Compound microbial agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological agents, and particularly discloses a compound microbial agent as well as a preparation method and application thereof. The compound microbial agent provided by the invention comprises compound bacillus, a carrier and a coating agent, the composite bacillus comprises bacillus velezensis, bacillus atrophaeus and bacillus firmus with the viable count ratio of (3-7): (1-4): 2; the viable count of the compound microbial agent is greater than or equal to 5 * 10 < 9 > cfu / g; the invention also provides a preparation method of the compound microbial agent. The preparation method comprises the following steps: strain fermentation, carrier adsorption, granulation and enveloping and low-temperature drying. The compound microbial agent provided by the invention can improve the soil structure, enhance the soil fertility and inhibit soil-borne diseases, so that the growth condition of crops is improved, and the quality and yield of the crops are improved.

Description

Technical Field

[0001] The present application relates to the technical field of biological preparations, and in particular to a composite microbial agent and a preparation method and application thereof. Background Art

[0002] Angelica sinensis is a common traditional Chinese medicinal herb with benefits such as nourishing blood, regulating menstruation, promoting blood circulation, and nourishing the liver and spleen. However, large-scale artificial cultivation of angelica sinensis suffers from severe continuous cropping problems, which affects both its yield and its medicinally active substances. Traditionally, chemical pesticides and fertilizers have been used to address these issues; however, these are prone to developing resistance and chemical residues. In recent years, with the development of green agriculture, microbial agents have become an increasingly important alternative to chemical pesticides and fertilizers due to their environmentally friendly, safe, and highly effective properties.

[0003] Currently, there are many types of microbial agents on the market, including single-species preparations, simple composite agents, and carrier-adsorbed agents. Among them, single-species preparations are agents that focus on a specific functional direction, while simple composite agents are physically mixed with two strains in the hope of integrating the functional advantages of different strains; carrier-adsorbed agents use carriers to carry bacteria, thereby improving the survival rate of bacteria to a certain extent. However, the above-mentioned agents are not perfect. Single-species preparations have a single function and a narrow disease prevention spectrum. Simple composite agents often have reduced effectiveness due to strain antagonism. Although carrier-adsorbed agents can increase the survival rate of bacteria, their release rate is uncontrollable. In addition, when used in the field, existing composite agents are greatly affected by environmental factors and have a significantly insufficient durability. Moreover, most agents lack multiple prevention and control mechanisms for soil-borne diseases, making it difficult to effectively deal with complex soil disease problems.

[0004] Therefore, there is an urgent need to develop a composite microbial agent with good symbiotic stability between microorganisms and long-lasting effects to improve the quality and yield of Chinese medicinal materials such as angelica. Summary of the Invention

[0005] In order to overcome the problems of existing microbial agents such as single function, antagonism of bacterial species or poor control effect of soil-borne diseases, the present application provides a composite microbial agent and its preparation method and application.

[0006] In the first aspect, the present application provides a composite microbial agent, which adopts the following technical solution: A composite microbial agent comprising a composite bacillus, a carrier and a coating agent; The composite Bacillus includes Bacillus velez, Bacillus atrophaeus and Bacillus firmus with a live cell count ratio of (3-7): (1-4): 2; The viable bacteria count of the composite microbial agent is ≥5×10 9 cfu / g.

[0007] The present application provides a composite microbial agent, wherein the three strains of Bacillus velez, Bacillus atrophaeus and Bacillus firmus used in the composite microbial agent are not antagonistic to each other and form a complementary relationship. On the one hand, they can synergistically form a biological barrier, inhibit or kill harmful pathogens and insect eggs in the soil, reduce the occurrence of pests and diseases, and improve the quality and yield of agricultural products; on the other hand, they can also decompose organic matter in the soil, release nutrients required by plants, generate organic substances such as humus, improve soil aggregate structure, improve soil fertility and water retention capacity, and improve the absorption and utilization rate of nutrients by crops, improve crop growth conditions, and improve the quality and yield of crops. In addition, the above-mentioned composite microbial agent is used in angelica planting management, which can also regulate the structure of soil microbial communities and alleviate continuous cropping obstacles. The carrier and the coating agent help to improve the survival rate of the bacteria in the composite microbial agent and can achieve slow release and long-term effect of the agent in the soil.

[0008] Optionally, the ratio of the viable cell counts of Bacillus velez, Bacillus atrophaeus and Bacillus firmus is (4-6):(2-3):2.

[0009] In some embodiments, the ratio of the viable cell counts of Bacillus Velezii, Bacillus atrophaeus and Bacillus firmus can be (3-4):3:2, (3-5):3:2, (3-6):3:2, (3-7):3:2, (4-5):3:2, (4-6):3:2, (4-7):3:2, (5-6):3:2, (5-7):3:2, (6-7):3:2, 5:(1-2):2, 5:(1-3):2, 5:(1-4):2, 5:(2-3):2, 5:(2-4):2 or 5:(3-4):2.

[0010] In a specific embodiment, the ratio of the viable cell counts of Bacillus velez, Bacillus atrophaeus and Bacillus firmus can also be 3:3:2, 4:3:2, 5:3:2, 6:3:2, 7:3:2, 5:1:2, 5:2:2 or 5:4:2.

[0011] Optionally, the carrier is a polyurethane-diatomaceous earth composite material with a pore size of 50-200 μm; and the coating agent is a mixture of pullulan and ε-polylysine.

[0012] The present application adopts a polyurethane-diatomaceous earth composite material as a carrier, which has the characteristics of high porosity and large specific surface area, so that the composite bacteria can be effectively adsorbed and stored inside the carrier, and supplied to the plant in a slow-release manner, thereby improving the utilization rate and durability of the composite microbial agent; In addition, the porous structure can provide a large number of attachment sites, which is conducive to the load and growth of microorganisms, which can not only enhance the biological activity of the agent, but also help promote the establishment and development of beneficial microbial communities in the soil. The present application adopts a mixture of pullulan and ε-polylysine as a coating agent, which can prevent the internal microorganisms from being adversely affected by the external environment (ultraviolet rays, drying) on ​​the one hand, and ensure the activity of the microorganisms; On the other hand, it can provide carbon sources and energy for microorganisms, maintain the balance of microbial flora, prevent the growth of harmful microorganisms, thereby providing a better living environment for beneficial microorganisms, promoting the growth and reproduction of beneficial microorganisms, and thus achieving soil improvement and crop quality and yield improvement.

[0013] Optionally, the preparation method of the polyurethane-diatomaceous earth composite material comprises the following steps: (1) Modifying diatomite with silane coupling agent KH550 to obtain silanized diatomite; (2) mixing the silanized diatomaceous earth and the pore-forming agent in a weight ratio of 1: (1.5-2.5) to form a uniform powder; (3) adding a chain extender and a catalyst to the preheated polyurethane prepolymer and reacting at a constant temperature for 1-2 hours; then adding the uniform powder to the prepolymer solution and stirring at a high speed of 1200-1500 rpm for 5-10 minutes to obtain a uniform slurry; the uniform slurry is injection molded, cured, washed with water, and vacuum dried to obtain a polyurethane-diatomaceous earth composite material; The weight ratio of the polyurethane prepolymer to the silanized diatomaceous earth is 100:(15-30).

[0014] Optionally, the weight ratio of the carrier to the coating agent is 10:(0.5-1); the weight ratio of the pullulan to ε-polylysine is 1:(0.5-0.8).

[0015] In some embodiments, the weight ratio of the carrier to the coating agent can be 10:(0.3-0.5), 10:(0.3-0.8), 10:(0.3-1), 10:(0.3-1.5), 10:(0.5-0.8), 10:(0.5-1), 10:(0.5-1.5), 10:(0.8-1), 10:(0.8-1.5), or 10:(1-1.5). In a specific embodiment, the weight ratio of the carrier to the coating agent can also be 10:0.3, 10:0.5, 10:0.8, 10:1 or 10:1.5.

[0016] In some embodiments, the weight ratio of pullulan to ε-polylysine can be 1:(0.5-0.6), 1:(0.5-0.7), 1:(0.5-0.8), 1:(0.5-1), 1:(0.6-0.7), 1:(0.6-0.8), 1:(0.6-1), 1:(0.7-0.8), 1:(0.7-1) or 1:(0.8-1).

[0017] In a specific embodiment, the weight ratio of the pullulan to ε-polylysine can also be 1:0.5, 1:0.6, 1:0.7, 1:0.8 or 1:1.

[0018] In a second aspect, the present application provides a method for preparing a composite microbial agent, comprising the following steps: Bacteria fermentation: Ferment the three strains separately until the number of viable bacteria is ≥1×10 12 cfu / mL, and then mixed according to the proportion to obtain a composite fermentation broth; Carrier adsorption: Mix the composite fermentation liquid and the carrier evenly and let it stand for adsorption for 2-3 hours; Granulation and coating: The carrier for adsorbing bacteria is placed in a granulator for granulation to form 2-3mm prefabricated particles; the prefabricated particles are then placed in a fluidized bed, and the diluted coating agent is sprayed onto the surface of the prefabricated particles to obtain coated particles.

[0019] Optionally, the dilution ratio of the coating agent is 5-10 times.

[0020] Optionally, the preparation method of the composite microbial agent further comprises low-temperature drying, specifically comprising the steps of drying the coated particles at 35±2° C. to a moisture content of ≤8%, to obtain a composite microbial agent with a particle size of 2-3 mm.

[0021] On the third aspect, the present application also provides the application of composite microbial agents in the cultivation of Chinese medicinal materials.

[0022] Optionally, the Chinese medicinal material is Angelica sinensis, and the dosage of the composite microbial agent is 10-20 kg / mu.

[0023] In summary, this application has the following beneficial effects: 1. This application uses Bacillus velezensis, Bacillus atrophaeus and Bacillus firmus with a live cell count ratio of (3-7): (1-4): 2 as a composite Bacillus, and prepares a composite microbial agent with excellent performance by adsorbing on a carrier and coating with a coating agent. The composite microbial agent can improve soil structure, enhance soil fertility and inhibit soil-borne diseases, thereby improving crop growth and increasing crop quality and yield.

[0024] 2. This application further controls the ratio of the number of live bacteria of Bacillus velezensis, Bacillus atrophaeus and Bacillus firmus within the range of (4-6): (2-3): 2, and selects a polyurethane-diatomaceous earth composite material with a pore size of 50-200 μm as a carrier, pullulan and ε-polylysine with a weight ratio of 1: (0.5-0.8) as a coating agent, and controls the weight ratio of the carrier to the coating agent within the range of 10: (0.5-1). The prepared composite microbial agent has a better use effect, can increase the yield of angelica to more than 600 kg / mu, reduce the incidence of paresthesia to less than 15%, increase the content of the medicinal active ingredient ferulic acid to more than 4.0 mg / g, and increase the content of the medicinal active ingredient ligustilide to more than 30.0 mg / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of the preparation method of the composite microbial agent provided in this application. DETAILED DESCRIPTION

[0026] The present application provides a composite microbial agent, comprising a composite Bacillus, a carrier and a coating agent; the composite Bacillus comprises Bacillus velezensis, Bacillus atrophaeus and Bacillus firmus with a viable cell count ratio of (3-7): (1-4): 2; the viable cell count of the composite microbial agent is ≥ 5×10 9 cfu / g. Furthermore, the ratio of the viable counts of Bacillus velezensis, Bacillus atrophaeus and Bacillus firmus is (4-6): (2-3): 2; The coating agent is a mixture of pullulan and ε-polylysine; the weight ratio of the carrier to the coating agent is 10:(0.5-1); and the weight ratio of the pullulan to ε-polylysine is 1:(0.5-0.8).

[0027] In the present application, the carrier is a polyurethane-diatomaceous earth composite material with a pore size of 50-200 μm. The preparation method of the polyurethane-diatomaceous earth composite material includes the following steps: (1) First, place the diatomaceous earth in an oven and dry it for 4-6 hours; dissolve the silane coupling agent KH550 in a 90-95% ethanol solution, adjust the pH to 4-5 with acetic acid, and hydrolyze for 30-60 minutes; then add the dried diatomaceous earth to the above solution, ultrasonically disperse it for 30-60 minutes, and continue stirring and reacting at 60-80°C for 2-4 hours. After centrifugation, filtration, washing, and vacuum drying, silanized diatomaceous earth is obtained; wherein the weight ratio of the diatomaceous earth to the silane coupling agent is 100:(1-5); (2) mixing silanized diatomaceous earth and ammonium bicarbonate pore-forming agent in a weight ratio of 1: (1.5-2.5), adding 4-6% of the total weight of acetone and performing ultrasonic dispersion to form a uniform powder; (3) The polyurethane prepolymer is heated to 55-70°C, and 1,4-butanediol chain extender is added under stirring, and the -NCO / -OH molar ratio of the prepolymer to 1,4-butanediol is controlled to be (1.05-1.2):1; then the catalyst DBTDL (1-3% of the weight of the prepolymer) is added dropwise, and the reaction is carried out at a constant temperature for 1-2 hours; then the uniform powder obtained in step (2) is added to the prepolymer solution, an appropriate amount of water is added, and the mixture is stirred at a high speed of 1200-1500 rpm for 5-10 minutes to obtain a uniform slurry; the slurry is injected into a mold and cured at 50-60°C for 24-30 hours; then the solid sample is washed with water to remove the pore-forming agent, and vacuum dried to obtain a polyurethane-diatomite composite material; wherein the weight ratio of the polyurethane prepolymer to the silanized diatomite is 100:(15-30); the pore size of the obtained polyurethane-diatomite composite material is 50-200 μm.

[0028] The preparation method of the composite microbial agent provided in this application comprises the following steps: (1) Fermentation: The three strains were fermented separately until the number of viable bacteria was ≥1×10 12 cfu / mL, and then mixed according to the proportion to obtain a composite fermentation broth; (2) Carrier adsorption: Mix the composite fermentation liquid and the carrier evenly and let it stand for adsorption for 2-3 hours; (3) Granulation and coating: The carrier adsorbing the bacteria is placed in a granulator for granulation to form 2-3 mm prefabricated granules; the prefabricated granules are then placed in a fluidized bed, and the coating agent diluted 5-10 times is sprayed onto the surface of the prefabricated granules to obtain coated granules; (4) Low-temperature drying: Dry the coated particles at 35±2°C to a moisture content of ≤8% to obtain a composite microbial agent with a particle size of 2-3 mm.

[0029] In the examples of the present application, the Bacillus velez is Bacillus velez BGB-89R or Bacillus velez LBSW21-04, the deposit number of Bacillus velez BGB-89R is CGMCC No. 28824, and the deposit number of Bacillus velez LBSW21-04 is CGMCC No. 24160; the Bacillus atrophaeus is Bacillus atrophaeus BGB-98R or Bacillus atrophaeus BGB-S1, the deposit number of Bacillus atrophaeus BGB-98R is CGMCC No. 27752, and the deposit number of Bacillus atrophaeus BGB-S1 is CGMCC No. 29366; Bacillus firmus is purchased from Shanghai Guyan Industrial Co., Ltd. with the product number GOY-JZ1072; the strains used in this application are all known strains disclosed in the prior art, and the strains, raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0030] The present application is further described in detail below with reference to the embodiments, performance testing and accompanying drawings.

[0031] Preparation Example 1 Preparation Example 1 provides a polyurethane-diatomaceous earth composite material, the preparation method of which includes the following steps: (1) First, place the diatomaceous earth in an oven and dry it for 5 hours. Then, dissolve 3g of silane coupling agent KH550 in 150mL of 90% ethanol solution, adjust the pH to 4.5 with acetic acid, and hydrolyze for 30 minutes. Then, add 100g of the dried diatomaceous earth to the above solution, ultrasonically disperse it for 30 minutes, and continue stirring at 70°C for 3 hours. After centrifugation, filtration, washing, and vacuum drying, silanized diatomaceous earth is obtained. (2) 20 g of silanized diatomaceous earth was mixed with 40 g of ammonium bicarbonate pore-forming agent, and 4-6% of the total weight of acetone was added for ultrasonic dispersion to form a uniform powder; (3) 100 g of isocyanate prepolymer WD-8100 was heated to 60°C, and 1,4-butanediol chain extender was added under stirring, and the -NCO / -OH molar ratio of the prepolymer to 1,4-butanediol was controlled to be 1.1:1; then 2% of the weight of the prepolymer catalyst DBTDL was added dropwise, and the reaction was carried out at a constant temperature for 2 hours; then the uniform powder obtained in step (2) was added to the prepolymer solution, an appropriate amount of water was added, and the mixture was stirred at a high speed of 1300 rpm for 10 minutes to obtain a uniform slurry; the slurry was injected into a mold and cured at 55°C for 24 hours; then the solid sample was washed with water to remove the pore-forming agent, and vacuum dried to obtain a polyurethane-diatomaceous earth composite material with a pore size of 100-150 μm.

[0032] Examples 1-8 Examples 1-8 each provide a composite microbial agent.

[0033] The difference between the above embodiments is that the ratio of the number of viable bacteria of each strain in the composite microbial inoculant is shown in Table 1 below.

[0034] The preparation method of the composite microbial agent provided in Examples 1-8 comprises the following steps: (1) Fermentation: Bacillus velezensis BGB-89R, Bacillus atrophaeus BGB-98R and Bacillus firmus GOY-JZ1072 were fermented and cultured until the viable cell count reached 1×10 12 cfu / mL, and then the three fermentation broths were mixed according to the ratio of viable bacterial counts shown in Table 1 (i.e., the volume ratio of each fermentation broth) to obtain a composite fermentation broth; (2) Carrier adsorption: 100 mL of the composite fermentation broth was mixed evenly with 100 g of the carrier (Preparation Example 1) and allowed to stand for adsorption for 2-3 h; (3) Granulation and coating: The carrier on which the bacteria were adsorbed was placed in a granulator for granulation to form prefabricated particles of 2-3 mm; the prefabricated particles were then placed in a fluidized bed, and 8 g of the coating agent was diluted with water to 50 mL and sprayed onto the surface of the prefabricated particles to obtain coated particles; the coating agent was a mixture of pullulan and ε-polylysine in a weight ratio of 1:0.7; (4) Low-temperature drying: Dry the coated particles at 35±2°C to a moisture content of ≤8%, obtaining a particle size of 1.5±0.3 mm and a viable cell count of ≥5×10 9 cfu / g of composite microbial agent.

[0035] Table 1 Ratio of viable bacteria count of each strain in the composite microbial inoculant provided in Examples 1-8 Example 9 Example 9 provides a composite microbial agent.

[0036] The difference between the above embodiment and embodiment 3 is that the coating agent is pullulan and ε-polylysine in a weight ratio of 1:0.5.

[0037] Example 10 Example 10 provides a composite microbial agent.

[0038] The difference between the above embodiment and embodiment 3 is that the coating agent is pullulan and ε-polylysine in a weight ratio of 1:0.6.

[0039] Example 11 Example 11 provides a composite microbial agent.

[0040] The difference between the above embodiment and embodiment 3 is that the coating agent is pullulan and ε-polylysine in a weight ratio of 1:0.8.

[0041] Example 12 Example 12 provides a composite microbial agent.

[0042] The difference between the above embodiment and embodiment 3 is that the coating agent is pullulan and ε-polylysine in a weight ratio of 1:1.

[0043] Example 13 Example 13 provides a composite microbial agent.

[0044] The difference between the above embodiment and embodiment 3 is that the amount of the coating agent used is 3 g, which is diluted to 20 mL.

[0045] Example 14 Example 14 provides a composite microbial agent.

[0046] The difference between the above embodiment and embodiment 3 is that the amount of the coating agent used is 5 g, which is diluted to 30 mL.

[0047] Example 15 Example 15 provides a composite microbial agent.

[0048] The difference between the above embodiment and embodiment 3 is that the amount of the coating agent used is 10 g, which is diluted to 60 mL.

[0049] Example 16 Example 16 provides a composite microbial agent.

[0050] The difference between the above embodiment and embodiment 3 is that the amount of the coating agent used is 15 g, which is diluted to 80 mL.

[0051] Example 17 Example 17 provides a composite microbial agent.

[0052] The difference between the above embodiment and embodiment 3 is that Bacillus velez BGB-89R is replaced by Bacillus velez LBSW21-04.

[0053] Example 18 Example 18 provides a composite microbial agent.

[0054] The difference between the above embodiment and embodiment 3 is that Bacillus atrophaeus BGB-98R is replaced by Bacillus atrophaeus BGB-S1.

[0055] Comparative Example 1 Comparative Example 1 provides a composite microbial agent.

[0056] The difference between the comparative example and Example 3 is that the ratio of the viable cell counts of Bacillus velezensis, Bacillus atrophaeus and Bacillus firmus is 1:1:1.

[0057] Comparative Example 2 Comparative Example 2 provides a composite microbial agent.

[0058] The difference between the comparative example and Example 3 is that Bacillus atrophaeus is replaced by Bacillus subtilis.

[0059] Comparative Example 3 Comparative Example 3 provides a composite microbial agent.

[0060] The difference between the comparative example and Example 3 is that Bacillus firmus is replaced by Bacillus subtilis.

[0061] Angelica Planting Experiment The growth promotion and disease control effects of the composite microbial agents provided in Examples 1-18 and Comparative Examples 1-3 were investigated, and the results are shown in Table 2 below.

[0062] (1) Test method: A continuous cropping obstacle soil where Angelica sinensis had been planted for three consecutive years was used as the test site, and weeding was carried out. The test site was divided into 22 areas (each area was 6 m × 5 m), of which 18 areas were basally treated with the composite microbial agent provided in Examples 1-18 (test areas 1-18), 3 areas were basally treated with the composite microbial agent provided in Comparative Examples 1-3 (control areas 1-3); and 1 area was not treated (blank control area).

[0063] In early April 2022, before planting angelica, apply a compound microbial agent at a rate of 15 kg / mu to the soil where Codonopsis pilosula is planted, and deep plowing and mixing should be carried out, with a rotary tillage depth of 35 cm. Transplant angelica seedlings with a height of 10-15 cm into the above-mentioned test field and plant them according to the specifications of 30-40 cm row spacing and 20-25 cm plant spacing. Water them in time after planting to keep the soil moist. During the planting period, timely weed control and pest and disease monitoring and control should be carried out; water should be applied every 7-10 days; 10 days after transplanting, apply 15 kg / mu of urea; and apply compound fertilizer and organic fertilizer appropriately during the growth period of angelica.

[0064] In mid-October 2024, the angelica in each experimental field was harvested, and the angelica yield, the incidence of angelica root numbness disease, and the contents of the medicinal active ingredients ferulic acid and ligustilide in the angelica roots in each experimental area were tested. The results are shown in Table 2 below.

[0065] Note: The yield of angelica refers to the fresh weight yield of angelica roots, converted to per mu; the incidence of angelica root paronychia refers to the ratio of the number of angelica plants with paronychia root disease in the experimental area to the total number of angelica plants in the experimental area; the contents of ferulic acid and ligustilide were detected by high performance liquid chromatography, and the specific method was referred to the angelica item in the "Chinese Pharmacopoeia". Ten groups were tested in each experimental area and the average value was taken.

[0066] Table 2 Results of yield and quality test of Angelica sinensis in each experimental area According to the test results in Table 2, the yield of angelica produced in the experimental areas 1-18 was 558.4-679.2 kg / mu, the incidence of paronychia was 12.4-18.5%, the content of the medicinal active ingredient ferulic acid was 3.713-4.817 mg / g, and the content of the medicinal active ingredient ligustilide was 26.593-34.261 mg / g; while the yield of angelica produced in the control areas 1-3 and the blank control area was only 315.3-472.5 kg / mu, the incidence of paronychia was 24.9-38.5%, the content of the medicinal active ingredient ferulic acid was 2.816-3.184 mg / g, and the content of the medicinal active ingredient ligustilide was 17.463-23.428 mg / g. Therefore, it is explained that the present application uses Bacillus velezensis, Bacillus atrophaeus and Bacillus firmus with a live bacteria count ratio of (3-7): (1-4): 2 as a composite Bacillus, and the resulting composite microbial agent can improve soil structure, enhance soil fertility and inhibit soil-borne diseases, thereby improving the growth of the Chinese medicinal material Angelica sinensis and improving the quality and yield of Angelica sinensis.

[0067] The test results of experimental areas 1-8 show that the yield of angelica produced in experimental areas 1, experimental areas 5-6 and experimental area 8 is 558.4-592.7 kg / mu, the incidence of paronychia is 15.9-18.2%, the content of the medicinal active ingredient ferulic acid is 3.713-4.007 mg / g, and the content of the medicinal active ingredient ligustilide is 26.593-29.090 mg / g; while the yield of angelica produced in experimental areas 2-4 and experimental area 7 is 632.7-679.2 kg / mu (≥600 kg / mu), the incidence of paronychia is 12.5-14.1% (≤15%), the content of the medicinal active ingredient ferulic acid is 4.328-4.817 mg / g (≥4.0 mg / g), and the content of the medicinal active ingredient ligustilide is 30.592-34.261 mg / g (≥30.0 ​​mg / g). Therefore, it is shown that the present application further controls the ratio of the number of live bacteria of Bacillus velez, Bacillus atrophaeus and Bacillus firmus within the range of (4-6): (2-3): 2, and the prepared composite microbial agent has better use effect and better effect on improving the quality and yield of Angelica sinensis.

[0068] The test results of test area 3 and test areas 9-12 show that the yield of angelica produced in test area 12 is 582.9 kg / mu, the content of the medicinal active ingredient ferulic acid is 3.846 mg / g, and the content of the medicinal active ingredient ligustilide is 32.654 mg / g; while the yield of angelica produced in test areas 3 and test areas 9-11 is 616.8-679.2 kg / mu (≥600 kg / mu), the content of the medicinal active ingredient ferulic acid is 4.179-4.817 mg / g (≥4.0 mg / g), and the content of the medicinal active ingredient ligustilide is 33.087-34.261 mg / g (≥33.0 mg / g). Therefore, it is shown that the present application further selects pullulan and ε-polylysine in a weight ratio of 1: (0.5-0.8) as coating agents, and the obtained composite microbial agent has better use effect and better effect on improving the quality and yield of Angelica sinensis.

[0069] The test results of test area 3 and test areas 13-16 show that with the increase in the amount of coating agent added, the yield of the obtained angelica sinensis shows a trend of first increasing and then remaining basically unchanged, the incidence of paronychia shows a trend of first decreasing and then increasing, and the contents of the medicinal active ingredients ferulic acid and ligustilide show a trend of first increasing and then decreasing; among them, the yield of angelica sinensis produced in test area 3 and test areas 14-15 is 638.6-679.2kg / mu (≥600kg / mu), the incidence of paronychia is 12.5-14.2% (≤15%), the content of the medicinal active ingredient ferulic acid is 4.383-4.817mg / g (≥4.0mg / g), and the content of the medicinal active ingredient ligustilide is 31.571-34.261mg / g (≥30.0mg / g). Therefore, it is shown that the present application further controls the weight ratio of the carrier to the coating agent within the range of 10:(0.5-1), and the prepared composite microbial agent has better use effect and better effect on improving the quality and yield of Angelica sinensis.

[0070] The test results of experimental areas 3 and 17-18 show that the yield of angelica produced in experimental area 17-18 is 573.9-591.0 kg / mu, the incidence of paronychia is 15.8-17.1%, the content of the medicinal active ingredient ferulic acid is 4.028-4.113 mg / g, and the content of the medicinal active ingredient ligustilide is 27.265-29.413 mg / g; while the yield of angelica produced in experimental area 3 is 679.2 kg / mu (≥600 kg / mu), the incidence of paronychia is 12.5% ​​(≤15%), the content of the medicinal active ingredient ferulic acid is 4.817 mg / g (≥4.0 mg / g), and the content of the medicinal active ingredient ligustilide is 34.261 mg / g (≥33.0 mg / g). Therefore, it is shown that the composite microbial agent prepared by using Bacillus velezensis BGB-89R, Bacillus atrophaeus BGB-98R and Bacillus firmus in this application has better use effect and better effect on improving the quality and yield of Angelica sinensis.

[0071] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A composite microbial agent, characterized in that: It includes a complex bacillus, a carrier and an encapsulating agent; The composite Bacillus includes Bacillus velez, Bacillus atrophaeus and Bacillus firmus with a live cell count ratio of (3-7): (1-4): 2; The viable bacteria count of the composite microbial agent is ≥5×10 9 cfu / g.

2. The composite microbial agent according to claim 1, characterized in that The ratio of the number of live bacteria of Bacillus velez, Bacillus atrophaeus and Bacillus firmus is (4-6): (2-3):

2.

3. The composite microbial agent according to claim 1, characterized in that The carrier is a polyurethane-diatomaceous earth composite material with a pore size of 50-200 μm; the coating agent is a mixture of pullulan and epsilon-polylysine.

4. The composite microbial agent according to claim 3, characterized in that The preparation method of the polyurethane-diatomaceous earth composite material comprises the following steps: (1) Using silane coupling agent KH550 to modify diatomite to obtain silanized diatomite; (2) Mixing the silanized diatomaceous earth and the pore-forming agent in a weight ratio of 1: (1.5-2.5) to form a uniform powder; (3) adding a chain extender and a catalyst to the preheated polyurethane prepolymer and reacting at a constant temperature for 1-2 hours; then adding the uniform powder to the prepolymer solution and stirring at a high speed of 1200-1500 rpm for 5-10 minutes to obtain a uniform slurry; the uniform slurry is injection molded, cured, washed with water, and vacuum dried to obtain a polyurethane-diatomaceous earth composite material; The weight ratio of the polyurethane prepolymer to the silanized diatomaceous earth is 100:(15-30).

5. The composite microbial agent according to claim 4, characterized in that The weight ratio of the carrier to the coating agent is 10:(0.5-1); the weight ratio of the pullulan to ε-polylysine is 1:(0.5-0.8).

6. The method for preparing the composite microbial agent according to any one of claims 1 to 5, characterized in that: The following steps are involved: Bacteria fermentation: Ferment the three strains separately until the number of viable bacteria is ≥1×10 12 cfu / mL, and then mixed according to the proportion to obtain a composite fermentation broth; Carrier adsorption: Mix the composite fermentation liquid and the carrier evenly and let it stand for adsorption for 2-3 hours; Granulation and coating: The carrier for adsorbing bacteria is placed in a granulator for granulation to form 2-3mm prefabricated particles; the prefabricated particles are then placed in a fluidized bed, and the diluted coating agent is sprayed onto the surface of the prefabricated particles to obtain coated particles.

7. The method for preparing the composite microbial agent according to claim 6, characterized in that: The dilution ratio of the coating agent is 5-10 times.

8. The method for preparing the composite microbial agent according to claim 7, characterized in that: The preparation method of the composite microbial agent also includes low-temperature drying, and the specific steps are: drying the coated particles at 35±2°C to a moisture content of ≤8%, to obtain a composite microbial agent with a particle size of 2-3 mm.

9. Use of the composite microbial agent according to any one of claims 1 to 5 in the cultivation of Chinese medicinal materials.

10. The use according to claim 9, characterized in that The Chinese medicinal material is angelica sinensis, and the dosage of the composite microbial agent is 10-20 kg / mu.

Images