening of urease inhibitor-producing strains

[0114] The above-mentioned test strains were tested according to the sample addition system shown in Table 2, with 3 replicates in each group. After incubation at 37 °C for 30 min, the urease activity inhibition rate at different reaction times was determined using the phenol-hypochlorous acid method. The reaction time was set at 30 min, 60 min and 120 min. The specific steps were carried out in accordance with the standard DB15 / T 1458-2018 "Determination of pH value, organic acid and ammonia nitrogen in silage", and the urease activity inhibition rate was calculated. The calculation formula is as follows:

[0115] Urease activity inhibition rate (%) = [1-(OD value of the experimental group-OD value of the experimental negative control group) / (OD value of the urease group-OD value of the urease negative control group)] × 100%.

[0116] Table 2 Sampling system for rescreening urease inhibitor producing strains

[0117]

[0118] Note: “-” means this solution is not used.

[0119] 1.5 Identification of strains

[0120] 1.5.1 Morphological identification

[0121] The strain with the highest urease activity inhibition rate was selected as the target strain, and its pure culture was inoculated into LB medium plates and cultured at 37°C for 20 h, and the colony morphology was observed.

[0122] 1.5.2 Molecular Biological Identification

[0123] The target strain was inoculated into fresh LB liquid medium and cultured for 20 h. The bacterial DNA was extracted using the kit of Tiangen Company, and the 16S rDNA sequence was amplified.

[0124] The primers used are universal primers:

[0125] 1492r: 5'-ggttaccttgttacgactt-3' (SEQ ID NO: 1);

[0126] 27f: 5'-agagttgatcctggctcag-3' (SEQ ID NO: 2).

[0127] The PCR reaction system (50 µL) was as follows: Mixture 25 µL (containing Taq DNA polymerase and dNTPs, Tiangen Biochemical Technology Co., Ltd.), upstream and downstream primers 1 µL each, template DNA 2 µL, and ultrapure water 21 µL.

[0128] The PCR amplification program was 94°C pre-denaturation for 5 min, 94°C denaturation for 1 min, 52°C annealing for 1 min, 72°C extension for 2 min, 25 cycles, and 72°C extension for 10 min. The PCR products were sent to Beijing Boshang Biotechnology Co., Ltd. for sequence determination.

[0129] 1.5.3 Phylogenetic analysis

[0130] Log in to GenBank, use Blast to search the 16S rDNA sequencing results, and download the 16S rDNA sequences of related genera and species. Use DNAMAN, DNAclub, MEGA3.1 and other software to perform homology analysis and construct a phylogenetic tree.

[0131] 2 Test results

[0132] 2.1 Rescreening of urease inhibitor-producing strains

[0133] Table 3 Results of urease activity inhibition rate of tested strains

[0134]

[0135] Note: Data in the same column with different lowercase letters indicate significant differences ( P <0.05, the same lowercase letters or no letters in the same column indicate no significant difference ( P >0.05).

[0136] From Table 3 and Figure 2 It can be seen that the urease activity inhibition rate of strain BLCC1-1219 was the highest at 30 min, 60 min and 120 min, which were 92.52%, 92.61% and 97.07% respectively, which were significantly higher than those of the other two strains ( P <0.05).

[0137] 2.2 Identification of BLCC1-1219 strain

[0138] 2.2.1 Morphological identification

[0139] The strain BLCC1-1219 was cultured at 37 °C for 20 h, and the colonies were round, colorless, raised, viscous, and had neat edges (e.g. Figure 3 ). Observed under an optical microscope, spores are lateral, Gram-positive, can become Gram-negative, and rod-shaped (e.g. Figure 4 ).

[0140] 2.2.2 Molecular biological identification

[0141] The electrophoresis results of the 16S rDNA PCR product of strain BLCC1-1219 showed that a band with good specificity was obtained at a molecular weight of about 1500 bp, which was consistent with the expected result, and the sequence was sequenced, and the sequence was shown in SEQ ID NO: 3. The sequence was compared with the 16S rDNA gene sequences of some strains registered on the website http: / / www.ncbi.nlm.nih.gov. The results showed that strain BLCC1-1219 was similar to Brevibacillus laterosporus ( Brevibacillus laterosporus The sequence homology of the model strain NRRL NRS-314 (KT363754.1) is 100% ( Figure 5 ), the strain BLCC1-1219 was identified as belonging to Brevibacillus laterosporus ( Brevibacillus laterosporus ).

[0142] Example 4: Determination of in vitro antibacterial properties of Brevibacillus laterosporus BLCC1-1219

[0143] 1 Materials and methods

[0144] 1.1 Test strains

[0145] The Brevibacillus laterosporus screened in Example 3 of the present invention ( Brevibacillus laterosporus )BLCC1-1219.

[0146] 1.2 Determination of in vitro antibacterial properties

[0147] 1.2.1 Preparation of fermentation supernatant

[0148] Brevibacillus laterosporus BLCC1-1219 was inoculated into LB liquid culture medium, cultured at 37°C and 180 r / min for 24 h, and then the fermentation broth was centrifuged at 5000 r / min for 10 min, and the supernatant was collected for later use.

[0149] 1.2.2 Indicator bacteria

[0150] Avian Escherichia coli BLCC8-0102, porcine Escherichia coli BLCC8-0001, Staphylococcus aureus BLCC8-0138, Salmonella Enteritidis BLCC8-0129, Aeromonas hydrophila BLCC8-0121, Vibrio alginolyticus BLCC8-0146, Vibrio parahaemolyticus BLCC8-0145 and Clostridium perfringens BLCC8-0044 are all preserved and provided by the Strain Resource Bank of the Science and Technology Innovation Center of Shandong Baolai Lilai Bioengineering Co., Ltd.

[0151] 1.2.3 Determination of antibacterial properties of Brevibacillus laterosporus BLCC1-1219

[0152] The antibacterial activity of Brevibacillus laterosporus BLCC1-1219 against indicator bacteria was determined by the punch method.

[0153] Escherichia coli BLCC8-0102, porcine Escherichia coli BLCC8-0001, Staphylococcus aureus BLCC8-0138, Salmonella enteritidis BLCC8-0129, and Aeromonas hydrophila BLCC8-0121 were inoculated into NB broth medium, cultured at 37 °C, 180 rpm for 12-18 h, and then adjusted to 1.0×10 7 CFU / mL for later use. At the same time, take 1 mL of diluted indicator bacteria and 17 mL of NA medium into the culture dish, mix well, and let it stand until it solidifies. Then use a hole puncher to punch holes, add 90 µL of fermentation supernatant, and place it in a 37 ℃ incubator for 12 h. Make 3 parallel plates for each sample.

[0154] Inoculate Clostridium perfringens BLCC8-0044 in liquid thioglycollate medium, incubate in a 42 ℃ incubator for 12-18 h, directly take 1 mL of the cultured Clostridium perfringens and 17 mL of iron sulfite agar into the culture dish, mix evenly, and let it stand until it solidifies. Then use a hole puncher to punch holes, add 90 µL of fermentation supernatant, place in a 37 ℃ incubator for anaerobically incubated for 12 h, and make 3 parallel plates for each sample.

[0155] Vibrio alginolyticus BLCC8-0146 and Vibrio parahaemolyticus BLCC8-0145 were inoculated into 2216E liquid medium, cultured at 37 °C and 180 rpm for 12-18 h, and then adjusted to 1.0×10 7 CFU / mL for later use. At the same time, take 1 mL of the diluted indicator bacteria and 17 mL of 2216E agar medium into the culture dish, mix well, and let it stand until it solidifies. Then use a hole puncher to make holes, add 90 µL of fermentation supernatant, place in a 37 ℃ incubator for 12 h, and make 3 parallel plates for each sample.

[0156] The size of the inhibition zone was measured with a vernier caliper and the antibacterial activity was evaluated.

[0157] 2 Test results

[0158] Table 4 In vitro antibacterial results of Brevibacillus laterosporus BLCC1-1219

[0159]

[0160] As shown in Table 4, Brevibacillus laterosporus BLCC1-1219 had an antibacterial effect on the eight pathogens tested, and had the best antibacterial effect on Vibrio parahaemolyticus with an inhibition zone diameter of 14.00 mm, followed by Salmonella enteritidis and avian Escherichia coli with inhibition zone diameters ≥ 13.50 mm.

[0161] Example 5: Application of Brevibacillus laterosporus BLCC1-1219 in reducing ammonia emissions

[0162] 1 Materials and methods

[0163] 1.1 Test strains

[0164] The Brevibacillus laterosporus screened in Example 3 of the present invention ( Brevibacillus laterosporus ) BLCC1-1219. Brevibacillus laterosporus BLCC1-0716 and BLCC1-1221 were provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Baolaililai Bioengineering Co., Ltd.

[0165] 1.2 Reagents and culture medium formulations

[0166] Sulfuric acid, ρ(H2SO4)=1.84 g / mL.

[0167] Sulfuric acid absorption solution, c(1 / 2H2SO4)=0.01 mol / L: Measure 2.7 mL of sulfuric acid and slowly add it into water, and dilute to 1L to prepare 0.1 mol / L stock solution, and dilute it 10 times before use.

[0168] Nessler's reagent: purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0169] Potassium sodium tartrate solution, ρ = 500g / L: Weigh 50 g of potassium sodium tartrate (KNaC4H6O6·4H2O), add 60 mL of distilled water, heat in a water bath to dissolve, and then cool and dilute to 100 mL.

[0170] LB liquid medium: peptone 10 g, yeast extract 5 g, sodium chloride 5 g, glucose 2 g, distilled water 1000 mL, pH 7.0, sterilized at 121 °C for 30 min.

[0171] 1.3 Preparation of fermentation broth

[0172] Firstly, the test strains BLCC1-0716, BLCC1-1219 and BLCC1-1221 were activated with LB solid slant and then inoculated into 200 mL LB liquid medium, respectively. The fermentation broth was obtained after fermentation at 37 °C and 180 r / min for 24 h.

[0173] 1.4 Application of Brevibacillus laterosporus in reducing fecal ammonia emissions

[0174] Accurately weigh 200 g of fresh chicken manure and evenly put it into a 1000 mL beaker. Pour the fermentation liquid of Bacillus laterosporus BLCC1-1219, BLCC1-0716, and BLCC1-1221 strains into the large beaker at a 20% inoculation volume (i.e. 40 mL) and mix evenly. Then put a 50 mL small beaker containing 40 mL of sulfuric acid absorption liquid into the large beaker. First, apply a layer of vaseline on the outer wall of the large beaker to ensure the fit and air tightness of the plastic wrap and the cup wall, and then seal the mouth of the large beaker with a double layer of plastic wrap to ensure that the generated ammonia will not leak into the atmosphere and can be absorbed by the sulfuric acid absorption liquid. The above is the experimental group, and each group has 3 replicates.

[0175] At the same time, a control group was set up. The feces of the control group was not inoculated with the fermentation liquid of the strain, but an equal amount of sterile water was added. The rest of the operations were the same as those of the experimental group.

[0176] Then put the sealed beaker into a biochemical incubator at a constant temperature of 30°C for 7 days. After the incubation, the relative ammonia content of the test group and the control group was determined in accordance with the National Environmental Protection Standard of the People's Republic of China HJ 533-2009 "Nessler's Reagent Spectrophotometric Method for Determination of Ammonia in Ambient Air and Waste Gas". By comparing the difference in ammonia production between the test group and the control group, it is determined whether the tested strain has the effect of reducing ammonia emissions from livestock and poultry manure.

[0177] 2 Test results

[0178] Table 5 Inhibitory effect of Brevibacillus laterosporus on ammonia emission in feces

[0179]

[0180] Note: Data in the same column with different lowercase letters indicate significant differences ( P <0.05, the same lowercase letters or no letters in the same column indicate no significant difference ( P >0.05).

[0181] As shown in Table 5, Brevibacillus laterosporus BLCC1-1219 has the best inhibitory effect on ammonia emission, with an inhibition rate of 82.65%.

[0182] In addition, using a similar method, the present embodiment also tested the removal effect of Brevibacillus laterosporus BLCC1-1219 on ammonia in urban sewage and the inhibitory effect on ammonia emissions during organic fertilizer composting, and the results showed a good effect of reducing ammonia emissions. These results show that Brevibacillus laterosporus BLCC1-1219 has a significant ammonia emission reduction effect in multiple fields such as livestock and poultry breeding, sewage treatment, and organic fertilizer production, and has broad application prospects.

[0183] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Brevibacillus laterosporus, which is named Brevibacillus laterosporus ( Brevibacillus laterosporus ) BLCC1-1219, which was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on September 29, 2024, with the deposit number CCTCC NO: M 20242118.

2. Use of the Brevibacillus laterosporus according to claim 1 in the preparation of a preparation for inhibiting urease activity.

3. Use of Brevibacillus laterosporus according to claim 1 in the preparation of antibacterial preparations.

4. The use according to claim 3, characterized in that: The antibacterial preparation is used to inhibit at least one of Vibrio parahaemolyticus, Salmonella enteritidis, avian Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus, Staphylococcus aureus, porcine Escherichia coli and Clostridium perfringens.

5. Use of Brevibacillus laterosporus according to claim 1 in preparing a preparation for reducing ammonia emission. A composition comprising the Brevibacillus laterosporus according to claim 1 .

7. A preparation comprising the Brevibacillus laterosporus according to claim 1 or the composition according to claim 6.

8. The preparation according to claim 7, characterized in that The preparation is a feed additive, a biological fertilizer, a biological pesticide, a biological deodorant, an environmental management agent or a soil improver.

9. A method for reducing ammonia emissions, comprising adding the Brevibacillus laterosporus according to claim 1 or the composition according to claim 6 or the preparation according to claim 7 to a substance to be treated, wherein the substance to be treated is a substance containing ammonia or capable of producing ammonia, including sewage or organic waste.

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