Sophora alopecuroide extract compound bio-organic fertilizer and preparation method thereof

Abstract

The invention belongs to the technical field of bio-organic fertilizer preparation, and relates to a sophora alopecuroide extract compound bio-organic fertilizer and a preparation method thereof, and the sophora alopecuroide extract compound bio-organic fertilizer comprises the following components by mass: 65-75 parts of plant straw powder, 5-15 parts of sophora alopecuroide extract, 0.05-0.1 part of tea polyphenol, 5-10 parts of a bacillus subtilis liquid, and 5-10 parts of an auxiliary additive. By adding the sophora alopecuroide extract and the tea polyphenol, the fermentation environment is improved, the dissolution of organic matters and trace elements in the organic fertilizer is promoted, the bioavailability of active ingredients in the organic fertilizer is remarkably improved, the fertilizer efficiency and disease resistance of the biological organic fertilizer are enhanced, and particularly, the morbidity of botrytis cinerea and potato late blight can be reduced; the use frequency of the pesticide is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of biological organic fertilizers, and relates to a Sophora alopecuroides extract compounded biological organic fertilizer and a preparation method thereof. Background Art

[0002] With the rapid development of modern agriculture, the problems of soil quality decline and environmental pollution have become increasingly prominent. Although traditional chemical fertilizers can increase crop yields, long-term use will lead to problems such as the destruction of soil structure, soil compaction, salinization, ecological balance disorder, and the decline of agricultural product quality. Due to its environmental protection and sustainable characteristics, organic fertilizer has become a research hotspot in the current agricultural field. However, the existing organic fertilizers still need to be improved in terms of fertilizer efficiency, pest and disease resistance ability, and production efficiency.

[0003] In the prior art, researchers have begun to add traditional Chinese medicines to fertilizers to improve their disease resistance ability and fertilizer efficiency. Sophora alopecuroides, as a traditional Chinese medicine, contains rich active ingredients such as alkaloids and flavonoids, and has significant insecticidal, antibacterial, and plant growth regulating effects. Chinese Patent CN102863294A discloses a Sophora alopecuroides and Cynanchum komarovii composite organic fertilizer and a preparation method thereof. The organic fertilizer is prepared from Sophora alopecuroides, Cynanchum komarovii, and straw (corn, wheat, rape, etc.) compost according to a certain ratio. Sophora alopecuroides, Cynanchum komarovii, and straw compost are mixed, granulated, dried, and packaged according to a certain ratio and sequence. Although this organic fertilizer improves the fertilizer efficiency, the dissolution rate of organic matter in traditional Chinese medicine is relatively low, resulting in low absorption rate of crops and poor disease resistance effect. Chinese Patent CN109095986A discloses a composite plant-derived water-soluble foliar fertilizer with a bactericidal function and a preparation method thereof. The formula includes 20-35 parts of plant active ingredient extract, 30-40 parts of nutrient elements, 3-5 parts of emulsifier, 3-5 parts of synergist, 2-4 parts of stabilizer, 15-20 parts of ethanol, and 7-12 parts of water; the plant active ingredient extract includes Folium Isatidis extract, Radix Isatidis extract, Flos Lonicerae extract, Radix Bupleuri extract, Cynanchum komarovii extract, Sophora alopecuroides extract, Allium sativum extract, Atropa belladonna extract, and Macleaya cordata extract; the nutrient elements include urea, potassium dihydrogen phosphate, EDTA chelated iron, EDTA chelated zinc, EDTA chelated boron, and ammonium molybdate. The ultrasonic-assisted extraction method is mainly used to extract plant active ingredients. Although the present invention improves the speed, duration, and disease resistance ability of the existing plant-derived water-soluble foliar fertilizer, the addition of inorganic fertilizers such as urea and potassium dihydrogen phosphate will cause soil compaction after long-term use, which is not conducive to plant growth.

[0004] Therefore, there is a need for a Sophora alopecuroides extract compounded biological organic fertilizer with excellent disease resistance effect and high fertilizer efficiency. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a sophora alopecuroides extract compound biological organic fertilizer and a preparation method thereof, wherein the sophora alopecuroides extract, tea polyphenols, organic fertilizer raw materials and Bacillus subtilis liquid are fermented and cultured to improve the fertilizer efficiency and disease resistance of the biological organic fertilizer.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] On one hand, the present invention provides a sophora alopecuroides extract compound biological organic fertilizer, the components of which are calculated by weight: 65-75 parts of plant straw powder, 5-15 parts of sophora alopecuroides extract, 0.05-0.1 parts of tea polyphenols, 5-10 parts of Bacillus subtilis liquid, and 5-10 parts of auxiliary additives.

[0008] Furthermore, the plant straw powder is selected from one or more of corn straw powder, sunflower straw powder, wheat straw powder and cotton straw powder.

[0009] Furthermore, the auxiliary additives are calculated in percentage by mass as follows: 50-70 wt % of humic acid and 30-50 wt % of amino acid.

[0010] Furthermore, the tea polyphenols were purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a specification of 100 mg.

[0011] Another aspect of the present invention provides a method for preparing a compound biological organic fertilizer of Sophora alopecuroides extract, comprising the following steps:

[0012] (1) crushing the whole plant of Sophora alopecuroides, pre-soaking it, extracting it in a 60% ethanol-water solution with a pH of 3.5 to 4.5 for 3 to 5 hours, repeating the extraction 3 to 4 times, filtering, combining the filtrate, and concentrating it to obtain a Sophora alopecuroides extract, wherein the mass ratio of the ethanol-water solution to the whole plant of Sophora alopecuroides is 5 to 7:1;

[0013] (2) mixing plant straw powder, bitter bean extract, tea polyphenols, and Bacillus subtilis liquid in proportion, fermenting in a container at 35-40° C. for 7-10 hours, and stirring intermittently for 20 minutes every 8 hours, and filtering to obtain a fermentation liquid;

[0014] (3) adding auxiliary additives to the fermentation liquid, mixing, granulating, and drying at 35-45° C. to obtain a compound biological organic fertilizer of the sophora alopecuroides extract.

[0015] Further, the 60% ethanol aqueous solution with a pH of 3.5 - 4.5 is adjusted in pH value using sulfuric acid. The pH value has a great influence on the extraction rate of alkaloids. The higher the pH value, the lower the extraction efficiency. An overly low pH value of the solvent can cause corrosion of equipment and bring inconvenience to subsequent treatment. Therefore, the pH value is 3.5 - 4.5. Since adding acid helps in the extraction of alkaloids, and the chloride ions in nitric acid and hydrochloric acid have strong corrosiveness, sulfuric acid is added during the extraction process to adjust the pH value.

[0016] Further, the preparation method of the Bacillus subtilis bacterial solution includes: 1) Resuscitation culture of Bacillus subtilis, using solid LB medium for culture, static culture at 37°C for 24 hours; 2) Inoculating the Bacillus subtilis colony into the first-stage liquid medium, shaking culture at 37°C, with a rotation speed of 150 - 200 r / min, and obtaining a liquid bacterial strain after overnight culture; 3) Inoculating the liquid bacterial strain into the second-stage fermentation medium, shaking culture at 37°C, with a rotation speed of 150 - 200 r / min, overnight, for scale-up culture to obtain the Bacillus subtilis bacterial solution; the content of Bacillus subtilis is 1 - 1.3 billion / g.

[0017] Further, the components of the LB medium are: 5 g of yeast extract, 10 g of tryptone, 10 g of sodium chloride, adding water to 1000 mL, pH = 7 - 7.5, autoclaving at 121°C for 20 min.

[0018] Further, the components of the first-stage liquid medium of Bacillus subtilis are: 5 g / L of molasses, 3 g / L of soybean cake powder, 2 g / L of corn starch, 1.0 g / L of Na2HPO4, 0.6 g / L of NH4NO3, 0.2 g / L of betaine, 0.1 g / L of MgSO4·7H2O, 0.5 g / L of CaCO3, 0.3 g / L of humic acid, pH = 7.5 - 8.0, adding water to 1000 mL, autoclaving at 121°C for 20 min.

[0019] Further, the components of the second-stage fermentation medium of Bacillus subtilis are: 8 g / L of molasses, 3 g / L of soybean cake powder, 5 g / L of corn starch, 4 g / L of corn starch, 1.5 g / L of Na2HPO4, 0.8 g / L of NH4NO3, 0.5 g / L of betaine, 0.2 g / L of MgSO4·7H2O, 1.0 g / L of CaCO3, 0.5 g / L of humic acid, 0.3 g / L of seaweed polysaccharide, pH = 7.5 - 8.0, dissolved oxygen content ≥ 30% saturation, adding water to 1000 mL, autoclaving at 121°C for 20 min.

[0020] The advantages and positive effects of the present invention are:

[0021] (1) The compound bio-organic fertilizer prepared by the present invention is obtained by extracting Sophora alopecuroides, and fermenting and culturing the extract of Sophora alopecuroides, tea polyphenols, organic fertilizer raw materials, and Bacillus subtilis bacterial liquid. By adding the extract of Sophora alopecuroides and tea polyphenols, the fermentation environment is improved, the dissolution of organic matter and trace elements in the organic fertilizer is promoted, the biological utilization rate of active ingredients in the organic fertilizer is significantly increased, the fertilizer efficiency and disease resistance of the bio-organic fertilizer are enhanced, and in particular, the incidence of Botrytis cinerea of tomatoes and Phytophthora infestans of potatoes can be reduced, and the use frequency of pesticides is decreased.

[0022] (2) The compound bio-organic fertilizer prepared by the present invention is fully water-soluble, has high fertilizer efficiency, can be used for sprinkler irrigation and drip irrigation, contains a high number of viable Bacillus subtilis bacteria, can activate the soil structure, reduce soil compaction, and the raw materials are of plant origin, non-toxic and harmless, meeting the development requirements of green agriculture. Detailed implementation mode

[0023] The present invention will be further described in detail below through specific examples. The following examples are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0024] Example 1

[0025] A compound bio-organic fertilizer with Sophora alopecuroides extract, the components of which are calculated by mass fraction: 70 parts of corn straw powder, 10 parts of Sophora alopecuroides extract, 0.1 part of tea polyphenols, 10 parts of Bacillus subtilis bacterial liquid, and 10 parts of auxiliary additive. The auxiliary additive includes 70wt% of humic acid and 30wt% of amino acid.

[0026] The preparation method steps are as follows:

[0027] (1) Crush the whole plant material of Sophora alopecuroides to 10mm, after pre-soaking, add it to a 60% ethanol-aqueous solution with a pH of 4 and extract for 5 hours, repeat the extraction 3 times, filter, combine the filtrates, and concentrate the filtrates to 1.5g / cm 3 , to obtain the extract of Sophora alopecuroides, and the mass ratio of the ethanol-aqueous solution to the whole plant material of Sophora alopecuroides is 6:1;

[0028] (2) The resuscitation culture of Bacillus subtilis is carried out using solid LB medium. The components of the LB medium are: 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride. Add water to 1000 mL, adjust the pH to 7 - 7.5, and sterilize at 121 °C under high-pressure steam for 20 min. Incubate statically at 37 °C for 24 hours, inoculate the Bacillus subtilis colonies into the first-stage liquid medium, and culture with shaking at 37 °C at a rotation speed of 180 r / min. After overnight culture, obtain the liquid strain. The components of the first-stage liquid medium of Bacillus subtilis are: 5 g / L of molasses, 3 g / L of soybean cake powder, 2 g / L of corn flour, 1.0 g / L of Na2HP04, 0.6 g / L of NH4N03, 0.2 g / L of betaine, 0.1 g / L of MgSO4·7H2O, 0.5 g / L of CaCO3, 0.3 g / L of humic acid, adjust the pH to 7.5 - 8.0, add water to 1000 mL, and sterilize at 121 °C under high-pressure steam for 20 min. Inoculate the liquid strain into the second-stage fermentation medium, culture with shaking at 37 °C at a rotation speed of 180 r / min overnight for scale-up culture to obtain the Bacillus subtilis bacterial liquid; the content of Bacillus subtilis is 10 - 1.3 billion / g. The components of the second-stage fermentation medium of Bacillus subtilis are: 8 g / L of molasses, 3 g / L of soybean cake powder, 5 g / L of corn flour, 4 g / L of corn flour, 1.5 g / L of Na2HP04, 0.8 g / L of NH4N03, 0.5 g / L of betaine, 0.2 g / L of MgSO4·7H2O, 1.0 g / L of CaCO3, 0.5 g / L of humic acid, 0.3 g / L of seaweed polysaccharide, adjust the pH to 7.5 - 8.0, the dissolved oxygen content ≥ 30% saturation, add water to 1000 mL, and sterilize at 121 °C under high-pressure steam for 20 min.

[0029] (4) Mix the corn straw powder, Sophora alopecuroides extract, tea polyphenols, and Bacillus subtilis bacterial liquid according to the above ratio, carry out fermentation culture at 39 °C in a container for 10 days, and use intermittent stirring, stirring for 20 minutes every 8 hours, and filter to obtain the fermentation broth;

[0030] (5) Add humic acid and amino acids to the fermentation broth, mix well, granulate, and dry at 38 °C to obtain the Sophora alopecuroides extract compound biological organic fertilizer.

[0031] Example 2

[0032] A Sophora alopecuroides extract compound biological organic fertilizer, the components of which are calculated by mass fraction: 65 parts of cotton straw powder, 15 parts of Sophora alopecuroides extract, 0.08 part of tea polyphenols, 10 parts of Bacillus subtilis bacterial liquid, and 10 parts of auxiliary additive. The components of the auxiliary additive are calculated by mass percentage: 70 wt% of humic acid and 30 wt% of amino acids.

[0033] The preparation method steps are as follows:

[0034] (1) Grind the whole Sophora alopecuroides material to 10 mm. After pre-soaking, add it to a 60% ethanol-aqueous solution with a pH of 5 and extract for 3 hours. Repeat the extraction 3 times, filter, combine the filtrates, and concentrate to 1.5 g / cm 3 , obtaining the Sophora alopecuroides extract. The mass ratio of the ethanol-aqueous solution to the whole Sophora alopecuroides material is 7:1;

[0035] (2) The resuscitation culture of Bacillus subtilis is carried out using solid LB medium. The components of the LB medium are: 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride. Add water to 1000 mL, adjust the pH to 7 - 7.5, and sterilize at 121 °C under high-pressure steam for 20 min. Incubate statically at 37 °C for 24 hours, inoculate the Bacillus subtilis colonies into the first-stage liquid medium, and culture with shaking at 37 °C at a rotation speed of 180 r / min. After overnight culture, obtain the liquid strain. The components of the first-stage liquid medium of Bacillus subtilis are: 5 g / L of molasses, 3 g / L of soybean cake powder, 2 g / L of corn flour, 1.0 g / L of Na2HPO4, 0.6 g / L of NH4NO3, 0.2 g / L of betaine, 0.1 g / L of MgSO4·7H2O, 0.5 g / L of CaCO3, 0.3 g / L of humic acid, adjust the pH to 7.5 - 8.0, add water to 1000 mL, and sterilize at 121 °C under high-pressure steam for 20 min. Inoculate the liquid strain into the second-stage fermentation medium, culture with shaking at 37 °C at a rotation speed of 180 r / min overnight for scale-up culture to obtain the Bacillus subtilis bacterial liquid; the content of Bacillus subtilis is 1 - 1.3 billion / g. The components of the second-stage fermentation medium of Bacillus subtilis are: 8 g / L of molasses, 3 g / L of soybean cake powder, 5 g / L of corn flour, 4 g / L of corn flour, 1.5 g / L of Na2HPO4, 0.8 g / L of NH4NO3, 0.5 g / L of betaine, 0.2 g / L of MgSO4·7H2O, 1.0 g / L of CaCO3, 0.5 g / L of humic acid, 0.3 g / L of seaweed polysaccharide, adjust the pH to 7.5 - 8.0, the dissolved oxygen content is ≥ 30% saturation, add water to 1000 mL, and sterilize at 121 °C under high-pressure steam for 20 min.

[0036] (4) Mix the cotton straw powder, Sophora alopecuroides extract, tea polyphenols, and Bacillus subtilis bacterial liquid according to the above ratios, carry out fermentation culture at 39 °C in a container for 10 days, and use intermittent stirring, stirring for 20 minutes every 8 hours, and filter to obtain the fermentation broth;

[0037] (5) Add humic acid and amino acids to the fermentation broth, mix well, granulate, and dry at 40 °C to obtain the compound biological organic fertilizer with Sophora alopecuroides extract.

[0038] Comparative Example 1

[0039] The difference from Example 1 is that tea polyphenols are not added.

[0040] Comparative Example 2

[0041] The difference from Example 1 is that the whole Sophora alopecuroides plant material before extraction is directly used.

[0042] Comparative Example 3

[0043] The difference from Example 1 is that no Sophora alopecuroides extract is added.

[0044] An experiment on promoting the growth of tomatoes and controlling Botrytis cinerea was carried out on the bio-organic fertilizers prepared in the examples and comparative examples.

[0045] This experiment was completed in a greenhouse. The test variety was Maofen 802. The test area was 3.6 mu, with 6 treatment groups, each treatment group being 0.2 mu, and each treatment group was repeated 3 times. The 6 treatment groups were Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, and traditional organic fertilizer respectively. After transplanting the tomatoes, the above-mentioned organic fertilizers were diluted 1000 times and sprayed or drip-irrigated on the leaves or roots once every 30 days, with uniform fertilization each time. After the tomatoes were mature and harvested, an investigation was carried out on Botrytis cinerea. The results showed that the growth rates of tomatoes in Example 1 and Example 2 were the fastest, and the incidence rates of Botrytis cinerea were 8.5% and 9.2% respectively; the growth rates of tomatoes in Comparative Example 1 and Comparative Example 2 were the second, and the incidence rates of Botrytis cinerea were 25.7% and 28.3% respectively; the growth rate of tomatoes in Comparative Example 3 was slightly worse than that in Comparative Example 2, and the incidence rate of Botrytis cinerea was 31%; the growth rate of the traditional organic fertilizer was the lowest, and the incidence rate of Botrytis cinerea was 35.6%, with a relatively high incidence rate.

[0046] An experiment on promoting the growth of potatoes and controlling Phytophthora infestans was carried out on the bio-organic fertilizers prepared in the examples and comparative examples.

[0047] This experiment was completed in a greenhouse. The test variety was Xisen No. 6. The test area was 3.6 mu, with 6 treatment groups, each treatment group being 0.2 mu, and each treatment group was repeated 3 times. The 6 treatment groups were Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, and traditional organic fertilizer respectively. After transplanting the potatoes, the above-mentioned organic fertilizers were diluted 1000 times and sprayed or drip-irrigated on the leaves or roots once every 30 days, with uniform fertilization each time. After the potatoes were mature and harvested, an investigation was carried out on Phytophthora infestans. The results showed that the growth rates of potatoes in Example 1 and Example 2 were the fastest, and the incidence rates of Phytophthora infestans were 6.3% and 7.1% respectively; the growth rates of potatoes in Comparative Example 1 and Comparative Example 2 were the second, and the incidence rates of Phytophthora infestans were 25.4% and 26.8% respectively; the growth rate of potatoes in Comparative Example 3 was slightly worse than that in Comparative Example 2, and the incidence rate of Phytophthora infestans was 27.7%; the growth rate of the traditional organic fertilizer was the lowest, and the incidence rate of Phytophthora infestans was 28.9%, with a relatively high incidence rate.

[0048] The fertilizer efficiency of the bio-organic fertilizers prepared in the examples and comparative examples was detected, as shown in Table 1.

[0049] Table 1 Detection results of the bio-organic fertilizers prepared in the examples and comparative examples

[0050]

[0051] According to the data in Table 1 and the experimental results of promoting growth and preventing diseases of tomatoes and potatoes, it can be seen that compared with the comparative examples, in the examples of the present invention, the content of organic matter, total nitrogen, phosphorus pentoxide and potassium chloride in the bio-organic fertilizer of the present invention has been significantly improved, and the growth promotion and disease resistance effects are both greatly improved compared with the comparative examples. This is because tea polyphenols can promote the secretion of cellulase by Bacillus subtilis during fermentation, and both tea polyphenols and Sophora alopecuroides extracts have antibacterial effects, inhibiting the growth of miscellaneous bacteria. The combined use of the two synergistically improves the fermentation environment, provides a favorable environment for the fermentation of Bacillus subtilis, promotes the full release of nutrient components in the organic fertilizer, improves the fertilizer efficiency and antibacterial and disease resistance ability of the bio-organic fertilizer, and the content of viable Bacillus subtilis in the organic fertilizer after fermentation is high. In Comparative Example 1, tea polyphenols were not added, and in Comparative Example 3, Sophora alopecuroides extract was not added. The content of organic matter, total nitrogen, phosphorus pentoxide and potassium chloride in the bio-organic fertilizer was relatively low, indicating that fermenting with tea polyphenols or Sophora alopecuroides extract alone did not have better fertilizer efficiency and disease resistance ability than the bio-organic fertilizer prepared by adding both of them at the same time, indicating that the two have a synergistic effect during the fermentation process. In Comparative Example 2, Sophora alopecuroides was not extracted, and the content of organic matter, total nitrogen, phosphorus pentoxide and potassium chloride in the bio-organic fertilizer was slightly higher than that in Comparative Example 3, but the overall effect was not as good as that of the examples. This is because after extraction, the Sophora alopecuroides extract contains more active substances, removing other substances that affect the fermentation environment, which is more conducive to improving the antibacterial performance of the bio-organic fertilizer.

[0052] The number of viable Bacillus subtilis in the bio-organic fertilizer prepared by the preparation method of the present invention is higher than that in the comparative examples, which can activate the soil structure, reduce soil compaction and promote plant growth.

[0053] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A biological organic fertilizer prepared from alopecuroides extract, characterized in that: The components thereof are calculated by weight: 65-75 parts of plant straw powder, 5-15 parts of sophora alopecuroides extract, 0.05-0.1 parts of tea polyphenols, 5-10 parts of bacillus subtilis liquid and 5-10 parts of auxiliary additives.

2. The compound biological organic fertilizer of Sophora alopecuroides extract according to claim 1, characterized in that: The plant straw powder is selected from one or more of corn straw powder, sunflower straw powder, wheat straw powder and cotton straw powder.

3. The compound biological organic fertilizer of Sophora alopecuroides extract according to claim 1, characterized in that: The auxiliary additive components are calculated in percentage by mass as follows: 50-70 wt % of humic acid and 30-50 wt % of amino acid.

4. The method for preparing the compound biological organic fertilizer of Sophora alopecuroides extract according to any one of claims 1 to 3, characterized in that: The steps include: (1) crushing the whole plant of Sophora alopecuroides, pre-soaking it, extracting it in a 60% ethanol aqueous solution with a pH of 3.5 to 4.5 for 3 to 5 hours, repeating the extraction 3 to 4 times, filtering, combining the filtrate, and concentrating it to obtain a Sophora alopecuroides extract, wherein the mass ratio of the ethanol-water solution to the whole plant of Sophora alopecuroides is 5 to 7:1; (2) mixing plant straw powder, bitter bean extract, tea polyphenols, and Bacillus subtilis liquid in proportion, fermenting and culturing in a container at 35-40° C. for 7-10 days, and stirring intermittently for 20 minutes every 8 hours, and filtering to obtain a fermentation liquid; (3) adding auxiliary additives to the fermentation liquid, mixing, granulating, and drying at 35-45° C. to obtain a compound biological organic fertilizer of the sophora alopecuroides extract.

5. The preparation method according to claim 4, characterized in that: The pH value of the 60% ethanol aqueous solution with a pH of 3.5 to 4.5 is adjusted using sulfuric acid.

6. The preparation method according to claim 4, characterized in that: The preparation method of Bacillus subtilis liquid comprises: 1) resuscitation culture of Bacillus subtilis, using solid LB culture medium for culture, and static culture at 37°C for 24 hours; 2) inoculating Bacillus subtilis colonies into a primary liquid culture medium, shaking culture at 37°C, with a rotation speed of 150-200r / min, and obtaining liquid strains after overnight culture; 3) inoculating the liquid strains into a secondary fermentation culture medium, shaking culture at 37°C, with a rotation speed of 150-200r / min, and culture overnight, and amplifying culture to obtain Bacillus subtilis liquid, wherein the content of Bacillus subtilis is 1 billion to 1.3 billion / g.

7. The preparation method according to claim 6, characterized in that: The LB medium contains 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride. Water is added to 1000 mL. The pH value is 7 to 7.

5. The medium is sterilized by high-pressure steam at 121° C. for 20 min.

8. The preparation method according to claim 6, characterized in that: The primary liquid culture medium of Bacillus subtilis comprises: 5 g / L molasses, 3 g / L bean cake powder, 2 g / L corn flour, 1.0 g / L Na2HP04, 0.6 g / L NH4N03, 0.2 g / L betaine, 0.1 g / L MgSO4·7H2O, 0.5 g / L CaCO3, 0.3 g / L humic acid, pH=7.5-8.0, water is added to 1000 mL, 121° C., and high-pressure steam sterilization is performed for 20 minutes.

9. The preparation method according to claim 6, characterized in that: The secondary fermentation medium of Bacillus subtilis comprises: 8 g / L molasses, 3 g / L bean cake powder, 5 g / L corn flour, 4 g / L corn flour, 1.5 g / L Na2HP04, 0.8 g / L NH4N03, 0.5 g / L betaine, 0.2 g / L MgSO4·7H2O, 1.0 g / L CaCO3, 0.5 g / L humic acid, 0.3 g / L seaweed polysaccharide, pH=7.5-8.0, dissolved oxygen content ≥30% saturation, water is added to 1000 mL, 121°C, and high-pressure steam sterilization is performed for 20 minutes.

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