A double-layered, double-strain microcapsule bacterial agent, its preparation method and its application

Abstract

This invention belongs to the field of microbial preparation technology, specifically relating to a double-layered microencapsulated bacterial agent, its preparation method, and its application. The preparation method of the double-layered microencapsulated bacterial agent includes the following steps: using a sodium alginate and xanthan gum composite solution as the inner matrix, a sodium alginate-xanthan gum monolayer microencapsulated bacterial agent is prepared using an emulsification-internal gelation method and an extrusion method; then, using the sodium alginate-xanthan gum monolayer microencapsulated bacterial agent, bacterial strain fermentation broth, sodium alginate solution, and calcium chloride solution as solidifying agents, a particle gelation method is used to prepare the double-layered microencapsulated bacterial agent. The double-layered microencapsulated bacterial agent prepared by the method provided by this invention achieves physical isolation and functional synergy between the two bacterial strains and possesses the characteristics of "rapid release in the outer layer and slow release in the inner layer," overcoming the problems of single bacterial agents having limited function, easy inactivation, and poor synergy in mixed bacterial agents.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, specifically relating to a double-layered, double-strain microcapsule bacterial agent, its preparation method, and its application. Background Technology

[0002] Microbial inoculants play a vital role in agriculture, environmental protection, and bioremediation. However, during storage and application, many microbial inoculants experience a sharp decline in viable bacterial counts due to environmental factors, making it difficult for them to establish stable colonies in the rhizosphere and severely impacting their efficacy. Traditional protection strategies (such as adding stress-resistant agents) often fail to simultaneously meet the dual requirements of physical barrier function and sustained-release release.

[0003] Microencapsulation technology offers a new approach to overcoming this bottleneck. By encapsulating microorganisms in a polymer matrix (such as sodium alginate-chitosan), environmental resistance can be significantly improved. For example, current microencapsulation technologies for microbial agents mainly involve encapsulating microorganisms in a single layer of polymer, such as sodium alginate-chitosan, and forming microcapsules through peremulsification or ionic cross-linking to provide a basic physical barrier. However, this results in a low viable cell count and limited environmental resistance. Some microencapsulation technologies also utilize composite wall materials for protection, such as starch / modified guar gum + protectants, and enhance stability through gelatinization or adsorption processes. However, these technologies are only suitable for synergistic strains without competitive relationships and are difficult to integrate with rhizosphere growth-promoting bacteria that exhibit antagonistic effects.

[0004] Current microencapsulation technologies for agricultural microbial agents cannot simultaneously satisfy the contradiction between high viable cell counts and long-term storage stability, nor can they effectively encapsulate competing microbial species, thus limiting practical application effectiveness. Therefore, developing a microencapsulation method that can achieve both high viable cell counts and long-term storage stability while effectively encapsulating competing microbial species to meet the application needs of complex rhizosphere environments is crucial for the sustainable development of the agricultural microbial preparation industry. Summary of the Invention

[0005] To address the contradiction between high viable cell count and long-term storage stability in existing agricultural microencapsulated bacterial agent preparation technologies, and the inability to effectively encapsulate competing bacterial strains, thus limiting practical application effectiveness, this invention provides a double-layer, double-strain microencapsulated bacterial agent, its preparation method, and its application. To achieve the above objectives, this invention adopts the following technical solution.

[0006] The first objective of this invention is to provide a method for preparing a double-layered, double-strain microcapsule bacterial agent, comprising the following steps:

[0007] Using a 1%~3% (w / v) sodium alginate and xanthan gum composite solution as the inner matrix, the fermentation broth of strain one is mixed with the sodium alginate and xanthan gum composite solution, calcium carbonate is added, and then a vegetable oil phase containing an emulsifier is added and mixed to form an emulsion; wherein, the mass ratio of the calcium carbonate to the volume ratio of the vegetable oil phase containing the emulsifier is 0.021g:50mL~100mL.

[0008] Glacial acetic acid was added to the emulsion to dissolve and break the emulsion of calcium carbonate, and sodium alginate-xanthan gum monolayer microcapsule bacterial agent was prepared by endogenous emulsification.

[0009] The sodium alginate-xanthan gum monolayer microcapsule bacterial agent was mixed with the fermentation broth of strain 2 and a sodium alginate solution with a mass-to-volume ratio of 3%~4% to obtain a composite solution. The composite solution was then dropped into a calcium chloride solution with a mass-to-volume ratio of 1.5% to undergo a cross-linking and solidification reaction. The two layers were bonded together by electrostatic interaction to form a three-dimensional network structure for the preparation of the double-layer double-bacterial microcapsule bacterial agent. The double-layer double-bacterial microcapsule bacterial agent was then obtained by filtration.

[0010] Among them, strain one and strain two are different species of strains.

[0011] The volume ratios of the fermentation broth of strain one, the sodium alginate and xanthan gum composite solution, the vegetable oil phase containing emulsifier, and the glacial acetic acid are 20 mL: 10 mL~20 mL: 50 mL~100 mL: 220 μL~440 μL, respectively.

[0012] The volume ratio of the sodium alginate-xanthan gum monolayer microcapsule bacterial agent, the fermentation broth of the second strain, the sodium alginate solution, and the calcium chloride solution is 20 mL: 10 mL~20 mL: 20 mL~40 mL: 50 mL~100 mL, respectively.

[0013] The present invention provides a method for preparing a double-layered, double-strain microcapsule bacterial agent. First, a sodium alginate and xanthan gum composite solution is used as the inner matrix, with strain one as the core strain. An emulsification-internal gelation method and an extrusion method are used to prepare a sodium alginate-xanthan gum monolayer microcapsule bacterial agent. Then, using the sodium alginate-xanthan gum monolayer microcapsule bacterial agent, the fermentation broth of strain two, sodium alginate solution, and calcium chloride solution as curing agents, a cross-linking and curing reaction occurs. The mixture is then filtered to obtain the double-layered, double-strain microcapsule bacterial agent. Strain one and strain two are different species of bacterial strains. Through the cooperation of the above steps, a two-step encapsulation process is achieved to prepare the double-layered, double-strain microcapsule bacterial agent, and to achieve physical isolation and functional synergy between the two different species of bacterial strains, exhibiting the characteristics of "rapid release from the outer layer and sustained release from the inner layer." This double-layer, double-strain microcapsule preparation method significantly improves the bacterial load and protective effect of functional bacteria in microcapsule preparations. It can solve the contradiction between high viable bacterial count and long-term storage stability that existing agricultural microcapsule preparation technologies cannot simultaneously meet. It can also achieve orderly release of mixed bacterial agents, prolong the overall action time of the agents, and significantly enhance the application effect, providing an innovative solution for the industrial production and field application of agricultural microbial preparations.

[0014] Preferably, in the sodium alginate and xanthan gum composite solution, the mass ratio of sodium alginate to xanthan gum is 9 to 1:1.

[0015] Preferably, the emulsifier-containing vegetable oil phase is sunflower seed oil containing Span80, and the mass ratio of Span80 to the volume of the sunflower seed oil is 0.5% to 1% (m / v).

[0016] Preferably, the conditions for mixing the vegetable oil containing emulsifier are: rotation speed 400 rpm to 600 rpm and time 15 min to 30 min.

[0017] Preferably, the method for preparing sodium alginate-xanthan gum monolayer microcapsule bacterial agent by endogenous emulsification includes the following steps:

[0018] Glacial acetic acid was added to the emulsion to carry out a curing reaction; after the curing reaction was completed, acetic acid buffer was added, and then centrifuged to obtain the sodium alginate-xanthan gum monolayer microcapsule bacterial agent.

[0019] Preferably, the centrifugation conditions are: rotation speed 5990 rpm to 6010 rpm and time 8 min to 10 min.

[0020] Preferably, the fermentation broth of strain one is the fermentation broth of Bacillus motility CS-06 or the fermentation broth of Bacillus atrophicus CT-05; the fermentation broth of strain two is the fermentation broth of Bacillus motility CS-06 or the fermentation broth of Bacillus atrophicus CT-05.

[0021] The OD of the fermentation broth of Bacillus CS-06 600 =2.5~3.0; OD of the fermentation broth of the Bacillus atrophus CT-05 600 =2.0~2.5.

[0022] A second objective of this invention is to provide a double-layered, double-bacterial microcapsule bacterial agent prepared by the aforementioned preparation method.

[0023] Preferably, the bacterial load of the double-layered, double-strain microcapsule agent is 1×10⁻⁶. 9 cfu / g ~1.48×10 9 cfu / g. After storage for 58-62 days, the viable count of Bacillus atrophus CS-06 in the double-layered microcapsule bacterial agent is 1×10⁻⁶. 7 cfu / g ~5.04×10 7 cfu / g, containing 1×10⁻⁶ viable bacteria of Bacillus motility CT-05. 7 cfu / g ~8.04×10 7 cfu / g.

[0024] A third objective of this invention is to provide the application of the double-layered microencapsulated bacterial agent in crop seed germination and seedling growth, wherein the double-layered microencapsulated bacterial agent is directly applied to the crop.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This invention provides a method for preparing a double-layered, double-strain microcapsule bacterial agent. The method first uses a sodium alginate and xanthan gum composite solution as the inner matrix, with strain one as the core strain, and prepares a sodium alginate-xanthan gum monolayer microcapsule bacterial agent using an emulsification-internal gelation method and an extrusion method. Then, using the sodium alginate-xanthan gum monolayer microcapsule bacterial agent, the fermentation broth of strain two, sodium alginate solution, and calcium chloride solution as solidifying agents, a cross-linking solidification reaction occurs, followed by filtration to obtain the double-layered, double-strain microcapsule bacterial agent. The fermentation broth of strain one and the fermentation broth of strain two are from different strains. Through the cooperation of the above steps, a two-step encapsulation preparation of the double-layered, double-strain microcapsule bacterial agent is achieved, as well as physical isolation and functional synergy between the two different strains, exhibiting the characteristics of "rapid release in the outer layer and slow release in the inner layer." This double-layer, double-strain microcapsule preparation method significantly improves the bacterial load and protective effect of functional bacteria in microcapsule preparations. It can solve the contradiction between high viable bacterial count and long-term storage stability that existing agricultural microcapsule preparation technologies cannot simultaneously meet. It can also achieve orderly release of mixed bacterial agents, prolong the overall action time of the agents, and significantly enhance the application effect, providing an innovative solution for the industrial production and field application of agricultural microbial preparations.

[0027] 2. The present invention provides a double-layer double-strain microcapsule bacterial agent. The two-step layer encapsulation improves the encapsulation efficiency of active bacteria and the stability of the inner strain, successfully achieving high-density encapsulation of microorganisms and providing stable activity assurance for subsequent applications.

[0028] 3. This invention provides a method for preparing a double-layered, double-strain microcapsule bacterial agent. This layered encapsulation design not only effectively reduces direct physical competition between the two strains but also achieves synergistic functional effects between the two strains.

[0029] 4. This invention involves mixing the fermentation broth of a bacterial strain with a composite solution of sodium alginate and xanthan gum, then adding calcium carbonate and sunflower seed oil containing Span 80, followed by stirring. Glacial acetic acid is then added to dissolve and demulsify the calcium carbonate, preparing single-layer microcapsules through endogenous emulsification. These single-layer microcapsules are then mixed with the fermentation broth of another bacterial strain and a sodium alginate solution, and calcium chloride solution is added dropwise. Through electrostatic interaction, they combine to form a three-dimensional network structure ("egg carton model"), thus preparing a double-layer dual-strain microcapsule agent. The product is then processed through filtration and freeze-drying. This invention's preparation method achieves stable survival and synergistic function of multiple bacterial strains, overcoming the shortcomings of existing technologies.

[0030] 5. The double-layer, double-strain microcapsule bacterial agent design in this invention enables a layered release pattern: the outer layer rapidly releases and colonizes to form a protective barrier, while the inner layer provides sustained supply. This characteristic effectively delays the release of the inner layer bacteria and provides an important means to control the encapsulation order, release order, and efficacy of the mixed bacterial agent according to application requirements. It is particularly suitable for encapsulating bacteria with low viable counts and rapid mortality rates in the inner layer, and encapsulating fast-growing, high-residue strains in the outer layer, further extending the overall action time of the bacterial agent and thus improving the application effect and stability of the mixed bacterial agent.

[0031] 6. After 58-62 days of storage, the viable bacterial count in both the outer and inner layers of the double-layered microcapsule agent showed a slow decreasing trend. The viable bacterial count of both types remained at 6.06 × 10⁻⁶. 6 CFU / g or higher. Attached Figure Description

[0032] Figure 1 These are images showing the surface morphology of sodium alginate-xanthan gum monolayer microcapsule bacterial agent and bilayer microcapsules encapsulating methylene blue simulated bacterial solution of strain CT-05 under an optical microscope, as described in this invention. Figure 1 In Figure 'a', the surface morphology of the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05 prepared in Example 1 is shown. Figure 1 In Figure b, the surface morphology of the bilayer microcapsules containing methylene blue simulated bacterial solution prepared in Comparative Example 2 is shown.

[0033] Figure 2The images shown are scanning electron microscope (SEM) images of the CT-05 strain sodium alginate-xanthan gum monolayer microcapsule bacterial agent and the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent (inner CT-05 outer CS-06) after lyophilization in this invention; wherein, Figure 2 In the image, 'a' is a scanning electron microscope image of the sodium alginate monolayer microcapsules prepared in Comparative Example 1 after lyophilization. Figure 2 b in the figure is a scanning electron microscope image of the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent prepared in Example 1 after lyophilization of the inner CT-05 and outer CS-06 layers.

[0034] Figure 3 This is a cross-sectional scanning electron microscope image of the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent after lyophilization, consisting of an inner CT-05 layer and an outer CS-06 layer, in Example 1 of this invention; wherein, Figure 3 In the image, 'a' is a cross-sectional scanning electron microscope image of the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent after lyophilization, consisting of an inner CT-05 layer and an outer CS-06 layer. Figure 3 b in the image is a high-magnification scanning electron microscope image of the cross-section of the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent after lyophilization, with inner layer CT-05 and outer layer CS-06.

[0035] Figure 4 This is a bar chart showing the encapsulation amount of the double-layer double-strain microcapsule bacterial agent in this invention; wherein, "CT-05" refers to the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05; "CS-06" refers to the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CS-06; "CT-05+CS-06" refers to the CT-05+CS-06 double-layer double-strain microcapsule bacterial agent with inner layer CT-05 and outer layer CS-06; "CS-06+CT-05" refers to the CS-06+CT-05 double-layer double-strain microcapsule bacterial agent with inner layer CS-06 and outer layer CT-05.

[0036] Figure 5 This is a release curve diagram of the double-layered, double-strain microcapsule bacterial agent of the present invention; wherein, Figure 5 In the figure, 'a' represents the release curve of the CS-06+CT-05 double-layer double-bacterial microcapsule bacterial agent prepared in Comparative Example 3, which has an inner layer of CS-06 and an outer layer of CT-05. Figure 5 In the figure, b is the release curve of the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent prepared in Example 1, with inner layer CT-05 and outer layer CS-06.

[0037] Figure 6 This is a storage performance test curve of the double-layered, double-strain microcapsule bacterial agent of the present invention; wherein, Figure 6 In the figure, 'a' represents the storage performance test curve of the CS-06+CT-05 double-layer double-bacterial microcapsule bacterial agent with inner layer CS-06 and outer layer CT-05 prepared in Comparative Example 3. Figure 6In the figure, b is the storage performance test curve of the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent with inner layer CT-05 and outer layer CS-06 prepared in Example 1.

[0038] Figure 7 This is a graph showing the effect of sodium alginate-xanthan gum monolayer microcapsule bacterial agent and double-layer dual-strain microcapsule bacterial agent on watermelon seed germination in this invention; wherein, "CT-05" refers to the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05; "CS-06" refers to the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CS-06; "CT-05+CS-06" refers to the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent with inner layer CT-05 and outer layer CS-06; "CS-06+CT-05" refers to the CS-06+CT-05 double-layer dual-strain microcapsule bacterial agent with inner layer CS-06 and outer layer CT-05.

[0039] Figure 8 This is a bar chart showing the effects of sodium alginate-xanthan gum monolayer microcapsule bacterial agent and double-layer dual-strain microcapsule bacterial agent on the growth of watermelon seedlings in this invention; wherein, "CT-05" refers to the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05; "CS-06" refers to the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CS-06; "CT-05+CS-06" refers to the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent with inner layer CT-05 and outer layer CS-06; "CS-06+CT-05" refers to the CS-06+CT-05 double-layer dual-strain microcapsule bacterial agent with inner layer CS-06 and outer layer CT-05. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0041] Example 1

[0042] A method for preparing a double-layered, double-strain microcapsule bacterial agent includes the following steps:

[0043] S1. Mix 10 mL of Bacillus atrophicus CT-05 fermentation broth with 20 mL of a 3.0% (w / v) sodium alginate and xanthan gum composite solution (sodium alginate: xanthan gum = 6:4). Then add 0.021 g of CaCO3 (Ca / SA mass ratio of 7% w / w), and homogenize thoroughly to obtain an aqueous phase mixture, which is used as the aqueous phase. Slowly add the above aqueous phase mixture to 50 mL of sunflower seed oil containing 0.5% (w / v) Span 80, and emulsify with magnetic stirring at 400 rpm for 15 min to form a stable oil-in-water (w / o) emulsion.

[0044] The preparation method of the fermentation broth of Bacillus atrophicus CT-05 is as follows: A single colony of Bacillus atrophicus CT-05 strain is picked using an inoculation loop and transferred to LB liquid medium for culture (28℃, 180 rpm). After the logarithmic phase, 1% (v / v) of the bacterial solution is added to fresh LB liquid medium and cultured at a constant temperature of 28℃ on a shaker at 180 rpm / min for 36 hours until the stationary phase is reached, thus obtaining the fermentation broth of Bacillus atrophicus CT-05. The CT-05 strain refers to Bacillus atrophicus CT-05, which was obtained by the team from the tomato rhizosphere soil in the previous stage. Bacillus atrophicus (… Bacillus atrophaeus CT-05 was deposited with the China General Microbiological Culture Collection Center on July 10, 2025, with accession number CGMCC NO.35173.

[0045] The method for preparing the sodium alginate and xanthan gum composite solution is as follows: Dissolve 1.8g of sodium alginate and 1.2g of xanthan gum in 100mL of pure water.

[0046] S2. Subsequently, 220 μL of glacial acetic acid was added dropwise to the oil-in-water (w / o) emulsion, and stirring was continued for 20 min to ensure that the calcium carbonate was fully dissolved and to trigger the internal gelation reaction, forming microcapsule beads of the bacterial strain. After the reaction was completed, a pH 5.5 acetate buffer was added for precise control to complete the dissolution of all calcium carbonate particles in the emulsion and the formation of all microcapsules. After the oil and water phases separated naturally and the microspheres precipitated, the upper oil phase was removed by suction. The lower aqueous phase was centrifuged (6000 rpm, 8 min), and the supernatant was discarded to obtain sodium alginate-xanthan gum monolayer microcapsule bacterial agent.

[0047] To ensure product purity, the obtained sodium alginate-xanthan gum monolayer microcapsule bacterial agent was washed twice with an equal volume of 0.9% (w / v) physiological saline to remove residual oil phase. The sodium alginate-xanthan gum monolayer microcapsule bacterial agent product was obtained and stored in physiological saline at 4°C for later use.

[0048] Among them, the sodium alginate-xanthan gum monolayer microcapsule bacterial agent is the CT-05 strain sodium alginate-xanthan gum monolayer microcapsule bacterial agent.

[0049] S3. Mix the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05 with the fermentation broth of Bacillus CS-06 at a volume ratio of 1:1. After mixing thoroughly on a magnetic stirrer, add the above mixture to a 4% (w / v) sodium alginate solution at a volume ratio of 1:1 and continue stirring until completely homogeneous to obtain a composite solution.

[0050] The preparation method of the fermentation broth of *Bacillus aggregatibacterium* CS-06 is as follows: A single colony of *Bacillus aggregatibacterium* CS-06 strain is picked with an inoculation loop and transferred to LB liquid medium for culture (28℃, 180 rpm). After the logarithmic phase, 1% (v / v) of the bacterial solution is added to fresh LB liquid medium and cultured on a shaker at 180 rpm / min for 36 hours under constant temperature conditions of 28℃ until the stationary phase is reached, thus obtaining the fermentation broth of *Bacillus aggregatibacterium* CS-06. The CS-06 strain refers to *Bacillus aggregatibacterium* CS-06, which was obtained by the team from the rhizosphere soil of tomatoes in the previous stage. *Bacillus aggregatibacterium* (… Bacillus mobilis CS-06 was deposited with the China General Microbiological Culture Collection Center on July 10, 2025, with accession number CGMCC No. 35174.

[0051] S4. A peristaltic pump was used to prepare bilayer microcapsules. The outlet pressure of the peristaltic pump was controlled at 0.1 MPa, and the composite solution was added dropwise to a 1.5% (w / v) CaCl2 solution (while maintaining slow stirring). After the addition was complete, cross-linking and curing continued at room temperature for 30 min. The obtained bilayer microcapsule agent was collected by filtration, pre-cooled overnight at -20°C, and then freeze-dried in a freeze dryer until completely freeze-dried, yielding the CT-05+CS-06 bilayer microcapsule agent with an inner layer of CT-05 and an outer layer of CS-06, denoted as CT-05+CS-06.

[0052] Comparative Example 1

[0053] This comparative example describes the preparation method of Bacillus atrophus CT-05 sodium alginate monolayer microcapsules. The difference between this method and S1 and S2 in Example 1 is that the sodium alginate and xanthan gum composite solution in step S1 is replaced with a sodium alginate solution.

[0054] Comparative Example 2

[0055] This comparative example describes the preparation method of double-layer microcapsules loaded with methylene blue simulated bacterial solution, which is the same as in Example 1, except that the fermentation broth of Bacillus atrophus CT-05 in step S1 is replaced with an equal amount of methylene blue solution with a concentration of 1 mol / L.

[0056] Comparative Example 3

[0057] The preparation of a double-layered double-strain microcapsule bacterial agent is the same as the preparation steps in Example 1, except that the CT-05 bacterial solution in step S1 is replaced with the CS-06 bacterial solution, and the CS-06 bacterial solution in step S3 is replaced with the CT-05, thus preparing the CS-06+CT-05 double-layered double-strain microcapsule bacterial agent with an inner layer of CS-06 and an outer layer of CT-05, denoted as CS-06+CT-05.

[0058] Comparative Example 4

[0059] This comparative example is a sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05, prepared by the same method as S1 and S2 in Example 1.

[0060] Comparative Example 5

[0061] This comparative example is a sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CS-06. The preparation method is the same as S1 and S2 of Example 1, except that in step S1, the bacterial solution is replaced with an equal amount of Bacillus motility CS-06 fermentation broth.

[0062] To illustrate the effectiveness of the preparation method of the double-layered double-strain microcapsule bacterial agent provided by the present invention, the following studies were conducted:

[0063] 1. Using optical microscopy, the morphology of the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05 prepared in Comparative Example 1 and the bilayer microcapsule loaded with methylene blue simulated bacterial solution prepared in Comparative Example 2 were characterized. The results are as follows: Figure 1 As shown.

[0064] Figure 1 Figure 'a' in Example 1 shows the effect of the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05. The results show that the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05 has a smooth surface, an intact capsule wall structure, and no rupture or aggregation was found. Figure 1 In Figure b, the bilayer microcapsules containing methylene blue simulated bacterial solution are characterized under the condition of staining with methylene blue simulated bacterial solution. Observation under an optical microscope revealed that the opaque part in the middle of the bilayer microcapsules containing methylene blue simulated bacterial solution is an embedded single-layer microcapsule. This result fully confirms the successful construction of the bilayer structure.

[0065] 2. Scanning electron microscopy was used to characterize the CT-05+CS-06 double-layer microcapsule bacterial agent (inner CT-05, outer CS-06) prepared in Example 1 and the sodium alginate single-layer microcapsule prepared in Comparative Example 1. The results are as follows: Figure 2As shown.

[0066] Figure 2 Figure 'a' in the diagram is an effect image of sodium alginate monolayer microcapsules. The results show that the sodium alginate monolayer microcapsules have a spherical structure with an average particle size of about 350 μm, and their surface is smooth and intact. Figure 2 Figure b is an effect diagram of the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent with inner CT-05 and outer CS-06. The results show that the CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent with inner CT-05 and outer CS-06 also has a spherical structure with a particle size range of 800μm~900μm. Its surface is generally intact, but the surface is relatively rough after freeze-drying and many protrusions appear.

[0067] 3. The CT-05+CS-06 double-layer double-bacterial microcapsule bacterial agent with inner CT-05 and outer CS-06 prepared in Example 1 was dissected, and the results are as follows: Figure 3 As shown.

[0068] Figure 3 The 'a' in the figure indicates that the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent with an inner CT-05 layer and an outer CS-06 layer exhibits a typical cheese-like porous structure. This suggests that the single-layer microcapsules embedded within the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent have small particle sizes and are uniformly distributed. This structural feature provides favorable conditions for the stable encapsulation and controlled release of microorganisms. Figure 3 Image b, using high-magnification imagery, clearly demonstrates the adhesion of bacteria within the concave structure. This indicates that the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CT-05 was successfully encapsulated in a double-layer dual-strain microcapsule bacterial agent.

[0069] 4. Under aseptic conditions, 1g of each of the following microcapsule preparations were accurately weighed: the CT-05+CS-06 double-layer microcapsule preparation with an inner CT-05 layer and an outer CS-06 layer (prepared in Example 1); the sodium alginate-xanthan gum monolayer microcapsule preparation of the CT-05 strain prepared in Comparative Example 4; the sodium alginate-xanthan gum monolayer microcapsule preparation of the CS-06 strain prepared in Comparative Example 5; and the CS-06+CT-05 double-layer microcapsule preparation with an inner CS-06 layer and an outer CT-05 layer (prepared in Comparative Example 3). Each microcapsule was placed in 50mL of phosphate buffer (pH 7.8, 0.1M) and continuously shaken until the microcapsules were completely broken and the encapsulated bacteria were fully released. The number of viable bacteria encapsulated in the microcapsules was then determined using a gradient dilution method. The results are shown below. Figure 4 .

[0070] The encapsulation amounts of sodium alginate-xanthan gum monolayer microcapsules for strain CT-05 and strain CS-06 were 1.8 × 10⁻⁶. 9cfu / g and 6.7×10 9 The higher encapsulation amount of the sodium alginate-xanthan gum monolayer microcapsule bacterial agent of strain CS-06 is due to the faster reproduction rate and higher bacterial density of Bacillus motility CS-06. Therefore, under the same encapsulation volume, the encapsulation amount of Bacillus motility CS-06 is higher than that of Bacillus atrophicus CS-06. The bacterial cell encapsulation amount of the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent with inner CT-05 and outer CS-06 is 7.16×10⁻⁶. 8 cfu / g and 3.5×10 9 The bacterial cell encapsulation amount of the CS-06+CT-05 double-layer dual-strain microcapsule bacterial agent, with an inner layer of CS-06 and an outer layer of CT-05, was 3.13 × 10⁻⁶ cfu / g. 8 cfu / g and 1.17×10 9 CFU / g, all different construction methods maintained a high bacterial encapsulation amount, but the method with inner layer CT-05 and outer layer CS-06 had a higher total bacterial load.

[0071] 5. The controlled-release performance of the CS-06+CT-05 double-layer dual-strain microcapsule bacterial agent with inner layer CS-06 and outer layer CT-05 prepared in Example 1 and the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent with inner layer CT-05 and outer layer CS-06 prepared in Comparative Example 3 is shown in the figure. Figure 5 .

[0072] Figure 5 In the figure, 'a' represents the release process of the CT-05+CS-06 double-layer dual-strain microcapsule bacterial agent, consisting of an inner CT-05 layer and an outer CS-06 layer. This process exhibits a significant graded release characteristic: the outer layer of *Bacillus motilityis* CS-06 is released initially at a slightly faster rate, while the inner layer of *Bacillus atrophicus* CS-06 is released continuously at a slower rate. Throughout the process, the release rate of *Bacillus motilityis* CS-06 is consistently faster than that of *Bacillus atrophicus* CS-06, reaching its maximum release value on day 5. Finally, on day 10, the viable count of *Bacillus motilityis* CS-06 released was 1.80 × 10⁻⁶. 9 CFU / g; while the inner layer of Bacillus atrophicus CS-06 reached equilibrium on day 8, and the viable count of Bacillus atrophicus CS-06 released on day 10 was 6.81 × 10⁻⁶. 8 cfu / g.

[0073] Figure 5Figure 'b' shows the release curve of the CT-05+CS-06 double-layer microcapsule bacterial agent, consisting of an inner CT-05 layer and an outer CS-06 layer. It also exhibits stratified characteristics, but the release rate changes differently: the outer Bacillus atrophicus CS-06 layer shows a faster release rate initially, which decreases significantly after day 4 and tends to reach equilibrium. Finally, on day 10, the viable count of Bacillus atrophicus CS-06 is 5.41 × 10⁻⁶. 8 cfu / g; while the release rate of Bacillus motility CS-06 from the inner layer slowed down, reaching equilibrium only on the 9th day, and finally the viable count of Bacillus motility CS-06 on the 10th day was 3.33 × 10⁻⁶. 9 cfu / g.

[0074] 6. The storage performance tests of the CT-05+CS-06 double-layer microcapsule bacterial agent with inner CT-05 and outer CS-06 prepared in Example 1 and the CT-05+CS-06 double-layer microcapsule bacterial agent with inner CT-05 and outer CS-06 prepared in Comparative Example 3 are shown in the figure. Figure 6 .

[0075] Figure 6 The storage results of the CT-05+CS-06 double-layer microcapsule bacterial agent (with an inner CT-05 layer and an outer CS-06 layer) showed that with increasing storage time, some of the Bacillus motility CS-06 cells in the outer layer became inactive, while the number of Bacillus atrophicus CS-06 cells in the inner layer changed less, showing a very slow decreasing trend and a smaller inactivation amount, demonstrating the advantage of double-layer encapsulation. After 60 days, the viable counts of Bacillus atrophicus CS-06 and Bacillus motility CS-06 were 5.04 × 10⁻⁶. 7 cfu / g and 8.43×10 7 cfu / g.

[0076] Figure 6 In the figure, 'b' represents the storage activity of the CT-05+CS-06 double-layer microcapsule bacterial agent with an inner CT-05 layer and an outer CS-06 layer. The viable cell counts of both the outer and inner layers showed a slow decreasing trend, but the loss of Bacillus motility CS-06 cells in the inner layer was relatively small. After 60 days, the viable cell counts of Bacillus atrophus CS-06 and Bacillus motility CS-06 were 6.06 × 10⁻⁶. 6 cfu / g and 8.05×10 7 cfu / g. This result indicates that the outer coating of the storage-resistant Bacillus motility strain CS-06 is more conducive to maintaining the total viable count.

[0077] Application Examples

[0078] Taking the sodium alginate-xanthan gum monolayer microcapsule inoculant of strain CT-05 (Comparative Example 4), the sodium alginate-xanthan gum monolayer microcapsule inoculant of strain CS-06 (Comparative Example 5), the CT-05+CS-06 double-layer microcapsule inoculant of inner CT-05 and outer CS-06 (Example 1), and the CS-06+CT-05 double-layer microcapsule inoculant of inner CS-06 and outer CT-05 (Comparative Example 3) as examples, the application effects on watermelon seed germination and seedling growth were tested. The specific application methods and results are as follows:

[0079] The flowerpots used in the experiment were thoroughly washed with clean water, then surface-sterilized with a 75% (v / v) ethanol solution, and allowed to air dry before use. Test soil was weighed and evenly spread in the flowerpots, and 10 sowing holes with a depth of 2 cm were pressed into the pots. According to the experimental design, 0.1 g of sodium alginate-xanthan gum monolayer microcapsule inoculant of strain CT-05 (Comparative Example 4), sodium alginate-xanthan gum monolayer microcapsule inoculant of strain CS-06 (Comparative Example 5), or 0.15 g of CT-05+CS-06 double-layer microcapsule inoculant of inner CT-05 and outer CS-06 (Comparative Example 1), and CS-06+CT-05 double-layer microcapsule inoculant of inner CS-06 and outer CT-05 (Comparative Example 3) were accurately weighed into each sowing hole as treatment groups. Untreated bare seeds were set up as a control group. Select uniformly sized watermelon seeds with intact seed coats (Zaojia 8424 watermelon, purchased from Yikou Seed Company), gently place them on the surface of the microcapsules, and press them down moderately to fix their position. Cover the seeds with soil until they are completely buried. Water with sterile water to keep the soil surface moist. Place the treated pots in a constant temperature and light incubator (30℃ for 14 hours of light, 15℃ for 10 hours of darkness, 60% humidity) for cultivation. Observe and record the germination status of each group of seeds daily, calculate the germination rate, and record until day 7. Each experiment was repeated three times. The germination rate was calculated using the following formula:

[0080] Germination rate = (Total number of germinated seeds / Number of seeds tested) × 100%;

[0081] In the above formula, the unit of germination rate is %.

[0082] Plant height is the result of vine length 30 days after watermelon sowing, and is measured directly with a ruler.

[0083] See results Figure 7Initially, the germination rate was as follows: control group (CK) > double-layer microcapsules > single-layer microcapsules. Compared with the control group, the microcapsule treatment group had a slower germination rate, but still maintained a high germination rate. This phenomenon may be attributed to the fact that the microcapsules occupy part of the soil space below the seed, preventing the seed from being completely enveloped by the soil, thus delaying the formation of a moist environment around the seed and slowing down the germination rate. However, after day 3 of the experiment, the germination rate of the microcapsule-treated groups increased significantly, reaching a high level on day 7. The germination rates of both the CT-05 and CS-06 strains treated with sodium alginate-xanthan gum single-layer microcapsules reached 90.00%. The germination rate of the CS-06+CT-05 double-layer microcapsule treatment group (with CS-06 inside and CT-05 outside) reached 99.97%, and the CT-05+CS-06 double-layer microcapsule treatment group (with CT-05 inside and CS-06 outside) reached 90.00%. This indicates that although microcapsules initially delay germination, they ultimately have no significant inhibitory effect on seed germination. The plant height of the single-layer microcapsule treatment group was significantly better than the control group on day 7, while the germination rate of the double-layer microcapsule treatment group was slower, but its post-germination growth rate was better than other groups. This result may be closely related to the sustained-release properties of the microcapsules: over time, the Bacillus motility strain CS-06 and Bacillus atrophicus strain CS-06 within the microcapsules are gradually released into the soil, continuously providing nutrients to the roots and thus promoting plant growth. Due to their stronger sustained-release properties, the double-layered microcapsules exhibit a more significant growth-promoting effect in the later stages.

[0084] The effects of sodium alginate-xanthan gum single-layer and double-layer microcapsule bacterial agents on watermelon seedling growth are shown in [reference]. Figure 8 The results showed that on day 3 after 7 days of germination, the control group seedlings grew the fastest, followed by the single-layer microcapsule group, while the double-layer microcapsule group grew the slowest. This may be due to the slow-release properties of the double-layer structure, resulting in less initial bacterial release. By day 5, the growth rate of the double-layer microcapsule group significantly increased, indicating that the outer layer of bacteria began to release and exert its effect, while the growth of the single-layer microcapsule group tended to stabilize. By day 7, the plant height of the double-layer microcapsule group significantly exceeded that of the single-layer microcapsule group and the control group. In contrast, the single-layer microcapsule group, due to the one-time release of bacteria, showed a weakened effect in the later stages. This result fully demonstrates the advantages of double-layer microcapsules in regulating the timing of bacterial release and enhancing growth-promoting effects.

[0085] In summary, due to its excellent sustained-release properties, the double-layered microencapsulated inoculant can continuously exhibit a stronger promoting effect during the post-germination growth stage, and has great potential as a long-lasting microbial inoculant for field use.

[0086] As can be seen from the above, the bacterial load of the double-layered, double-strain microcapsule bacterial agent provided by the present invention is 1.48 × 10⁻⁶.9 After 60 days of storage, the viable counts of Bacillus atrophus CS-06 and Bacillus motility CT-05 were 5.04 × 10⁻⁶ cfu / g. 7 cfu / g, 8.04×10 7 The cfu / g significantly improved the survival time and stability of functional bacteria; the application of double-layer double-strain microcapsule inoculant can significantly promote the germination of watermelon seeds and the growth of seedlings.

[0087] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0088] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A method for preparing a double-layered, double-strain microcapsule bacterial agent, characterized in that, Includes the following steps: Using a sodium alginate and xanthan gum composite solution as the inner matrix, the fermentation broth of strain one is mixed with the sodium alginate and xanthan gum composite solution, calcium carbonate is added, and then a vegetable oil phase containing an emulsifier is added and mixed to form an emulsion; wherein, the mass ratio of the calcium carbonate to the volume ratio of the vegetable oil phase containing the emulsifier is 0.021 g: 50 mL~100 mL; Glacial acetic acid was added to the emulsion to dissolve and break the emulsion of calcium carbonate, and sodium alginate-xanthan gum monolayer microcapsule bacterial agent was prepared by endogenous emulsification. The sodium alginate-xanthan gum monolayer microcapsule bacterial agent was mixed evenly with the fermentation broth of strain 2 and sodium alginate solution to obtain a composite solution; the composite solution was dripped into calcium chloride solution, and after cross-linking and solidification reaction, it was filtered to obtain the double-layer double-bacterial microcapsule bacterial agent; Wherein, strain one and strain two are different species of strains; strain one is Bacillus atrophus (… Bacillus atrophaeus CT-05; the second strain is Bacillus motilityis CS-06; The volume ratios of the fermentation broth of strain one, the sodium alginate and xanthan gum composite solution, the vegetable oil phase containing emulsifier, and the glacial acetic acid are 20 mL: 10 mL~20 mL: 50 mL~100 mL: 220 μL~440 μL, respectively. The volume ratio of the sodium alginate-xanthan gum monolayer microcapsule bacterial agent, the fermentation broth of the second strain, the sodium alginate solution, and the calcium chloride solution is 20 mL: 10 mL~20 mL: 20 mL~40 mL: 50 mL~100 mL, respectively.

2. The preparation method according to claim 1, characterized in that, In the sodium alginate and xanthan gum composite solution, the mass ratio of sodium alginate to xanthan gum is 9~1:

1.

3. The preparation method according to claim 1, characterized in that, The emulsifier-containing vegetable oil phase is sunflower seed oil containing Span80, and the mass ratio of Span80 to the volume of the sunflower seed oil is 0.5% to 1%.

4. The preparation method according to claim 1, characterized in that, The conditions for mixing the vegetable oil containing emulsifier are: rotation speed 400 rpm to 600 rpm and time 15 min to 30 min.

5. The preparation method according to claim 1, characterized in that, The method for preparing sodium alginate-xanthan gum monolayer microcapsule bacterial agent by endogenous emulsification includes the following steps: Glacial acetic acid was added to the emulsion to carry out a curing reaction; after the curing reaction was completed, acetic acid buffer was added, and then centrifuged to obtain the sodium alginate-xanthan gum monolayer microcapsule bacterial agent.

6. The preparation method according to claim 5, characterized in that, The centrifugation conditions are: rotation speed 5990 rpm to 6010 rpm, time 8 min to 10 min.

7. The preparation method according to claim 1, characterized in that, The fermentation broth of strain one is the fermentation broth of Bacillus atrophus CT-05; the fermentation broth of strain two is the fermentation broth of Bacillus motility CS-06. The OD of the fermentation broth of Bacillus CS-06 600 =2.5~3.0; OD of the fermentation broth of the Bacillus atrophus CT-05 600 =2.0~2.

5.

8. The double-layered, double-bacterial microcapsule bacterial agent prepared by the preparation method according to claim 1.

9. The double-layered, double-strain microcapsule bacterial agent according to claim 8, characterized in that, The bacterial load of the double-layered, double-strain microcapsule agent is 1×10⁻⁶. 9 cfu / g ~1.48×10 9 cfu / g.

10. The application of the double-layered, double-strain microcapsule inoculant according to claim 8 in crop seed germination and seedling growth, characterized in that, The double-layered, double-strain microcapsule inoculant is applied directly to crops.

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