Preparation method of compound microbial agent and lucid ganoderma cultivation soil improvement method

Abstract

The invention discloses a preparation method of a compound microbial agent, which comprises the following steps: respectively inoculating bacillus subtilis and bacillus amyloliquefaciens into an LB solid culture medium for culturing to obtain single colonies; single colonies are selected and transferred into an LB liquid culture medium to be subjected to shake culture, and bacterium liquid is obtained; inoculating the two bacterial solutions into an LB liquid culture medium, and culturing to obtain a mixed bacterial solution; centrifuging the mixed bacteria liquid to obtain a mixed bacteria culture; mixing the mixed bacteria culture with gamma-polyglutamic acid according to a mass ratio of 3: 1-5: 1 to obtain a mixed material; paving the mixed material on a sterile tray, and transferring to a vacuum drying box, wherein the moisture content is 1t; 20%, so as to obtain the compound microbial agent. After the compound microbial agent is spread on the surface of the soil, ploughing is conducted, and the dosage of the compound microbial agent is 25-50 kg / mu. The gamma-polyglutamic acid is adopted to embed beneficial microorganisms, and after the gamma-polyglutamic acid is applied to the soil, the variety and number of harmful microorganisms in the soil can be reduced. The aqueous extract of the gamma-polyglutamic acid has certain water retention capacity and ideal release effect in the covering soil, and can continuously and obviously improve the biological performance of the soil.

Description

Technical Field

[0001] The present invention relates to the technical field of edible fungus cultivation, and in particular to a method for preparing a composite microbial agent. The present invention also relates to a method for improving ganoderma cultivation soil using the composite microbial agent. Background Art

[0002] The edible fungus industry, a traditional agricultural advantage in my country, has flourished and become a crucial component of China's modern industrial structure. However, in recent years, the growth of the industry has also been accompanied by the emergence of continuous cropping problems, which can lead to yield reductions. Continuous cropping refers to the phenomenon of cultivating two or more crops of the same or closely related species on the same plot of land under normal management, resulting in decreased yield and quality, increased pest and disease infestations, and low economic returns.

[0003] Edible fungi, as an industry that is nutritious and healthy, can be used as both medicine and food, and can adjust the dietary structure in the "big food concept", are becoming more and more popular among consumers, and the demand for edible fungi products is increasing. However, soil-covered cultivation of edible fungi is affected by continuous cropping problems, and their yield and quality are declining. How to scientifically prevent and control continuous cropping problems of edible fungi has become a key common problem that some edible fungi cultivation varieties urgently need to solve. The prevention and control of continuous cropping problems of crops generally uses crop rotation, intercropping and interplanting to weaken viral pests and continuous cropping problems, and balances the soil nutrient composition by utilizing the different characteristics of different crops in absorbing nutrients during growth and development. Different crops produce different allelopathic substances, which can also alleviate and hinder the reproduction of pathogenic microorganisms, and achieve a balanced composition of the cultivated soil microbial population. However, for the cultivation of Ganoderma lucidum in mountainous areas, this method is difficult to implement because mountainous areas are not suitable for growing other crops. Summary of the Invention

[0004] The present invention aims to provide a method for preparing a composite microbial agent that can reduce the types and numbers of harmful microorganisms in the soil, effectively alleviating the continuous cropping problem during Ganoderma lucidum cultivation. The present invention also provides a method for improving Ganoderma lucidum cultivation soil.

[0005] In order to achieve the above technical purpose, the technical solution of the present invention is:

[0006] A method for preparing a composite microbial agent comprises the following steps:

[0007] (1) Bacillus subtilis and Bacillus amyloliquefaciens were inoculated into LB solid medium respectively and cultured at 34-37°C for 24 h to obtain single colonies.

[0008] (2) Select a single colony and transfer it to LB liquid culture medium. Culture it at 34-37°C and 180-200 rpm with shaking for 12 h to obtain a bacterial suspension.

[0009] (3) The Bacillus subtilis culture liquid and the Bacillus amyloliquefaciens culture liquid were inoculated into LB liquid culture medium at a volume ratio of 1:1, with the total inoculation amount being 2% to 3% of the culture medium volume; the culture temperature was 34 to 37°C, the culture speed was 180 to 200 rpm, and the culture time was 36 to 40 h to obtain a mixed culture liquid.

[0010] (4) The mixed bacterial solution was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded to obtain a mixed bacterial culture.

[0011] (5) The mixed bacterial culture and γ-polyglutamic acid are mixed in a mass ratio of 3:1 to 5:1, placed in a sterile mixer, and stirred at 30 rpm for 20 minutes to ensure that the bacteria are evenly coated with γ-polyglutamic acid to obtain a mixed material.

[0012] (6) Spread the mixed material on a sterile tray with a thickness of ≤5 mm, transfer it to a vacuum drying oven, and dry it at 50-56°C for 3-5 hours until the moisture content is <20%, thereby obtaining a composite microbial agent.

[0013] LB liquid medium formula: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, distilled water to 1 L, natural pH (pH 6.8-7.1 after sterilization).

[0014] A method for improving soil for cultivating ganoderma lucidum comprises the following steps:

[0015] After spreading the compound microbial agent on the soil surface, plowing is carried out. The dosage of the compound microbial agent is 25-50kg / mu.

[0016] This invention uses γ-polyglutamic acid to encapsulate beneficial microorganisms. Applying it to soil can reduce the types and numbers of harmful microorganisms in the soil. The aqueous extract of γ-polyglutamic acid has a certain water retention capacity and a relatively ideal release effect when applied to the soil, significantly and sustainably improving the biological properties of the soil.

[0017] The present invention adopts beneficial microorganisms and their fermentation products to improve the flora in the soil environment, adopts biological methods to fundamentally alleviate the number and types of harmful microorganisms in continuous cropping obstacles, is environmentally friendly, and can improve soil utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 This is a picture of Bacillus subtilis colonies on a plate.

[0020] Figure 2 This is a colony image of Bacillus amyloliquefaciens. DETAILED DESCRIPTION

[0021] LB liquid medium formula: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, distilled water to 1 L, natural pH (pH 6.8-7.1 after sterilization).

[0022] Example 1

[0023] A method for preparing a composite microbial agent comprises the following steps:

[0024] (1) Inoculate Bacillus subtilis and Bacillus amyloliquefaciens into LB solid culture medium respectively, and culture at 34℃ for 24 hours to obtain single colonies. Figure 1 As shown, the colony of Bacillus amyloliquefaciens is shown in Figure 2 shown.

[0025] (2) Select a single colony and transfer it to LB liquid culture medium. Culture it at 34°C and 180 rpm with shaking for 12 h to obtain a bacterial suspension.

[0026] (3) The Bacillus subtilis culture liquid and the Bacillus amyloliquefaciens culture liquid were inoculated into LB liquid culture medium at a volume ratio of 1:1, and the total inoculation amount was 2% of the culture medium volume; the culture temperature was 34°C, the culture speed was 180 rpm, and the culture time was 36 h to obtain a mixed culture liquid.

[0027] (4) The mixed bacterial solution was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded to obtain a mixed bacterial culture.

[0028] (5) The mixed bacterial culture and γ-polyglutamic acid were mixed in a mass ratio of 3:1, placed in a sterile mixer, and stirred at 30 rpm for 20 min to ensure that the bacteria were evenly coated with γ-polyglutamic acid to obtain a mixed material.

[0029] (6) The mixed material was spread flat on a sterile tray with a thickness of ≤5 mm, and then transferred to a vacuum drying oven; dried at 50°C for 3 h until the moisture content was <20%, thereby obtaining a composite microbial agent.

[0030] Example 2

[0031] A method for preparing a composite microbial agent comprises the following steps:

[0032] (1) Bacillus subtilis and Bacillus amyloliquefaciens were inoculated into LB solid culture medium respectively and cultured at 37°C for 24 h to obtain single colonies.

[0033] (2) Select a single colony and transfer it to LB liquid culture medium. Culture it at 37°C and 200 rpm with shaking for 12 h to obtain a bacterial suspension.

[0034] (3) The Bacillus subtilis culture liquid and the Bacillus amyloliquefaciens culture liquid were inoculated into LB liquid culture medium at a volume ratio of 1:1, with the total inoculation amount being 3% of the culture medium volume; the culture temperature was 37°C, the culture speed was 200 rpm, and the culture time was 40 h to obtain a mixed culture liquid.

[0035] (4) The mixed bacterial solution was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded to obtain a mixed bacterial culture.

[0036] (5) The mixed bacterial culture and γ-polyglutamic acid were mixed in a mass ratio of 5:1, placed in a sterile mixer, and stirred at 30 rpm for 20 min to ensure that the bacteria were evenly coated with γ-polyglutamic acid to obtain a mixed material.

[0037] (6) The mixed material was spread flat on a sterile tray with a thickness of ≤5 mm, and transferred to a vacuum drying oven; dried at 56°C for 5 h until the moisture content was <20%, thereby obtaining a composite microbial agent.

[0038] Example 3

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

[0040] (1) Inoculate Bacillus subtilis and Bacillus amyloliquefaciens into LB solid culture medium respectively, and culture at 35°C for 24 hours to obtain single colonies. Figure 1 As shown, the colony of Bacillus amyloliquefaciens is shown in Figure 2 shown.

[0041] (2) Select a single colony and transfer it to LB liquid culture medium. Shake and culture at 35 and 190 pm for 12 h to obtain a bacterial solution.

[0042] (3) The Bacillus subtilis culture liquid and the Bacillus amyloliquefaciens culture liquid were inoculated into LB liquid culture medium at a volume ratio of 1:1, and the total inoculation amount was 3% of the culture medium volume; the culture temperature was 35, the culture speed was 19 rpm, and the culture time was 38 h to obtain a mixed culture liquid.

[0043] (4) The mixed bacterial solution was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded to obtain a mixed bacterial culture.

[0044] (5) The mixed bacterial culture and γ-polyglutamic acid were mixed in a mass ratio of 4:1, placed in a sterile mixer, and stirred at 30 rpm for 20 min to ensure that the bacteria were evenly coated with γ-polyglutamic acid to obtain a mixed material.

[0045] (6) The mixed material was spread flat on a sterile tray with a thickness of ≤5 mm, and then transferred to a vacuum drying oven; dried at 53°C for 4 hours until the moisture content was <20% to obtain a composite microbial agent.

[0046] Example 4

[0047] A method for improving soil for Ganoderma lucidum cultivation comprises the following steps: spreading the composite microbial agent obtained in Example 1 on the soil surface, followed by tillage, wherein the amount of the composite microbial agent used is 50 kg / mu.

[0048] Example 5

[0049] A method for improving soil for Ganoderma lucidum cultivation comprises the following steps: spreading the composite microbial agent obtained in Example 2 on the soil surface, followed by tillage, wherein the amount of the composite microbial agent used is 30 kg / mu.

[0050] Example 6

[0051] A method for improving soil for Ganoderma lucidum cultivation comprises the following steps: spreading the composite microbial agent obtained in Example 3 on the soil surface, followed by tillage, wherein the amount of the composite microbial agent used is 4 kg / mu.

[0052] The above embodiments do not limit the present invention in any way, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for preparing a composite microbial agent, characterized in that: The following steps are involved: (1) Bacillus subtilis and Bacillus amyloliquefaciens were inoculated into LB solid medium and cultured to obtain single colonies; (2) Select a single colony and transfer it to LB liquid medium for shaking culture to obtain a bacterial solution; (3) inoculating the Bacillus subtilis culture liquid and the Bacillus amyloliquefaciens culture liquid into LB liquid culture medium at a volume ratio of 1:1, culturing at a temperature of 34-37°C, a culture speed of 180-200 rpm, and a culture time of 36-40 h to obtain a mixed culture liquid; (4) After centrifuging the mixed bacterial solution, discard the supernatant to obtain a mixed bacterial culture; (5) mixing the mixed bacterial culture with γ-polyglutamic acid in a mass ratio of 3:1 to 5:1 to obtain a mixed material; (6) The mixed material is spread on a sterile tray and transferred to a vacuum drying oven with a moisture content of <20% to obtain a composite microbial agent.

2. The method for preparing a composite microbial agent according to claim 1, wherein: LB liquid medium formula: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, distilled water to 1 L, natural pH.

3. The method for preparing a composite microbial agent according to claim 1, wherein: In step (3), the total inoculation amount is 2% to 3% of the volume of the culture medium.

4. The method for preparing a composite microbial agent according to claim 1, wherein: In step (5), the drying temperature is 50-56° C., and the drying time is 3-5 hours.

5. A method for improving soil for growing Ganoderma lucidum, comprising the following steps: The composite microbial agent obtained by the preparation method of the composite microbial agent according to any one of claims 1 to 4 is spread on the soil surface and then plowed. The dosage of the composite microbial agent is 25-50 kg / mu.

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