Compound microbial inoculant of synergistic chinese medicine and bacillus megaterium for planting
By combining the medicinal herb Bacillus megaterium with the plant-growing compound microbial agent, a staged fermentation and cross-linking process with medicinal polyphenols is used to transform the active ingredients of the medicinal herbs into small-molecule growth-promoting substances. This solves the problems of low utilization rate of large molecules of medicinal herbs and poor activity stability of the microbial agent, and achieves a multi-level synergistic effect of the microbial agent in the soil.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHAOQING JUNCHUANG HUINONG AGRICULTURAL DEVELOPMENT CO LTD
- Filing Date
- 2026-05-09
- Publication Date
- 2026-08-04
AI Technical Summary
Existing technologies that combine Chinese herbal extracts with microbial agents have problems such as low absorption and utilization rates of macromolecular components of Chinese herbal medicines and a significant decrease in the number of live bacteria in the microbial agents during long-term storage.
A compound microbial agent for planting, which combines the Chinese herbal medicine Bacillus megaterium with the Chinese herbal medicine Bacillus megaterium, is used. Through a co-fermentation process of staged relay fermentation of the microbial strains and in-situ cross-linking of Chinese herbal medicine polyphenols, the active ingredients of Chinese herbal medicine are efficiently converted into small molecule growth-promoting active ingredients. The microcapsule protective structure is formed by using chitosan oligosaccharide cross-linking carrier.
It significantly improves the synergistic effect of Chinese medicine raw materials, solves the problem of poor stability of long-term coexistence between Chinese medicine components and live bacteria, and endows the microbial agent with multi-level functions such as slow release of chitosan oligosaccharide in soil to induce resistance, continuous carbon supply and calcium supplementation, and synergistic phosphorus solubilization and antibacterial activity.
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Figure CN122498521A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural microbial inoculants, and in particular to a compound inoculant for planting made by combining the traditional Chinese medicine Bacillus subtilis and Bacillus megaterium. Background Technology
[0002] Improving crop quality and yield is a core requirement for ensuring food security and increasing farmers' income. Microbial agents, due to their multiple functions of improving soil, inhibiting diseases, and promoting growth, have become important inputs in green agriculture. Bacillus strains, due to their spore production, strong resistance, and ability to secrete growth-promoting substances and antibacterial active ingredients, are the most widely used functional bacteria in the field of microbial agents. However, the functions of a single strain are limited. Combining multiple functional bacteria and introducing natural synergistic ingredients to enhance the overall effect is an important direction for current technological development.
[0003] Traditional Chinese medicine contains abundant bioactive substances. Ferulic acid in Ligusticum chuanxiong, ligustilide in Angelica sinensis, and cinnamaldehyde in Cinnamomum cassia have all been proven to have physiological activities such as promoting root growth and resisting stress. Introducing Chinese medicinal materials into a microbial inoculant system and utilizing the metabolism of the strains to transform them into more efficient small molecule active substances can theoretically achieve "synergistic effect of microorganisms and medicines".
[0004] There are existing reports on the combination of traditional Chinese medicine extracts and microbial agents, but most of them are prepared separately and then physically mixed, which has the following shortcomings: The macromolecular components of traditional Chinese medicine have not undergone biotransformation, resulting in low absorption and utilization rates by the root system and limited synergistic effects. The volatile oils and phenolic components of traditional Chinese medicine have an inhibitory effect on live bacteria, and the number of live bacteria in the inoculant decreases significantly during long-term storage.
[0005] Therefore, it is necessary to design a compound microbial agent for planting, which combines the effects of the traditional Chinese medicine Bacillus subtilis with Bacillus megaterium. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a compound microbial agent for planting, which combines the traditional Chinese medicine Bacillus subtilis and Bacillus megaterium, thus solving the problems mentioned in the background section.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: A compound microbial agent for planting, which combines the Chinese herb Bacillus subtilis and Bacillus megaterium, includes a mixed bacterial solution and a synergistic carrier; The mixed bacterial solution consists of Bacillus subtilis fermentation broth and Bacillus megaterium fermentation broth, with a volume ratio of Bacillus subtilis fermentation broth to Bacillus megaterium fermentation broth of 1:0.8-1.2. The enhancing carrier is a concentrated extract of fermented Chinese medicine that has been cross-linked with chitosan oligosaccharide and Chinese herbal polyphenols.
[0008] Furthermore, the total number of viable bacteria in the mixed bacterial solution is ≥2 billion / ml.
[0009] Furthermore, the medicinal materials of the synergistic carrier are Ligusticum chuanxiong, Angelica sinensis, Cinnamomum cassia, and Eriobotrya japonica.
[0010] Furthermore, the chitosan oligosaccharide-Chinese herbal polyphenol cross-linking modification is formed by covalent cross-linking of chitosan oligosaccharide and Chinese herbal polyphenols under enzyme catalysis via Schiff base reaction.
[0011] The preparation steps of the above-mentioned compound microbial agent for planting, which combines the Chinese herbal medicine *Bacillus subtilis* and *Bacillus megaterium*, are as follows: Step S1: Mix and pulverize the chuanxiong, angelica, cinnamon twig, and loquat leaf, then sieve them. Add the carbon source, nitrogen source, inorganic salt, natural organic acid composition, and water, stir evenly, adjust the pH to 6.8-7.0, raise the temperature to 105-115℃, sterilize for 20-30 minutes, and cool to 28-30℃ to obtain the fermentation culture medium. Step S2: Inoculate the Bacillus megaterium seed culture into the fermentation medium, rotate at 120-150 rpm, aeration rate of 0.5-0.8 vvm, ferment for 24-36 h to obtain the first stage fermentation broth; Step S3: Heat the first-stage fermentation broth to 35-37℃, inoculate with Bacillus subtilis seed liquid, and slowly add micronized mineral carrier. Ferment at 180-200 rpm and 0.8 vvm for 7-8 hours, then continue fermentation at 180-200 rpm and 0.3-0.5 vvm for 30-42 hours to obtain the second-stage fermentation broth. Step S4: Add chitosan oligosaccharide to the second-stage fermentation broth, stir at 80-120 rpm for 4-6 minutes, add polyphenol oxidase activator, heat to 35-37℃, stir at 80-100 rpm, react for 8-12 hours, and the reaction is complete to obtain cross-linked fermentation broth. Step S5: Add the camellia oil microemulsion, xanthan gum, and sodium benzoate to the cross-linked fermentation broth, homogenize, and fill to obtain a compound microbial agent for planting, which is a combination of Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
[0012] Furthermore, in step S1, the mass ratio of the composition of Ligusticum chuanxiong, Angelica sinensis, Cinnamomum cassia, Eriobotrya japonica, carbon source, nitrogen source, inorganic salt, and natural organic acid to water is 20-30:20-30:15-25:25-35:5-8:3-5:3-5:3-4:800-1000.
[0013] Furthermore, the natural organic acid composition described in step S1 is composed of citric acid and malic acid, and the mass ratio of citric acid to malic acid is 2:1-2; The mass ratio of Bacillus megaterium seed culture to fermentation medium in step S2 is 5-8:1000.
[0014] Furthermore, in step S3, the mass ratio of Bacillus subtilis seed liquid, micronized mineral carrier, and first-stage fermentation broth is 5-8:4-6:1000; The micronized mineral carrier mentioned in step S3 is composed of light calcium carbonate and oyster shell ultrafine powder, and the mass ratio of light calcium carbonate to oyster shell ultrafine powder is 1:0.25-0.5, and the fineness of both is 800-1250 mesh.
[0015] Furthermore, in step S4, the mass ratio of chitosan oligosaccharide, polyphenol oxidase activator, and second-stage fermentation broth is 1-2:0.04-0.07:1000; The molecular weight of the chitosan oligosaccharide mentioned in step S4 is 1000-3000 Da, the degree of deacetylation is ≥90%, and the amount added is 0.1%-0.2% based on the mass of the fermentation broth.
[0016] Furthermore, in step S5, the mass ratio of the camellia oil microemulsion, xanthan gum, sodium benzoate, and cross-linked fermentation broth is 5-8:0.5-1:0.3-0.5:1000.
[0017] Compared with existing technologies, the advantages of this invention are as follows: This invention integrates a co-fermentation process involving staged relay fermentation of microorganisms and in-situ cross-linking of polyphenols from traditional Chinese medicine. This process combines the efficient conversion of active ingredients in medicinal herbs, the spore-inducing protection of functional bacteria, and the cascade synergistic effect after soil application into a unified technical solution. Compared with existing technologies, this invention achieves the targeted biotransformation of macromolecular substances in traditional Chinese medicine into small-molecule growth-promoting active ingredients, significantly improving the synergistic efficiency of medicinal raw materials. It also solves the problem of poor long-term stability of medicinal components and live bacteria. Furthermore, it endows the microbial agent with multi-level functions in soil, including slow-release chitosan oligosaccharide-induced resistance, continuous carbon and calcium supply, and synergistic phosphorus solubilization and antibacterial activity. This represents a technological leap from production to application and has broad application prospects in the field of green agriculture. Attached Figure Description
[0018] Figure 1 This is a flowchart illustrating the preparation method of the compound microbial agent for planting, which combines the traditional Chinese medicine Bacillus megaterium with the herb Bacillus megaterium, as proposed in this invention. Detailed Implementation
[0019] Reference Figure 1 To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments.
[0020] The sources and properties of some of the raw materials used in this invention are as follows: The Bacillus subtilis used in the fermentation broth of Bacillus subtilis in this invention is Bacillus subtilis, which is disclosed in the invention patent with announcement number "CN121294217A" and title "A Bacillus subtilis and its application", and the accession number is CGMCCNO:29256. The Bacillus megaterium used in the fermentation broth of this invention is Bacillus megaterium HP-3, which is disclosed in the invention patent with authorization announcement number "CN118421520A" and title "A Bacillus megaterium HP-3 and its application", and accession number CCTCCNO.M20221791.
[0021] Example 1: Preparation of a compound microbial agent for planting, combining the traditional Chinese medicine *Bacillus subtilis* and *Bacillus megaterium*. S1: Mix and pulverize 2000g of Ligusticum chuanxiong, 3000g of Angelica sinensis, 1500g of Cinnamomum cassia, and 3500g of Eriobotrya japonica leaves, then sieve them. Add 500g of carbon source, 500g of nitrogen source, 300g of inorganic salt, 200g of citric acid, 200g of malic acid, and 100,000g of water. Stir well, adjust the pH to 6.8-7.0, heat to 105℃, sterilize for 30 minutes, and cool to 28℃ to obtain the fermentation medium. S2: Inoculate 500g of Bacillus megaterium seed liquid into 100,000g of fermentation medium, rotate at 150rpm, aeration rate of 0.5vvm, ferment for 36h to obtain the first stage fermentation broth; S3: Heat 100,000g of the first-stage fermentation broth to 35℃, inoculate with 800g of Bacillus subtilis seed liquid, and slowly add 300g of light calcium carbonate and 75g of oyster shell ultrafine powder. Ferment at 200rpm and 0.8vvm for 7 hours, then continue fermenting at 200rpm and 0.3vvm for 42 hours to obtain the second-stage fermentation broth. S4: Add 100g of chitosan oligosaccharide to 100,000g of the second-stage fermentation broth, stir at 120rpm for 4min, add 7g of polyphenol oxidase activator, heat to 35℃, stir at 100rpm for 8h, and the reaction is complete to obtain cross-linked fermentation broth. S5: Add 500g of camellia oil microemulsion, 100g of xanthan gum and 30g of sodium benzoate to 100,000g of cross-linked fermentation broth, homogenize and fill to obtain a compound microbial agent for planting, which is a combination of Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
[0022] Example 2: Preparation of a compound microbial agent for planting, combining the traditional Chinese medicine *Bacillus subtilis* and *Bacillus megaterium*. S1: Mix and pulverize 2500g of Ligusticum chuanxiong, 2500g of Angelica sinensis, 2000g of Cinnamomum cassia, and 3000g of Eriobotrya japonica leaves, then sieve them. Add 650g of carbon source, 400g of nitrogen source, 400g of inorganic salt, 200g of citric acid, 150g of malic acid, and 100,000g of water. Stir well, adjust the pH to 6.8-7.0, heat to 110℃, sterilize for 25 minutes, and cool to 29℃ to obtain the fermentation medium. S2: Inoculate 650g of Bacillus megaterium seed liquid into 100,000g of fermentation medium, rotate at 135rpm, aeration rate of 0.65vvm, ferment for 30h to obtain the first stage fermentation broth; S3: Heat 100,000g of the first-stage fermentation broth to 36℃, inoculate with 650g of Bacillus subtilis seed liquid, and slowly add 300g of light calcium carbonate and 115g of oyster shell ultrafine powder. Ferment at 190rpm and 0.8vvm for 7.5h, then continue fermenting at 190rpm and 0.4vvm for 36h to obtain the second-stage fermentation broth. S4: Add 150g of chitosan oligosaccharide to 100,000g of the second-stage fermentation broth, stir at 100rpm for 5min, add 5.5g of polyphenol oxidase activator, heat to 36℃, stir at 90rpm for 10h, and the reaction is complete to obtain cross-linked fermentation broth. S5: Add 650g of camellia oil microemulsion, 75g of xanthan gum and 40g of sodium benzoate to 100,000g of cross-linked fermentation broth, homogenize and fill to obtain a compound microbial agent for planting, which is a combination of Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
[0023] Example 3: Preparation of a compound microbial agent for planting, combining the traditional Chinese medicine *Bacillus subtilis* and *Bacillus megaterium*. S1: Mix and pulverize 3000g of Ligusticum chuanxiong, 2000g of Angelica sinensis, 2500g of Cinnamomum cassia and 2500g of Eriobotrya japonica leaves, then sieve them. Add 800g of carbon source, 300g of nitrogen source, 500g of inorganic salt, 200g of citric acid, 100g of malic acid and 100000g of water, stir well, adjust the pH to 6.8-7.0, heat to 115℃, sterilize for 20min, and cool to 30℃ to obtain the fermentation medium. S2: Inoculate 800g of Bacillus megaterium seed liquid into 100,000g of fermentation medium, rotate at 120rpm, aeration rate of 0.8vvm, ferment for 24h to obtain the first stage fermentation broth; S3: Heat 100,000g of the first-stage fermentation broth to 37℃, inoculate with 500g of Bacillus subtilis seed liquid, and slowly add 300g of light calcium carbonate and 150g of oyster shell ultrafine powder. Ferment at 180rpm and 0.8vvm for 8 hours, then continue fermenting at 180rpm and 0.5vvm for 30 hours to obtain the second-stage fermentation broth. S4: Add 200g of chitosan oligosaccharide to 100,000g of the second-stage fermentation broth, stir at 80rpm for 6min, add 4g of polyphenol oxidase activator, heat to 37℃, stir at 80rpm for 12h, and the reaction is complete to obtain cross-linked fermentation broth. S5: Add 800g of camellia oil microemulsion, 50g of xanthan gum, and 50g of sodium benzoate to 100,000g of cross-linked fermentation broth, homogenize, and fill to obtain a compound microbial agent for planting, which is a combination of Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
[0024] Comparative Example 1: Compared with Example 1, this comparative example only reduced the amount of Bacillus megaterium seed solution in step S2 from 500g to 100g. All other steps and parameters are the same, and will not be repeated here. The final result is a compound microbial agent for planting, which combines the Chinese herbal medicine Bacillus megaterium with the Chinese herbal medicine Bacillus megaterium.
[0025] Comparative Example 2: This comparative example differs from Example 1 only in that chitosan oligosaccharide was not added in step S4; all other steps and parameters are the same. This comparative example will not be repeated here. The final product is a compound microbial agent for planting, which combines the Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
[0026] Comparative Example 3: Compared with Example 1, this comparative example only replaces 300g of light calcium carbonate and 75g of oyster shell ultrafine powder in step S3 with an equal mass of 375g of diatomaceous earth. All other steps and parameters are the same, and will not be repeated here. The final product is a compound microbial agent for planting, which combines the Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
[0027] Comparative Example 4: Compared with Example 1, this comparative example only increases the fermentation temperature of step S2 from 28°C to 40°C. All other steps and parameters are the same, and will not be repeated here. The final product is a compound microbial agent for planting, which combines the Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
[0028] Performance testing: Effective viable bacteria count test 1. Referring to GB 20287-2006 "Agricultural Microbial Inoculants" standard, 10.0 g of sample was weighed under aseptic conditions and added to 90 mL of sterile water. The mixture was shaken at 180 rpm for 30 min to obtain 10 g of sample. -1 Diluent; 2. Using a sterile pipette, take 1 mL of the above diluent and add it to 9 mL of sterile water. Perform serial dilutions of 10-fold to a final volume of 10. -8 ; 3. Select 10 -6 10 -7 10 -8 Three dilutions were prepared, with 0.1 mL of each sample spread onto LB solid medium plates, and three replicates were made for each dilution. The plates were incubated upside down at 30°C for 48 h. 4. After the culture is completed, collect plates with colony counts between 30 and 300 and calculate the effective viable count. The results are expressed as 100 million CFU / mL.
[0029] Table 1 Results of Effective Viable Bacteria Count Test
[0030] Seed germination index test (effect on promoting root growth and seedling development) 1. Refer to GB / T 3543.4-1995 "Grain Germination Test Procedure for Crop Seeds" standard, select uniform and plump wheat or cucumber seeds, disinfect the surface with 1% sodium hypochlorite solution for 10 minutes, and rinse 3 times with sterile water. 2. Dilute the sample 500 times with sterile water, take 5 mL and add it to a petri dish lined with double-layered filter paper. Place 20 seeds evenly in each dish. Use sterile water as a blank control. Each group has 3 replicates. 3. Place the petri dishes in a 25℃ constant temperature incubator and incubate in the dark for 72 hours; 4. After cultivation, measure the root length and shoot length of each seed, and calculate the germination index GI = (root length of treatment group × germination rate) / (root length of control group × germination rate) × 100%, and take the average value.
[0031] Table 2 Seed germination index test results
[0032] Antibacterial activity test (resistance to soil-borne diseases) 1. Prepare PDA culture plates according to NY / T 1156.2-2006 "Guidelines for Indoor Bioassay Tests of Pesticides - Fungicides Part 2: Plate Method for Inhibition of Mycelial Growth of Pathogenic Fungi". 2. Take 100 μL of a spore suspension of Streptomyces scabies or Rhizoctonia solani (concentration 1×10⁻⁶). 8 (CFU / mL), evenly coated onto a PDA plate; 3. Place a sterile Oxford cup in the center of the plate, inject 200 μL of the original sample solution, and use sterile water as a blank control. Each group has 3 replicates, and incubate at 28℃ for 5-7 days. 4. Measure the diameter of the inhibition zone with vernier calipers (measure twice using the cross method and take the average value), and calculate the inhibition diameter ratio = inhibition zone diameter / pathogen growth diameter × 100%.
[0033] Table 3 Results of antibacterial activity test
[0034] Field yield increase test 1. Referring to the standard NY / T 206-2005 "Technical Specifications for Field Trials of Agricultural Microbial Inoculants", select potato or tomato fields with obvious continuous cropping obstacles and divide them into plots (each plot is 20-30m²). 2 ), randomized block design, with 3 replicates per group; 2. Dilute the sample 1:600 and apply it to the roots during crop transplanting. Apply 30 mL per plant. Use an equal volume of water as a blank control. Perform routine fertilization and management. 3. During the harvest period, the yield of all plants in each plot was measured and the yield data was recorded; 4. Calculate the yield increase rate = (treatment group yield - control group yield) / control group yield × 100%, and take the average of each replication. The unit is .
[0035] Table 4. Results of Field Yield Increase Tests
[0036] Data Analysis: As can be seen from Tables 1-4, the compound microbial agent for planting prepared by the present invention, which combines the Chinese herbal medicine Bacillus subtilis and Bacillus megaterium, has better live bacteria survival ability, stronger root promotion and seedling strengthening effect, more prominent antibacterial activity against soil-borne diseases, and more significant field yield increase. In contrast, in Comparative Example 1, the amount of Bacillus megaterium seed solution inoculated was significantly reduced from 500g to 100g, resulting in insufficient pre-degradation of the macromolecules of traditional Chinese medicine in the first stage. This led to a significant decrease in the number of effective viable bacteria, germination index, and field yield. The reason for this was that the insufficient number of Bacillus megaterium prevented the full secretion of phytase and phosphatase at the low temperature of 28℃. Phytate-complexed phosphorus in the traditional Chinese medicines Angelica sinensis and Ligusticum chuanxiong was not effectively released, resulting in a reduction in the available available phosphorus for Bacillus subtilis. At the same time, the macromolecules of traditional Chinese medicine (ferulic acid, ligustilide, etc.) were not pre-degraded into small phenolic acid frameworks with ortho-dihydroxyl groups, resulting in a severe lack of chitosan oligosaccharide-traditional Chinese medicine polyphenol cross-linking substrate in step S4. The microcapsule protective structure was not fully formed, and the viable bacteria attenuated rapidly during the storage of the inoculum. After application to the soil, the slow release and immune induction functions of chitosan oligosaccharide were simultaneously weakened, ultimately resulting in a decrease in root-promoting effect and yield increase. Comparative Example 2, due to the complete removal of chitosan oligosaccharide, suffered from the dual loss of microcapsule protective structure and long-term soil immune induction function. Its germination index and field yield increase rate decreased the most among all comparative examples. The number of effective viable bacteria also decreased due to the lack of cross-linking protection. This is because chitosan oligosaccharide is the core skeleton material that undergoes Schiff base covalent cross-linking with the polyphenols of traditional Chinese medicine in step S4. Without the addition of chitosan oligosaccharide, it is impossible to form a three-dimensional network microcapsule structure to encapsulate the Bacillus cells. During storage, the inoculant is directly exposed to dissolved oxygen and the volatile oil environment of traditional Chinese medicine, which accelerates the inactivation of spores and reduces the survival rate of viable bacteria. After being applied to the soil, due to the lack of chitosan oligosaccharide slow-release precursor, it is impossible to continuously activate the systemic acquired resistance signaling pathway (SAR) in the crop. The crop's self-defense ability against soil-borne pathogens is not enhanced. At the same time, the lack of cross-linking network for the slow-release protection of small molecule phenolic acids in traditional Chinese medicine causes the small molecules to diffuse and be lost rapidly in the soil. It is impossible to form a continuous chemotactic signal and beneficial bacteria colonization induction effect in the rhizosphere, resulting in a significant weakening of the root-promoting and seedling-strengthening effects and yield increase. Comparative Example 3, due to the replacement of light calcium carbonate and oyster shell ultrafine powder with an equal mass of diatomaceous earth, resulted in the disappearance of the CO2 slow-release spore-promoting effect during fermentation and the loss of the calcium replenishment function in the soil. The most significant decrease was in the number of effective viable bacteria, and the antibacterial activity was also affected to some extent. This is because calcium carbonate / oyster shell ultrafine powder has a dual function in step S3: firstly, when Bacillus subtilis metabolizes and produces acid, causing the system pH to drop to 5.8-6.2, calcium carbonate slowly dissolves and releases Ca2+. 2+CO2 microbubbles, in the fermentation broth, form localized micro-aerobic / anaerobic alternating microzones, which act as a mild environmental stress signal to stimulate Bacillus subtilis to initiate spore differentiation prematurely. Diatomaceous earth, being an inert silicate, lacks the ability to neutralize acids and bases and cannot generate CO2 stress signals, resulting in a significant decrease in spore formation rate and a drop in the number of viable bacteria at the end of the inoculant. Secondly, after being applied to the soil, calcium carbonate / oyster shell microparticles continuously and slowly release CO2 under the action of rhizosphere organic acids, providing a rhizosphere carbon fertilizer effect and supplementing exchangeable calcium. This, combined with the phosphorus solubilization effect of Bacillus megaterium, forms a synergistic absorption of calcium and phosphorus. Diatomaceous earth does not possess this function, resulting in the lack of a long-term synergistic effect mechanism in the soil. In Comparative Example 4, increasing the fermentation temperature of the first stage in step S2 from 28℃ to 40℃ suppressed the high-temperature growth and metabolism of Bacillus megaterium. This resulted in the largest decrease in the effective viable cell count, germination index, and inhibition diameter ratio. The optimal growth temperature range for Bacillus megaterium is 28-30℃, and raising it to 40℃ exceeded its tolerance limit. Under high-temperature conditions, the secretion of phytase and phosphatase in Bacillus megaterium decreased sharply, essentially halting the pre-degradation process of large molecules in traditional Chinese medicine. Active components such as ferulic acid in Ligusticum chuanxiong and Angelica sinensis could not be converted into small hydroxyl molecules that could be used for subsequent cross-linking, causing the entire synergistic chain to break down in the first stage. Furthermore, high temperature could also cause partial thermal death of Bacillus megaterium. The contents released from the rupture of residual cells in the fermentation broth interfered with the subsequent growth of Bacillus subtilis. Simultaneously, due to the lack of readily available phosphorus and small phenolic acid precursors produced in the first stage, Bacillus subtilis lacked metabolic substrates and co-stimulatory factors in step S3, affecting its ability to produce spores and antimicrobial peptides, ultimately leading to a comprehensive deterioration in the overall performance of the bacterial agent.
[0037] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.
Claims
1. A compound microbial agent for planting, consisting of the herb *Bacillus megaterium* and *Bacillus megaterium*, characterized in that... Includes mixed bacterial culture and synergistic carrier; The mixed bacterial solution consists of Bacillus subtilis fermentation broth and Bacillus megaterium fermentation broth, with a volume ratio of Bacillus subtilis fermentation broth to Bacillus megaterium fermentation broth of 1:0.8-1.
2. The enhancing carrier is a concentrated extract of fermented Chinese medicine that has been cross-linked with chitosan oligosaccharide and Chinese herbal polyphenols.
2. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 1, is characterized in that... The total number of viable bacteria in the mixed bacterial solution is ≥2 billion / ml.
3. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 1, is characterized in that... The medicinal materials used in the synergistic carrier are Ligusticum chuanxiong, Angelica sinensis, Cinnamomum cassia, and Eriobotrya japonica.
4. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 1, is characterized in that... The chitosan oligosaccharide-Chinese herbal polyphenol cross-linking modification is formed by covalent cross-linking of chitosan oligosaccharide and Chinese herbal polyphenols under enzyme catalysis via Schiff base reaction.
5. The compound microbial agent for planting, formulated with *Bacillus megaterium* and *Hypericum megaterium* according to any one of claims 1-4, is characterized in that... The preparation steps are as follows: Step S1: Mix and pulverize the chuanxiong, angelica, cinnamon twig, and loquat leaf, then sieve them. Add the carbon source, nitrogen source, inorganic salt, natural organic acid composition, and water, stir evenly, adjust the pH to 6.8-7.0, raise the temperature to 105-115℃, sterilize for 20-30 minutes, and cool to 28-30℃ to obtain the fermentation culture medium. Step S2: Inoculate the Bacillus megaterium seed culture into the fermentation medium, rotate at 120-150 rpm, aeration rate of 0.5-0.8 vvm, ferment for 24-36 h to obtain the first stage fermentation broth; Step S3: Heat the first-stage fermentation broth to 35-37℃, inoculate with Bacillus subtilis seed liquid, and slowly add micronized mineral carrier. Ferment at 180-200 rpm and 0.8 vvm for 7-8 hours, then continue fermentation at 180-200 rpm and 0.3-0.5 vvm for 30-42 hours to obtain the second-stage fermentation broth. Step S4: Add chitosan oligosaccharide to the second-stage fermentation broth, stir at 80-120 rpm for 4-6 minutes, add polyphenol oxidase activator, heat to 35-37℃, stir at 80-100 rpm, react for 8-12 hours, and the reaction is complete to obtain cross-linked fermentation broth. Step S5: Add the camellia oil microemulsion, xanthan gum, and sodium benzoate to the cross-linked fermentation broth, homogenize, and fill to obtain a compound microbial agent for planting, which is a combination of Chinese herbal medicine Bacillus subtilis and Bacillus megaterium.
6. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 5, is characterized in that... The mass ratio of the combination of Ligusticum chuanxiong, Angelica sinensis, Cinnamomum cassia, Eriobotrya japonica leaf, carbon source, nitrogen source, inorganic salt, and natural organic acid to water in step S1 is 20-30:20-30:15-25:25-35:5-8:3-5:3-5:3-4:1000.
7. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 5, is characterized in that... The natural organic acid composition described in step S1 consists of citric acid and malic acid, and the mass ratio of citric acid to malic acid is 2:1-2. The mass ratio of Bacillus megaterium seed culture to fermentation medium in step S2 is 5-8:1000.
8. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 5, is characterized in that... The mass ratio of Bacillus subtilis seed liquid, micronized mineral carrier and first-stage fermentation broth in step S3 is 5-8:4-6:1000; The micronized mineral carrier mentioned in step S3 is composed of light calcium carbonate and oyster shell ultrafine powder, and the mass ratio of light calcium carbonate to oyster shell ultrafine powder is 1:0.25-0.5, and the fineness of both is 800-1250 mesh.
9. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 5, is characterized in that... The mass ratio of chitosan oligosaccharide, polyphenol oxidase activator, and second-stage fermentation broth in step S4 is 1-2:0.04-0.07:1000; The molecular weight of the chitosan oligosaccharide mentioned in step S4 is 1000-3000 Da, the degree of deacetylation is ≥90%, and the amount added is 0.1%-0.2% based on the mass of the fermentation broth.
10. The compound microbial agent for planting, a combination of *Bacillus megaterium* and *Hypericum megaterium* as described in claim 5, is characterized in that... In step S5, the mass ratio of camellia oil microemulsion, xanthan gum, sodium benzoate and cross-linked fermentation broth is 5-8:0.5-1:0.3-0.5:1000.