A composite microbial agent and its preparation method and application

Abstract

The present invention belongs to the field of microbial control technology, and specifically relates to a composite microbial agent, a preparation method thereof, and an application thereof. The preparation method of the composite microbial agent comprises: (1) activating Bacillus megaterium, Bacillus marineus N6‑2, and Lactobacillus plantarum A37 respectively to obtain activation solutions of each strain; mixing the activation solutions of each strain to prepare a mixed bacterial solution; (2) inoculating the mixed bacterial solution into a seed culture medium to prepare a seed solution; (3) inoculating the seed solution into a fermentation culture medium for fermentation, and then drying and crushing to obtain a composite microbial agent. The composite microbial agent prepared by the present invention increases the total number of effective viable bacteria by optimizing the composition of the culture medium. After using the composite microbial agent, the content of organic matter, available nitrogen, available phosphorus, and available potassium in the soil can be significantly increased, soil fertility can be improved, thereby promoting tomato growth, and improving tomato yield and quality, and has broad application prospects.

Description

Technical Field

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

[0002] During the growth of crops and vegetables, long-term excessive application of chemical fertilizers can lead to a series of problems, including soil compaction, acidification, reduced organic matter content, and degradation of soil communities. These problems, in turn, affect crop and vegetable quality and restrict the soil's ability to sustain growth. Therefore, the search for novel fertilizers to replace chemical fertilizers has become a hot topic and a challenge in the field of agricultural resources and environment. Against this backdrop, microbial fertilizers have emerged.

[0003] As a green and environmentally friendly agricultural input, microbial agents have received widespread attention in recent years. By introducing beneficial microorganisms, microbial agents can improve the structure of soil microbial communities, promote the conversion and release of nutrients such as nitrogen, phosphorus, and potassium in the soil, and improve soil fertility. At the same time, some microorganisms also have antibacterial and insecticidal properties, which can effectively prevent and control crop diseases and reduce the use of chemical pesticides. However, existing microbial agents still have some problems in practical application, such as low strain activity, poor adaptability, and single function, resulting in unstable effects and difficulty in meeting the diverse needs of modern agricultural production.

[0004] In the preparation process of microbial agents, the activity and quantity of the strains are the key factors that determine their effectiveness. Traditional microbial fermentation technology often has difficulty in ensuring the efficient proliferation and activity maintenance of the strains, especially in large-scale production, where the activity and stability of the strains are easily affected by factors such as culture medium composition and fermentation conditions. In addition, the functions of microbial agents based on a single strain are limited, making it difficult to simultaneously achieve multiple goals such as soil improvement, disease control, and crop growth promotion. Therefore, the development of a composite microbial agent that optimizes the strain combination and fermentation process to increase the number of effective live bacteria in the agent and give it multiple functions is of great practical significance. Summary of the Invention

[0005] In order to fill the deficiencies of the prior art, one of the objectives of the present invention is to provide a method for preparing a composite microbial agent.

[0006] A second object of the present invention is to provide a composite microbial agent prepared by the above preparation method.

[0007] The third object of the present invention is to provide the use of the above-mentioned composite microbial agent in promoting the growth of crops.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A method for preparing a composite microbial agent comprises the following steps:

[0010] (1) Activating Bacillus megaterium, Bacillus marineus N6-2, and Lactobacillus plantarum A37 separately to obtain activation solutions of each strain; mixing the activation solutions of each strain to prepare a mixed bacterial solution;

[0011] (2) inoculating the mixed bacterial solution obtained in step (1) into a seed culture medium to prepare a seed solution;

[0012] (3) The seed liquid obtained in step (2) is inoculated into a fermentation medium for fermentation, and then dried and crushed to obtain a composite microbial agent.

[0013] Furthermore, the seed culture medium in step (2) comprises the following components by mass fraction: 1.5-2.5% lactose, 0.5-1.5% yeast extract, 0.5-1.5% magnesium sulfate, 0.2-1.0% trisodium fructose 1,6-diphosphate, and the balance water.

[0014] Furthermore, the fermentation medium in step (3) comprises the following components in mass fractions: 0.1-1.5% starch, 0.5-2% soybean cake powder, 0.5-2% ammonium sulfate, 0.5-1.0% peptone, 0.3-0.5% yeast powder, 0.1-0.3% calcium carbonate, 0.5-2.5% potassium dihydrogen phosphate, 0.5-1.5% 6-amino-3-methylpurine, and the balance water.

[0015] Furthermore, the ratio of the effective bacterial concentrations of Bacillus megaterium, Bacillus marineus N6-2 and Lactobacillus plantarum A37 in the mixed bacterial solution in step (1) is 1:(1-3):(1-3).

[0016] Furthermore, the total effective bacterial concentration of the composite microbial agent is ≥10 9 CFU / g.

[0017] Furthermore, the inoculation amount of the seed liquid in step (3) is 4-6%.

[0018] Furthermore, in step (3), the fermentation temperature is 25-35° C., and the fermentation time is 25-40 h.

[0019] A composite microbial agent is prepared according to the method described above.

[0020] The application of the above-mentioned composite microbial agent in promoting the growth of crops.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0022] The present invention provides a method for preparing a composite microbial agent. By adding trisodium 1,6-fructose diphosphate and 6-amino-3-methylpurine to the seed culture medium and fermentation medium, respectively, the effective viable counts of Bacillus megaterium, Bacillus marineus N6-2, and Lactobacillus plantarum A37 in the composite microbial agent are increased. When applied to soil, the composite microbial agent significantly increases the content of organic matter, available nitrogen, available phosphorus, and available potassium in the soil, improving soil fertility, thereby promoting tomato growth, and increasing tomato yield and quality. The agent has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of the composite microbial agent prepared in the present invention on tomato yield. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained from commercial channels.

[0025] The accession number of the Bacillus megaterium of the present invention is CGMCC No. 3770, the accession number of the marine Bacillus N6-2 is CCTCC NO: M2014586, and the accession number of the Lactobacillus plantarum A37 is CCTCC NO: M2019559.

[0026] Example 1

[0027] A method for preparing a composite microbial agent comprises the following steps:

[0028] (1) Bacillus megaterium, marine Bacillus N6-2, and Lactobacillus plantarum A37 were activated separately to obtain activation solutions of each strain; the activation solutions of each strain were mixed to prepare a mixed bacterial solution; the ratio of the number of live bacteria of Bacillus megaterium, marine Bacillus N6-2, and Lactobacillus plantarum A37 in the mixed bacterial solution was 1:2:2, and the number of live bacteria of Bacillus megaterium in the mixed bacterial solution was 1×10 8 CFU / g.

[0029] (2) The mixed bacterial solution obtained in step (1) was transferred to a seed culture medium containing the following components by mass fraction: 2.0% lactose, 1.0% yeast extract, 1.0% MgSO4, 0.6% trisodium fructose 1,6-diphosphate, and the remainder water. It was placed in a shaker at 30°C and cultured at 200 rpm until the OD 600 The pH value is about 5 to obtain seed liquid.

[0030] (3) The seed liquid obtained in step (2) was inoculated with a fermentation medium containing the following components by mass fraction at a rate of 5%: 0.8% starch, 1.2% soybean meal, 1.2% ammonium sulfate, 0.7% peptone, 0.4% yeast powder, 0.2% calcium carbonate, 1.5% potassium dihydrogen phosphate, 1.0% 6-amino-3-methylpurine, and the balance water. The medium was placed in a shaker at 30°C and fermented at 200 rpm for 35 hours. The fermentation culture was dried at 38°C, crushed, and passed through a 200-mesh sieve to obtain a composite microbial agent; the total number of viable bacteria in the composite microbial agent was ≥10 9 CFU / g.

[0031] This embodiment also provides a composite microbial agent obtained by the above preparation method.

[0032] Example 2

[0033] A method for preparing a composite microbial agent comprises the following steps:

[0034] (1) Bacillus megaterium, marine Bacillus N6-2, and Lactobacillus plantarum A37 were activated separately to obtain activation solutions of each strain; the activation solutions of each strain were mixed to prepare a mixed bacterial solution; the ratio of the number of live bacteria of Bacillus megaterium, marine Bacillus N6-2, and Lactobacillus plantarum A37 in the mixed bacterial solution was 1:1:1, and the number of live bacteria of Bacillus megaterium in the mixed bacterial solution was 1×10 8 CFU / g.

[0035] (2) The mixed bacterial solution obtained in step (1) was transferred to a seed culture medium containing the following components by mass fraction: 1.5% lactose, 0.5% yeast extract, 0.5% magnesium sulfate, 0.2% trisodium fructose 1,6-diphosphate, and the remainder water. It was placed in a shaker at 25°C and cultured at 200 rpm until the OD 600 At around 4, seed liquid is obtained.

[0036] (3) The seed liquid obtained in step (2) was inoculated with a fermentation medium containing the following components by mass fraction at a 4% inoculum volume: 0.1% starch, 0.5% soybean meal, 0.5% ammonium sulfate, 0.5% peptone, 0.3% yeast powder, 0.1% calcium carbonate, 0.5% potassium dihydrogen phosphate, 0.5% 6-amino-3-methylpurine, and the balance water. The medium was placed in a shaker at 25°C and fermented at 200 rpm for 25 hours. The fermentation culture was dried at 38°C, crushed, and passed through a 200-mesh sieve to obtain a composite microbial agent; the total number of viable bacteria in the composite microbial agent was ≥10 9 CFU / g.

[0037] This embodiment also provides a composite microbial agent obtained by the above preparation method.

[0038] Example 3

[0039] A method for preparing a composite microbial agent comprises the following steps:

[0040] (1) After activating Bacillus megaterium, Bacillus marineus N6-2, and Lactobacillus plantarum A37 separately, the activation solutions of the respective strains were mixed to prepare a mixed bacterial solution; the ratio of the number of live bacteria of Bacillus megaterium, Bacillus marineus N6-2, and Lactobacillus plantarum A37 in the mixed bacterial solution was 1:3:3, and the number of live bacteria of Bacillus megaterium in the mixed bacterial solution was 1×10 8 CFU / g.

[0041] (2) The mixed bacterial solution obtained in step (1) was transferred to a seed culture medium containing the following components by mass fraction: 2.5% lactose, 1.5% yeast extract, 1.5% magnesium sulfate, 1.0% trisodium fructose 1,6-diphosphate, and the remainder water. It was placed in a shaker at 35°C and cultured at 200 rpm until the OD 600 At around 6, seed liquid is obtained.

[0042] (3) The seed liquid obtained in step (2) was inoculated with a fermentation medium containing the following components by mass fraction at an inoculum rate of 6%: 1.5% starch, 2% soybean meal, 2% ammonium sulfate, 1.0% peptone, 0.5% yeast powder, 0.3% calcium carbonate, 2.5% potassium dihydrogen phosphate, 1.5% 6-amino-3-methylpurine, and the balance water. The medium was placed in a shaker at 35°C and fermented at 200 rpm for 45 hours. The fermentation culture was dried at 38°C, crushed, and passed through a 200-mesh sieve to obtain a composite microbial agent; the total number of viable bacteria in the composite microbial agent was ≥10 9 CFU / g.

[0043] This embodiment also provides a composite microbial agent obtained by the above preparation method.

[0044] Comparative Example 1

[0045] The difference between Comparative Example 1 and Example 1 is that trisodium fructose 1,6-diphosphate is omitted from the seed culture medium in step (2).

[0046] Comparative Example 2

[0047] The difference between Comparative Example 1 and Example 1 is that 6-amino-3-methylpurine is omitted from the fermentation medium in step (3).

[0048] Comparative Example 3

[0049] The difference between Comparative Example 3 and Example 1 is that in step (1), Bacillus marineus N6-2 and Lactobacillus plantarum A37 are omitted, and the amount of Bacillus megaterium is adjusted to the sum of the three.

[0050] Comparative Example 4

[0051] The difference between Comparative Example 4 and Example 1 is that Lactobacillus plantarum A37 is omitted in step (1), while the amounts of Bacillus marineus N6-2 and Bacillus megaterium are the same as those in Example 1.

[0052] Comparative Example 5

[0053] The difference between Comparative Example 5 and Example 1 is that Bacillus marineus N6-2 is omitted in step (1), while the amounts of Lactobacillus plantarum A37 and Bacillus megaterium are the same as those in Example 1.

[0054] Test Example 1

[0055] The number of viable bacteria of different composite microbial agents

[0056] 10 g of each composite microbial agent prepared in Examples 1-3 and Comparative Examples 1-5 was weighed and added to 100 mL of sterile water. The mixture was allowed to stand for 20 minutes and then thoroughly shaken on a rotary shaker at 20 rpm for 30 minutes to prepare a bacterial suspension. Each of these bacterial suspensions was then serially diluted, and the total number of viable cells in each composite microbial agent was determined by the smear plate count method. The results are shown in Table 1.

[0057] Table 1 Determination of viable bacteria count of composite microbial agents

[0058]

[0059] As shown in Table 1, the total number of effective viable bacteria in the composite microbial inoculants prepared in Examples 1-3 of the present invention is higher than that in Comparative Examples 1-2. This indicates that the two nutrients, trisodium fructose-1,6-diphosphate and 6-amino-3-methylpurine, play a very important role in microbial fermentation and can significantly increase the total number of effective viable bacteria in the composite microbial inoculant.

[0060] Test Example 2

[0061] Effects of composite microbial agents on soil properties

[0062] Alkaline farmland soil samples were selected as research subjects for the experiment. The collected soil samples were air-dried, ground, and sieved, and divided into a control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group. The composite microbial inoculants prepared in Examples 1-3 and Comparative Examples 1-5 were mixed with the soil at a concentration of 8 g / kg soil. No composite microbial inoculant was added to the control group. After mixing, the soil was placed in a culture dish, with approximately 300 g of soil per dish. The inoculated soil was cultured at 28°C and 60% relative humidity for one month. After the incubation period, random soil samples were collected weekly to determine soil organic matter using the potassium dichromate volumetric method described in NY / T 52, "Determination of Soil Organic Matter." Available nitrogen was determined using NY / T 53, "Determination of Soil Alkaline Nitrogen." Available phosphorus was determined using the Olsen method (sodium bicarbonate extraction followed by molybdenum antimony colorimetry). Available potassium was determined using ammonium acetate extraction followed by flame photometry. All experiments were repeated three times. The results are shown in Table 2.

[0063] Table 2 Nitrogen fixation, phosphorus solubility and potassium of composite microbial agents

[0064]

[0065] As shown in Table 2, compared with Comparative Examples 1-5, the composite microbial agents prepared in Examples 1-3 of the present invention can significantly increase the content of organic matter, available nitrogen, available phosphorus, and available potassium in the soil, improve soil nutrients, and thus enhance soil fertility, which helps promote plant growth. Among them, the composite microbial agent prepared in Example 1 has the most significant effect in improving soil nutrients and enhancing soil fertility.

[0066] Test Example 3

[0067] Field Experiments of Composite Microbial Agents

[0068] A field experiment of the composite microbial agent was carried out in a tomato greenhouse. The greenhouse covers an area of 8 mu and is divided into Example 1 Group, Example 2 Group, Example 3 Group, Comparative Example 1 Group, Comparative Example 2 Group, Comparative Example 3 Group, Comparative Example 4 Group, and Comparative Example 5 Group. Each group covers an area of 1 mu and planted 1,800 tomato seedlings. Before transplanting the tomato seedlings, the composite microbial agent prepared in Examples 1-3 and Comparative Examples 1-5 was diluted with water at 1 kg / mu and sprayed on the field, and then rotary tillage was carried out; each group was managed uniformly, and after waiting for the tomatoes to mature, the total yield of each group of tomatoes was measured. The results are as follows: Figure 1 Tomatoes were harvested after 90-100 days of growth. 20 fruits were selected from each group and their soluble sugar content, soluble protein, vitamin C, titratable acid, lycopene and other indicators were measured. The results are shown in Table 3.

[0069] Table 3 Effects of composite microbial agents on tomato fruit quality

[0070]

[0071] Depend on Figure 1 As shown in Table 3, compared with Comparative Examples 1-5, the composite microbial agents prepared in Examples 1-3 of the present invention increased tomato yield and enhanced the content of soluble sugars, soluble protein, vitamin C, and lycopene. This indicates that the composite microbial agents prepared in the present invention can significantly promote tomato growth, improving both yield and quality. Among them, the composite microbial agent prepared in Example 1 was the most effective in promoting tomato growth and improving both yield and quality.

[0072] Further analysis shows that compared with Example 1, the seed culture medium of Comparative Example 1 omits trisodium 1,6-diphosphate fructose; the fermentation culture medium of Comparative Example 2 omits 6-amino-3-methylpurine; in step (1) of Comparative Example 3, marine Bacillus N6-2 and Lactobacillus plantarum A37 are omitted, and the amount of Bacillus megaterium is adjusted to the sum of the three; in step (1) of Comparative Example 4, Lactobacillus plantarum A37 is omitted, while the amounts of marine Bacillus N6-2 and Bacillus megaterium are consistent with those in Example 1; in step (1) of Comparative Example 5, marine Bacillus N6-2 is omitted, while the amounts of Lactobacillus plantarum A37 and Bacillus megaterium are consistent with those in Example 1. The tomato quality of Comparative Examples 1-5 all declined, indicating that trisodium fructose-1,6-diphosphate and 6-amino-3-methylpurine play a crucial role in microbial fermentation. By increasing the total number of viable bacteria in the composite microbial inoculant, they further boost the soil's organic matter, available nitrogen, available phosphorus, and available potassium content, thereby promoting tomato growth and improving yield and quality. The combined presence of Bacillus megaterium, Bacillus marineus N6-2, and Lactobacillus plantarum A37 in the composite microbial inoculant effectively promotes tomato growth and improves yield and quality.

[0073] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A method for preparing a composite microbial agent, characterized in that: The following steps are involved: (1) Activating Bacillus megaterium, Bacillus marineus N6-2, and Lactobacillus plantarum A37 respectively to obtain activation solutions of each strain; The activated solutions of the strains were mixed to prepare a mixed bacterial solution; the accession number of the Bacillus megaterium is CGMCC No. 3770; the accession number of the marine Bacillus N6-2 is CCTCC NO: M2014586; and the accession number of the Lactobacillus plantarum A37 is CCTCC NO: M2019559; (2) inoculating the mixed bacterial solution obtained in step (1) into a seed culture medium to prepare a seed solution; the seed culture medium comprises the following components by mass fraction: 1.5-2.5% lactose, 0.5-1.5% yeast extract, 0.5-1.5% magnesium sulfate, 0.2-1.0% trisodium fructose 1,6-diphosphate, and the balance water; (3) The seed liquid obtained in step (2) is inoculated into a fermentation medium for fermentation, and then dried and crushed to obtain a composite microbial agent; the fermentation medium comprises the following components in mass fractions: 0.1-1.5% starch, 0.5-2% soybean cake powder, 0.5-2% ammonium sulfate, 0.5-1.0% peptone, 0.3-0.5% yeast powder, 0.1-0.3% calcium carbonate, 0.5-2.5% potassium dihydrogen phosphate, 0.5-1.5% 6-amino-3-methylpurine, and the balance is water.

2. The method for preparing a composite microbial agent according to claim 1, characterized in that: The ratio of the effective bacterial concentrations of Bacillus megaterium, Bacillus marineus N6-2 and Lactobacillus plantarum A37 in the mixed bacterial solution in step (1) is 1:(1-3):(1-3).

3. The method for preparing a composite microbial agent according to claim 1, characterized in that: The total effective bacterial concentration of the composite microbial agent is ≥10 9 CFU / g.

4. The method for preparing a composite microbial agent according to claim 1, characterized in that: The inoculation amount of the seed solution in step (3) is 4-6%.

5. The method for preparing a composite microbial agent according to claim 1, characterized in that: The fermentation temperature in step (3) is 25-35°C, and the fermentation time is 25-40h.

6. A composite microbial agent, characterized in that: Prepared according to the method according to any one of claims 1 to 5.

7. Use of the composite microbial agent according to claim 6 in promoting the growth of crops.

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