Preparation method of agricultural microbial fertilizer containing plant extract

Abstract

The application discloses a kind of plant extract-containing agricultural microbial fertilizer preparation method, and the application relates to the field of agricultural microbial fertilizer preparation, method includes: gradient stress directional domestication is carried out to functional strain, and domestication qualified strain with tolerance to plant extract effective component is obtained, domestication qualified strain is embedded with prebiotic in sodium alginate and modified starch crosslinking framework to form inner core particle, plant extract effective component is loaded in low methoxyl pectin to form coating slurry, and it is coated on the surface of inner core particle by fluidized bed bottom spray, form core-shell double-layer particle, and the fertilizer is prepared, the application is relieved by strain tolerance modification The antagonistic contradiction between strain and extract, and through the time-space decoupling of shell fast release clearing field and inner core slow release colonization, the survival rate and field colonization rate of strain are improved.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial fertilizer preparation technology, specifically to a method for preparing agricultural microbial fertilizer containing plant extracts. Background Technology

[0002] Agricultural microbial fertilizers use biocontrol and growth-promoting strains such as Bacillus and Pseudomonas as core active ingredients. They achieve disease prevention and growth promotion effects through antagonistic metabolites produced after colonization of plant rhizosphere and the induction of systemic resistance. Plant extract active ingredients, such as matrine and strychnine extract, have the characteristics of natural source, low residue, and broad-spectrum antibacterial and insecticidal properties. In recent years, they have been widely used in combination with microbial fertilizers to form an integrated fertilizer-pesticide product system.

[0003] In the prior art solution closest to this invention, functional strains are usually first fermented to obtain bacterial powder or bacterial liquid, and then the effective components of plant extracts are made into concentrated liquid or dry powder. Subsequently, the bacterial powder and plant extracts are directly mixed in proportion, and after adding carriers and stabilizers, they are made into granular or powdered products through processes such as extrusion granulation, drying or coating. In this solution, the functional strains and plant extracts are in the same physical phase, separated without a barrier layer, and the two are in contact, mixed and released synchronously.

[0004] Because the active ingredients in plant extracts have broad-spectrum antibacterial activity, functional strains are directly mixed with extracts without tolerance treatment. The extracts continue to act on the strain cells, resulting in a significant decrease in the number of viable bacteria during storage and a short product shelf life. At the same time, plant extracts and functional strains are released and act on the rhizosphere environment simultaneously. Functional strains are forced to compete with native pathogens and the residual antibacterial effect of extracts in the unfavorable environment where the rhizosphere has not yet been cleared, resulting in extremely low field colonization rates. Current technologies lack structured design methods to decouple and regulate the two processes of disease suppression and strain colonization in time and space, making it difficult for plant extracts and functional strains to form a temporal synergy, thus limiting the effectiveness of industrial applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that in the preparation and application of existing agricultural microbial fertilizers containing plant extracts, there is a compatibility-antagonism contradiction between the active ingredients of plant extracts and functional strains, which leads to a decrease in the number of viable bacteria during the storage period.

[0006] Meanwhile, existing products lack the ability to decouple and regulate the two processes of disease suppression and field clearing and strain colonization in time and space, resulting in low field colonization rate of functional strains and difficulty in achieving synergistic effects between plant extracts and functional strains.

[0007] To address the aforementioned technical problems, this invention provides a method for preparing agricultural microbial fertilizer containing plant extracts, comprising the steps of fermenting and culturing functional strains, and preparing granular fertilizer by combining the effective components of the plant extracts with the fermentation products of the functional strains. The method also includes the following steps:

[0008] The functional strains were subjected to gradient stress acclimatization to obtain qualified acclimatized strains. Specifically, this included determining the initial minimum inhibitory concentration of the functional strains against the effective components of the target plant extracts.

[0009] Based on the initial minimum inhibitory concentration, a concentration gradient sequence containing multiple subgenerations was designed, wherein the concentration of the effective component of the target plant extract in the culture medium of each subgeneration is lower than the measured minimum inhibitory concentration of the current strain and higher than the acclimatization concentration of the previous subgeneration.

[0010] The functional strains were inoculated into concentration gradient culture media for corresponding passages and cultured generation by generation.

[0011] After each preset number of subcultures is completed, the minimum inhibitory concentration of the current generation strain is determined and the tolerance index is calculated.

[0012] Once the tolerance index reaches a preset threshold, the strain is inoculated into a culture medium that does not contain the effective components of the target plant extract and continuously passaged to verify the stability of the tolerance trait and obtain the domesticated qualified strain.

[0013] The domesticated strains were inoculated into a fermenter for high-density fermentation and expansion culture to obtain a domesticated bacterial suspension.

[0014] Sodium alginate, modified starch, the domesticated bacterial suspension and prebiotics are mixed to prepare a core granulation slurry, so that sodium alginate and calcium ions in the core granulation slurry cross-link and solidify to form core particles that encapsulate the domesticated qualified bacterial strains and the prebiotics.

[0015] Low-methoxyl pectin was mixed with the effective components of the target plant extract that had been extracted and concentrated to prepare a shell coating slurry;

[0016] A fluidized bed bottom spray coating process is used to spray the outer shell coating slurry onto the surface of the fluidized suspended core particles and dry it into a film, forming core-shell bilayer particles with a degradation rate of the outer shell layer greater than that of the core layer.

[0017] The core-shell double-layer particles are dried, inspected, and packaged to obtain the agricultural microbial fertilizer.

[0018] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows:

[0019] 1. This invention enables functional strains to acquire specific tolerance to the effective components of target plant extracts through gradient stress-oriented domestication, fundamentally alleviating the compatibility-antagonism contradiction between functional strains and plant extracts, and significantly improving the survival rate of viable bacteria during storage.

[0020] 2. This invention constructs a core-shell bilayer particle structure with a degradation rate of the outer shell layer greater than that of the inner shell layer, thereby achieving temporal and spatial decoupling between the rapid release and clearing of plant extracts and the slow-release colonization of functional strains, creating a non-competitive colonization window for functional strains.

[0021] 3. The synergistic encapsulation of prebiotics and domesticated strains in the core layer of this invention provides a dedicated carbon source for functional strains, further enhancing their rhizosphere colonization advantage;

[0022] Furthermore, this invention establishes an overall parameter system that links fermentation, domestication, and fluidized bed bottom spray double-layer coating processes, which is beneficial for achieving batch stability of product quality under continuous industrial production conditions. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the overall preparation process of agricultural microbial fertilizer in an embodiment of the present invention.

[0024] Figure 2 This is a flowchart of the strain gradient stress acclimatization process in an embodiment of the present invention;

[0025] Figure 3 This is a schematic diagram of the differential release process of bilayer particles in an embodiment of the present invention. Detailed Implementation

[0026] The following is in conjunction with the appendix Figure 1-3 The specific embodiments of the present invention will be further described below. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0027] The technical solution of the present invention will be further described below with reference to a specific embodiment, which does not constitute a limitation on the scope of protection of the present invention.

[0028] In this embodiment, a biocontrol-functional Bacillus strain was selected as the acclimatization target, and matrine was selected as the effective component of the target plant extract. First, the preserved strain was inoculated into a conventional liquid culture medium and activated until the logarithmic growth phase. The initial minimum inhibitory concentration (MIC) of this strain against matrine was determined using the two-fold dilution method to be 800 mg / L of culture medium, denoted as [missing information]. .

[0029] by Design a concentration gradient sequence based on the baseline, with the initial sublethal concentration taken as... Seventy percent, or 560 mg per liter of culture medium, with a generational concentration escalation factor of 70%. Take 0.1 and determine the target concentration for each generation using the following formula:

[0030] ;

[0031] In the formula, Indicates the first Target concentration of matrine in subculture medium;

[0032] Indicates the initial sublethal concentration;

[0033] Indicates the intergenerational concentration increase coefficient;

[0034] The passage number is a positive integer. The strain is inoculated into the corresponding concentration gradient of the culture medium and cultured at 33 degrees Celsius and 180 rpm until the late logarithmic growth stage. Then it is transferred to the next concentration gradient of fresh culture medium and a total of 20 passages are completed.

[0035] After every 5 subcultures, the minimum inhibitory concentration (MIC) of the current generation strain was determined. And calculate the tolerance index using the following formula:

[0036] ;

[0037] In the formula, Indicates tolerance index;

[0038] Indicates The measured minimum inhibitory concentration of matrine against the domesticated strains;

[0039] This represents the initial minimum inhibitory concentration (MIC) of the strain before domestication, measured after 20 generations of domestication. Reaching 4.2 times the preset target threshold, the strain was passaged for 8 generations in conventional culture medium without matrine, and the minimum inhibitory concentration was re-determined. It remained above 4.0 times, and the tolerance trait did not regress. After reviewing the functional characteristics such as antagonistic spectrum and spore production ability, it was confirmed that the original biocontrol and growth-promoting functional characteristics of the domesticated strain remained unchanged, and it was determined to be a qualified domesticated strain.

[0040] The domesticated strains were inoculated into a fermenter, and high-density fermentation was carried out by controlling the appropriate carbon-nitrogen source ratio, dissolved oxygen, temperature, and pH. After fermentation, the cell concentration reached 2×10^9 cells per milliliter of culture medium. The domesticated bacterial suspension was obtained by centrifugation and concentration.

[0041] Sodium alginate (2% by mass), modified starch (3% by mass), the obtained domesticated bacteria suspension, and oligochitosan prebiotic (1% by mass) were mixed and stirred evenly to form a core granulation slurry. The slurry was then dropped into a calcium chloride crosslinking and curing liquid (2% by mass) to allow sodium alginate and calcium ions to undergo ionic crosslinking and curing, forming spherical core particles. After washing and drying, the core particle semi-finished product was obtained.

[0042] Low-methoxyl pectin was dissolved and prepared into a coating matrix slurry with a mass concentration of 3%. The extracted and concentrated matrine effective component was added to make the mass concentration of matrine effective component in the slurry reach 5%. After stirring evenly, the outer shell coating slurry was formed.

[0043] The core particles are fed into a fluidized bed bottom spray coating equipment. Under conditions of an inlet air temperature of 40 degrees Celsius, a spray rate of 15 grams per minute, an atomization pressure of 0.2 MPa, and a suitable fluidization air velocity, the outer shell coating slurry is sprayed from the bottom of the equipment upwards onto the surface of the fluidized suspended core particles, allowing them to adhere evenly and dry rapidly to form a film, creating a core-shell double-layer particle. During the spraying process, the inlet air temperature is controlled between 38 and 42 degrees Celsius, the spray rate is controlled between 13 and 17 grams per minute, and the atomization pressure is controlled within the range of 0.18 MPa to 0.22 MPa. For core particles with larger particle sizes, the spraying time is appropriately extended and the spray rate is reduced. For core particles with higher moisture content, the outer shell drying temperature is appropriately increased to ensure the stability of the core-shell structure and batch consistency.

[0044] The obtained core-shell double-layer particles are dried at low temperature until the moisture content meets the storage requirements. After passing the tests for viable bacteria count, shell thickness uniformity, and disintegration release performance, they are graded, screened, and packaged to obtain the finished agricultural microbial fertilizer of this embodiment.

[0045] The finished product obtained in this embodiment was compared with the control sample prepared by direct mixing and granulation according to the existing technology. After being stored at room temperature for 6 months, the viable bacteria retention rate of the finished product in this embodiment reached more than 85%, while the viable bacteria retention rate of the control sample was less than 40%.

[0046] The two samples were applied to potted soil respectively, and the number of rhizosphere functional strains colonized was detected after 30 days. The field colonization rate of the product in this embodiment reached more than 15% of the inoculum amount, while the field colonization rate of the control sample was less than 1% of the inoculum amount. The above results show that the product obtained by the preparation method of the present invention has significantly improved in terms of storage stability and field colonization effect compared with the existing technical solutions.

[0047] The above release process can also be described and the process parameters optimized using the following equation:

[0048] ;

[0049] In the formula, This indicates the time from when the microcapsule comes into contact with moisture until... The cumulative amount of active ingredients released at any given time;

[0050] This indicates the total amount of active ingredients loaded in the microcapsules;

[0051] That is The cumulative release score over time;

[0052] The release rate constant is related to the material structure and porosity, and is the pectin material in the outer shell layer. The value is greater than that of the core layer cross-linked framework material. This value ensures that the outer shell is released before the core;

[0053] Indicates the release time from contact with moisture;

[0054] The release index is used to characterize the type of release mechanism. It can be controlled by adjusting the degree of pectin methoxylation and the ratio of sodium alginate to modified starch, so that the outer shell and inner shell exhibit the expected differentiated release characteristics.

[0055] Those skilled in the art should understand that the strains, plant extracts, proportions of each component, and process parameters in the above embodiments are merely illustrative examples. Without departing from the technical concept of this invention, the use of other biocontrol or growth-promoting strains, other effective components of plant extracts, and other material proportions and process parameters consistent with the technical concept of this invention are all within the scope of protection of this invention.

[0056] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, any modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing agricultural microbial fertilizer containing plant extracts, comprising the steps of fermenting and culturing functional strains, and preparing granular fertilizer by combining the effective components of the plant extracts with the fermentation products of the functional strains, characterized in that, It also includes the following: Functional strains were subjected to gradient stress-directed acclimatization to obtain qualified acclimatized strains, including: The initial minimum inhibitory concentration of the functional strain against the effective components of the target plant extract was determined. Based on the initial minimum inhibitory concentration, a concentration gradient sequence containing multiple subgenerations was designed, wherein the concentration of the effective component of the target plant extract in the culture medium of each subgeneration is lower than the measured minimum inhibitory concentration of the current strain and higher than the acclimatization concentration of the previous subgeneration. The functional strains were inoculated into concentration gradient culture media for corresponding passages and cultured generation by generation. After each preset number of subcultures is completed, the minimum inhibitory concentration of the current generation strain is determined and the tolerance index is calculated. Once the tolerance index reaches a preset threshold, the strain is inoculated into a culture medium that does not contain the effective components of the target plant extract and continuously passaged to verify the stability of the tolerance trait and obtain the domesticated qualified strain. The domesticated strains were inoculated into a fermenter for high-density fermentation and expansion culture to obtain a domesticated bacterial suspension. Sodium alginate, modified starch, the domesticated bacterial suspension and prebiotics are mixed to prepare a core granulation slurry, so that sodium alginate and calcium ions in the core granulation slurry cross-link and solidify to form core particles that encapsulate the domesticated qualified bacterial strains and the prebiotics. Low-methoxyl pectin was mixed with the effective components of the target plant extract that had been extracted and concentrated to prepare a shell coating slurry; A fluidized bed bottom spray coating process is used to spray the outer shell coating slurry onto the surface of the fluidized suspended core particles and dry it into a film, forming core-shell bilayer particles with a degradation rate of the outer shell layer greater than that of the core layer. The core-shell double-layer particles are dried, inspected, and packaged to obtain the agricultural microbial fertilizer.

2. The method according to claim 1, characterized in that, The initial sublethal concentration corresponding to the initial minimum inhibitory concentration is 60% to 80% of the initial minimum inhibitory concentration, and the intergenerational concentration increment coefficient in the concentration gradient sequence ranges from 0.05 to 0.

15.

3. The method according to claim 1, characterized in that, The preset threshold is when the tolerance index reaches 3 to 5 times.

4. The method according to claim 1, characterized in that, The functional strain is a Bacillus strain, and the effective component of the target plant extract is matrine.

5. The method according to claim 1, characterized in that, The core particles are prepared by dripping the core granulation slurry into a cross-linking curing liquid containing calcium ions, so that sodium alginate and calcium ions undergo ionic cross-linking and curing to form spherical particles, which are then washed and dried to obtain the core particles.

6. The method according to claim 1, characterized in that, The prebiotic is oligochitosan.

7. The method according to claim 1, characterized in that, The temperature for the successive subculturing is 30 to 37 degrees Celsius.

8. The method according to claim 1, characterized in that, In the fluidized bed bottom spray coating process, by controlling the inlet air temperature to be 38°C to 42°C, the spray rate to be 13g to 17g per minute, and the atomization pressure to be 0.18MPa to 0.22MPa, the shell coating time and spray rate are determined according to the particle size of the core particles, and the shell drying temperature is determined according to the moisture content of the core particles.

9. An agricultural microbial fertilizer with a core-shell double-layer microcapsule structure, comprising a core layer and an outer shell layer, characterized in that: The core layer uses a sodium alginate and modified starch cross-linked network as a framework, and encapsulates domesticated qualified strains and prebiotics obtained through gradient stress directional domestication as described in claim 1. The outer shell layer covers the outer surface of the core layer, uses low-methoxyl pectin as a matrix, and is loaded with effective components of plant extracts; the degradation rate of the outer shell layer is greater than that of the core layer.

10. The agricultural microbial fertilizer according to claim 9, characterized in that, The prebiotic is oligochitosan, and both the core layer and the core-shell bilayer microcapsule structure are spherical particles.

Images