Microbial agent and application thereof in pepper planting

Abstract

The invention discloses a microbial agent and application thereof in pepper planting. According to the microbial agent, bacillus subtilis and bacillus megatherium are subjected to independent liquid fermentation, auxiliary materials and a carrier are added, and the microbial agent is prepared for pepper planting. Wherein in the liquid fermentation process, waste liquid obtained after trehalose production is adopted as one of production raw materials for high-density fermentation, the bacterial colony number of the final liquid fungicide is larger than or equal to 1.4 * 10 < 9 > cfu / mL, the content of polyglutamic acid is larger than or equal to 32.5 g / L, and the titer of the antibacterial peptide is larger than or equal to 600 U / mL. The microbial agent can be applied to pepper planting, the pepper growth cycle is short, the leaf surfaces are emerald green and dense, the acre yield of the pepper can be remarkably increased, the vitamin C content in the pepper is remarkably increased to 967.5 mg / Kg, and the soluble sugar content in the pepper is remarkably increased to 3.43%.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a microbial inoculant and its application in pepper cultivation. Background Art

[0002] A microbial inoculant is a kind of live fertilizer. It mainly relies on a large number of beneficial microorganisms, and uses their vigorous reproduction and metabolism to convert substances and continuously form beneficial metabolites. At the same time, wild-type microorganisms can be obtained through a series of experiments such as separation, purification, orthogonalization, and amplification to obtain dominant strains, and then the dominant strains are used to produce and ferment through biotechnology. Microbial inoculants have strong degradation ability, can effectively improve soil fertility, have broad development potential, and are also widely used in pollution treatment, agricultural production, food fermentation, water purification, improving soil fertility, etc. At the same time, they can increase crop yields, improve the quality of agricultural products, enhance the drought resistance, cold resistance, lodging resistance, and disease prevention ability of crops, improve the utilization rate of chemical fertilizers, and improve soil nutrients.

[0003] The wastewater from trehalose production mainly comes from ion-exchanged water, barrel washing water, workshop floor flushing water, condensate water, etc., and has the characteristics of high concentration, complex composition, large fluctuations in discharge concentration and water volume. And biological methods need to be used to degrade organic matter in the treatment of such industrial wastewater. Bacillus subtilis is widely used in animal feed, plant disease resistance, environmental remediation, food fermentation, etc., and its application as a microbial growth-promoting inoculant is less, but it is developing rapidly. It is a typical aerobic microbial inoculant and has the following advantages: first, it has no destructive effect on soil structure; second, it is non-toxic and harmless to humans and animals and is safe to use; third, it can effectively and continuously improve soil fertility; fourth, the production cost is relatively low; fifth, the self-renewal and reproducibility of microorganisms can reduce its repeated application.

[0004] There is no precedent in current reports for preparing a microbial inoculant using the wastewater from the production process of Bacillus subtilis, Bacillus megaterium and trehalose. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of a microbial inoculant in pepper cultivation. This microbial inoculant uses the wastewater generated during the production and fermentation of trehalose as the production raw material, replaces the carbon source and water in the fermentation for use, and at the same time, the trehalose wastewater can be used as a protective agent after the fermentation is completed to extend the storage time of the microbial inoculant;

[0006] The present invention can also solve the problem of environmental pollution caused by the industrial wastewater generated during trehalose production, reduce pollution and protect the environment, and achieve the advantages of waste utilization.

[0007] The microbial inoculant of the present invention can play a promoting role during the growth process of peppers.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A microbial inoculum for pepper cultivation, which is obtained by fermenting a culture medium with trehalose wastewater by microorganisms. The fermentation product contains a large amount of probiotics, polyglutamic acid and antibacterial peptides; the microorganisms are at least one of Bacillus subtilis and Bacillus megaterium.

[0010] The number of colonies in the microbial inoculum is ≥ 1.4×10 9 cfu / mL, the content of polyglutamic acid is ≥ 32.5 g / L, and the titer of antibacterial peptides is ≥ 600 U / mL.

[0011] The trehalose wastewater fermentation medium is composed of 1-10 g / L of sodium glutamate, 0.1-2 g / L of disodium hydrogen phosphate, 0.1-2 g / L of ammonium sulfate, 0.1-1 g / L of magnesium sulfate, 0.1-1 g / L of trace element mixture, 0.1-1 g / L of amino acid mixture, and supplemented with trehalose wastewater.

[0012] The temperature of the fermentation is 25°C - 40°C, the aerobic fermentation period is 24h - 60h, and the aeration ratio is 1 vvm.

[0013] The fermentation time is preferably 30 - 36h.

[0014] The fermentation can be carried out by conventional fermentation or fed-batch fermentation. The fermentation temperature is 25°C - 40°C, the aerobic fermentation period is 24h - 60h, and feeding is carried out in the middle of fermentation. The feed is 5% - 10% of the treated trehalose waste liquid and 5% - 10% of sodium glutamate; the fermentation product contains probiotics, polyglutamic acid and antibacterial peptides.

[0015] The trace element mixture is composed of calcium chloride and ferrous sulfate mixed in a mass ratio of (1 - 10):(1 - 5). Further preferably, calcium chloride and ferrous sulfate are mixed in a mass ratio of (1 - 3):1, and more preferably, calcium chloride and ferrous sulfate are mixed in a mass ratio of 2:1. The addition concentration of trace elements is preferably 0.4 g / L.

[0016] The amino acid mixture is composed of glutamic acid, lysine and arginine mixed in a mass ratio of (1 - 10):(1 - 5):(1 - 5), preferably mixed in a mass ratio of 1:2:1 or 2:1:1 of glutamic acid, lysine and arginine. The addition concentration of the amino acid mixture is preferably 0.4 g / L.

[0017] The trehalose wastewater is obtained by separating and filtering the wastewater generated in the production and processing of trehalose and adjusting the pH to 7.0 - 8.0.

[0018] The trehalose fermentation medium is preferably: sodium glutamate 2 g / L, disodium hydrogen phosphate 0.6 g / L, ammonium sulfate 0.9 g / L, magnesium sulfate 0.3 g / L, a mixed solution of trace elements (calcium chloride: ferrous sulfate mass ratio is 2:1) 0.4 g / L, a mixed solution of amino acids (glutamic acid, lysine and arginine mass ratio 2:1:1) 0.4 g / L, and the trehalose wastewater is added to make up the volume.

[0019] A method for preparing a microbial inoculant for pepper cultivation, comprising the following steps:

[0020] 1) Preparation of trehalose wastewater medium: Take the wastewater generated in the production and processing of trehalose, carry out separation and filtration, and adjust the pH of the wastewater to 7.0 - 8.0;

[0021] 2) Plate culture and seed liquid culture of the microorganisms are carried out respectively, and then the microorganisms are inoculated into the trehalose wastewater fermentation medium for fermentation culture. After fermentation is completed, the fermentation broth is mixed according to different ratios to form the microbial inoculant.

[0022] The application of the above microbial inoculant for pepper cultivation in pepper cultivation.

[0023] Peppers are cultivated in three different ways: drip irrigation, spraying or watering.

[0024] The microorganisms can be various common Bacillus subtilis and Bacillus megaterium. In one embodiment of the present invention, the Bacillus subtilis and Bacillus megaterium used have preservation numbers CGMCC No. 22878 and CGMCC No. 1.7397 respectively, and both are purchased from the China General Microbiological Culture Collection Center.

[0025] A method for preparing a microbial inoculant for pepper cultivation, more specifically, the steps are as follows:

[0026] (1) Plate and liquid culture of strains

[0027] LB solid medium for Bacillus subtilis: peptone 10 g, yeast powder 5 g, sodium chloride 10 g, agar 20 g, add distilled water to 1000 mL, sterilize at 115 °C for 20 min;

[0028] Primary seed liquid liquid medium for Bacillus subtilis: peptone 10 g, yeast powder 5 g, sodium chloride 10 g, add distilled water to 1000 mL, sterilize at 115 °C for 20 min;

[0029] Secondary seed liquid liquid medium for Bacillus subtilis: glucose 10 g, peptone 8 g, yeast powder 2 g, sodium chloride 10 g, add distilled water to 1000 mL, sterilize at 110 °C for 20 min;

[0030] Bacillus subtilis is first cultured on a solid medium in a petri dish at a culture temperature of 37°C and a fermentation time of 18 h. Then, it is inoculated into a liquid medium at an inoculation amount of 2% (V / V) as the primary seed liquid, with a culture temperature of 35°C and a fermentation time of 24 h. Subsequently, it is inoculated into a liquid medium at an inoculation amount of 3% (V / V) as the secondary seed liquid, with a culture temperature of 35°C and a fermentation time of 29 h.

[0031] LB solid medium for Bacillus megaterium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, add distilled water to 1000 mL, sterilize at 115°C for 20 min;

[0032] Liquid medium for the primary seed liquid of Bacillus megaterium: 10 g of glucose, 5 g of peptone, 5 g of yeast extract, 1 g of magnesium chloride, 1 g of dipotassium hydrogen phosphate, add distilled water to 1000 mL, sterilize at 110°C for 20 min;

[0033] Liquid medium for the secondary seed liquid of Bacillus megaterium: 15 g of glucose, 6 g of peptone, 2 g of yeast extract, 3 g of beef extract, 1 g of magnesium chloride, 1 g of dipotassium hydrogen phosphate, add distilled water to 1000 mL, sterilize at 110°C for 20 min;

[0034] Bacillus megaterium is first cultured on a solid medium in a petri dish at a culture temperature of 35°C and a fermentation time of 20 h. Then, it is inoculated into a liquid medium at an inoculation amount of 2.5% (V / V) as the primary seed liquid, with a culture temperature of 35°C and a fermentation time of 24 h. Subsequently, it is inoculated into a liquid medium at an inoculation amount of 4% (V / V) as the secondary seed liquid, with a culture temperature of 35°C and a fermentation time of 28 h.

[0035] The number of colonies in the microbial inoculant ≥ 1.4×10 9 cfu / mL, the content of polyglutamic acid ≥ 32.5 g / L, and the titer of antibacterial peptide ≥ 600 U / mL.

[0036] 2) Take the wastewater generated in the production and processing of trehalose, and perform separation and filtration (use a tubular centrifuge to separate the bacterial cells from the wastewater generated in the trehalose production process to prevent a large amount of production bacterial cells from remaining in the waste liquid), and adjust the pH of the wastewater to 7.0 - 8.0;

[0037] 3) Inoculate the seed liquids of two different microorganisms (Bacillus subtilis, Bacillus megaterium) into the fermentation medium at an inoculation amount of 1% - 10% respectively. The fermentation temperature is 25°C - 40°C, the aerobic fermentation period is 24 h - 60 h, and feeding is carried out in the middle of fermentation. The feedings are 5% - 10% of the treated trehalose waste liquid and 5% - 10% of monosodium glutamate. The fermentation products contain probiotics, polyglutamic acid, and antibacterial peptides.

[0038] The trehalose waste liquid treated in step 2) is sterilized at 115°C for 30 min and cooled to below 25°C for standby.

[0039] The fermentation medium is composed of 1 - 10 g / L of sodium glutamate, 0.1 - 2 g / L of disodium hydrogen phosphate, 0.1 - 2 g / L of ammonium sulfate, 0.1 - 1 g / L of magnesium sulfate, 0.1 - 1 g / L of trace element mixture, 0.1 - 1 g / L of amino acid mixture, and supplemented with trehalose wastewater.

[0040] The microbial inoculum is a mixture of one or more of Bacillus subtilis and Bacillus megaterium after fermentation. The mixture is prepared according to the mass ratio of (1 - 10):(1 - 10).

[0041] The application of the said microbial inoculum in pepper planting.

[0042] Peppers are planted in any one of the three different ways of drip irrigation, spraying, and watering. The spraying amount is 3% (the dilution multiple is adding 30 mL of microbial inoculum to 1 L of tap water).

[0043] The fermentation medium of the present invention contains components of trehalose production and processing wastewater. It not only realizes the reuse of waste resources, reduces environmental pollution caused by waste materials, greatly reduces production costs, but also can extend the preservation time of the microbial inoculum. Trehalose production wastewater contains a large amount of trehalose, amino acids, etc., which can provide carbon sources and nitrogen sources for fermentation production. At the same time, it can also promote the content of polyglutamic acid and antibacterial peptides. Moreover, the fermentation process is simple and the fermentation cycle is short. In terms of production cost, the price is significantly reduced.

[0044] Bacillus subtilis is a non - pathogenic bacterium widely existing in nature, harmless to humans and animals, and is often used as a biocontrol bacterium. Bacillus megaterium can secrete growth regulators that promote plant growth. At the same time, it can assist in binding with non - leguminous plants to form root nodules, thereby improving the nitrogen utilization rate of crops. The present invention uses Bacillus subtilis and Bacillus megaterium to produce polyglutamic acid, probiotics, and antibacterial peptides by liquid fermentation using trehalose production and processing wastewater to prepare a microbial inoculum, and its application in pepper planting has excellent effects. Using the wastewater in the trehalose production process as a fermentation raw material in the high - density fermentation process can, to a certain extent, replace carbon sources such as glucose, xylose, and starch, thereby reducing production costs. At the same time, its microbial inoculum has a promoting effect on pepper planting, meeting the demand for resource utilization and having a market prospect. Therefore, we propose a new method for planting peppers with microbial inoculum.

[0045] The microbial fermentation inoculant in the present invention can not only increase the per-acre yield of chili peppers, but also improve the quality of chili peppers. At the same time, it can utilize the waste liquid after trehalose production, turning waste into treasure, realizing the reuse of waste resources, reducing environmental pollution, and achieving economic and ecological value. The microbial inoculant in the present invention has low production cost and no pollution; high fertilizer efficiency, easy to absorb, and high utilization rate; uniform nutrients, and can achieve the effects of water and fertilizer conservation, yield increase and efficiency improvement. It has good market prospects and high industrial application value at home and abroad.

[0046] The present invention has the following advantages:

[0047] 1. The microbial inoculant is prepared by the combined production of Bacillus subtilis and Bacillus megaterium and used for planting chili peppers. Compared with the traditional chili pepper planting method, the growth situation is significantly better, the single fruit rate is high, and the disease rate is low.

[0048] 2. Compared with the traditional liquid fermentation method, the fermentation of this strain can utilize the processing wastewater in the trehalose production process to produce inoculant, polyglutamic acid and antibacterial peptides; the trehalose production wastewater contains a large amount of trehalose, amino acids, etc., which can provide carbon sources and nitrogen sources for fermentation production, and at the same time can also promote the content of polyglutamic acid and antibacterial peptides. At the same time, the fermentation process is simple and the fermentation cycle is short. In terms of production cost, the price is significantly reduced.

[0049] 3. The microbial inoculant can be directly sprayed after being diluted with clear water, and has good planting effect on chili peppers and an efficient and energy-saving production method; there is no need to increase production cost, realizing pollution-free production, safety and environmental protection. Specific implementation mode

[0050] Through the following embodiments, the present invention can be better understood. Then, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and their results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0051] LB solid medium for Bacillus subtilis: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 20 g of agar, add distilled water to 1000 mL, sterilize at 115 °C for 20 min;

[0052] Liquid medium for the first-stage seed liquid of Bacillus subtilis: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, add distilled water to 1000 mL, sterilize at 115 °C for 20 min;

[0053] Liquid medium for the second-stage seed liquid of Bacillus subtilis: 10 g of glucose, 8 g of peptone, 2 g of yeast powder, 10 g of sodium chloride, add distilled water to 1000 mL, sterilize at 110 °C for 20 min;

[0054] LB solid medium for Bacillus megaterium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, add distilled water to 1000 mL, sterilize at 115 °C for 20 min;

[0055] Liquid medium for the first-stage seed liquid of Bacillus megaterium: 10 g of glucose, 5 g of peptone, 5 g of yeast extract, 1 g of magnesium chloride, 1 g of dipotassium hydrogen phosphate, add distilled water to 1000 mL, sterilize at 110 °C for 20 min;

[0056] Liquid medium for the second-stage seed liquid of Bacillus megaterium: 15 g of glucose, 6 g of peptone, 2 g of yeast extract, 3 g of beef extract, 1 g of magnesium chloride, 1 g of dipotassium hydrogen phosphate, add distilled water to 1000 mL, sterilize at 110 °C for 20 min;

[0057] Example 1

[0058] In this example, the effects of different trace element ratios on the single fermentation of two strains were investigated. Bacillus subtilis and Bacillus megaterium were cultured on a solid medium in a petri dish, and then cultured in a shake flask with a liquid medium. Then, they were inoculated into the liquid medium according to their respective inoculation amounts as the first-stage seed liquid, and then inoculated into the liquid medium according to their respective inoculation amounts as the second-stage seed liquid. Then, the respective seed liquids (the inoculation amount of Bacillus subtilis was 3% and the inoculation amount of Bacillus megaterium was 4%) were respectively inoculated into the fermentation medium for fermentation experiments. Fermentation medium: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.1 - 1 g / L of trace element mixture, 0.5 g / L of amino acid mixture, supplemented with trehalose wastewater. Mix and stir, sterilize at 115 °C for 20 minutes. Inoculate into the prepared fermentation medium, at a temperature of 37 °C, aerobic fermentation for 24 h. The effects of the mass ratios of calcium chloride to ferrous sulfate in the trace element mixture of 1:1, 1:2, 2:1, 1:3, 3:1, 1:5 and 5:1 on fermentation were investigated; after determining the optimal ratio of trace element metal ions, the effects of the concentrations of the trace element mixture of 0 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L on fermentation were investigated, and the colony count, polyglutamic acid and antibacterial peptide in the liquid fermentation broth were measured. The results are shown in Table 1 and Table 2.

[0059] Table 1 Effects of different trace element ratios on the fermentation broth of strains

[0060]

[0061]

[0062] Table 2 Effects of different concentrations of trace element mixture on the colony count in the fermentation broth of two strains

[0063]

[0064] Example 2

[0065] In this example, the effects of different amino acid element ratios on the separate fermentation of two strains were investigated. Bacillus subtilis and Bacillus megaterium were cultured on a solid medium in a petri dish, and then cultured in a liquid medium in a shaking flask. Then, they were inoculated into the liquid medium according to their respective inoculation amounts as the primary seed liquid, and then inoculated into the liquid medium according to their respective inoculation amounts as the secondary seed liquid. Then, the respective seed liquids (the inoculation amount of Bacillus subtilis was 3% and the inoculation amount of Bacillus megaterium was 4%) were respectively inoculated into the fermentation medium for fermentation experiments. Fermentation medium: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate was 2:1), 0.1 - 1 g / L of an amino acid mixed solution, and supplemented with trehalose wastewater. Mix and stir, sterilize at 115°C for 20 minutes. Inoculate into the prepared fermentation medium, at a temperature of 37°C, carry out aerobic fermentation for 24 h. The effects of different amino acids (mass ratio of glutamic acid, lysine and arginine) in the amino acid mixed solution with ratios of 1:1:1, 1:1:2, 1:2:1, 2:1:2, 2:1:1 on fermentation were investigated; after determining the optimal ratio of different amino acid contents, the effects of concentrations of 0 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L on fermentation were investigated, and the colony count, polyglutamic acid and antibacterial peptides in the liquid fermentation broth were measured. The results are shown in Table 3 and Table 4.

[0066] Table 3 Effects of different amino acid masses on the fermentation of two strains

[0067]

[0068] Table 4 Effects of different amino acid mixed solution concentrations on the fermentation of two strains

[0069]

[0070] Example 3

[0071] In this example, the influence of fermentation time on the fermentation situation was investigated. Bacillus subtilis and Bacillus megaterium were cultured on a solid medium in petri dishes, then cultured in a liquid medium in shake flasks. Then, they were inoculated into the liquid medium according to their respective inoculation amounts as the primary seed liquid, and then inoculated into the liquid medium according to their respective inoculation amounts as the secondary seed liquid. Then, their respective seed liquids (the inoculation amount of Bacillus subtilis was 3% and the inoculation amount of Bacillus megaterium was 4%) were respectively inoculated into the fermentation medium for fermentation experiments. Fermentation medium: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with trehalose wastewater. Mix and stir, sterilize at 115 °C for 20 minutes. Inoculate into the prepared fermentation medium, at a temperature of 37 °C, aerobic fermentation for 18 - 60 h. Measure the colony count, polyglutamic acid and antibacterial peptides in the liquid fermentation broth, and the results are shown in Table 5.

[0072] Table 5 Influence of different fermentation times on the fermentation of strains

[0073]

[0074] Example 4

[0075] In this example, the influence of the mixing volume ratio of different strains was investigated. Bacillus subtilis and Bacillus megaterium were cultured on a solid medium in petri dishes, then cultured in a liquid medium in shake flasks. Then, they were inoculated into the liquid medium according to their respective inoculation amounts as the primary seed liquid, and then inoculated into the liquid medium according to their respective inoculation amounts as the secondary seed liquid. Then, their respective seed liquids (the inoculation amount of Bacillus subtilis was 3% and the inoculation amount of Bacillus megaterium was 4%) were respectively inoculated into the fermentation medium for fermentation experiments. Fermentation medium: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with trehalose wastewater. Mix and stir, sterilize at 115 °C for 20 minutes. Inoculate into the prepared fermentation medium, at a temperature of 37 °C, aerobic fermentation for 36 h. After the fermentation of the fermentation broth was completed, the two fermentation broths were mixed and proportioned. The mixing volume ratios of the Bacillus subtilis and Bacillus megaterium bacterial solutions were (1:1), (1:2), (2:1), (1:3), (3:1) respectively. Measure the colony count, polyglutamic acid and antibacterial peptide content of the mixed bacterial solution, and the results are shown in Table 6.

[0076] Table 6 Influence of different mixing volume ratios of bacterial solutions on the colony count, polyglutamic acid and antibacterial peptides

[0077] Mixing volume ratio Viable count, cfu / mL Polyglutamic acid content, g / L Antibacterial peptide titer, U / mL 1:1 <![CDATA[2.12×10 9 > 26.9 809.5 1:2 <![CDATA[2.34×10 9 > 33.7 853.3 2:1 <![CDATA[2.78×10 9 > 52.8 907.6 1:3 <![CDATA[1.99×10 9 > 24.6 812.4 3:1 <![CDATA[1.78×10 9 > 42.5 673.2

[0078] Example 5

[0079] In this example, the effects of different fermentation media on fermentation were studied. Bacillus subtilis and Bacillus megaterium were cultured on solid medium in petri dishes, and then cultured in shake flasks with liquid medium. Then, they were inoculated into liquid medium according to their respective inoculation amounts as the primary seed liquid, and then inoculated into liquid medium according to their respective inoculation amounts as the secondary seed liquid. Then, the respective seed liquids (the inoculation amount of Bacillus subtilis was 3% and the inoculation amount of Bacillus megaterium was 4%) were respectively inoculated into the fermentation medium for fermentation experiments.

[0080] Fermentation medium ①: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with trehalose wastewater. Mix and stir, and sterilize at 115 °C for 20 minutes.

[0081] Fermentation medium ②: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with rice flour wastewater. Mix and stir, and sterilize at 115 °C for 20 minutes.

[0082] Fermentation medium ③: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with tap water. Mix and stir, and sterilize at 115 °C for 20 minutes.

[0083] Fermentation medium ④: 2 g / L of sodium glutamate, 10 g / L of glucose, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with tap water. Mix and stir, and sterilize at 115 °C for 20 minutes.

[0084] Inoculate into the above-prepared fermentation medium at a temperature of 37°C and carry out aerobic fermentation for 36 h. After the fermentation of the fermentation broth is completed, mix and proportion the two fermentation broths. The volume ratio of the Bacillus subtilis and Bacillus megaterium broths is 2:1. Measure the colony count, polyglutamic acid and antibacterial peptide contents of the mixed bacterial liquid, and the results are shown in Table 7.

[0085] Table 7 Effects of different fermentation media on colony count, polyglutamic acid and antibacterial peptide

[0086] Fermentation medium Viable count, cfu / mL Polyglutamic acid content, g / L Antibacterial peptide titer, U / mL ① <![CDATA[2.89×10 9 > 53.8 912.5 ② <![CDATA[2.25×10 9 > 29.5 823.6 ③ <![CDATA[1.23×10 8 > 12.6 134.7 ④ <![CDATA[1.01×10 9 > 15.3 235.8

[0087] Example 6

[0088] In this example, the effects of different treatment methods (drip irrigation, spraying and watering, where the spraying amount is 3% (the dilution multiple is 1 L of tap water added with 30 mL of microbial inoculum)) on the cultivation of chili peppers were investigated. Bacillus subtilis and Bacillus megaterium were cultured on a solid medium in a petri dish, and then cultured in a shaking flask with a liquid medium. Then, they were inoculated into the liquid medium according to their respective inoculation amounts as the primary seed liquid, and then inoculated into the liquid medium according to their respective inoculation amounts as the secondary seed liquid. Then, the respective seed liquids (the inoculation amount of Bacillus subtilis is 3% and the inoculation amount of Bacillus megaterium is 4%) were respectively inoculated into the fermentation medium for fermentation experiments. Fermentation medium ①: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), and supplemented with trehalose wastewater. Mix and stir, and sterilize at 115°C for 20 minutes. Fermentation medium ②: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), and supplemented with tap water. Mix and stir, and sterilize at 115°C for 20 minutes. Inoculate into the prepared fermentation medium at a temperature of 37°C and carry out aerobic fermentation for 36 h. After fermentation is completed, the fermentation broths are mixed and used according to the volume ratio of Bacillus subtilis and Bacillus megaterium broths of 2:1 respectively for chili pepper cultivation. The vitamin C content and soluble sugar content of the chili peppers change significantly, and the experimental results are shown in Table 8.

[0089] Table 8 Effects of different treatment methods on chili pepper cultivation

[0090] Treatment method Vitamin C (mg / Kg) Soluble content (%) ① Medium drip irrigation 967.5 5.43 ① Medium spraying 834.2 3.92 ① Medium watering 712.9 2.31 ② Medium drip irrigation 812.3 4.22 ② Medium spraying 723.4 3.78 ② Medium watering 711.2 3.56

[0091] Example 7

[0092] In this example, different vegetable plantings were investigated. Bacillus subtilis and Bacillus megaterium were cultured on a solid medium in petri dishes, then cultured in a liquid medium in shake flasks. Then, they were inoculated into the liquid medium according to their respective inoculation amounts as the primary seed liquid, and then inoculated into the liquid medium according to their respective inoculation amounts as the secondary seed liquid. Then, the respective seed liquids (the inoculation amount of Bacillus subtilis was 3% and the inoculation amount of Bacillus megaterium was 4%) were respectively inoculated into the fermentation medium for fermentation experiments. Fermentation medium ①: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with trehalose wastewater. Fermentation medium ②: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate is 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine is 2:1:1), supplemented with tap water. Mix and stir, sterilize at 115 °C for 20 minutes. Inoculate into the prepared fermentation medium, at a temperature of 37 °C, carry out aerobic fermentation for 36 h. The mixing volume ratios of the Bacillus subtilis and Bacillus megaterium bacterial solutions were 2:1 respectively, and the spraying amount was 3% (the dilution multiple was 30 mL of microbial inoculant added with 1 L of tap water for drip irrigation of vegetables). The experimental results are shown in Table 9.

[0093] Table 9 Drip irrigation planting effect of microbial inoculant on different vegetables

[0094]

[0095]

[0096] Example 8

[0097] In this example, the cultivation of chili peppers was investigated. Bacillus subtilis and Bacillus megaterium were cultured on a solid medium in petri dishes, and then cultured in a liquid medium in shake flasks. Subsequently, they were inoculated into the liquid medium according to their respective inoculation amounts as the primary seed liquid, and then inoculated into the liquid medium according to their respective inoculation amounts as the secondary seed liquid. Then, the respective seed liquids (the inoculation amount of Bacillus subtilis was 3% and the inoculation amount of Bacillus megaterium was 4%) were respectively inoculated into the fermentation medium for fermentation experiments. Fermentation medium: 2 g / L of sodium glutamate, 0.6 g / L of disodium hydrogen phosphate, 0.9 g / L of ammonium sulfate, 0.3 g / L of magnesium sulfate, 0.4 g / L of a mixed solution of trace elements (mass ratio of calcium chloride to ferrous sulfate was 2:1), 0.4 g / L of a mixed solution of amino acids (mass ratio of glutamic acid, lysine and arginine was 2:1:1), and supplemented with trehalose wastewater. Mix and stir, sterilize at 115 °C for 20 minutes. Inoculate into the prepared fermentation medium, at a temperature of 37 °C, carry out aerobic fermentation for 36 h. The volume ratios of the Bacillus subtilis and Bacillus megaterium bacterial solutions were 2:1 respectively, and an antibacterial test against chili pepper pathogens was carried out, and the antibacterial effects were significantly different. The experimental results are shown in Table 10.

[0098] Table 10 Antibacterial effects of microbial inoculants against chili pepper pathogens

[0099] Pepper pathogen Inhibition zone (mm) Phytophthora capsici 32.6±0.2 Colletotrichum capsici 33.3±0.2

[0100] Example 9

[0101] In this example, the shape of chili peppers in chili pepper cultivation was investigated. The above-mentioned conventional cultivation + drip irrigation of microbial inoculants, conventional cultivation + drip irrigation of common chili pepper cultivation fertilizers (phosphorus and potassium fertilizers), and conventional cultivation + drip irrigation of clear water were used for antibacterial tests against chili pepper pathogens, and the antibacterial effects were significantly different. The chili pepper cultivation time was 6 months. The incidence of chili pepper cultivation and the plant height, number of fruits per plant, single fruit weight, etc. of chili peppers during the harvesting period were investigated. The tests are shown in Tables 11 and 12.

[0102] Table 11 Incidence of chili pepper cultivation under different treatment methods

[0103]

[0104]

[0105] Table 12 Conditions of chili pepper growth shape under different treatment methods

[0106]

[0107] The above has introduced in detail the technical solutions provided by the embodiments of the present invention. Specific examples are used herein to elaborate on the principles and implementation manners of the embodiments of the present invention. The description of the above embodiments is only applicable to helping understand the principles of the embodiments of the present invention. At the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A microbial inoculant for pepper cultivation, characterized in that, The microbial inoculum is obtained by fermenting a trehalose wastewater fermentation medium with microorganisms, and the fermentation product contains a large amount of probiotics, polyglutamic acid and antibacterial peptides; the microorganisms are at least one of Bacillus subtilis and Bacillus megaterium.

2. The microbial inoculum for pepper cultivation according to claim 1, wherein, The number of colonies in the microbial inoculum ≥ 1.4×10 9 cfu / mL, the content of polyglutamic acid ≥ 32.5 g / L, and the titer of antibacterial peptide ≥ 600 U / mL.

3. A microbial inoculant for pepper cultivation according to claim 1, characterized in that, The trehalose wastewater fermentation medium is composed of 1-10 g / L of sodium glutamate, 0.1-2 g / L of disodium hydrogen phosphate, 0.1-2 g / L of ammonium sulfate, 0.1-1 g / L of magnesium sulfate, 0.1-1 g / L of a trace element mixture, 0.1-1 g / L of an amino acid mixture, and the trehalose wastewater is added to make up the volume.

4. The microbial inoculum for pepper cultivation according to claim 1, characterized in that, The temperature of the fermentation is 25 °C to 40 °C, the aerobic fermentation period is 24 h to 60 h, and the aeration ratio is 1 vvm.

5. The microbial inoculum for pepper cultivation according to claim 3, characterized in that, The trace element mixture is composed of calcium chloride and ferrous sulfate mixed in a mass ratio of (1-10):(1-5).

6. The microbial inoculum for pepper cultivation according to claim 3, wherein The amino acid mixture is composed of glutamic acid, lysine and arginine mixed in a mass ratio of (1-10):(1-5):(1-5).

7. A microbial inoculant for pepper cultivation according to claim 3, characterized in that, The trehalose wastewater is obtained by separating and filtering the wastewater generated during the production and processing of trehalose and adjusting the pH to 7.0-8.

0.

8. The preparation method of the microbial inoculum for pepper cultivation according to any one of claims 1-7, characterized in that, It includes the following steps: 1) Preparation of the trehalose wastewater medium: Take the wastewater generated during the production and processing of trehalose, carry out separation and filtration, and adjust the pH of the wastewater to 7.0-8.0; 2) The microorganisms are respectively subjected to plate culture and seed liquid culture and then inoculated into the trehalose wastewater fermentation medium for fermentation culture. After fermentation is completed, the fermentation broth is mixed in different proportions to form the microbial inoculum.

9. Application of the microbial inoculum for pepper planting according to any one of claims 1-7 in pepper planting.

10. The application according to claim 9, characterized in that, Plant peppers in three different ways: drip irrigation, spraying or watering.

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