A strain of Bacillus velez and its application in preparing water-soluble microbial agents

Abstract

The invention relates to a strain of Bacillus Velez and application thereof in preparing a water-soluble microbial agent, belonging to the technical field of microorganisms. The Bacillus Velez has the functions of producing exopolysaccharides, promoting soil aggregate formation, degrading avermectin, hydrolyzing proteins, producing indoleacetic acid, promoting tomato plant growth, and promoting enzyme activity of tomato rhizosphere soil enzymes. The water-soluble microbial agent prepared based on the strain not only has the effects of improving soil aggregate structure and promoting tomato plant growth, but also can accelerate the temperature rise of avermectin fermentation waste residue composting, extend the high temperature period and shorten the composting cycle, thereby facilitating the harmless treatment and resource utilization of the avermectin fermentation waste residue.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Bacillus velezensis and application thereof in preparing a water-soluble microbial agent. Background Art

[0002] With the rapid development of biotechnology and integrated water-fertilization technologies, the application of microbial agents is becoming increasingly widespread. Water-soluble microbial agents combine functional bacteria with soluble organic matter and can be applied via drip irrigation or flushing. They improve soil quality, promote crop growth, and enhance crop disease resistance. Currently, the development of water-soluble microbial agents that combine functional bacteria with soluble organic matter and are suitable for integrated water-fertilization has become a research hotspot.

[0003] Currently, the primary raw materials for the production of water-soluble microbial inoculants used in drip irrigation or flushing applications are water-soluble organic materials, such as fulvic acid, amino acids, molasses, and wood vinegar. However, the limited availability and high prices of these raw materials have hindered their large-scale promotion and application. Fermentation or enzymatic hydrolysis can be used to convert water-insoluble organic materials into water-soluble organic materials for the production of water-soluble microbial inoculants, demonstrating broad development potential. However, relatively little research has been conducted on this topic.

[0004] Abamectin fermentation waste is a byproduct of the antibiotic fermentation industry. Rich in proteins, amino acids, carbohydrates, trace elements, and residual mycelium, it is nutritious and inexpensive, making it an ideal raw material for the production of water-soluble microbial agents using fermentation or enzymatic methods. However, the residual avermectin in avermectin fermentation waste presents significant challenges for its comprehensive utilization. During enzymatic hydrolysis, avermectin cannot be hydrolyzed and remains in the hydrolyzate, posing a potential risk. During fermentation hydrolysis, avermectin's inhibitory effect on the fermenting microorganisms can hinder the fermentation process. Furthermore, if avermectin is not effectively degraded by the microorganisms, it will also remain in the hydrolyzate. Therefore, when utilizing avermectin fermentation waste, it is essential to select microorganisms that can effectively degrade avermectin. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a strain of Bacillus velezensis and its application in preparing a water-soluble microbial agent.

[0006] The technical solutions of the present invention are as follows:

[0007] A strain of Bacillus velezinis ( Bacillus velezensis ) NKBV-01, deposited on November 6, 2024, at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32500.

[0008] The 16S rDNA sequence of the Bacillus velezensis NKBV-01 is shown in SEQ ID NO.1.

[0009] The Velez Bacillus NKBV-01 is used in producing extracellular polysaccharides, promoting soil aggregate formation, degrading avermectin, hydrolyzing protein, producing indoleacetic acid, promoting tomato plant growth, and promoting enzyme activity of tomato rhizosphere soil enzymes.

[0010] Preferably, the tomato rhizosphere soil enzymes include urease, catalase, and sucrase.

[0011] A water-soluble microbial agent comprises the Bacillus Velez subtilis NKBV-01.

[0012] Preferably, the water-soluble microbial agent also includes low-value fish hydrolysate dry powder and kason.

[0013] Further preferably, the water-soluble microbial agent comprises the following components: 75-85 parts by weight of Bacillus Velez NKBV-01 fermentation broth, 14.85-24.95 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.05-0.15 parts by weight of kasone.

[0014] More preferably, the water-soluble microbial agent comprises the following components: 80 parts by weight of Bacillus Velez NKBV-01 fermentation broth, 19.9 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.1 parts by weight of kasone.

[0015] The preparation method of the water-soluble microbial agent comprises the following steps: adding low-value fish enzymatic hydrolysate dry powder and kason to Bacillus velez NKBV-01 fermentation liquid, and mixing them evenly to obtain the water-soluble microbial agent.

[0016] Preferably, the preparation method of the low-value fish hydrolysate dry powder comprises the following steps: using low-value fish meal as a substrate, preparing a slurry with a substrate mass percentage of 8% to 12%, heating the slurry at 80 to 90°C for 10 to 20 minutes to achieve protein denaturation; after the slurry is cooled, adding a composite protease at a ratio of 1% to 3% of the substrate mass percentage, and then enzymolyzing at 50 to 55°C for 3 to 5 hours; after the enzymolysis is completed, heating to 80 to 90°C and maintaining for 10 to 20 minutes to inactivate the enzyme, and then lowering the temperature to below 55°C; performing solid-liquid separation to obtain a filtrate, concentrating the filtrate to a solid mass percentage of ≥25%, and spray drying to obtain a low-value fish hydrolysate dry powder.

[0017] Preferably, the composite protease comprises alkaline protease and neutral protease, and the mass ratio of the alkaline protease to the neutral protease is 1:1.

[0018] More preferably, the enzyme activity of the alkaline protease is 2×10 5 U / g, the enzyme activity of the neutral protease is 1×10 5 U / g. The definition and test method of activity unit refer to the national standard GB / T 23527-2009.

[0019] Preferably, the process parameters of the spray drying are: liquid temperature 50-55°C, inlet temperature 180-200°C, and outlet temperature 70-80°C.

[0020] Preferably, the method for preparing the Bacillus Velez subtilis NKBV-01 fermentation broth comprises the following steps:

[0021] (1) Inoculate Bacillus velezensis NKBV-01 onto a slant of LB solid culture medium and culture at 35-37°C for 20-24 hours to obtain an activated strain;

[0022] (2) Inoculate the activated strain into LB liquid medium and culture with shaking at 35-37°C and 200-220 rpm for 20-24 hours to obtain seed solution;

[0023] (3) Inoculate the seed liquid into LB liquid medium at a volume ratio of 1% to 3%, control the ventilation volume at 5 to 10 L / min, and culture under shaking conditions of 35 to 37°C and 200 to 220 rpm for 20 to 24 hours to obtain the bacterial liquid;

[0024] (4) Inoculate the bacterial liquid into the fermentation medium at a volume ratio of 2% to 5%, control the ventilation volume at 300 to 400 L / min, and culture at 35 to 37°C and 200 to 220 rpm for 48 to 72 hours; remove the residue to obtain the Bacillus velezensis NKBV-01 fermentation liquid.

[0025] Preferably, the fermentation medium in step (4) comprises the following components: 130 g / L of avermectin fermentation waste dry powder, 0.5 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, and 0.25 g / L of neutral protease, and the initial pH value is 6.5-7.5.

[0026] More preferably, the enzyme activity of the neutral protease is 1×10 5 U / g. The definition and test method of activity unit refer to the national standard GB / T 23527-2009.

[0027] Preferably, the method for preparing the fermentation medium comprises the following steps: after the above components are formulated, enzymatic hydrolysis is performed at 50-55° C. for 120-180 min; and then heating and sterilizing at 121° C. for 20-30 min to obtain the fermentation medium.

[0028] Preferably, the content of live bacteria of Bacillus velez NKBV-01 in the fermentation broth is (1.5-2.5)×10 10 cfu / mL.

[0029] Preferably, the viable bacteria content of Bacillus velezensis NKBV-01 in the water-soluble microbial agent is ≥5×10 9 cfu / g, and the content of water-soluble organic matter is ≥150g / L.

[0030] The water-soluble microbial agent is used in promoting the growth of tomato plants and harmlessly treating avermectin fermentation waste residue.

[0031] Preferably, the method for applying the water-soluble microbial agent in promoting the growth of tomato plants is: mixing the water-soluble microbial agent with water in a weight ratio of 1:200-1000 and then drip irrigation or flushing.

[0032] Preferably, the application method of the water-soluble microbial agent in the harmless treatment of avermectin fermentation waste residue is: mixing the water-soluble microbial agent with water in a weight ratio of 1:10 to 20, spraying the mixture on the surface of the avermectin fermentation waste residue compost, and stirring the mixture thoroughly.

[0033] Beneficial effects of the present invention:

[0034] (1) The present invention provides a strain of Bacillus velezinis NKBV-01, which has the functions of producing exopolysaccharides, promoting soil aggregate formation, degrading avermectin, hydrolyzing protein, producing indoleacetic acid, promoting tomato plant growth, and promoting the enzyme activity of tomato rhizosphere soil enzymes; the water-soluble microbial agent prepared based on the strain not only has the effect of improving soil aggregate structure and promoting tomato plant growth, but also can accelerate the temperature rise of avermectin fermentation waste residue composting, prolong the high temperature period and shorten the composting cycle.

[0035] (2) The present invention utilizes fermentation strains of avermectin fermentation waste residue and uses low-value fish hydrolysate as the main raw material to prepare water-soluble microbial agents, which not only helps to reduce production costs and broaden the source of raw materials, but also helps to promote the harmless treatment and resource utilization of avermectin fermentation waste residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the colony morphology of strain NKBV-01.

[0037] Figure 2 This is the bacterial morphology of strain NKBV-01.

[0038] Figure 3 is the phylogenetic tree of strain NKBV-01.

[0039] Figure 4 This is the temperature change during the composting process in Application Example 2. DETAILED DESCRIPTION

[0040] The following is explained in conjunction with specific embodiments:

[0041] Source of experimental materials:

[0042] Low-value fish meal: purchased from Xindeli Biotechnology Co., Ltd., Tai'an City, Shandong Province, China, and is the waste material of fish meal production.

[0043] Example 1: Isolation and screening of Bacillus velezensis NKBV-01

[0044] The samples were collected from the compost of avermectin fermentation waste residue in Ancheng Town, Pingyin County, Jinan City, Shandong Province, China. The specific separation and screening methods were as follows: 10 g of compost sample was weighed, 100 mL of sterile water was added, and the sample was placed on a constant temperature shaker at 35°C and 180 r / min for 30 minutes to obtain an extract.

[0045] (1) Screening of avermectin-degrading strains: The above-mentioned extract was inoculated into LB liquid culture medium containing 200 mg / L avermectin, and cultured at 35°C and 180 r / min for 3 days; the culture medium was then transferred to LB liquid culture medium containing 400 mg / L, 600 mg / L, and 800 mg / L avermectin in turn, and cultured for 3 days respectively to obtain enriched culture; the enriched culture was appropriately diluted, and then spread on LB solid culture medium with a concentration of 1 / 10 containing 800 mg / L avermectin, and cultured at 35°C until a single colony grew; the single colony was separated to obtain strains with different colony morphologies, and then inoculated into culture medium with avermectin as the sole carbon source, and cultured at 35°C and 180 r / min for 24 to 72 h. The strain that could make the culture medium turbid was picked out, i.e., the strain that could degrade avermectin was obtained and stored for future use.

[0046] The LB liquid culture medium comprises the following components: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 1000 mL of distilled water; pH 7.0, and sterilization at 121° C. for 20 min.

[0047] The LB solid medium containing 800 mg / L avermectin at a concentration of 1 / 10 is a medium in which the concentration of each nutrient is 1 / 10 of that of a conventional LB solid medium, and contains 800 mg / L avermectin; the specific components are: 1 g of peptone, 0.5 g of yeast extract, 1 g of NaCl, 800 mg of avermectin, 20 g of agar, and 1000 mL of distilled water.

[0048] The components of the culture medium with avermectin as the sole carbon source are: (NH4)2SO42g, MgSO40.2g, NaH2PO40.5g, CaCl20.1g, K2HPO40.5g, avermectin800mg, and distilled water1000mL.

[0049] (2) Screening of extracellular polysaccharide-producing strains: The strains that can degrade avermectin screened above were inoculated into the polysaccharide-producing fermentation medium and cultured at 35°C and 220 r / min for 48 h to obtain the fermentation broth; the fermentation broth was centrifuged at 5000 r / min for 15 min, the supernatant was taken, and 95% (v / v) ethanol solution with a volume three times that of the supernatant was added, mixed evenly and allowed to stand at 4°C for 2 h; if flocculent precipitates were produced, it was determined that the strain was capable of producing extracellular polysaccharides, and the more flocculent precipitates, the stronger the strain's ability to produce extracellular polysaccharides.

[0050] The polysaccharide-producing fermentation medium has the following formula: NaNO3 0.5 g / L, K2HPO4 0.5 g / L, MgSO4 0.5 g / L, NaCl 10 g / L, sucrose 25 g / L, yeast extract 3 g / L, peptone 3 g / L, and pH 7.0-7.2.

[0051] Through the above isolation and screening work, we finally screened out a strain that has the characteristics of degrading avermectin and producing extracellular polysaccharides, and named it "NKBV-01".

[0052] Example 2: Identification of Bacillus velezensis NKBV-01

[0053] The colony morphology of the strain NKBV-01 obtained by the above screening on LB solid medium is as follows: Figure 1 As shown, the colonies are round, milky white, and opaque; the bacterial morphology of strain NKBV-01 is as follows Figure 2 As shown, the bacterial body is rod-shaped and has the ability to form spores.

[0054] In addition, the physiological and biochemical characteristics of strain NKBV-01 were identified, and the identification results were as follows: catalase test+; methyl red test+; starch hydrolysis test+; denitrification test+; ammonia production test+; lactose fermentation test-; sucrose fermentation test+; xylose fermentation test+; arabinose test+; salicin test-; melibiose fermentation test+; rhamnose fermentation test-; mannitol fermentation test+; glucose fermentation test+ (+, positive; -, negative).

[0055] Genomic DNA of strain NKBV-01 was extracted and amplified by PCR using universal 16S rDNA primers. The amplification conditions were: 95°C pre-denaturation for 3 minutes; 94°C denaturation for 1 minute, 55°C annealing for 1 minute, and 72°C extension for 1.5 minutes, for a total of 30 cycles; and 72°C extension for 10 minutes. The amplified 16S rDNA gene sequence was sequenced, and the sequencing results are shown in SEQ ID NO. 1. The obtained 16S rDNA sequence was compared with existing sequences in the GenBank database, and multiple sequence homology analysis was performed to construct a phylogenetic tree. The results are shown in Figure 1. Figure 3 shown by Figure 3 It can be seen that the strain NKBV-01 obtained by the above screening is Bacillus velezensis The strain psa-3 Bacteria is relatively close in evolutionary distance. Combined with the physiological and biochemical characteristics of the strain, it was identified as "Bacillus velezinis ( Bacillus velezensis )".

[0056] Bacillus velez ( Bacillus velezensis ) NKBV-01, deposited on November 6, 2024, at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32500.

[0057] Example 3: Determination of beneficial properties of Bacillus velezinis NKBV-01

[0058] (1) Ability of Bacillus velezensis NKBV-01 to produce exopolysaccharides

[0059] The strain NKBV-01 was inoculated into LB liquid culture medium and cultured with shaking at 35°C and 200 r / min for 16 h to obtain seed liquid; the seed liquid was inoculated into polysaccharide fermentation medium at a volume ratio of 5%, and cultured with shaking at 35°C and 200 r / min for 60 h to obtain fermentation liquid; the fermentation liquid was centrifuged at 5000 r / min for 15 min, 10 mL of supernatant was taken, 40 mL of anhydrous ethanol was added, and the mixture was mixed evenly and allowed to stand at 4°C for 2 h to precipitate polysaccharides; the mixture was centrifuged at 5000 r / min for 15 min, the supernatant was removed, and the precipitate was dissolved with 4 mL of distilled water to obtain a crude polysaccharide extract, and the polysaccharide content was determined by the phenol-sulfuric acid method.

[0060] The experimental results showed that the polysaccharide content in the above-mentioned polysaccharide crude extract reached 1.62 g / L, which indicated that Bacillus velezensis NKBV-01 had a strong ability to produce extracellular polysaccharides.

[0061] (2) Effects of Bacillus velezensis NKBV-01 and its exopolysaccharides on soil aggregate formation

[0062] The strain NKBV-01 was inoculated into LB liquid culture medium and cultured with shaking at 35°C and 200 r / min for 16 h to obtain seed liquid; the seed liquid was inoculated into polysaccharide fermentation medium at a volume ratio of 5%, and cultured with shaking at 35°C and 200 r / min for 60 h to obtain fermentation liquid.

[0063] The fermentation broth was divided into two parts, one of which was normal fermentation broth, and the other was sterilized at high temperature and high pressure at 121°C for 30 minutes to obtain inactivated fermentation broth; 20 mL each of polysaccharide-producing fermentation medium (control group), inactivated fermentation broth and normal fermentation broth were respectively aspirated and added into a conical flask containing 200 g of soil (passed through a 0.25 mm sieve), mixed evenly, and cultured at room temperature for 45 days. During this period, deionized water was regularly added to keep the soil surface moist; 3 replicates were set for each treatment group, and the proportion of water-stable aggregates (>0.25 mm) in the soil was determined.

[0064] The results are shown in Table 1 below, where different letters represent significant differences (P < 0.05).

[0065] Table 1. Proportions of water-stable aggregates in different treatment groups

[0066]

[0067] As shown in Table 1, compared with the control group, the proportion of water-stable aggregates in the soil treated with inactivated fermentation broth and the normal fermentation broth increased by 33.06% and 62.33%, respectively. Compared with the inactivated fermentation broth group, the proportion of water-stable aggregates in the soil treated with the normal fermentation broth increased by 21.99%. These results indicate that Bacillus velezensis NKBV-01 and its exopolysaccharide production significantly promote the formation of soil aggregates.

[0068] (3) Degradation ability of Bacillus velezensis NKBV-01 on avermectin

[0069] The strain PAPM-LX25 was inoculated into the avermectin fermentation waste residue culture medium and cultured at 35°C and 200 r / min for 72 h to obtain the culture solution. The initial concentration of avermectin in the avermectin fermentation waste residue culture medium and the residual avermectin concentration in the culture solution were determined by liquid chromatography, and the degradation rate of avermectin by the strain PAPM-LX25 was calculated.

[0070] The preparation method of the avermectin fermentation waste residue culture medium is as follows: after drying the avermectin fermentation waste residue, crush it to 200 mesh, weigh 10g of the powder, add 100mL of distilled water, adjust the pH value to 6.5, and sterilize at 121°C for 15 minutes. The avermectin fermentation waste residue is solid waste generated during the avermectin fermentation production process, and its organic matter content is 75.86%, the nitrogen, phosphorus, and potassium contents are 3.62%, 1.35%, and 0.63%, respectively. The avermectin residue is 2.857mg / g, and the moisture content is 4.82%.

[0071] The experimental results showed that the degradation rate of avermectin by Bacillus Velez PAPM-LX25 could reach 96.87%.

[0072] (4) Protein degradation ability of Bacillus velezensis NKBV-01

[0073] The strain NKBV-01 was inoculated onto a solid medium containing skim milk powder and cultured at 35°C for 48 hours. The diameters of the clear zone and colony formations were measured, and the clear zone to colony diameter ratio (HC) was calculated. The results showed that the clear zone formed by the strain NKBV-01 on the medium had a diameter of 18.7 mm and the colony diameter was 4.6 mm, with an HC value of 4.07.

[0074] The components of the skimmed milk powder solid culture medium are as follows: 3.0 g skimmed milk powder, 1 g glucose, 3.2 g agar, 200 mL deionized water, pH 7.0, and high-temperature sterilization at 108° C. for 15 min.

[0075] The experimental results showed that Bacillus Velez PAPM-LX25 has excellent protein hydrolysis ability, which helps the strain to hydrolyze avermectin fermentation waste, helps the strain to grow and reproduce in the soil, and decompose and transform organic nutrients in the soil.

[0076] (5) Ability of Bacillus velezensis NKBV-01 to produce indoleacetic acid

[0077] The strain NKBV-01 was inoculated into LB liquid medium containing 200 mg / L L-tryptophan and cultured at 35°C and 200 rpm for 3 days to obtain a bacterial suspension. 50 μL of the suspension was dripped onto a white ceramic plate and mixed with 50 μL of Salkowski colorimetric solution. A Salkowski colorimetric solution containing 50 mg / L IAA was also added as a positive control. The white ceramic plate was placed in the dark at room temperature for 30 minutes, and the color change was observed. A red color indicates that the strain is capable of secreting indoleacetic acid.

[0078] The components of the Salkowski colorimetric solution are as follows: 450 mL of 35% HClO solution and 1 mL of 0.5 mol / L FeCl3 solution.

[0079] The experimental results showed that the ceramic plate turned dark red after the NKBV-01 bacterial suspension was added, indicating that the Bacillus velezensis NKBV-01 strain had excellent indoleacetic acid production ability.

[0080] Example 4: Preparation of water-soluble microbial agent

[0081] 1. Preparation of low-value fish enzymatic hydrolysate dry powder

[0082] Low-value fish meal is used as a substrate to prepare a feed liquid with a substrate mass percentage of 10%. After stirring evenly, the feed liquid is heated at 85°C for 15 minutes to achieve moderate protein denaturation; after the feed liquid is cooled to below 55°C, a composite protease is added at a ratio of 2% of the substrate mass percentage, and then enzymatic hydrolysis is carried out at 52.5°C for 5 hours; after the enzymatic hydrolysis is completed, the temperature is raised to 85°C and maintained for 15 minutes to inactivate the enzyme, and then the temperature is lowered to below 55°C; solid-liquid separation is carried out through a plate and frame filter to obtain a clarified filtrate, the filtrate is concentrated to a solid mass percentage of 25%, and spray-dried to obtain a low-value fish enzymatic hydrolysate dry powder.

[0083] The composite protease comprises alkaline protease and neutral protease, the mass ratio of the alkaline protease to the neutral protease is 1:1; the enzyme activity of the alkaline protease is 2×10 5 U / g, the enzyme activity of the neutral protease is 1×10 5 U / g. The definition and test method of activity unit refer to the national standard GB / T 23527-2009.

[0084] The process parameters of the spray drying are: liquid temperature 50-55°C, inlet temperature 180-200°C, and outlet temperature 70-80°C.

[0085] 2. Preparation of Bacillus Velezii NKBV-01 Fermentation Broth

[0086] (1) Inoculate Bacillus velezensis NKBV-01 on the slant of a LB solid culture tube and culture at 37°C for 24 hours to obtain an activated strain;

[0087] (2) Use an inoculation loop to inoculate the activated strain into a flask containing LB liquid medium, and culture it at 37°C and 200 rpm for 24 hours to obtain seed liquid;

[0088] (3) Inoculate the seed liquid into a 10 L seed tank containing 8 L of LB liquid medium at a volume ratio of 3%, control the ventilation volume at 8 L / min, and culture under shaking conditions of 37°C and 220 rpm for 20 h to obtain the seed tank bacterial liquid;

[0089] (4) The bacterial liquid from the seed tank was inoculated into a 1000 L fermentation tank containing 700 L fermentation medium at a volume ratio of 5%, and the ventilation volume was controlled at 350 L / min. The culture was carried out at 35 °C and 220 rpm for 60 h. After the culture was completed, the residue was removed using a 60-mesh vibrating screen, and the suspension that could pass through the vibrating screen was collected, namely the fermentation liquid of Bacillus velezensis NKBV-01, in which the viable bacterial content of strain NKBV-01 was 2.0 × 10 10 cfu / mL.

[0090] The components of the fermentation medium in step (4) are as follows: 130 g / L of avermectin fermentation waste dry powder, 0.5 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, and 0.25 g / L of neutral protease. The initial pH value is 6.5-7.5; the enzyme activity of the neutral protease is 1×10 5 U / g, the definition and detection method of the activity unit refer to the national standard GB / T 23527-2009. After the above components are prepared, the fermentation medium is obtained by enzymatic hydrolysis at 50°C for 120 minutes and sterilization at 121°C for 20 minutes.

[0091] The preparation method of the avermectin fermentation waste residue dry powder is as follows: drying the avermectin fermentation waste residue and then crushing it to 200 mesh. The avermectin fermentation waste residue is solid waste generated during the avermectin fermentation production process, and has an organic matter content of 75.86%, nitrogen, phosphorus, and potassium contents of 3.62%, 1.35%, and 0.63%, respectively. The avermectin residue is 2.857 mg / g, and the moisture content is 4.82%.

[0092] 3. Preparation of water-soluble microbial agents

[0093] Take 80 parts by weight of Bacillus Velez NKBV-01 fermentation liquid, 19.9 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.1 part by weight of kason; slowly add the low-value fish enzymatic hydrolysate dry powder and kason to the Bacillus Velez NKBV-01 fermentation liquid, and continue stirring until the mixture is uniform and no lumpy particles are visible, to obtain a water-soluble microbial agent.

[0094] Wherein, the content of live bacteria of Bacillus Velez NKBV-01 in the water-soluble microbial agent is ≥5×10 9 cfu / g, and the content of water-soluble organic matter is ≥150g / L.

[0095] Application Example 1: Application of water-soluble microbial agents in promoting the growth of tomato plants

[0096] This experiment was carried out in Ancheng Town, Pingyin County, Shandong Province. The specifications of the flower pots used were 20 cm in upper inner diameter, 14 cm in lower inner diameter, and 16 cm in height. Each pot was filled with 3 kg of soil, and the crop planted in the pots was tomato.

[0097] Treatment groups T1, T2, and CK were set up, with 20 replicates per group. In treatment group T1, 15 mL of the Bacillus velezensis NKBV-01 fermentation broth prepared in Example 4 was applied to each pot of soil; in treatment group T2, 15 mL of the water-soluble microbial agent prepared in Example 4 was applied to each pot of soil; and in control group CK, 15 mL of the fermentation medium described in Example 4 was applied to each pot of soil. Application was by flushing with water, with 5 mL applied each time. The solution was diluted 200-fold by weight with water and then slowly poured into the pot. The first application was on the 7th day after transplanting the tomatoes, and then every 10 days, for a total of 3 applications.

[0098] On the 60th day after transplanting, tomato plant height, stem diameter, fresh weight, and rhizosphere soil enzyme activities were measured. Plant height and stem diameter were measured with a tape measure and vernier caliper, respectively; fresh weight was determined by weighing. Rhizosphere soil enzyme activities were determined using the following methods: urease activity was determined using the sodium phenolate colorimetric method, catalase activity was determined using potassium permanganate titration, and sucrase activity was determined using the DNS colorimetric method.

[0099] The experimental results are shown in Tables 2 and 3, respectively. Different letters in the table represent significant differences (P < 0.05).

[0100] Table 2. Effects of water-soluble microbial agents on tomato plant growth

[0101]

[0102] As shown in Table 2, compared with the control group CK, treatment groups T1 and T2 showed different degrees of improvement in the plant height, stem diameter and fresh weight of tomato plants, indicating that Bacillus Velez-Nebels NKBV-01 can promote the growth of tomato plants; compared with treatment group T1, the increase rate of plant height, stem diameter and fresh weight of treatment group T2 was 16.18%, 20.00% and 21.94% respectively.

[0103] It can be seen that the water-soluble microbial agent prepared with Bacillus Velez subtilis NKBV-01 as the main ingredient has a significant promoting effect on the growth of tomato plants.

[0104] Table 3. Effects of water-soluble microbial agents on enzyme activities in tomato rhizosphere soil

[0105]

[0106] As shown in Table 3, compared with the control group CK, the activities of urease, catalase and sucrase in the tomato rhizosphere soil in treatment groups T1 and T2 were significantly increased, indicating that Bacillus Velezii NKBV-01 can significantly promote the enzyme activity of tomato rhizosphere soil enzymes; compared with treatment group T1, the increase rate of urease activity in the tomato rhizosphere soil in treatment group T2 was 18.12%, the increase rate of catalase activity was 13.33%, and the increase rate of sucrase activity was 6.23%.

[0107] It can be seen that the water-soluble microbial agent prepared with Bacillus Velez NKBV-01 as the main ingredient can significantly promote the enzyme activity of tomato rhizosphere soil enzymes.

[0108] Application Example 2: Application of Water-Soluble Microbial Agents in the Harmless Treatment of Avermectin Fermentation Waste Residue

[0109] This experiment was conducted at Shandong Zuotianshi Biotechnology Co., Ltd. (Ancheng Town, Pingyin County, Shandong Province). The composting container used was a foam box with internal dimensions of 60 cm in length, 45 cm in width, 41 cm in height, and a wall thickness of 5 cm. The composting raw materials consisted of 25% avermectin fermentation waste residue and 75% edible fungus bran in a mass ratio. The loading capacity of each box was 40 kg, and the initial moisture content was 55%.

[0110] Treatment group T, control group CK1, and control group CK2 were set up. Treatment group T was inoculated with the water-soluble microbial agent prepared in Example 4, control group CK1 was not inoculated with any agent, and control group CK2 was inoculated with a commercially available organic material composting agent. The inoculum dosage for each group was 0.1% (mass percentage). The inoculum was added to 20 times its weight of water, stirred evenly, sprayed on the compost surface, and thoroughly stirred. The compost was turned every 1-2 days for the first 50 days of composting, and every 5 days thereafter to provide oxygen and maintain a uniform state of the compost.

[0111] Among them, the avermectin fermentation waste residue is solid waste generated during the avermectin fermentation production process, with an organic matter content of 75.86%, nitrogen, phosphorus and potassium contents of 3.62%, 1.35% and 0.63% respectively, an avermectin residue of 2.857 mg / g, and a moisture content of 4.82%.

[0112] Among them, the edible fungus bran is a by-product of the cultivation process of Enoki mushrooms. Its organic matter content is 71.35%, and the contents of nitrogen, phosphorus and potassium are 1.52%, 2.38% and 1.21% respectively (all based on dry matter), and the moisture content is 56.75%.

[0113] Among them, the commercially available organic material composting agent is produced by Shandong Zuotianshi Biotechnology Co., Ltd., with an effective live bacterial count of 1 billion cfu / g. The bacterial species in the composting agent include Bacillus subtilis, Bacillus licheniformis, Aspergillus niger and yeast.

[0114] The test results are as follows Figure 4 As shown. Figure 4 It can be seen that the compost temperature of the control group CK1, CK2 and the treatment group T reached 50°C on the 10th day, the 10th day and the 4th day, respectively, and the high temperature duration was 10 days, 10 days and 14 days, respectively. This shows that the water-soluble microbial agent provided by the present invention can significantly accelerate the decomposition process of avermectin fermentation waste residue, extend the high temperature period and shorten the composting cycle, which has positive significance for the harmless treatment of avermectin fermentation waste residue, improving the composting efficiency of avermectin fermentation waste residue and improving product quality.

Claims

1. A strain of Bacillus velezinoffii ( Bacillus velezensis ) NKBV-01, deposited on November 6, 2024, at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32500.

2. The use of the Bacillus Velezii NKBV-01 according to claim 1 in producing exopolysaccharides, promoting soil aggregate formation, degrading avermectin, hydrolyzing proteins, producing indoleacetic acid, promoting tomato plant growth, and promoting enzyme activity in tomato rhizosphere soil; wherein, The tomato rhizosphere soil enzymes are urease, catalase and sucrase.

3. A water-soluble microbial agent, characterized in that: The invention comprises 75-85 parts by weight of the Bacillus Velez NKBV-01 according to claim 1, 14.85-24.95 parts by weight of low-value fish enzymatic hydrolysate dry powder and 0.05-0.15 parts by weight of kason.

4. The water-soluble microbial agent according to claim 3, wherein The invention comprises 80 parts by weight of the Bacillus Velez NKBV-01 according to claim 1, 19.9 parts by weight of low-value fish enzymatic hydrolysate dry powder and 0.1 part by weight of kason.

5. The water-soluble microbial agent according to claim 3, wherein The live bacteria content of Bacillus velezensis NKBV-01 is ≥5×10 9 cfu / g, and the content of water-soluble organic matter is ≥150g / L.

6. The method for preparing the water-soluble microbial agent according to claim 3, characterized in that: The method comprises the following steps: adding low-value fish enzymatic hydrolysate dry powder and kason into Bacillus velez NKBV-01 fermentation liquid, and mixing them evenly to obtain a water-soluble microbial agent.

7. The preparation method according to claim 6, wherein The preparation method of the low-value fish enzymatic hydrolysate dry powder comprises the following steps: using low-value fish meal as a substrate to prepare a feed liquid with a substrate mass percentage of 8% to 12%, heating the feed liquid at 80 to 90° C. for 10 to 20 minutes to achieve protein denaturation; after the feed liquid is cooled, adding a composite protease at a ratio of 1% to 3% of the substrate mass percentage, and then performing enzymolysis at 50 to 55° C. for 3 to 5 hours; after the enzymatic hydrolysis is completed, heating to 80 to 90° C. and maintaining for 10 to 20 minutes to inactivate the enzyme, and then reducing the temperature to below 55° C.; performing solid-liquid separation to obtain a filtrate, concentrating the filtrate to a solid mass percentage of 25% or more, and spray drying to obtain the low-value fish enzymatic hydrolysate dry powder.

8. The preparation method according to claim 7, wherein The composite protease comprises alkaline protease and neutral protease, and the mass ratio of the alkaline protease to the neutral protease is 1:

1.

9. The preparation method according to claim 8, wherein The enzyme activity of the alkaline protease is 2×10 5 U / g, the enzyme activity of the neutral protease is 1×10 5 U / g, its activity unit definition and detection method refer to the national standard GB / T23527-2009.

10. The preparation method according to claim 7, wherein The process parameters of the spray drying are: liquid temperature 50-55°C, inlet temperature 180-200°C, and outlet temperature 70-80°C.

11. The preparation method according to claim 6, wherein The preparation method of the Bacillus Velez subtilis NKBV-01 fermentation broth comprises the following steps: (1) Inoculate Bacillus velezensis NKBV-01 onto a slant of LB solid culture medium and culture at 35-37°C for 20-24 hours to obtain an activated strain; (2) Inoculate the activated strain into LB liquid medium and culture with shaking at 35-37°C and 200-220 rpm for 20-24 hours to obtain seed solution; (3) Inoculate the seed liquid into LB liquid medium at a volume ratio of 1% to 3%, control the ventilation volume at 5 to 10 L / min, and culture under shaking conditions of 35 to 37°C and 200 to 220 rpm for 20 to 24 hours to obtain the bacterial liquid; (4) Inoculate the bacterial liquid into the fermentation medium at a volume ratio of 2% to 5%, control the ventilation volume at 300 to 400 L / min, and culture at 35 to 37°C and 200 to 220 rpm for 48 to 72 hours; remove the residue to obtain the Bacillus velezensis NKBV-01 fermentation liquid.

12. The preparation method according to claim 11, wherein The fermentation medium in step (4) comprises the following components: 130 g / L of avermectin fermentation waste dry powder, 0.5 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, and 0.25 g / L of neutral protease, with an initial pH value of 6.5-7.

5.

13. The preparation method according to claim 12, wherein The enzyme activity of the neutral protease is 1×10 5 U / g. The definition and test method of activity unit refer to the national standard GB / T 23527-2009.

14. The preparation method according to claim 6, wherein The content of live bacteria of Bacillus velez NKBV-01 in the fermentation liquid of Bacillus velez NKBV-01 is (1.5~2.5)×10 10 cfu / mL.

15. Use of the water-soluble microbial agent according to claim 3 in promoting the growth of tomato plants and in harmless treatment of avermectin fermentation waste residue.

16. The use according to claim 15, characterized in that The method for applying the water-soluble microbial agent to promote the growth of tomato plants is as follows: mixing the water-soluble microbial agent with water in a weight ratio of 1:200 to 1000 and then drip irrigation or flushing application; The application method of the water-soluble microbial agent in the harmless treatment of avermectin fermentation waste residue is as follows: after mixing the water-soluble microbial agent with water in a weight ratio of 1:10-20, spraying the mixture on the surface of avermectin fermentation waste residue compost and stirring the mixture thoroughly.

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